Expression Of H1 Research Articles

Expression analyses of three members of the AtPHO1 family reveal differential interactions between signaling pathways involved in phosphate deficiency and the responses to auxin, cytokinin, and abscisic acid

The PHO1 protein is involved in loading inorganic phosphate (Pi) to the root xylem. Ten genes homologous to AtPHO1 are present in the Arabidopsis thaliana (L.) Heyn genome. From this gene family, transcript levels of only AtPHO1, AtPHO1;H1 and AtPHO1;H10 were increased by Pi-deficiency. While the up-regulation of AtPHO1;H1 and AtPHO1;H10 by Pi deficiency followed the same rapid kinetics and was dependent on the PHR1 transcription factor, phosphite only strongly suppressed the expression of AtPHO1;H1 and had a minor effect on AtPHO1;H10. Addition of sucrose was found to increase transcript levels of both AtPHO1 and AtPHO1;H1 in Pi-sufficient or Pi-deficient plants, but to suppress AtPHO1:H10 under the same conditions. Treatments of plants with auxin or cytokinin had contrasting effect depending on the gene and on the Pi status of the plants. Thus, while both hormones down-regulated expression of AtPHO1 independently of the plant Pi status, auxin and cytokinin up-regulated AtPHO1;H1 and AtPHO1;H10 expression in Pi-sufficient plants and down-regulated expression in Pi-deficient plants. Treatments with abscisic acid inhibited AtPHO1 and AtPHO1;H1 expression in both Pi-sufficient and Pi-deficient plants, but increased AtPHO1;H10 expression under the same conditions. The inhibition of expression by abscisic acid of AtPHO1 and AtPHO1;H1, and of the Pi-starvation responsive genes AtPHT1;1 and AtIPS1, was dependant on the ABI1 type 2C protein phosphatase. These results reveal that various levels of cross talk between the signal transduction pathways to Pi, sucrose and phytohormones are involved in the regulation of expression of the three AtPHO1 homologues.

Histamine H4 receptor expression is elevated in human nasal polyp tissue

Altered histamine metabolism is thought to be involved in the pathomechanism of nasal polyposis characterized by local eosinophil infiltration. The present study was performed to determine whether histamine receptors play a role in the effect of histamine in nasal polyp tissue. The findings suggest that the expression of H1 and H4 receptors is elevated in polyp tissue (p=0.045; p<0.001), while the level of H2 and H3 receptors is not increased significantly. The elevation of H1 and H4 receptors' expression may indicate that the histamine related mechanisms are preferentially mediated through H1 and H4 histamine receptors in the polyp tissue. Simultaneously with increased H4 receptor expression, the concentration of eosinophil cationic protein (ECP) was increased significantly in polyp tissue (p=0.002). One may speculate that the H4 receptor mediated histamine effects have a role in eosinophil accumulation and activation in inflammatory diseases of the nasal and paranasal sinus mucosa, such as nasal polyposis.

Reduction of Calponin h1 expression in human colon cancer blood vessels

Aims Calponin h1 (CN) is a differentiation marker of smooth muscle cells that has been reported to be down-regulated in the blood vessels of several human tumors. In this study, we examined CN expression in blood vessels in relation to the clinical and pathological features of colon cancer tissue samples. Methods Fifty-six patients who had undergone colectomy for colon cancer were examined. To assess patients' disease-free survival, those who had metastasis at the time of surgical operation were excluded. Immunohistochemistry was performed by the indirect immunoperoxidase method, using serial sections made from formalin fixed and paraffin embedded tissue blocks. Results We found that the expression of vascular CN in the peripheral region of colon cancer tissues was significantly reduced in association with tumor progression, lymphatic invasion, vascular invasion and recurrence. This reduction of CN indicated not only a decrease of pericytes and/or smooth muscle cells in tumor vessels, but also the immaturity of those cells, since CN down-regulation occurred even in α-smooth muscle actin-positive cells. The down-regulation of CN in vessels in the peripheral region of tumor tissues was inversely associated with the expression of VEGF (vascular endothelial growth factor), seemingly advantageous to angiogenesis. Conclusion The down-regulation of CN expression in colon cancer vasculature evaluated by immunohistochemistry may be useful in conjunction with conventional staging procedures to predict more reliable outcome and to select therapeutic treatment.

