30-kD Protein Research Articles

Glia Maturation Factor Beta: A Novel Neuro-Impairment Prediction Factor in Toxoplasmosis.

Toxoplasma gondii, a neurotropic protozoan, infects up one to third of the world population. The parasite can invade a wide variety of nucleated cells but preferably glial cells. Glia maturation factor β (GMFβ), a 17KD protein expressed at high levels in the central nervous system is predominantly related to neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Multiple sclerosis. We aimed to determine the expression level of GMFβ and its relation to other pro-inflammatory factors (IL33, SDF1, and CCL2) on T. gondii infected human neuroblastoma cell line. The human neuroblastoma (SK_NMC C535) cell line was infected by 5×106 (1:1 ratio). The supernatant was collected after cell lysis and centrifugation. Total RNA was extracted using the Yekta Tajhiz RNA extraction kit. cDNA was synthesized based on RevertAid First Strand cDNA Synthesis Kit manufacturer's protocol (Parstous, cDNA synthesis kit, Iran). The specificity of each primer pair (GMFβ, IL33, SDF1, and CCL2) was provided by NCBI BLAST. Gene expression level was measured using Real-Time PCR. All experiments were conducted at the Hamadan University of Medical Sciences, western Iran in 2022. The GMFβ increased significantly up to 1.35-fold (P=0.007). The increase in GMFβ expression in neuroblastoma cells was consistent with the increase in pro-inflammatory factors (CCL2 (0.47), IL33 (0.152) and, SDF1 (1.33)). GMFβ upregulation can be a novel indicator of the destruction of nerve cells.

Quercetin-Induced Enhancement of Nasal Epithelial Cells' Ability to Produce Clara Cell 10-kD Protein In Vitro and In Vivo.

Background: Quercetin, a polyphenolic flavonoid found in various plants and foods, is known to have antioxidant, antiviral and anticancer effects. Although quercetin is well known to exert anti-inflammatory and anti-allergic effects, the precise mechanisms by which quercetin favorably modifies the clinical status of allergic diseases, such as allergic rhinitis (AR), remain unclear. The present study examined whether quercetin could modulate the production of the endogenous anti-inflammatory molecule, Clara cell 10-kD protein (CC10), in vitro and in vivo. Methods: Human nasal epithelial cells (1 × 105 cells/mL) were stimulated with 20 ng/mL of tumor necrosis factor-alpha (TNF) in the presence of quercetin for 24 h. CC10 levels in culture supernatants were examined by ELISA. Sprague Dawley rats were sensitised with toluene 2,4-diisocyanate (TDI) by intranasal instillation of 10% TDI in ethyl acetate at a volume of 5.0 μL once daily for five days. This sensitisation procedure was repeated after an interval of two days. The rats were treated with different dosages of quercetin once daily for five days starting on the 5th day following the second sensitization. Nasal allergy-like symptoms induced by the bilateral application of 5.0 μL of 10% TDI were assessed by counting sneezing and nasal-rubbing behaviours for 10 min immediately after the TDI nasal challenge. The levels of CC10 in nasal lavage fluids obtained 6 h after TDI nasal challenge were examined using ELISA. Results: The treatment of cells with low doses of quercetin (<2.5 μM) scarcely affected TNF-induced CC10 production from nasal epithelial cells. However, the ability of nasal epithelial cells to produce CC10 after TNF stimulation significantly increased on treatment with quercetin doses (>5.0 μM). The oral administration of quercetin (>25 mg/kg) for five days significantly increased the CC10 content in nasal lavage fluids and attenuated the nasal symptoms induced by the TDI nasal challenge. Conclusions: Quercetin inhibits AR development by increasing the ability of nasal epithelial cells to produce CC10.

