Reverse Transcriptase Chain Reaction Research Articles

A Novel Approach to detect COVID-19 from chest X-ray images using CNN

In light of the present COVID-19 pandemic, it is important to consider the worth of human life, prosperity, and quality of life while also realizing that it is difficult to restrict case spread and mortality. One of the most difficult challenges for practitioners is identifying individuals who are COVID19-infected and isolating patients to stop COVID transmission. Therefore, identifying the covid19 infection is important. For the detection of COVID-19, a 4-6-hour reverse transcriptase chain reaction is used. Chest X-rays provide us with a different method for detecting Coronavirus early in the disease phase. We detected properties from chest X-ray scans and divided them into three categories with VGG16 as well as ResNet50 deep learning algorithms: COVID-19, normal, and viral pneumonia. To test the model's accuracy in specialized cases, we injected them with 15153 scans. The average COVID-19 case detection accuracy for the ResNet50 model is 91.39%, compared to 89.34% for the VGG16 model. However, a larger dataset is required when using deep learning to identify COVID-19. It accurately detects situations, which is the desired outcome.

Investigation of Group A Rotavirus G10, G12 Genotypes Emerging in Patients with Acute Gastroenteritis in a Tertiary Care Hospital

Rotaviruses are the most common cause of viral gastroenteritis with the highest mortality and morbidity rates in children aged 0-5 years. The aim of this study was to determine the frequency of rotavirus infection in patients whose stool samples were sent to microbiology laboratory to investigate the etiology of diarrhea, to investigate the rotavirus genotypes that are common in our region and G10, G12 genotypes that have recently become common in the world. Fecal samples of 476 patients aged between 0-92 years who applied between November 2016 and February 2018 were studied via immunochromatographic rapid test and enzyme-linked immunosorbent assay (ELISA) methods. ELISA positive samples were studied by nested reverse transcriptase chain reaction (RT-PCR) and genotyped by agarose gel electrophoresis. Rotavirus was found positive in 18.3% and 17% of stool samples by immunochromatographic test and ELISA, respectively. All ELISA positive samples were also detected as positive by RT-PCR. 18.5% of female patients and 15.7% of male patients were found to be positive and rotavirus positivity was not statistically significant between genders. The frequency of rotavirus in different age groups was 23.5% (6-12 years), 17.3% (13-24 months) and 16% (25-36 months). It was determined that rotavirus cases were most common in the spring. G1, G2, G3, G4, G9, G10, and G12 were detected in 37%, 7.4%, 16.1%, 6.2%, 9.9%, 2.5%, 26% of the samples, respectively. G12 was the most common genotype after G1. The most common G and P genotype combination was G1P[8] (17.2%). This was followed by G12P[8] (11.11%) and G3P[8] (11.11%). P[8] (53%) was found to be the dominant P genotype. In this study, it was observed that rotavirus, which is the cause of childhood diarrhea, can also be encountered in advanced ages and even new genotypes that infect humans worldwide may also be the causative agents. Therefore, we concluded that it is important to investigate new genotypes such as G10 and G12 in molecular epidemiological studies.

Adaptation strategy for Jambal Catfish (Pangasius djambal) to stress the aquatic environment

Oxygen is a major element in the metabolic process of cells in an aquatic animal’s body. Water oxygen solubility fluctuations will affect the metabolic process. Acute oxygen deficiency conditions can cause physiological changes in the body of fish. The research aims to analyze morphometric characters and genetic characters as environmental pressure. Test fish used in this research are 24.21+ 0.96 cm jambal fish (P.djambal) with 223.42 + 31.23 g. The treatment provided is an environment with different oxygen solubility, i.e., low oxygen (hypoxia), high oxygen (normoxia), and high fluctuating conditions naturally as control. The observed variables include the main parameters: the extent of gills filaments, the hemoglobin levels in the blood, and the expression of Lactate Dehydrogenase (LDH) genes. Supporter parameters are growth and mortality as well as water quality parameters. LDH gene expression analysis on jambal catfish (P.djambal) using Reverse Transcriptase Chain Reaction (RT-PCR) method. An analysis shows the morphometric characters of jambal catfish living in a hypoxia environment (P < 0.05). The genetic character of jambal catfish (P.djambal) shows that the hypoxic environment of gene expression LDH is better than fish living in a normoxia and natural environment. Gene LDH indicates the strategy of jambal catfish (P.djambal) adaptation used in the selection program to produce jambal catfish (P.djambal) resistant to the hypoxic environment.