Prolonged Histamine Deficiency in Histidine Decarboxylase Gene Knockout Mice Affects Leydig Cell Function

The present study focuses on histaminergic regulation of Leydig cell physiology, since limited information is available so far. To evaluate the dependency of Leydig cells on histamine (HA), we performed experiments using highly purified Leydig cells in culture, isolated from wild type (WT) and histidine decarboxylase (Hdc) gene knockout (HDC KO)-so HA-deprived-mice. HDC KO Leydig cells showed lower basal and human choriogonadotropin (hCG)-induced testosterone production compared to WT Leydig cells, presumably due to altered P450scc gene (Cyp11a1) expression levels. Moreover, in HDC KO cells, hCG did not increase basal expression levels of HA H1 and H2 receptor genes, while the hormone showed a significant inducing effect in WT cells. Based on these findings, we propose that prolonged HA deficiency in HDC KO mice affects various aspects of Leydig cell physiology, most importantly the response to hCG, providing definite evidence that HA plays a role as direct modulator of Leydig cell function and steroid synthesis in the testis. Also, the results presented herein constitute the first molecular evidence for the expression of HA H1 and H2 receptor subtypes in isolated Leydig cells.

Immunotherapeutic Effect of Concholepas Hemocyanin in the Murine Bladder Cancer Model: Evidence for Conserved Antitumor Properties Among Hemocyanins

We determined the antitumor properties of a newly available hemocyanin obtained from the Chilean gastropod Concholepas concholepas (Biosonda Corp., Santiago, Chile) in a syngeneic heterotopic mouse bladder carcinoma model. Since keyhole limpet hemocyanin (Pierce, Rockford, Illinois) is used increasingly in biomedicine as a carrier for vaccines and an immunotherapeutic agent for bladder transitional cell carcinoma, there is a growing interest in finding new substances that share its potent immunomodulatory properties. Considering that keyhole limpet hemocyanin and Concholepas concholepas hemocyanin differ significantly, it was not possible to predict a priori the antitumor properties of Concholepas concholepas hemocyanin. C3H/He mice were primed with Concholepas concholepas hemocyanin before subcutaneous implantation of mouse bladder tumor-2 cells. Treatment consisted of a subcutaneous dose of Concholepas concholepas hemocyanin (1 mg or 100 mug) at different intervals after implantation. Keyhole limpet hemocyanin and phosphate buffered saline served as positive and negative controls, respectively. In addition, experiments were designed to determine which elements of the immune response were involved in its adjuvant immunostimulatory effect. Mice treated with Concholepas concholepas hemocyanin showed a significant antitumor effect, as demonstrated by decreased tumor growth and incidence, prolonged survival and lack of toxic effects. These effects were similar to those achieved with keyhole limpet hemocyanin. We found that each hemocyanin increased natural killer cell activity but the effect of Concholepas concholepas hemocyanin was stronger. Analysis of serum from treated mice showed an increased interferon-gamma and low interleukin-4, which correlated with antibody isotypes, confirming that hemocyanins induce a T helper type 1 cytokine profile. To our knowledge our results are the first demonstration of the antitumor effect of a hemocyanin other than keyhole limpet hemocyanin. They suggest that this is an ancient conserved immunogenic mechanism shared by those hemocyanins that is able to enhance T helper type 1 immunity and lead to antitumor activity. Therefore, Concholepas concholepas hemocyanin may be an alternative candidate for providing safe and effective immunotherapy for human superficial bladder cancer.

Functional characterization of histamine receptor subtypes in a human bronchial epithelial cell line

Histamine is a well-known mediator eliciting a broad range of responses in different cell types. Four different subtypes of G protein-coupled histamine receptors (H1-H4) have been cloned and pharmacologically characterized. However, involvement of the different histamine receptor subtypes in immunomodulatory functions of bronchial epithelium has only been investigated marginally. The expression and function of histamine receptor subtypes on the human bronchial epithelial cell line BEAS-2B was analyzed by PCR, intracellular Ca++ -measurements and ELISA. We show mRNA expression of the histamine receptor subtypes H1, H2, and H3, but not H4 in the human bronchial epithelial cell line BEAS-2B. Using intracellular Ca++ -measurements, we demonstrated functional expression of the H1 and H3 receptors. To characterize the biological properties of histamine in airway epithelial biology, we also investigated its effects on cytokine secretion by BEAS-2B cells. Thereby, we were able to show up-regulation of the proinflammatory mediators IL-6 and CXCL8/ IL-8 via activation of the H1, H2 and H3 receptor subtypes. The Th1 cytokines CXCL9/MIG and CXCL10/IP-10 and the chemokine CCL5/RANTES were regulated in a distinct manner: Whereas histamine inhibited the IFN-gamma/TNF-alpha-induced secretion of MIG via the histamine receptor subtypes H1, H2, and H3, the histamine-induced suppression of RANTES was due to activation of the H2 and H3 receptors, while reduction of cytokine-triggered IP-10 secretion was mediated only by triggering the H2 receptor. In summary our data provide evidence that histamine released during allergic lung diseases exerts regulatory influence on airway epithelial cells.