Abstract 3743: circPMS1 is a pro-metastatic circular RNA in melanoma

Abstract Deregulated gene expression is a major driver of melanoma metastasis, a process that results in the majority of melanoma-related deaths. Importantly, these alterations are not limited to protein-coding mRNAs, but also include noncoding RNAs. Despite many recent studies implicating circular RNAs (circRNAs) in cancer development, it is unknown if their deregulation contributes to melanoma metastasis. To identify circRNAs with putative roles in melanomagenesis, we performed RNA sequencing on a panel of melanoma and melanocyte cell lines. 21 differentially expressed circRNAs were validated by qPCR and Sanger sequencing of the backsplice junction. Further analyses of the ratio of the circular and cognate linear RNA transcripts and expression in a larger panel of cell lines identified circPMS1 as a circRNA that is upregulated in melanoma through enhanced backsplicing of the linear PMS1 mRNA. Using transposon-mediated delivery, we examined the effects of stable circPMS1 overexpression on melanoma cells. While proliferation and focus formation were not impacted, migration and invasion were enhanced by circPMS1. Moreover, tail vein injection of circPMS1 overexpressing melanoma cells increased lung metastasis. In addition to this transplant model, we generated the first genetically engineered mouse model of melanoma overexpressing a pro-tumorigenic circRNA. These mice are currently being analyzed. Mechanistically, we find that circularization is required for the pro-migratory effects of the circPMS1 overexpression construct. Additionally, circPMS1 harbors the canonical PMS1 start codon, a predicted ORF spanning over the backsplice junction, and a predicted IRES sequence. Indeed, inserting a flag-tag in the overexpression construct revealed circPMS1 to be translated into a truncated 25kD PMS1 protein as well as a 20kD protein likely arising from an in-frame downstream start codon. Importantly, these truncated circPMS1 proteins are endogenously expressed and increased in melanoma compared to melanocytes while the full-length PMS1 protein is evenly expressed. These findings suggest that circPMS1 promotes melanoma metastasis, at least in part, by coding for truncated circPMS1 proteins. Further work will establish the functions of the circPMS1-encoded proteins and their roles in driving melanoma progression and metastasis. Citation Format: Nicol Mecozzi, Olga Vera, Florian Karreth. circPMS1 is a pro-metastatic circular RNA in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3743.

Elarekibep (PRS-060/AZD1402), a new class of inhaled Anticalin medicine targeting IL-4Ra for type 2 endotype asthma

T2 endotype asthma is driven by IL-4 and IL-13 signaling via IL-4Ra, which is highly expressed on airway epithelium, airway smooth muscle and immunocytes in the respiratory mucosa, suggesting potential advantages of an inhalable antagonist. Lipocalin 1 (Lcn1), a 16kD protein abundant in human periciliary fluid, has a robust drug-like structure well-suited to protein engineering, but has never been used to make an inhaled "Anticalin" protein therapeutic. To re-engineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile. Lcn1 was systematically modified by directed protein mutagenesis yielding a high affinity, slowly dissociating, long-acting full antagonist of IL-4Ra designated 'PRS-060' with properties analogous to dupilumab, competitively antagonizing IL-4Ra dependent cell proliferation, mucus induction and eotaxin expression in vitro. As PRS-060 displayed exquisite specificity for human IL-4Ra, with no cross-reactivity to rodents or higher primates, we created a new triple-humanized mouse model substituting hIL-4Ra, hIL-4, and hIL-13 at their correct syntenic murine loci to model clinical dosing. Inhaled PRS-060 strongly suppressed acute allergic inflammation indices in triple humanized mice with a duration of action longer than its bulk clearance suggesting it may act locally in the lung. Lcn1 can be re-engineered into the Anticalin antagonist PRS-060, exemplifying a new class of inhaled topical, long-acting therapeutic with potential to treat T2 endotype asthma.

Brassica napus Drought-Induced 22-kD Protein (BnD22) Acts Simultaneously as a Cysteine Protease Inhibitor and Chlorophyll-Binding Protein.

Class II water-soluble chlorophyll proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins that bind with chlorophyll (Chl) and its derivatives. The physiological function of WSCPs is still unclear, but it is assumed to be involved in stress responses, which is likely related to their Chl-binding and protease inhibition (PI) activities. Yet, the dual function and simultaneous functionality of WSCPs must still be better understood. Here, the biochemical functions of Brassica napus drought-induced 22-kDa protein (BnD22), a major WSCP expressed in B. napus leaves, were investigated using recombinant hexahistidine-tagged protein. We showed that BnD22 inhibited cysteine proteases, such as papain, but not serine proteases. BnD22 was able to bind with Chla or Chlb to form tetrameric complexes. Unexpectedly, BnD22-Chl tetramer displays higher inhibition toward cysteine proteases, indicating (i) simultaneous Chl-binding and PI activities and (ii) Chl-dependent activation of PI activity of BnD22. Moreover, the photostability of BnD22-Chl tetramer was reduced upon binding with the protease. Using three-dimensional structural modeling and molecular docking, we revealed that Chl binding favors interaction between BnD22 and proteases. Despite its Chl-binding ability, the BnD22 was not detected in chloroplasts but rather in the endoplasmic reticulum and vacuole. In addition, the C-terminal extension peptide of BnD22, which cleaved off post-translationally in vivo, was not implicated in subcellular localization. Instead, it drastically promoted the expression, solubility and stability of the recombinant protein.