Crustacean hyperglycemic hormone of Portunus trituberculatus: evidence of alternative splicing and potential roles in osmoregulation.

The crustacean hyperglycemic hormone (CHH) gene of Portunus trituberculatus (Pt-CHH) consists of four exons and three introns spanning 3849bp in size and generating two mature mRNA, Pt-CHH1, and Pt-CHH2. The primary gene transcript produces a cDNA encoding for the putative Pt-CHH2 from exons 1, 2, 3, and 4 and an alternative transcript encodes for a putative Pt-CHH1 peptide from exons 1, 2, and 4. A promoter fragment of about 3kb was obtained by genomic walking. The tissue-specific expression pattern is examined by reverse transcriptase chain reaction, and the results show that Pt-CHH1 is detected in the eyestalk, brain, muscle, and blood. However, Pt-CHH2 is detected in the ganglia thoracalis and gill. The results indicate that the expression of Pt-CHH2 in the gill might suggest a potential role in osmoregulation. The Pt-CHH transcript level in the gill increases when the crab is exposed to low salinity. The injection of dsRNA for Pt-CHH causes a significant reduction in Pt-CHH2 transcript level and the activity of Na+/K+-ATPase, and carbonic anhydrase (CA) show a serious decrease. In conclusion, this study provides molecular evidence to support the osmoregulatory function of Pt-CHH2.

Molecular pathotyping and phylogenesis of the first Newcastle disease virus strain isolated from backyard chickens in Qatar.

The first Newcastle disease virus (NDV) field strain (NDV-QC08), previously isolated and identified in Qatar, was further subjected to molecular characterization and phylogenic analysis at passage levels two. Initial amplification and detection of the complementary DNA (cDNA) fragment of the NDV RNA amnioallantoic fluid (AAF) extract demonstrated the positive virus identity using real-time reverse transcriptase chain reaction (RRT-PCR). Partial sequence of the totally amplified 216 bp of the functional cleavage region encoding the F gene retrieved (112)R-R-Q-K-R-F(117) amino acid motif indicating the velogenic nature of the strain, and confirming the previously investigated in-vivo pathotyping. Phylogenic analysis and genotypic clustering studies classified the strain under NDV subgenotype VIId.

Delayed ethanol elimination and enhanced susceptibility to ethanol-induced hepatosteatosis after liver resection.