Escherichia coli-based expression of functional novel DNA-binding histone H1 from Carassius auratus

Histones are DNA-binding proteins that assist in DNA packaging and protection. Here, we, for the first time, cloned a novel histone H1 cDNA (638 bp) from the goldfish, Carassius auratus. Sequencing revealed that this histone H1 shared 68.1% amino acid identity and 73.9% similarity with that from the rainbow trout, Salmo gairdneri. We successfully expressed a full-length recombinant version (∼20 kDa) and a truncated C-terminal fragment (∼6 kDa; 61 amino acids) of this histone H1 as a partially soluble form using a maltose binding protein (MBP) fusion strategy in an Escherichia coli expression system. Our results revealed that both these recombinant histone H1 versions had DNA binding and protection functions, and MBP fusion system was an effective way to produce biological functional recombinant histone proteins in E. coli. This novel recombinant histone H1 protein and/or its C-terminal peptide could be used as potential mediators for efficacious gene delivery.

Differential Expression of Two Flavonoid 3′-Hydroxylase cDNAs Involved in Biosynthesis of Anthocyanin Pigments and 3-Deoxyanthocyanidin Phytoalexins in Sorghum

Three unique sorghum flavonoid 3'-hydroxylase (F3'H) cDNAs (SbF3'H1, SbF3'H2 and SbF3'H3) were discovered through bioinformatics analysis. Their encoded proteins showed >60% identity to the Arabidopsis TT7 (F3'H) protein. Overexpression of SbF3'H1 or SbF3'H2 restored the ability of tt7 mutants to produce 3'-hydroxylated flavonoids, establishing their roles as functional F3'H enzymes. In sorghum mesocotyls, SbF3'H1 expression was involved in light-specific anthocyanin accumulation while SbF3'H2 expression was involved in pathogen-specific 3-deoxyanthocyanidin synthesis. No SbF3'H3 expression was detected in all tissues examined. The sorghum mesocotyls represent a good system for investigation of differential regulation of F3'H genes/alleles responding to different external stimuli.

Human subjects without peanut allergy demonstrate T cell–dependent, T H2-biased, peanut-specific cytokine and chemokine responses independent of T H1 expression

Peanut allergy is a major cause of anaphylaxis. Regulation of immune responses to peanut allergen, particularly why sensitization does not usually progress to allergic reactions, is not well investigated. Most studies focus exclusively on serologic responses and individuals with peanut allergy. We sought to determine the existence, prevalence, and nature of peanut-specific, T cell-dependent cytokine and chemokine responses of adults who eat peanut without having symptoms. We developed systems to examine specific immunity in peanut-tolerant individuals who had (1) negative histories and negative peanut skin test responses, (2) negative histories and positive peanut skin test responses, and (3) clinically apparent peanut allergy. After primary culture of PBMCs restimulated with whole peanut extract, we quantified responses characteristic of TH1 (IFN-gamma and CXCL10) and TH2-like immunity (IL-5, IL-13, CCL17, and CCL22) using ultrasensitive ELISAs. Antigen-presenting cell costimulatory requirements (CD4, HLA-DR, CD80/86, and cytotoxic T lymphocyte-associated antigen 4 [CTLA4] Ig) were determined. T cell-dependent, peanut-specific IL-5, IL-13, and CCL22 were common in peanut-tolerant individuals, regardless of whether they had positive or negative skin test responses. These were blocked by anti-CD4 and were dependent on CD28/CD86 costimulation. None of the 70 individuals studied had demonstrable IFN-gamma or CXCL10 responses to peanut. All demonstrated TH1 and TH2 responses to the ubiquitous recall antigen streptokinase. Qualitatively similar and quantitatively increasing peanut-specific TH2 responses in the consistent absence of putatively protective TH1 immunity were found in both peanut-tolerant individuals and those with peanut allergy. The continuum of responses between individuals with negative and individuals with positive skin test results, rather than TH1 versus TH2 bias, might be important in peanut allergy.