Acetyl-L-carnitine improves erectile function in bilateral cavernous nerve injury rats via promoting cavernous nerve regeneration.

Neurogenic erectile dysfunction (NED) caused by cavernous nerve (CN) injury is a typical complication after pelvic surgery, which lacks efficient treatments. Acetyl-L-carnitine (ALCAR) has been proven to promote nerve repair. To investigate the effect and potential mechanism of ALCAR in the treatment of NED. Thirty-two rats were randomly divided into bilateral CN injury (BCNI) group, BCNI + lower-dose ALCAR (50mg/kg/day) group, BCNI + higher-dose (100mg/kg/day) group, and sham-operated group. Erectile function was assessed 14 days after daily intraperitoneal injection of ALCAR or placebo. The penile tissues were gathered for subsequent histological and molecular biological analysis. Rat Schwann cell (SC) line S16 was used to verify the mechanism of ALCAR in vitro. We found that the erectile function of the rats in the BCNI group was severely impaired, which was improved considerably in both BCNI+ALCAR-LD and BCNI+ALCAR-HD groups. Also, we observed decreased smooth muscle and increased collagen content in the corpus cavernosum in the BCNI group. The expressions of fibrosis markers transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), and Smad 2/3 were significantly up-regulated in the BCNI group. The above changes were alleviated after the administration of lower and higher-dose ALCAR. Meanwhile, the nitric oxide (NO)/cyclic guanosine monophosphate pathway (cGMP) was promoted and the Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) pathway was inhibited in the corpus cavernosum of BCNI rats after ALCAR treatment, accompanied by increased neuronal nitric oxide synthase (nNOS) and down-regulated tyrosine hydroxylase (TH). In vitro, ALCAR promoted the migration and proliferation of SC and increased the expression of 22-kD peripheral myelin protein and nerve growth factor (NGF). Further, rats treated with ALCAR had high expression of ATF3 and S100 in the distal nerve tissues of the CN extrusion site. ALCAR could promote nerve repair and regeneration, inhibit penile fibrosis, and improve penile erection by promoting the proliferation and migration of SC and the secretion of NGF. Our study confirms that ALCAR may be a potential treatment strategy for NED.

The MAP Kinase Phosphatase MKP-1 Modulates Neurogenesis via Effects on BNIP3 and Autophagy.

Inherited and acquired defects in neurogenesis contribute to neurodevelopmental disorders, dysfunctional neural plasticity, and may underlie pathology in a range of neurodegenerative conditions. Mitogen-activated protein kinases (MAPKs) regulate the proliferation, survival, and differentiation of neural stem cells. While the balance between MAPKs and the family of MAPK dual-specificity phosphatases (DUSPs) regulates axon branching and synaptic plasticity, the specific role that DUSPs play in neurogenesis remains unexplored. In the current study, we asked whether the canonical DUSP, MAP Kinase Phosphatase-1 (MKP-1), influences neural stem cell differentiation and the extent to which DUSP-dependent autophagy is operational in this context. Under basal conditions, Mkp-1 knockout mice generated fewer doublecortin (DCX) positive neurons within the dentate gyrus (DG) characterized by the accumulation of LC3 puncta. Analyses of wild-type neural stem cell (NSC) differentiation in vitro revealed increased Mkp-1 mRNA expression during the initial 24-h period. Notably, Mkp-1 KO NSC differentiation produced fewer Tuj1-positive neurons and was associated with increased expression of the BCL2/adenovirus E1B 19-kD protein-interacting protein 3 (BNIP3) and levels of autophagy. Conversely, Bnip3 knockdown in differentiated Mkp-1 KO NSCs reduced levels of autophagy and increased neuronal yields. These results indicate that MKP-1 exerts a pro-neurogenic bias during a critical window in NSC differentiation by regulating BNIP3 and basal autophagy levels.

Associations Between Smoking and Systemic Lupus Erythematosus-Related Cytokines and Chemokines Among US Female Nurses.