To investigate ethanol-induced hepatic steatosis after liver resection and the mechanisms behind it. First, the preliminary examination was performed on 6 sham-operated (Sham) and 30 partial hepatectomy (PH) male Wistar rats (8-wk-old) to evaluate the recovery of the liver weight and liver function after liver resection. PH rats were sacrificed at the indicated time points (4, 8, and 12 h; 1, 3, and 7 d) after PH. Second, the time point for the beginning of the chronic ethanol exposure (1 wk after sham- or PH-operation) was determined based on the results of the preliminary examination. Finally, pair-feeding was performed with a controlled diet or with a 5-g/dL ethanol liquid diet for 28 d in another 35 age-matched male Wistar rats with a one-week recovery after undergoing a sham- (n = 15) or PH-operation (n = 20) to evaluate the ethanol-induced liver injury after liver resection. Hepatic steatosis, liver function, fatty acid synthase (Fas) gene expression level, the expression of lipid metabolism-associated enzyme regulator genes [sterol regulatory element binding protein (Srebp)-1 and peroxisome proliferator-activated receptor (Ppar)-α], the mediators that alter lipid metabolism [plasminogen activator (Pai)-1 gene expression level and tumor necrosis factor (Tnf)-α production], and hepatic class-1 alcohol dehydrogenase (Adh1)-associated ethanol elimination were investigated in the 4 groups based on histological, immunohistochemical, biochemical, Western blotting, reverse transcriptase chain reaction, and blood ethanol concentration analyses. The relevant gene expression levels, liver weight, and liver function were assessed before and 1 wk after surgery to determine the subject's recovery from the liver resection using the rats that had been subjected to the preliminary examination. In the PH rats, ethanol induced marked hepatic steatosis with impaired liver functioning, as evidenced by the accumulation of fatty droplets within the hepatocytes, the higher increases in their hepatic triglyceride and blood alanine aminotransferase and blood aspartate aminotransferase levels after the 28-d pair-feeding period. The Sham-ethanol rats, not the PH-ethanol rats, demonstrated the up-regulation of Srebp-1 and the down-regulation of Ppar-α mRNA expression levels after the 28-d pair-feeding period. The 28-d ethanol administration induced the up-regulation of Pai-1 gene expression level and an overproduction of TNF-α in the Sham and the PH rats; however, the effect was more significant in the PH rats. The PH-ethanol rats (n = 4) showed higher residual blood ethanol concentrations than did the Sham-ethanol rats (n = 6) after a 5-h fast (0.66 ± 0.4 mg/mL vs 0.2 ± 0.1 mg/mL, P < 0.05); these effects manifested without up-regulation of Adh1 gene expression, which was present in the Sham-ethanol group after the 28-d pair-feeding period. One week after the liver resection, the liver weight, function, the gene expression levels of Fas, Srebp-1, Ppar-α, Pai-1 and Tnf-α recovered; however, the Adh1 gene expression did not recover in rats. Desensitization to post-hepatectomy ethanol treatment and slow recovery from PH in Adh1 gene expression enhanced the susceptibility to ethanol-induced hepatic steatosis after PH in rats.

APPLICATION OF THE REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION FOR DETECTION AND IDENTIFICATION OF THE INFECTIOUS PANCREATIC NECROSIS VIRUS OF RAINBOW TROUT (ONCORHYNCHUS MYKISS)

Purpose: To develop a diagnostic test system for detection and identification of infectious pancreatic necrosis virus of trout based on the reverse transcriptase polymerase chain reaction (RT-PCR). Materials and methods: Viruses: infectious pancreatic necrosis virus, reference strain – Аb (8.0 lg TCID/cm3); Ukrainian isolates – VF-11(6.8 lg TCID/ cm3), VF-08 (6.2 lg TCID/cm3), viral hemorrhagic septicemia virus of trout (VHSV), strain «DH4p101» [AY546581] (8.0 lg TCID/cm3). Finite cell line of fish: FHM (fathead minnow peduncle) and RTG (rainbow trout gonads). For detection and identification of infectious pancreatic necrosis virus of trout, two pairs of oligonucleotide primers were used. For search and analysis of gene homology of VP2 and NS proteins of infectious pancreatic necrosis virus, we used the BLAST 2.0 software of the National Center for Biotechnological Information (NCBI), USA. Constructing and matching of the primers were performed with the use of the «Vector NTI Advanced v.11» software package (Invitrogen, USA). Findings: For selecting effecting primers for PCR, we performed comparative study of nucleotide sequences of IPNV isolate genome of different genotypes. As a result, for the work we synthesized oligonucleotide primers, which flank VP2 protein gene sequence. The size of the amplified DNA fragment is 630 p.n. Additionally, we used oligonucleotide primers specific for the nucleoprotein gene (NS). The size of the amplified DNA fragment is 204 p.n. When checking the specificity of the polymerase chain reaction with matched primers as a matrix, we used RNA samples of the reference IPNV strains and isolates isolated from rainbow trout fingerlings from different fish farms of Ukraine. Conclusions: The developed diagnostic test system based on the reverse transcriptase chain reaction (RT-PCR) is highly specific and sensitive for detection and identification of IPNV virus in the materials of various origins.

Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived autoserum

ObjectiveOsteogenesis in the bone defect at the site of an alveolar cleft is important to enable patients with cleft lip and palate to acquire dental articulation. The presence of umbilical cord-derived mesenchymal stem cells has been reported. In this study, we used autoserum derived from the umbilical cord blood (UCB) of neonates in an attempt to examine the osteoblastic differentiation potential of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) in nude mice. Materials and methodsUCB, hydroxyapatite, and rhBMP were used as the supply source of autoserum, scaffold, and osteoinductive growth factor, respectively. MSCs, obtained from Wharton's jelly and cultured for 3–4weeks to induce their differentiation into osteoblasts, were implanted subcutaneously into the dorsum of male nude mice for 6weeks before the assessment by real-time reverse transcriptase chain reaction of osteoblast marker expression. ResultsUCB-derived autoserum was a viable source for the culture and implantation of UC-MSCs. The osteoblastic differentiation potential of UC-MSCs was demonstrated in nude mice by performing immunohistochemical staining and by the presence of osteoblast marker expression. ConclusionsOur results confirm the osteogenic potential of UC-MSCs and provide basic evidence for the realization of regenerative medicine using autologous tissues.

Expression and biological-clinical significance of hTR, hTERT and CKS2 in washing fluids of patients with bladder cancer

Backgroundat present, pathogenesis of bladder cancer (BC) has not been fully elucidated. Aim of this study is to investigate the role of human telomerase RNA (hTR), human telomerase reverse transcriptase (hTERT) and CDC28 protein kinase regulatory subunit 2 (CKS2) in bladder carcinogenesis and their possible clinical significance;Methodsthe transcript levels of hTR, hTERT and CKS2 were quantified by Real time reverse transcriptase chain reaction in exfoliated cells from bladder washings of 36 patients with BC and 58 controls. The statistical significance of differences between BC bearing patients and control groups, in the general as well as in the stratified analysis (superficial or invasive BC), was assessed by Student's t test. Non parametric Receiver Operating Characteristics analysis (ROC) was performed to ascertain the accuracy of study variables to discriminate between BC and controls. The clinical value of concomitant examination of hTR, hTERT and CKS2 was evaluated by logistic regression analysis;Resultsa significant decrease in hTR and a significant increase in hTERT or CKS2 gene expression were found between BC bearing patients and controls, as well as in the subgroups analysis. The area under the curve (AUC) indicated an average discrimination power for the three genes, both in the general and subgroups analysis, when singularly considered. The ability to significantly discriminate between superficial and invasive BC was observed only for hTR transcript levels. A combined model including hTR and CKS2 was the best one in BC diagnosis;Conclusionsour results, obtained from a sample set particularly rich of exfoliated cells, provide further molecular evidence on the involvement of hTR, hTERT and CKS2 gene expression in BC carcinogenesis. In particular, while hTERT and CKS2 gene expression seems to have a major involvement in the early stages of the disease, hTR gene expression, seems to be more involved in progression. In addition, our findings suggest that the studied genes have a clinical role in discriminating between BC and controls in the general as well as in the stratified analysis, when singularly considered. A combined model improved over the single marker BC diagnosis.

Effects of short chain fatty acids on effector mechanisms of neutrophils

Short chain fatty acids (SCFAs) are metabolic by products of anaerobic bacteria fermentation. These fatty acids, despite being an important fuel for colonocytes, are also modulators of leukocyte function. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate, and butyrate) on function of neutrophils, and the possible mechanisms involved. Neutrophils obtained from rats by intraperitoneal lavage 4 h after injection of oyster glycogen solution (1%) were treated with non toxic concentrations of the fatty acids. After that, the following measurements were performed: phagocytosis and destruction of Candida albicans, production of ROS (O(2)(*-), H(2)O(2), and HOCl) and degranulation. Gene expression (p47(phox) and p22(phox)) and protein phosphorylation (p47(phox)) were analyzed by real time reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. Butyrate inhibited phagocytosis and killing of C. albicans. This SCFA also had an inhibitory effect on production of O(2)(*-), H(2)O(2), and HOCl by neutrophils stimulated with PMA or fMLP. This effect of butyrate was not caused by modulation of expression of NADPH oxidase subunits (p47(phox) and p22(phox)) but it was in part due to reduced levels of p47(phox) phosphorylation and an increase in the concentration of cyclic AMP. Acetate increased the production of O(2)(*-) and H(2)O(2) in the absence of stimuli but had no effect on phagocytosis and killing of C. albicans. Propionate had no effect on the parameters studied. These results suggest that butyrate can modulate neutrophil function and thus could be important in inflammatory neutrophil-associated diseases.