Immunodetection of GLUT1, p63 and phospho‐histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinization

Immunodetection of GLUT1, p63 and phospho-histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinizationAims : To examine invasive head and neck squamous carcinomas for expression of GLUT1, a glucose transporter and marker of increased glucose uptake, glycolytic metabolism and response to tissue hypoxia; p63, a p53 homologue that is a marker of the undifferentiated proliferative basaloid phenotype; and phospho-histone H1, a marker of activation of the cell cycle-promoting cyclin-dependent kinases 1 and 2. Methods : Routinely processed slides from 34 invasive squamous carcinomas, including 25 with intraepithelial components, were immunostained with anti-GLUT1 (Chemicon), anti-p63 (4A4, Santa Cruz), and antiphospho-histone H1 (monoclonal 12D11). Results : In keratinizing carcinomas, all three markers were most commonly immunodetected peripherally, with loss of expression in central keratinized zones. In contrast, in non-keratinizing carcinomas, p63 and phospho-histone H1 expression was most commonly observed throughout tumour nests and anti-GLUT1 stained in a pattern suggestive of hypoxia-induced expression ('antistromal' staining), in which cells at the tumour-stromal interface were GLUT1- and cells in central, perinecrotic zones showed progressive induction of GLUT1. Intraepithelial components also displayed basal and 'antibasal' GLUT1 staining patterns, homologous to the pro- and antistromal patterns in invasive carcinoma; basal patterns in intraepithelial lesions appeared to be more predictive of keratinizing invasive carcinoma and antibasal intraepithelial staining more predictive of non-keratinizing poorly differentiated carcinomas. Conclusions : Keratinizing and non-keratinizing squamous carcinomas differ in expression patterns of GLUT1, p63 and phospho-histone H1. In the former, all three markers were typically suppressed in conjunction with keratinization; in the latter, GLUT1 expression was more likely to occur in a hypoxia-inducible pattern and expression of p63 and phospho-histone H1 was unsuppressed. GLUT1 expression patterns in intraepithelial lesions may be predictive of the differentiation status of the associated invasive carcinoma.

Human Medullary Thyroid Carcinoma: A Source and Potential Target for Relaxin‐Like Hormones

We investigated the expression of H1, H2 relaxin and INSL-3, mRNA and protein, and LGR7 and LGR8 transcripts in human C-cell hyperplasia, primary medullary thyroid carcinoma (MTC) tissues, MTC metastases, and the human MTC-TT and mouse MTC-M cell lines. Relaxin-like peptide hormones were detected in C-cell hyperplasia and in MTC tissues, but were absent in human normal parafollicular C-cells of benign goiter tissues. In contrast to calcitonin, mRNA, and immunoreactive protein, no differences in the expression of relaxin and INSL3 were observed in MTC tissues of different pTNM classification or between primary and metastatic MTC tissues studied. All MTC tissues constitutively expressed LGR7 and LGR8 transcripts. Thus, relaxin-like hormones appear to be present early during C-cell hyperplasia and potentially functional relaxin/INSL3 ligand-receptor systems are present in human MTC tissues and in MTC cell lines.

Mouse protein arrays from a T H1 cell cDNA library for antibody screening and serum profiling

The mouse is the premier genetic model organism for the study of disease and development. We describe the establishment of a mouse T helper cell type 1 (T H1) protein expression library that provides direct access to thousands of recombinant mouse proteins, in particular those associated with immune responses. The advantage of a system based on the combination of large cDNA expression libraries with microarray technology is the direct connection of the DNA sequence information from a particular clone to its recombinant, expressed protein. We have generated a mouse T H1 expression cDNA library and used protein arrays of this library to characterize the specificity and cross-reactivity of antibodies. Additionally, we have profiled the autoantibody repertoire in serum of a mouse model for systemic lupus erythematosus on these protein arrays and validated the putative autoantigens on highly sensitive protein microarrays.