Smoking has been associated with increased systemic lupus erythematosus (SLE) risk, but the biologic basis for this association is unknown. Our objective was to investigate whether women's smoking was positively associated with SLE-associated proinflammatory chemokines/cytokines (stem cell factor [SCF], B lymphocyte stimulator [BLyS], interferon-γ-inducible 10-kd protein [IP-10], and interferon-α); or negatively associated with antiinflammatory cytokine interleukin-10 (IL-10); and whether associations were modified by SLE-related autoantibody status. The Nurses' Health Study (NHS, n = 121,700) and NHSII (n = 116,429) cohorts were begun in 1976 and 1989. In 1988-1990 (NHS) and 1996-1999 (NHSII), ~25% of participants donated blood samples. We identified 1,177 women without SLE with banked samples, and we tested by enzyme-linked immunoassay (ELISA) for chemokines/cytokines as well as anti-Sm, anti-Ro/SSA, anti-La/SSB, and anti-RNP. Antinuclear antibodies (ANAs) were detected by HEp-2 cell indirect immunofluorescence, and anti-double-stranded DNA antibodies and were assayed by ELISA. Smoking was assessed until blood draw. Separate tobit and linear regression analyses, adjusted for potential confounders, modeled associations between smoking and log-transformed chemokine/cytokine concentrations. Analyses were stratified by autoantibody status. Effect estimates were calculated as ratios of geometric means expressed as percentage differences. Among the 15% of current/recent versus 85% of past/never smokers, BLyS levels were 8.7% higher (P < 0.01) and were 24% higher (P < 0.0001) among those who were ANA positive. Current/recent smokers had IL-10 concentrations 46% lower (P < 0.01) than past/never smokers; each 10 pack-years of smoking was associated with a 17% decrease in IL-10 level (P < 0.001). Smoking was not associated with IP-10 or SCF. Elevated BLyS and lower IL-10 levels among current smokers, particularly among ANA-positive women, may be involved in SLE pathogenesis.

Roles of Clara cell 10-kD protein and type 2 innate lymphoid cells in allergic rhinitis

ABSTRACT This study examined the potential roles of CC10 (Clara cell 10-kD protein) and ILC2s (type 2 innate lymphoid cells) in allergic rhinitis (AR). After ovalbumin was used to construct the AR model, microarray analysis was performed to reveal the key differentially expressed genes. The phenotypic changes of nasal mucosa were examined by H&E staining. Western blot analysis, qRT-PCR, ELISA and immunohistochemistry were performed to identify the levels of cytokines. The lineage markers (CD127 and CD117) of ILC2s were detected using immunofluorescence. The microarray analysis and qRT-PCR results showed that CC10 overexpression inhibited the expression of A20, BAFF, and IL-4 R in vivo. Also, CC10 overexpression was found to ameliorate the damage of nasal mucosa in AR mice. Investigations revealed that the ILC2s were activated in AR mice and AR patients with high levels of IgE, IgG1, IL-4, IL-5, IL-13, IL-25, and IL-33. Moreover, CD127+ was found to activate ILC2s. However, CC10 overexpression suppressed the activation of ILC2s. In conclusion, this research suggested that CC10 could suppress the activation of ILC2s to attenuate the damage of nasal mucosa and that CD127+ may be a biomarker of the activation of ILC2s in AR mice and AR patients.

New insights into the proteins interacting with the promoters of silkworm fibroin genes

The silkworm, Bombyx mori, is a silk-producing insect that has contributed greatly to human society. The silk gland of B. mori is a specialized organ responsible for synthesizing silk fibroin and sericin proteins under control of numerous factors. However, which factors are involved in direct silk protein synthesis regulation remains largely unknown. We report the identification of promoter-interacting proteins (PIPs) necessary for the regulation of genes encoding fibroin proteins, including the fibroin heavy chain (fibH), fibroin light chain (fibL), and a 25-kD polypeptide protein (P25). In the fourth larval molting stage (M4) or day 5 fifth-instar larvae (L5D5), a total of 198, 292, and 247 or 330, 305, and 460 proteins interacting with the promoter region of fibH, fibL and P25, respectively, were identified from the posterior silk gland by DNA pull-down combined with mass spectrometry. Many PIPs were particularly involved in ribosome- and metabolism-related pathways. Additionally, 135 and 212 proteins were identified as common PIPs of fibH, fibL and P25 in M4 and L5D5, respectively. Among all PIPs, we identified 31 potential transcription factors, such as Y-box and poly A-binding proteins, which play roles in nucleotide binding, ATP binding, or protein folding. This study provides the first in-depth profile of proteins interacting with fibroin gene promoters and contributes to a better understanding of silk protein synthesis regulation.