Expression of Growth factors during Osseointegration of Titanium Implant ; TGF-beta, IGF-I, BMP2, BMP4

Osseointegration is a result of bone formation and bone regeneration process, which take place at the interface between bone and implant and biologic determinants such as cytokine, growth factors, bone matrix proteins play an important role in osseointegration. The purpose of this study is to compare the expressoin of TGF-beta, IGF-I, BMP2, BMP4 during osseointegration. We designed an experimental group which was inserted with a RBM surface titanium implants and machined surface, and compared with a control group which had a simple bone cavity and normal bone. Titanium implants were placed into tibia of 8 rabbits. We compared the expression of TGF-beta, IGF-I, BMP2, BMP4 using RT_PCR (reverse transcriptase chain reaction)analysis in day 3,7,14 and 28 of implant insertion. According to the results, growth factors of experimental groups were more expressed than control groups. Among experimental groups, expression of TGF-beta, IGF-I, BMP4 of BMP group had tedency to increase more at 14th, 28th days than Machined surface group. Therefore, our results suggest that TGF-beta, IGF-I, BMP4 are expressed within the bone around the implant and more increased around rough surface implants while osseointegration occurs after dental implant insertion.

Aquaporin 1 is expressed in the human endometrium during normal cycle and increases after mifepristone treatment

Aquaporin-1 (AQP1) is involved in the angiogenesis and structural modifications of microvessels and possibly also in the pathogenesis of idiopathic menhorrhagia, where a reduced AQP1 expression is seen in the endometrium. Mifepristone treatment induces reduced menstrual bleeding and amenorrhea and also has a direct effect on endometrial arterioles. Administered with gestagen-only contraceptive methods, antiprogestins improve the bleeding pattern. The objective of this study was to evaluate the AQP1 expression in endometrial blood vessels during normal cycle and after mifepristone treatment. Localization and expression of AQP1 was determined using immunohistochemistry and reverse transcriptase chain reaction (RT-PCR) in 43 biopsies from human endometrium taken during a normal cycle and after mifepristone treatment. AQP1 expression in human endometrial vessels is not cycle dependent and is stronger in capillaries and arteries than in veins. After mifepristone treatment the staining intensity was increased, but not the number of stained vessels. The presence of AQP1 was also confirmed using RT-PCR. The changes in AQP1 expression could contribute to the reduced bleeding seen following mifepristone treatment and could be an effect of either antagonizing progesterone or cortisol.

MicroRNA profiles of malignantly transformed cells induced by anti-benzo-a-pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide

To screen microRNA (miRNA) profiles of malignantly transformed cells induced by anti-benzo-a-pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide (BPDE) and to look for miRNAs which is expressed differently between malignantly transformed cells and normal human bronchial epithelial cells 16HBE. Experimental group was the malignantly transformed 16HBE which was induced by cultured with final concentration 2.0 micromol/L of BPDE which was dissolved in dimethyl sulphoxide. The control group was 16HBE that was cultured with minimal essential medium including dimethyl sulphoxide. 327 miR-NAs were tested be-tween those two groups with miRNA microarray analysis. MiR-10a that was down expressed and miR-320 that was overexpressed were selected to be validated by miRNA specific quantitative real-time reverse transcriptase chain reaction (miR qRT-PCR). 327 human miRNAs were tested with miRNA microarray analysis. 55 miRNAs were found expressing differently between those two groups and of which 46 were overexpressed and 9 were down expressed. Some data were validated by quantitative RT-PCR. miRNAs expressed significantly between malignantly transformed 16HBE and normal cells and this helps us look for unique miRNAs of malignantly transformed cells induced by BPDE, but there should have more sufficient evidences to prove their functions in malignant cells.

