Prevalence of tri- and tetraantennary glycans of humanα 1-acid glycoprotein in release of macrophage inhibitor of interleukin-1 activity

Based on the affinity for concanavalin A (Con A), human ₁-acid glycoprotein (AGP) can be separated by chromatography on Con A-Sepharose gel into three variants: Con A unreactive AGP, Con A weakly reactive AGP, and Con A strongly reactive AGP. When exposed to native AGP or to its glycan variants, murine peritoneal macrophages released a factor that inhibited the interleukin-1 (IL-1) proliferative activity as measured in terms of the thymocyte comitogenic assay. Con A unreactive AGP, which contains tri- and tetraantennary glycans and no biantennae, proved to be more effective than Con A weakly and Con A strongly reactive variants, which contain one and two diantennary glycans, respectively. The inhibitory effect was not a function of the negative charge related to the sialyl residues and was not mediated by the maanosyl-fucosyl receptor.     

Kinetics of LFA-1 Mediated Adhesion of Human Neutrophils to ICAM-1—Role of E-Selectin Signaling Post-Activation

LFA-1 and Mac-1 are the two integrins involved in the arrest and firm adhesion of neutrophils. LFA-1 plays a role in the early stage of cell arrest while Mac-1 stabilizes firm adhesion. Here, we further elucidated the kinetics of LFA-1 activation and its role in mediating neutrophil adhesion to ICAM-1 in the presence of E-selectin interaction. We confirm that LFA-1 activation to high affinity is transient in nature, decaying back to low affinity within 1 min after chemotactic stimulation. However, we show for the first time that this downshift in LFA-1 affinity does not return back to the low affinity state when E-selectin interaction is present and active, but rather E-selectin signals an intermediate LFA-1 conformation through PI3-Kinase that maintains an intermediate level of neutrophil firm adhesion. We further show that this E-selectin signaling is capable of returning LFA-1 to the intermediate affinity conformation outside the 1-min window previously reported for LFA-1 functionality. While our work confirms a role for PI3-Kinase in neutrophil firm adhesion, we show that PI3-Kinase may not be important in the initial transition from rolling to firm arrest (i.e. LFA-1 shift from low to high affinity conformation occurring within seconds of chemotactic stimulation).     

Asd-homothallism of Saccharomyces cerevisiae: identification of asd1-1 as an allele of sir4 and detection of α-specific suppressors of it

Asd-homothallism of Saccharomyces cerevisiae involves a life cycle characterized by a non-mating phenotype and endomitotic diploidization. The former trait is determined by a single mutation, asd1-1. This mutation was mapped between hom2 and lys4 on the right arm of chromosome IV and was complemented by the cloned SIR4 gene. Therefore, we conclude that asd1-1 is an allele of sir4-11 and renamed it sir4-11. Endomitotic diploidization of asd-homothallism is caused by the collaboration of three to four mutations including sir4-11. In the course of this study, we detected -specific suppressors of sir4-11.     

BRCA1 – Conductor of the Breast Stem Cell Orchestra: The Role of BRCA1 in Mammary Gland Development and Identification of Cell of Origin of BRCA1 Mutant Breast Cancer

Breast cancer treatment has been increasingly successful over the last 20 years due in large part to targeted therapies directed against different subtypes. However, basal-like breast cancers still represent a considerable challenge to clinicians and scientists alike since the pathogenesis underlying the disease and the target cell for transformation of this subtype is still undetermined. The considerable similarities between basal-like and BRCA1 mutant breast cancers led to the hypothesis that these cancers arise from transformation of a basal cell within the normal breast epithelium through BRCA1 dysfunction. Recently, however, a number of studies have called this hypothesis into question. This review summarises the initial findings which implicated the basal cell as the cell of origin of BRCA1 related basal-like breast cancers, as well as the more recent data which identifies the luminal progenitor cells as the likely target of transformation. We compare a number of key studies in this area and identify the differences that could explain some of the contradictory findings. In addition, we highlight the role of BRCA1 in breast cell differentiation and lineage determination by reviewing recent findings in the field and our own observations suggesting a role for BRCA1 in stem cell regulation through activation of the p63 and Notch pathways. We hope that through an increased understanding of the BRCA1 role in breast differentiation and the identification of the cell(s) of origin we can improve treatment options for both BRCA1 mutant and basal-like breast cancer subgroups.     