Urinary Neutrophil Gelatinase-associated Lipocalin: As an Early Marker for Diabetic Nephropathy

Background: Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Diagnostic marker to detect diabetic nephropathy at early stage is important as early intervention can slow the loss of kidney function and reduce adverse outcomes. Neutrophil gelatinase-associated lipocalin (NGAL) is a small (25-kd) protein that belongs to the lipocalin protein family. Urinary NGAL (u-NGAL) levels have been demonstrated to be a very promising marker especially in acute kidney disease. Objective: To measure urinary neutrophil gelatinase-associated lipocalin (NGAL) as an early marker for diagnosis of diabetic nephropathy in type 2 diabetes mellitus (T2DM) patients. Patients and Methods: The study was conducted on 84 subjects, from (18- 80 years old), 45 (53.5%) were males and 39 (46.4%) were females, with mean age (58.18 ± 13.98). They were collected from Endocrinology and Nephrology Clinic after a written consent was taken from the patients at Zagazig University Hospital. Results: Urinary NGAL is higher in diabetic patients compared with non-diabetic controls with high significant difference (p < 0.0001). In our result we found a positive correlation between NGAL and the duration of DM (r =0.279, p=0.027). There was a positive correlation between NGAL level and HbA1C, with significant difference (r =0.778, p =0.000) in patients groups. Our results showed a positive correlation between NGAL and FPG (r= 0.562) (p < 0.001) and 2hPPPG (r=0.687) (p < 0.001). There was a positive correlation between NGAL level and albumin-creatinine ratio (Alb/Cr) with significant difference (r =0.888, p < 0.001) in patients groups. Conclusion: There was a high significant difference in the level of urinary NGAL in diabetic patients than control subjects. Urine NGAL had a positive correlation with albumin-creatinine ratio, duration of DM, HbA1c.

Predictive Significance of Serum Interferon-Inducible Protein Score for Response to Treatment in Systemic Sclerosis-Related Interstitial Lung Disease.

Response to immunosuppression is highly variable in systemic sclerosis (SSc)-related interstitial lung disease (ILD). This study was undertaken to determine whether a composite serum interferon (IFN)-inducible protein score exhibits predictive significance for the response to immunosuppression in SSc-ILD. Serum samples collected in the Scleroderma Lung Study II, a randomized controlled trial of mycophenolate mofetil (MMF) versus cyclophosphamide (CYC), were examined. Results were validated in an independent observational cohort receiving active treatment. A composite score of 6 IFN-inducible proteins IFNγ-inducible 10-kd protein, monokine induced by IFNγ, monocyte chemotactic protein 2, β2 -microglobulin, tumor necrosis factor receptor type II, and macrophage inflammatory protein 3β) was calculated, and its predictive significance for longitudinal forced vital capacity percent predicted measurements was evaluated. Higher baseline IFN-inducible protein score predicted better response over 3 to 12 months in the MMF arm (point estimate = 0.41, P = 0.001) and CYC arm (point estimate = 0.91, P = 0.009). In contrast, higher baseline C-reactive protein (CRP) levels were predictive of a worse ILD course in both treatment arms. The predictive significance of the IFN-inducible protein score and CRP levels remained after adjustment for baseline demographic and clinical predictors. During the second year of treatment, in which patients in the CYC arm were switched to placebo, a higher IFN-inducible protein score at 12 months showed a trend toward predicting a worse ILD course (point estimate = -0.61, P = 0.068), while it remained predictive of better response to active immunosuppression in the MMF arm (point estimate = 0.28, P = 0.029). The predictive significance of baseline IFN-inducible protein score was replicated in the independent cohort (rs = 0.43, P = 0.028). A higher IFN-inducible protein score in SSc-ILD is predictive of better response to immunosuppression and could potentially be used to identify patients who may derive the most benefit from MMF or CYC.

Aryl hydrocarbon receptor promotes lipid droplet biogenesis and metabolic shift in respiratory Club cells.

Club cells are critical in maintaining airway integrity via, in part, secretion of immunomodulatory Club cell 10kd protein (CC10) and xenobiotic detoxification. Aryl hydrocarbon receptor (AhR) is important in xenobiotic metabolism, but its role in Club cell function is unclear. To this end, an AhR ligand, 6-formylindolo[3,2-b]carbazole (FICZ, 10nM) was found to induce, in a ligand and AhR-dependent manner, endoplasmic reticulum stress, phospholipid remodeling, free fatty acid and triglyceride synthesis, leading to perilipin 2-dependent lipid droplet (LD) biogenesis in a Club cell-like cell line, NL20. The increase in LDs was due, in part, to the blockade of adipose triglyceride lipase to LDs, while perilipin 5 facilitated LDs-mitochondria connection, leading to the breakdown of LDs via mitochondrial β-oxidation and acetyl-coA generation. In FICZ-treated cells, increased CC10 secretion and its intracellular association with LDs were noted. Administration of low (0.28ng), medium (1.42ng), and high (7.10ng) doses of FICZ in C57BL/6 mice significantly enhanced lipopolysaccharide (LPS, 0.1μg)-induced airway inflammation, mucin secretion, pro-inflammatory cytokines and CC10 in the bronchoalveolar lavage fluids, as compared to those seen in mice receiving LPS alone, suggesting the importance of AhR signaling in controlling the metabolic homeostasis and functions of Club cells.