IL-23 P19 Expression Induced by IL-17 and IL-1β in Rheumatoid Arthritis Synovial Mononuclear Cells

Interleukin-23 (IL-23) is a novel pro-inflammatory cytokine which has been implicated to play a pathogenic role in rheumatoid arthritis (RA). This study was undertaken to investigate the IL-23 inductive activity of the proinflammatory cytokine IL-17, IL-1β and tumor necrosis factor (TNF-α) in RA synovial fluid mononuclear cells (SFMC). Expression of IL-23p19, IL-17, IL-1β and TNF-α in joint was examined by immunohistochemistry (IHC) of patients with RA and osteoarthritis (OA). The effects of IL-17 and IL-1β on expression of IL-23p19 in human SFMC from RA patients were determined by reverse transcriptase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). IL-23p19 was expressed in the RA fibroblast like synoviocyte (FLS), but not from OA FLS. Similar to the protein expression, IL-23p19 mRNA could be detected by RT-PCR in RA SFMC. IL-17 and IL-1β could induce RA SFMC to produce the IL-23p19. The effects of IL-17 were much stronger than IL-1β or TNF-α. These responses were observed in a doseresponsive manner. In addition, IL-17 or IL-1β neutralizing antibody down-regulated the expression of IL-23p19 induced by LPS in RA-SFMC. Our results demonstrate that IL-23p19 is overexpressed in RA synovium and IL-17 and IL-1β appears to upregulate the expression of IL-23p19 in RA-SFMC.

Glutamine supplementation prevents exercise‐induced neutrophil apoptosis and reduces p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression

We have previously shown that a single session of exercise induces DNA fragmentation, mitochondrial membrane depolarization, increases expression of pro-apoptotic genes (bax and bcl-xS) and decreases expression of anti-apoptotic genes (bcl-xL) in rat neutrophils. Glutamine supplementation had a protective effect in the apoptosis induced by a single session of exercise. The mechanism involved in the effect of single session of exercise to induce apoptosis was investigated by measuring expression of p53 and caspase 3 and phosphorylation of p38 mitogen-activated protein kinases (MAPK) and cJun NH(2)-terminal kinase (JNK) in neutrophils from rats supplemented or not with glutamine. Exercise was carried out on a treadmill for 1 h and the rats were killed by decapitation. Neutrophils were obtained by intraperitoneal (i.p.) lavage with PBS, 4 h after injection of oyster glycogen solution. Glutamine supplementation (1g per Kg b.w.) was given by gavage 1 h before the exercise session. Gene expression and protein phosphorylation were then analyzed by reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. A single session of exercise increased p38 MAPK and JNK phosphorylation and p53 and caspase 3 expression. Glutamine supplementation partially prevented the increase in p38 MAPK and JNK phosphorylation and p53 expression, and fully abolished the increase in caspase 3 expression. Thus, neutrophil apoptosis induced by a single session of exercise is accompanied by increased p53 and caspase 3 expression and p38 MAPK and JNK phosphorylation. Glutamine supplementation prevents these effects of exercise and reduces apoptosis.