Expression of sialylated carbohydrate antigens and nm23-H1 gene in prognosis of breast cancer

To assess the significance of expression of sialylated carbohydrate antigens and nm23-H1 gene in metastasis and prognosis of breast cancer. Tissue specimens from 102 cases of primary breast cancer were stained with antibodies against sialyl Lewis A (SleA) and salyl Lewis X (SleX), and nm23-H1 proteins by immunohistochemical methods. Of the 102 cases, the positive cases of SleA and SleX were 24.5% (25/102) and 59.89% (61/102),respectively; the reduced expression of nm23-H1 was showed in 37.3% (38/102) of the cases. The positive expression of SleX and the reduced expression of nm23-H1 gene were significantly associated with lymph node involvement. Among the 100 patients who underwent curative surgery, the disease-free survival rate was significantly correlated with nm23-H1 and SleX expression, respectively,but not with SleA expression. In multivariate analysis using Cox regression model, combination assay of nm23 H1 and SleX expression emerged as independent prognostic factors. These results suggest that nm23-H1 gene and SleX may be involved in the metastatic process in human breast cancer, and immunohistochemical detection of SleX and nm23-H1 may be used as a biologic marker of prognosis.

Calponin h1 Expression in Renal Tumor Vessels: Correlations With Multiple Pathological Factors of Renal Cell Carcinoma

We determined whether the architecture of renal tumor vessels is immunohistochemically different from that of normal renal vessels and related to the various pathological factors that affect prognosis of renal cell carcinoma (RCC). A total of 52 cases of primary RCC were selected. Tissues from radical nephrectomy specimens were stained with antibody to alpha-smooth muscle actin (alpha-SMA) and calponin h1. Immunostaining was evaluated semiqualitatively as 0-no staining to 3+-strong staining. Tumor cell proliferation was observed using proliferating marker Ki-67. Data were statistically compared with pathological factors, such as tumor size, histological pattern, growth pattern, cell type, nuclear grade, pathological stage and presence or absence of venous invasion. In normal renal tissues smooth muscle cells of the blood vessels showed strong immunoreactions with antibody to calponin h1 and alpha-SMA. Although alpha-SMA antibody showed similar strong immunoreactions in all types of renal tumor vessels, we observed qualitative alterations in the expression of calponin h1 in different types of RCCs. Strong to moderate immunoreactions with calponin h1 were observed in tumors with expansive growth and an alveolar pattern. Small tumors without venous invasion and chromophobe cell carcinomas also showed strong to moderate expression of calponin h1. Weak or absent expression of calponin h1 was observed significantly in infiltrating tumors, sarcomatous type, large, high grade and high stage tumors associated with significantly higher proliferating indexes. Our results strongly suggest that the renal tumor vessels are immunohistochemically different from normal renal vessels in respect to calponin h1 expression. We speculate that due to the decrease in or absence of calponin h1 tumor vessels do not develop adequate maturity to maintain vascular integrity. In addition, the distribution of calponin h1 significantly correlated with multiple pathological factors of RCC. Therefore, calponin h1 expression in renal tumor vessels could be a new, important pathological factor in RCC.

235RETINOID-DEPENDENT MRNA EXPRESSION IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