The Modulated Expression of Mo5, a Human Myelomonocytic Plasma Membrane Antigen

Mo5 is a 94-kd protein antigen expressed by human peripheral blood monocytes, neutrophils, and by all bone marrow myeloperoxidase-positive myeloid precursors (promyelocytes, myelocytes, metamyelocytes, and bands). Mo5 is borne by the malignant cells of 74% of patients (N = 27) with acute monocytic leukemia (French-American-British [FAB] group M4, M5), and 50% of patients (N = 38) with acute granulocytic leukemia (FAB M1, M2, and M3). Nonmyeloid cells in peripheral blood and bone marrow are Mo5-negative. The surface expression of Mo5 by myeloid cells is modulated by several experimental conditions: Exposure of neutrophils to calcium ionophore (1 mumol/L, 37 degrees C, ten minutes) under conditions resulting in degranulation of specific granules produces a three- to fourfold increase in the plasma membrane density of Mo5 antigen. This suggests that, in neutrophils, there is an intracellular pool of Mo5 antigen, which may be associated with specific granules, and that granule-associated Mo5 is translocated to the plasma membrane upon degranulation. Conversely, incubation of monocytes, neutrophils, U-937, and Mo5-positive leukemia cells in medium containing anti-Mo5 monoclonal antibody results in a significant decrease in surface Mo5 expression. This loss of surface Mo5 is a rapid, temperature-dependent process (occurring within 30 minutes at 37 degrees C) that is produced by divalent anti-Mo5 immunoglobulin [F(ab')2 but not F(ab)]. After down-modulation, Mo5 is reexpressed by monocytes within 48 hours. Mo5 is therefore a human myelomonocytic differentiation antigen whose expression is modulated up or down depending on the nature of extracellular stimuli.

Regulation of monocyte migration by amphoterin (HMGB1)

AbstractAmphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-γ (IFN-γ)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5′-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium. (Blood. 2004;104:1174-1182)

Clinical significance of CC16 and IL-12 in bronchoalveolar lavage fluid of various stages of silicosis.

Identification of novel biomarkers for silicosis could be helpful for disease diagnosis and pathophysiological mechanism exploration. Our study aims to investigate the Clara cell secretory 16-kd protein (CC16) and interleukin-12 (IL-12) levels in bronchoalveolar lavage fluid (BALF) in patients with silicosis at various stages. The enzyme-linked immunosorbent assay (ELISA) double antibody sandwich method was used to determine the CC16 and IL-12 in BALF levels from 79 patients with silicosis of various stages. Correlation analyses were performed between CC16 and IL-12 levels, and lung function and cytological counts in patients with silicosis at various stages. There were no significant differences in the BALF recovery volume, the number of cells, percentages of macrophages and lymphocytes in the alveolar lavage fluid of patients with silicosis in different stages (P>0.05); the percentage of neutrophils in stage I and stage II were higher than the control group (P<0.05) with statistically significant differences. The CC16 in BALF levels in stage I and II silicosis groups lower than the control group and stage III silicosis group with statistically significant differences (P<0.05), whereas CC16 levels in stage II silicosis group are higher than the stage I group (P<0.01). The IL-12 levels were higher than the control group (P<0.01), and the IL-12 levels in stage II and III silicosis group was higher than the stage I silicosis group (P<0.01). With the increase of the length of dust service, the CC16 and IL-12 levels decreased and showed a positive correlation between these indexes (correlation coefficient r=0.559, P<0.01). In addition, CC16 silicosis patient levels were positively correlated with FEV1/FVC and VCmax (r=0.242, 0.257; both P<0.05); IL-12 levels were negatively correlated with FEV1 and VC max (r=-0.250, -0.483; both P<0.05). The CC16 and IL-12 levels may have a specific reference value for the early diagnosis of silicosis and the assessment of lung function.

Proteomic and ultrastructural analysis of Clara cell and type II alveolar epithelial cell-type lung cancer cells.