Down-regulation of DMBT1 gene expression in human oral squamous cell carcinoma

Deleted in malignant brain tumors 1 (DMBT1) gene was recently isolated on chromosome 10q25.3-26.1 and has been proposed as a putative candidate tumor suppressor for brain, esophageal, gastric, colorectal, and lung cancer. However, little is known about the association of DMBT1 with oral squamous cell carcinoma (OSCC). To study the role of DMBT1 gene in OSCC oncogenesis, we examined 9 OSCC derived cell lines and 45 primary OSCC tissue specimens with respective normal tissues. Semi-quantitative reverse transcriptase chain reaction (RT-PCR) analysis revealed down-regulation or deletion of DMBT1 expression in all of the 9 cell lines and in 18 (40%) of 45 primary OSCC tissues. Additionally, 57 OSCC tissue specimens were examined by immunohistochemical staining of protein showing down-regulation of DMBT1 protein in 31 (56.1%) of the 57 primary OSCC tissue specimens. To assess restoration of DMBT1 expression by demethylation of promoter region, the 9 cell lines were treated with 5-aza-2-deoxycytidine (5-Aza-C), one of the DNA demethylating agents. Six (66.7%) of 9 cell lines demonstrated restoration of DMBT1 expression after 5-Aza-C treatment. These results suggest that DMBT1 gene is involved in OSCC oncogenesis and/or progression and that methylation of promoter region is one of the important mechanisms suppressing the DMBT1 gene expression.

Expression and regulation of the prointerleukin-1alpha processing enzymes calpain I and II in the rat testis.

Interleukin-1alpha (IL-1alpha) is constitutively expressed in an age- and stage-dependent manner by rat Sertoli cells. However, the mechanism of regulation of IL-1alpha is unclear in testis. We studied this regulation at the level of the enzyme calpain, a potential regulator that cleaves 32 kDa proIL-1alpha to produce mature 17 kDa IL-1alpha. Both calpain I and II were found to cleave recombinant rat testis 32proIL-1alpha in vitro. A temporary age-related increase in messenger RNA (mRNA) levels of calpain I was found in testis of 20- and 25-day-old rats, coinciding with important events of spermatogenesis and a gradual increase in IL-1alpha, while calpain II expression was constant. In response to lipopolysaccharide (LPS), calpain I protein levels were down-regulated in the seminiferous tubules, while calpain II was less affected. By contrast, the liver after LPS treatment showed up-regulated calpain I and II immunoreactive protein and reverse transcriptase chain reaction (RT-PCR) signal. Depleting Leydig cells by ethane 1,2-dimethane sulphonate treatment resulted in down-regulated calpain I mRNA and protein expression, whereas calpain II remained unchanged. In summary, there is a differential expression of calpain I and II under pathological conditions induced either by endotoxin stimuli or Leydig cell depletion, which may produce a differential effect on IL-1alpha processing.

Role of aquaporin-4 water channel in the development and integrity of the blood-brain barrier.

In this study, we have investigated the expression of aquaporin 4 during blood-brain barrier development in the optic tectum of chick embryos and newly hatched chicks, by means of western-blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and freeze-fracture and high-resolution immunogold electron microscopy. In the optic tecta of day-14 embryos, western blot analysis revealed an approx. 30 kDa band, immunoreactive for aquaporin-4, which was increased in day-20 embryos and in chicks. Semi-quantitative reverse transcriptase chain reaction experiments showed that there was already a high level of aquaporin-4 mRNA in day-9 embryos as well as in the subsequent stages and in newly hatched chicks. Immunohistochemically, reactivity for aquaporin-4 was detected in the optic tectum of day-14 embryos; similar results were obtained in telencephalon and cerebellum. Ultrastructurally, the microvessels of the tectum showed immunoreactivity for aquaporin-4 on the astroglial endfeet, which discontinuously surrounded endothelial cells joined by immature tight junctions. In the tectum, telencephalon and cerebellum of 20-day embryos and chicks, aquaporin-4 strongly labeled the ependymal cells and the subpial glial membranes, as well as the bodies and processes of astroglial cells. A continuous aquaporin-4 staining was found around the microvessel endothelial cells, which were sealed off from one another by extensive tight junctions. A complete astrocytic sheath, labeled by anti-aquaporin-4 gold particles, enveloped the endothelium-pericyte layer. Orthogonal arrays of particles were observed on fractured astrocytic membranes, starting from embryonic day 14 when the aquaporin-4 immunogold staining revealed clusters of gold particles, often forming square or rectangular clusters. The results showed that aquaporin-4 expression and organization of the intramembrane particles in orthogonal arrays followed the same temporal sequence. Finally, the lipopolysaccharide, a substance that induces blood-brain barrier disruption, determines a remarkable reduction in aquaporin-4 labeling, expressed by a few aquaporin-4 gold particles attached on swollen perivascular glial membranes. All these data show that aquaporin-4 expression occurs in the chick embryonic brain, in parallel with maturation and functioning of the blood-brain barrier and suggest that there is a close relationship between water transport regulation and brain development.