As a transcription factor, retinoic acid (RA) can activate or silence a wide number of genes, thus inducing differentiation in cell systems and playing a role in cell cycle regulation. However, little is known of RA-dependent gene expression in the oocyte. Bovine oocytes and cumulus cells express most RA receptors, and the presence of 9-cis-RA during in vitro maturation (IVM) is beneficial to oocyte development (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). The present work analyzes the relative abundance of various developmentally important gene transcripts in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were manipulated in defined medium with polyvinyl-alcohol (DM-PVA). Those COCs undergoing prematuration were cultured for 24h in DM-PVA with 25μM roscovitine. For IVM, some prematured COCs were cultured for 24h in DM-PVA containing pFSH, LH and E2. Incubations were made at 39°C in an atmosphere of 5% CO2 in air and high humidity. Within experiments, COCs were cultured with nM 9-cis-RA 5, in 1% ethanol (both as vehicle and inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Using Real Time PCR (10 oocytes per group) (Rizos et al., 2003 Biol. Reprod. 68, 236) we examined the relative mRNA expression of genes involved in protection against free oxygen radicals (Mn-superoxide dismutase, MnSOD), glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH) and cell cycle events (Cyclin B1 and H1). Data (of 4 replicates) were analyzed by ANOVA and Duncan test (P&amp;lt;0.05). Regarding immature oocytes, prematuration in 1% ethanol increased cyclin B1 expression and decreased cyclin H1, while 9-cis-RA increased G6PDH. Maturation without additives increased cyclin B1 and G6PDH, but decreased cyclin H1 and MnSOD expression;; opposite trends were observed under increasing ethanol dosages (3% and 5%). Maturation with 1% ethanol or 9-cis-RA enhanced cyclin B1 and G6PDH, while reducing cyclin H1 and MnSOD expressions. The presence of 9-cis-RA during both prematuration and maturation processes tended to show more prominent effects than the ones observed when it was present only during prematuration or maturation alone. In our study, in presence of 9-cis-RA during both prematuration and maturation processes, the expression of cyclin B1 and G6PDH tended to increase, while cyclin H1 and MnSOD tended to decrease. However, the differences with the control group without additives were not significant. Our study during both prematuration and maturation processes show that beneficial effects of RA on oocyte developmental competence may not be related to the alteration of mRNA expression of the four genes analyzed. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175; 2003-05783).

Immunohistochemical markers in differential diagnosis of endometrial stromal sarcoma and cellular leiomyoma

Objective. Distinction of endometrial stromal sarcoma (ESS) from benign smooth muscle proliferations like cellular leiomyoma (CL) is sometimes problematic. The purpose of this study was to evaluate the potential utility of a panel of antibodies in the differential diagnosis of ESS and CL. Methods. Using a standard streptavidin–biotin method, the expression of desmin, alpha smooth muscle actin (SMA), calponin h1, h-caldesmon, estrogen receptor (ER), progesterone receptor (PR), CD10, CD44v3, proliferating cell nuclear antigen (PCNA), and mast cells (MCs) were evaluated in 26 cases of ESS (21 low grade, 5 high grade), 25 CL (17 common CL, 8 highly CL), 25 myometria, and 25 endometria. Results. Among ESS, 20 of 26, 17 of 26, 9 of 26, 12 of 26, 14 of 26, and 22 of 26 were positive for expression of desmin, SMA, calponin h1, ER, PR, and CD10, respectively, while only 2 of 26 were positive for CD44v3 and all were entirely negative for h-caldesmon. Of CL, all were positive for SMA, calponin h1, PR, and CD44v3; 24 of 25, 24 of 25, and 19 of 25 were positive for desmin, h-caldesmon, and ER, respectively, whereas 1 of 25 focally marked with antibodies to CD10. There was no significant difference of PCNA expression between ESS and CL, although the ESS cases tended to have higher values. The MC counts were significantly higher in the CL group than in the ESS group ( P < 0.01). When using the cut-off value of seven MCs per HPF to distinguish ESSs from CLs, the sensitivity and specificity of this cut-off value were 92.9% and 100%, respectively. Conclusions A panel of h-caldesmon, CD10, and CD44v3 should be used and will distinguish ESS from CL in most cases. In addition, counting the number of MCs might be useful as part of a multivariate approach to the differential diagnosis of them. But the biological function of MC and CD44v3 in these tumors is worthy of further investigation.

H1 and H2 Histamine Receptors Are Absent on Langerhans Cells and Present on Dermal Dendritic Cells

Human monocyte-derived dendritic cells (MoDC) have both histamine H1 and H2 receptors and can induce CD86 expression by histamine. Nevertheless, it has not been reported whether human epidermal Langerhans cells (LC) have histamine receptors or not. In this study, using RT-PCR, we investigated the expression of H1 and H2 receptor mRNA on DC with the features of LC (LC-like DC) that were generated in vitro from peripheral blood monocytes, LC derived from CD34+ hematopoietic progenitor cells, and LC obtained from human epidermis. We compared the histamine-induced CD86 expression among these cells. In contrast to MoDC, LC and LC-like DC did not express H1 or H2 receptors. In addition, they could not augment the CD86 expression by histamine. Interestingly, when transforming growth factor-beta1 (TGF-beta1) was added to the culture of MoDC, the expression of H1 and H2 receptors and the histamine-induced CD86 expression were abrogated in a concentration-dependent fashion. Finally, in the assessment of the cell surface expression of histamine receptors using fluorescence-labeled histamine, histamine could bind to MoDC and dermal dendritic cells obtained from the skin, whereas there was no specific binding of histamine to LC-like DC or LC obtained from the skin. These data suggest that LC do not express either H1 or H2 receptors, mainly because of the effect of TGF-beta1. This made a striking contrast with the expression of the functional H1 and H2 receptors on MoDC and dermal dendritic cells.