BackgroundCurrently, the identification of Clara cell and type II alveolar epithelial cell-type cancer cells requires electron microscopy, which is a time-consuming and expensive process involving a complicated tissue sampling procedure. The aim of this study was to identify unique biomarkers for Clara cell and type II alveolar epithelial cell-type lung cancer cells, respectively, with proteomic profiling.MethodsSix human lung adenocarcinoma cell lines (A549, NCI-H358, NCI-H1650, HCC827, NCI-H1395, and NCI-H1975) were investigated for their ultrastructural characteristics. The differentially expressed proteins (DEPs) were screened between NCI-H358 cells (Clara cell type) and A549 cells (type II alveolar epithelial cell type) using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS), and then they were validated by western blot. The protein expression levels of endoplasmic reticulum oxidoreductin 1-α (ERO1L), Clara cell 10-kD protein (CC10), and surfactant protein C (SP-C) were also determined in the six cell lines assayed.ResultsNCI-H358 cells featured Clara cell differentiation; A549, NCI-H1975, and HCC827 cells had characteristics of type II alveolar epithelial cells; and NCI-H1395 and NCI-H1650 cells had no differentiation characteristics of any lung adenocarcinoma cell type. Five DEPs including ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), cytokeratin 19 (CK19), cytokeratin 8 (CK8), ERO1L, and peroxiredoxin 2 (PRDX2) between NCI-H358 and A549 cells were identified for further validation; however, none of them showed suitability as an effective biomarker. Similarly, CC10 and SP-C were not appropriate biomarkers.ConclusionsCytological subtypes of NCI-H1975 and HCC827 cells were identified, but no promising biomarker was discovered in the present study.

Isolation of Staphylococcus xylosus from Urinary Tract Infection in Al-Diwaniya City, Iraq

One hundred diuresis samples were collected from patients with urinary tract infection from Al-Diwaniyah General Teaching Hospital in Al-Diwaniyah city for the period from January 1, 2019, to August 1, 2019, as 35 isolates belonging to the sex of streptococcus were isolated and with an isolation rate of 35% of the total 100 diuresis samples, The yield of 20 isolates to the negative staphylococcus aureus was tested for cocaine, and 5 isolates were of type S. xylosus, with an isolation rate of 25% of the total negative staphylococci for testing cocaine, and by 5% of the total of the reagent samples. These biochemical bacteria were diagnosed using the API Staph system. The clinical stages of the infection are in the subacute or chronic phase without clear symptoms. Streptococcus unproductive streptococcus resistant and anti-life antibody novobiocin, especially S. saprophyticus, are most common in urinary tract infections in immunocompetent patients, and mild infection in women is accompanied by dysuria. Young women between the ages of 16-16 years old to 42%. It affects the ureter in men. It can cause cystitis, pyelonephritis and glomerulonephritis. This group possesses a high affinity to epithelial cells, and this comes from the participation of different proteins such as a 160-Kd protein with Hemagglutinin and Fibriller protein, and it is symbolized by 95-Kd, which is found in 98% of clinical isolates.

The Arabidopsis Protein CGL20 Is Required for Plastid 50S Ribosome Biogenesis.

Biogenesis of plastid ribosomes is facilitated by auxiliary factors that process and modify ribosomal RNAs (rRNAs) or are involved in ribosome assembly. In comparison with their bacterial and mitochondrial counterparts, the biogenesis of plastid ribosomes is less well understood, and few auxiliary factors have been described so far. In this study, we report the functional characterization of CONSERVED ONLY IN THE GREEN LINEAGE20 (CGL20) in Arabidopsis (Arabidopsis thaliana; AtCGL20), which is a Pro-rich, ∼10-kD protein that is targeted to mitochondria and chloroplasts. In Arabidopsis, CGL20 is encoded by segmentally duplicated genes of high sequence similarity (AtCGL20A and AtCGL20B). Inactivation of these genes in the atcgl20ab mutant led to a visible virescent phenotype and growth arrest at low temperature. The chloroplast proteome, pigment composition, and photosynthetic performance were significantly affected in atcgl20ab mutants. Loss of AtCGL20 impaired plastid translation, perturbing the formation of a hidden break in the 23S rRNA and causing abnormal accumulation of 50S ribosomal subunits in the high-molecular-mass fraction of chloroplast stromal extracts. Moreover, AtCGL20A-eGFP fusion proteins comigrated with 50S ribosomal subunits in Suc density gradients, even after RNase treatment of stromal extracts. Therefore, we propose that AtCGL20 participates in the late stages of the biogenesis of 50S ribosomal subunits in plastids, a role that presumably evolved in the green lineage as a consequence of structural divergence of plastid ribosomes.