Fine Needle Aspiration May Shed Breast Cells into Peripheral Blood as Determined by RT-PCR

Objective: A diagnostic test applying reverse-transcriptase chain reaction (RT-PCR) assay targeted against cytokeratin 19 (CK19), cytokeratin 20 (CK20) and the β-subunit of human chorionic gonadotropin (β-hCG) mRNAs was used to evaluate the impact of fine needle aspiration (FNA) on breast cell shedding into peripheral blood. Methods: The sensitivity of this assay was based on the different degree of admix of MCF-7 breast cancer cell line with HL-60 leukemic cell line. For blood samples of 24 cases with benign breast diseases and 20 cases with malignant ones, 5 ml of peripheral blood was drawn before and within 10 min after puncture. Total RNA was extracted from peripheral blood mononuclear (PBMN) cells; β-actin was used to assess the quality of cDNA. RT-PCR products were run in ethidium bromide gel and observed under ultraviolet. RT-PCR products for β-hCG were digested with Sty I endonuclease to confirm the specificity. Results: The sensitivity of RT-PCR assay was 1 MCF-7 cell in 10⁵ HL-60 cells for CK19 and CK20, and 1 in 10⁶ for β-hCG. For 24 benign cases, none of the pre- FNA samples was positive for CK20 and β-hCG, and 3 cases (12.5%) were positive for CK19. As for 20 malignant cases, 1 pre-FNA sample was positive for all three markers and 2 other samples were positive for CK19. After aspiration, 3/21 benign cases and 1/17 malignant case with pre-FNA negative signals became positive for CK19, while 3/19 malignant cases with pre-FNA negative signals were converted to a positive result for CK20 and β-hCG. Of 6 pre-FNA positive cases, all cases remained positive for the respective marker. Conclusion: FNA to breast tumor may cause hematogenous dissemination of breast cells.

Combined detection of breast cancer micrometastases in the lymph nodes and bone marrow using reverse-transcriptase chain reaction and southern hybridization

Objective: The presence of lymph nodes and bone marrow micrometastases of patients with breast carcinoma by immunohistochemistry (IHC) methods has been strongly correlated to early recurrence and shorter overall survival. The aim of this study was to detect micrometastases in matched sample pairs of lymph nodes and the bone marrow of primary breast cancer patients using a more sensitive method, and compare with other clinical parameters. Methods: Cytokeratin 19 (CK-19) gene mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization. Human breast cancer cell line T47D was mixed with bone marrow cells at different proportions. The positive detection rate was compared among RT-PCR, Southern blotting and IHC methods. Results: Cytokeratin 19 gene was expressed in all 6 positive control samples, while the expression wasn't seen in 18 negative control samples. CK-19 IHC positive cells were detected at a dilution of one T47D cell in 5 xl0 5 bone marrow cells, while the sensitivity detected by PCR and Southern blot hybridization was at l:5X10 4 and 1:10 6 , respectively. In the samples from the 35 patients, we found CK-19 positive cells in 2 cases (5.7%) by IHC. CK-19 gene expression signal was detected in 14/35 (40%) by RT-PCR, and 17/35 (48.6%) by southern blotting. Four cases were micrometastases positive both in lymph node and bone marrow (11.4%). There was no correlation between CK-1 9 detection and other clinical parameters. Conclusion: combined detection of micrometastases in lymph node and bone marrow by RT-PCR and Southern blotting, using CK-19 as a biological marker, is a highly sensitive method for breast cancer.