Cerebral small-vessel disease protein HTRA1 controls the amount of TGF-β1 via cleavage of proTGF-β1

Cerebral small-vessel disease is a common disorder in elderly populations; however, its molecular basis is not well understood. We recently demonstrated that mutations in the high-temperature requirement A (HTRA) serine peptidase 1 (HTRA1) gene cause a hereditary cerebral small-vessel disease, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HTRA1 belongs to the HTRA protein family, whose members have dual activities as chaperones and serine proteases and also repress transforming growth factor-β (TGF-β) family signaling. We demonstrated that CARASIL-associated mutant HTRA1s decrease protease activity and fail to decrease TGF-β family signaling. However, the precise molecular mechanism for decreasing the signaling remains unknown. Here we show that increased expression of ED-A fibronectin is limited to cerebral small arteries and is not observed in coronary, renal arterial or aortic walls in patients with CARASIL. Using a cell-mixing assay, we found that HTRA1 decreases TGF-β1 signaling triggered by proTGF-β1 in the intracellular space. HTRA1 binds and cleaves the pro-domain of proTGF-β1 in the endoplasmic reticulum (ER), and cleaved proTGF-β1 is degraded by ER-associated degradation. Consequently, the amount of mature TGF-β1 is reduced. These results establish a novel mechanism for regulating the amount of TGF-β1, specifically, the intracellular cleavage of proTGF-β1 in the ER.     

ATP depletion induces translocation of STIM1 to puncta and formation of STIM1–ORAI1 clusters: translocation and re-translocation of STIM1 does not require ATP

Depletion of the endoplasmic reticulum (ER) calcium store triggers translocation of stromal interacting molecule one (STIM1) to the sub-plasmalemmal region and formation of puncta—structures in which STIM1 interacts and activates calcium channels. ATP depletion induced the formation of STIM1 puncta in PANC1, RAMA37, and HeLa cells. The sequence of events triggered by inhibition of ATP production included a rapid decline of ATP, depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and a slow calcium leak from the ER followed by formation of STIM1 puncta. STIM1 puncta induced by ATP depletion were co-localized with clusters of ORAI1 channels. STIM1–ORAI1 clusters that developed as a result of ATP depletion were very poor mediators of Ca²⁺ influx. Re-translocation of STIM1 from puncta back to the ER was observed during total ATP depletion. We can therefore conclude that STIM1 translocation and re-translocation as well as formation of STIM1–ORAI1 clusters occur in an ATP-independent fashion and under conditions of PI(4,5)P₂ depletion. Michael Chvanov and Ciara M. Walsh are considered as equal first authors.     

Construction of a Mammary-specific Expression Vector of Humanα-defensin-1 (HNP-1) Gene

1 IntroductionDefensins, also called human neutrophil peptides(HNP), are small cationic peptides with broad antimicrobial activity~([1]). Human defensins are highly abundant in the cytoplasmic granules of polymorphonuclear neutrophils. Alpha-defensin-1 is an important mediator in either innate immunity or anti-infection. It can be developed to be an ideal new type antibiotic and may provide a better solution for the present situation of extensive antibiotics-resistence. It is difficult to ac…     

Cloning of a phospholipase C-δ1 of rabbit skeletal muscle

Summary The phospholipase C isoform responsible for the increase in the total myoplasmic inositol 1,4,5-trisphosphate concentration during tetanic contraction of isolated skeletal muscle and its mechanism of activation is not known. We have cloned and sequenced a phospholipase C cDNA of rabbit skeletal muscle coding for a protein of 745 amino acids with a molecular mass of 84 440 kDa. The deduced amino acid sequence exhibits the phospholipase C-specific domains X and Y which according to current knowledge very likely represent the catalytic centre of the enzyme. An overall sequence homology of 88% to the phospholipase C-1 of rat brain suggests that the encoded protein represents a phospholipase C-1 isoform of rabbit skeletal muscle. Northern blot analysis shows, that this phospholipase C- is dominantly expressed in skeletal muscle, less strongly in smooth muscle (uterus) and lung and weakly in heart, kidney and brain. In the N-terminal part of the primary structure a consensus sequence for a canonical EF-hand Ca²⁺ binding domain can be identified together with a short positively charged motif which recently has been suggested to be essential for the binding of phosphatidylinositol 4,5-bisphosphate. If these two domains which are unique for phospholipase C- are sufficient in establishing a mechanism for the activation of the enzyme, inositol 1,4,5-trisphosphate formation in skeletal muscle could be the consequence of an increase in myoplasmic Ca²⁺.     