Prognostic Significance of Reduced BCAS4 expression in Bone Marrow Cells of Patients with Myelodysplastic Syndromes

Deletion of long arm of chromosome 20 (del(20q)) is commonly observed in myelodysplastic syndromes (MDS). Reduced expression of genes located within the common deleted region (CDR) of del(20q) due to haploinsufficiency may play a role in molecular pathogenesis of MDS. In the previous study, we examined expression of genes located within the CDR which we determined using array-CGH, in bone marrow mononuclear cells in MDS patients with or without del(20q), indicating that BCAS4 expression was significantly reduced in bone marrow cells in MDS patients with or without del(20q). The BCAS4 gene, which was identified as a fusion transcript expressed in MCF7 cells, encodes 23kD protein. Although function of BCAS4 protein remains unclear, it could be a member of "cappuccino" family, which belong to lysosome-related organelles. Abnormality of genes encoding lysosome-related organelles cause variety of congenital disorders, including the Hermansky-Pudlak syndromes, which is characterized by oculocutaneous albinism and bleeding tendency due to platelet dysfunction as a result of lysosome abnormalities. In the present study we investigated clinical implication of BCAS4 expression level in MDS patients. Mononuclear cells separated from bone marrow samples taken at the time of MDS diagnosis were used for analysis. Written informed consent was obtained from patients before study. To analyze BCAS4 expression, quantitative RT-PCR was performed using cDNA from mononuclear cells as template by the TaqMan probe method (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems). Samples from 103 MDS patients, 64 males and 39 females with median age of 67 years (range: 20-91 years), with (n=14) or without (n=89) del(20q), were examined in the present study. Patients were classified as RCUD (n=12), RCMD (n=55), RARS (n=9), RAEB-1 (n=10), and RAEB-2 (n=13), according to WHO 2008 classification, and in RAEB-T (n=4) according to FAB classification. They also were categorized in four IPSS risk groups, low risk (n=30), intermediate-1 risk (n=46), intermediate-2 risk (n=18), and high risk (n=9). There was no significant difference in relative BCAS4 expression level between patients with del(20q) and those without del(20q), and among WHO subtypes. Higher IPSS risk groups (INT-2 and High) showed trend in association with reduced BCAS4 expression compared with lower IPSS risk groups (Low and INT-1) (P=0.104). We analyzed impact of BCAS4 expression on overall survival (OS). Based on BCAS4 expression level, 103 patients were divided into four groups, highest (Q1), intermediate (Q2, Q3), and lowest (Q4) quartiles. The Kaplan-Meier analysis demonstrated that Q4 showed significantly worse OS compared with remaining quartiles (Q1-Q3) (log-rank test, P=0.0031). The estimated 2-year OS rates in Q1-3 group and Q4 group were 75.1% and 48.9%, respectively. According to the COX proportional hazards model, univariate analysis showed lower BCAS4 expression (Q4 vs Q1-Q3) was associated with worse OS (hazard ratio 3.43, 95%CI 1.89-6.11, P=0.0001) as well as older age (65 years or older vs less than 65 years), and higher IPSS risk groups (INT-2 and High vs Low and INT-1). Multivariate analysis indicated that lower BCAS4 expression showed trend for association with worse OS (hazard ratio 1.90, 95%CI 0.96-3.64, P=0.0651) by analyzing with two variables (older age and higher IPSS groups). Next, we investigated whether OS is predicted by combination of three variables, BCAS4 expression level, IPSS risk groups, and age at diagnosis. We defined lower BCAS4 expression (Q4), higher IPSS (INT-2 and High), and older age (65 years or older), as risk factors. The Kaplan-Meier analysis showed that survival curves were well separated according to number of risk factors (0, 1, and 2 or more) (P&lt;0.0001). The estimated 1-year, 2-year, and 5-year survival rates were 100%, 100%, and 86.5% in patients without risk factor, 75%, 70.2%, and 51.7% in patients with one risk factor, and 54%, 34.3%, and 11.4% in patients with two or more risk factors. The present study demonstrated that reduced BCAS4 expression is associated with inferior clinical outcome, indicating that BCAS4 expression level could be a useful prognostic marker in MDS, especially by combination with IPSS risk and patients age at diagnosis. Disclosures Tanaka: Bristol-Myers Squibb: Research Funding.