Reduced expression of actin-binding proteins, h-caldesmon and calponin h1, in the vascular smooth muscle inside melanoma lesions: an adverse prognostic factor for malignant melanoma.

The structural integrity of the blood vessels such as small arteries and veins is studied less frequently in malignant tumours than is angiogenesis. Objectives To clarify the characteristics of small arteries and small veins of melanoma lesions. We immunohistochemically investigated various types of melanocytic tumours using antibodies specific for endothelial and vascular smooth muscle cells, and analysed the relationship between the expression of these molecules in the blood vessels and the biological characteristics of the tumours. Formalin-fixed, paraffin-embedded sections of 15 cases of benign melanocytic tumours and 64 cases of malignant melanomas were investigated. Significant suppression of expression of h-caldesmon (h-CD) and calponin h1 (CNh1) was observed in the blood vessels of malignant melanomas compared with both benign melanocytic tumours and normal tissues. In particular, the level of h-CD expression was inversely correlated with the frequency of metastasis and positively correlated with the survival rate in patients with malignant melanoma. These findings suggest that alterations of the tumour vessels are an important factor for the prognosis of malignant melanoma, and that suppression of h-CD and CNh1 in the blood vessels in malignant melanoma reflects a structural fragility of the vessels, leading to their easy penetration by tumour cells. Defective expression of these molecules is likely to be an important marker for metastatic potential and for poor prognosis of melanoma.

Differential cytoplasmic and whole cellular expression of histone H1 within the avian bursa of Fabricius

Bursal anti-steroidogenic peptide (BASP), purified from the chicken bursa of Fabricius (BF), has been previously demonstrated to be a potent and efficacious inhibitor of steroid hormone biosynthesis from chicken ovarian, and both mammalian and avian adrenal cells in vitro. Other studies have demonstrated that BASP can markedly reduce avian and mammalian mitogen-stimulated lymphocyte proliferation. Recent studies have indicated that BASP has a structural and functional relationship with histone H1. Immunohistochemical studies using a monoclonal antibody, which is known to recognize a common histone H1 epitope from several plant and animal species identified the protein within the cytoplasm and nucleus of distinct cells within both the cortex and medulla of all BF follicles. Additionally, epithelial cells within the BF expressed the protein strongly in the cytoplasm with reduced nuclear staining. In contrast, the same antibody did not recognize the protein in thymus of the same animals. The differential expression of histone H1 immunoreactivity within selected cells of the BF may support a previous proposed role of histone H1 in extranuclear and extracellular signaling in chickens and possibly other species.

Family of Shuttle Vectors for Ruminal Bacteroides

A family of shuttle plasmids was constructed for genetic transformation of Escherichia coli and of ruminal Bacteroides strains AR20 and AR29. Plasmids were based on the replicon from Bacteroides plasmid pBI191 and were designed for studies of chromosomal integration (pBA), for the identification and study of Bacteroides gene promoters (pPPR) and for the expression of heterologous genes in Bacteroides (pBAC). Electroporation efficiency of Bacteroides was up to 10⁵ transformants/µg plasmid, depending on the source of the DNA. The largest plasmid, pBA, was maintained at approximately 8 copies per cell in AR20 and did not measurably alter in vitro growth of transformed cells. In the current work, pBA did not integrate into the chromosomes of AR20 or AR29. The ability of plasmid pPPR to select promoter sequences was demonstrated by removal and replacement of promoters that activate the clindamycin resistance gene. The suitability of pBAC for expression of heterologous genes was demonstrated by expression of the Moraxella species fluoroacetate dehalogenase gene H1 to give intracellular activity of 7 nmol fluoride released/min/mg soluble protein in AR20 and 4 nmol/min/mg in AR29. Spontaneous loss of pBAC under non-selective conditions was 0.11–0.165% per generation, significantly less than loss of the native Bacteroides plasmid pBI191, which was lost at 0.53% per generation.