Expression of cyclooxygenase-1 (COX-1) in labial salivary glands of Sjögren's syndrome

COX plays an important role in inflammatory diseases such as rheumatoid arthritis. To determine the role of COX in Sjögren's syndrome (SS), we examined COX expression in the salivary glands of SS patients. We examined 15 patients with SS and two normal subjects. Labial salivary gland tissue samples were analysed immunohistochemically using anti‐COX‐1 and COX‐2 antibodies. All biopsy samples from 15 patients with SS were stained for COX‐1. In contrast, COX‐1 immunostaining was not detected in normal salivary gland tissues. Co‐expression of COX‐1 and CD68 was confirmed by mirror section technique and double antibody immunostaining. This finding indicated that COX‐1‐expressing cells in SS salivary glands were infiltrating macrophages. In contrast to COX‐1 staining, only a little COX‐2 immunostaining was observed in salivary gland tissues from SS patients. These data suggest that COX‐1 expression on infiltrating macrophages may contribute to the inflammatory process of salivary glands in SS.     

Discoordinate expression of IL-1α and IL-1β in interfacial membranes of failed joint prostheses

Bone resorption following either cemented or uncemented total hip replacement has been implicated as an important etiologic factor in aseptic loosening of prostheses, the most frequent cause of clinical failure. Interleukin-1 (IL-1), collagenase and prostaglandin E₂ are considered to play key roles in pathological bone resorption. We have measured the actual levels and quantified the genes coding for several cytokines [IL-1, IL-1, IL-4, IL-6, platelet-derived growth factors (PDGF), transforming growth factor- (TGF) and tumor necrosis factor- (TNF)] in interfacial membranes obtained from cemented or uncemented loosened joint replacements. IL-1, IL-6 and TNF were barely detectable in the interfacial membranes either at protein or mRNA levels, while IL-1 and TGF were found to be expressed at the highest levels in freshly isolated tissues. However, the expression of IL-1 increased 10–1000-fold either in isolated cells or explant cultures of interfacial membranes within 24 h. The expression of other cytokines, measured directly in tissue or cells, did not suggest a discoordinate expression of bone-resorbing cellular mediators.     

Mapping of the functional domains of the α 4 protein of herpes simplex virus 1

Summary Truncated 4 genes were introduced into BHK tk^– cells along with the neomycin phosphotransferase gene, that confers resistance to the eukaryotic antibiotic G 418, driven by the HSV-1 tk promoter ( tk^–neo^r). Stably transformed cell lines were obtained and studied for the ability of the resident truncated 4 genes to regulate the expression of the tk^–neo^r, and for the ability of the truncated 4 polypeptides to localize to the nuclei of transformed cells. The results indicated that the domain(s) for gene induction and for nuclear localization of the 4 protein are located within the N-terminal 288 amino acids of the protein.     

HLA-DRB1 and -DQB1 alleles in the Brazilian population of the northeastern region of the state of São Paulo

The HLA-DRB1 and -DQB1 alleles in 161 healthy unrelated individuals, including Caucasians, Blacks and Mulattos (mixed Caucasian and Black), from the Northeastern region of the state of S?o Paulo, Brazil were analysed. The 36 different DRB1 alleles detected included not only common Caucasian alleles, but also DRB1*0411, 0807 and 1402, typical of Amerindians, and DRB1*0302, 1503, and 0804, typical of African American Blacks.     

Association of IL-1β, IL-1Ra and FABP1 gene polymorphisms with the metabolic features of polycystic ovary syndrome

Background

Polycystic ovary syndrome (PCOS), a highly prevalent endocrinopathy is currently being designated as chronic low grade inflammatory state. IL-1β, IL-1Ra and FABP1 are critical mediators of inflammatory processes and are speculated to play a role in the pathogenesis of PCOS. The aim of this study was to study the association of IL-β, IL-1Ra and FABP1 gene polymorphisms with PCOS and related metabolic features.

Subjects

95 PCOS and 45 age matched healthy control subjects were enrolled in this study.

Methods

Polymorphism in genes IL-1β, IL-1Ra and FABP1 was studied by PCR, PCR–RFLP and sequencing methods, respectively. Hormonal and lipid profiles were evaluated for all the subjects.

Results

Hormonal and lipid profiles showed significant differences between PCOS and control subjects. Allele and genotype frequencies of IL-1β, IL-1Ra and FABP1 gene polymorphisms did not vary between the control and PCOS group. However, T allele of C[-511]T variant of IL-1β, allele II in intron 2 of IL-1Ra and A allele of A/G variant of FABP1 (rs2197076) showed significant association with many metabolic features associated with PCOS.

Conclusions

Polymorphism in genes encoding cytokines and proteins involved in lipid metabolism can provide insights into the genetics of the disease and may contribute to assess the associated risk of cardiovascular diseases (CVD), dyslipidemia and metabolic syndrome (MetS) associated with PCOS.

     

Studies of the Mechanism of Development of “Deprivation” Potentiation of Population Responses of Neurons in Field CA1 of Living Hippocampal Slices

Experiments on rat hippocampal slices were performed with testing of the synaptic connections of Schaffer collaterals and neurons in field CA1 to study the effects of interrupting low-frequency test stimulation (0.05 Hz) on the amplitude of population spikes. These studies demonstrated a correlation between the duration of pauses in stimulation (form 10 to 120 min) and increases in the amplitude of spikes (on average by 30–100% of baseline response magnitude). This deprivation potentiation was additive and could persist for long periods of time (testing was up to 1 h). Preliminary induction of long-term post-tetanic potentiation, which prevented the subsequent development of the late, but not the short-term phase, for 1–3 h, led to suppression of the development of deprivation potentiation after a 60-min pause in stimulation. Similar results were obtained in experiments using 20 M polymyxin B, which blocks protein kinase C and the PKC-dependent phase of long-term post-tetanic potentiation; this is evidence supporting the previously advanced hypothesis that the development of deprivation potentiation and the late, PKC-dependent phase of long-term post-tetanic potentiation share common mechanisms, associated with people phosphorylation.     

Study of hypoxia-induced expression of HIF-1α in retina pigment epithelium

It was shown that the expression of HIF-1α in retinal pigment epithelium increased under hypoxic conditions. Eight hours after the start of hypoxic exposure, the expression of HIF-1α reached the peak and sustained after 24-hour hypoxia. However, the morphology of PRE cells began to change and the expression of HIF-1α decreased after long-term (48-hour) hypoxia. Hypoxia-induced increase in the level of HIF-1α in RPE. It can be an important step in choriodal neovascularization. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 3, pp. 293–297, March, 2007     

Loss of β1-integrin-deficient cells during the development of endoderm-derived epithelia

β1-Integrins (β1) represent cell surface receptors which mediate cell–matrix and cell–cell interactions. Fässler and Meyer described chimeric mice containing transgenic cells that express the LacZ gene instead of the β1 gene. They observed β1-negative cells in all germ layers at embryonic day E 8.5. Later in development, using a glucose phosphate isomerase assay of homogenized tissue samples, high levels of transgenic cells were found in skeletal muscle and gut, low levels in lung, heart, and kidney and none in the liver and spleen (Fässler and Meyer 1995). In order to study which cell types require β1 during development of the primitive gut including its derivatives, chimeric fetuses containing 15 to 25% transgenic cells were obtained at days E 14.5 and E 15.5. They were LacZ (β-galactosidase) stained “en bloc” and cross-sectioned head to tail. In esophagus, trachea, lung, stomach, hindgut, and the future urinary bladder, we observed various mesoderm-derived β1-negative cells (e.g. fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells) but no β1-negative epithelial cells. Since the epithelia of lung, esophagus, trachea, stomach, hindgut, and urinary bladder are derived from the endodermal gut tube, we hypothesize that β1 is essential for the development and/or survival of the epithelia of the fore- and hindgut and its derivatives.     

Analysis of CDKN1A polymorphisms: markers of cancer susceptibility?

The CDKN1A (TP21) gene encodes a 21-kD protein that is a critical downstream mediator of wild-type TP53 and an important regulator of the cell cycle. Failure in the function of this gene would be expected to result in abnormal cell proliferation and transformation. Tumor-associated mutations of the coding region of the TP21 are rare. On the other hand, some TP21 polymorphisms have been identified and characterized by single base substitutions. In the present study, we investigated the potential role of TP21 gene polymorphisms in skin, head, and neck tumorigenesis. A total of 261 samples were examined by polymerase chain reaction single-strand conformational analysis, and one mutation at codon 31 and four polymorphisms in exons 2 (codon 55) and 3 [nucleotide (nt)590] and in promoter region (nt2298) were identified. In conclusion, this investigation confirmed the rarity of mutations in this gene, arguing against a role for TP21 mutations in skin, head, and neck cancers. Also, our results show significant differences in nt2298 allele frequencies between normal individuals and skin malignant tumors (P < 0.05). The results suggest that this polymorphism affects TP21 transactivator binding and may be important during the pathogenesis of skin cancer.