Theα isoform of protein kinase C inhibits histamine-stimulated adenylate cyclase activity in a particulate fraction of the human gastric cancer cell line HGT-1

The isoform of protein kinase C responsible for the inhibition of histamine-stimulated adenylate cyclase by the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), has been investigated in a particulate fraction prepared from the human gastric cancer cell line HGT-1. The and isoforms of protein kinase C were detected in HGT-1 cells and in a 40,000 × g particulate fraction by immunoblotting procedures. The inhibitory effect of TPA on histamine-stimulated adenylate cyclase was enhanced by the presence of Ca²⁺, but decreased in a concentrationdependent manner by anti-peptide antibody to protein kinase C, but not to protein kinase C. Addition of Ca²⁺ and TPA to the 40,000 × g particulate fraction stimulated the phosphorylation of the protein kinase C substrate myelin basic peptide 4–14. Protein kinase C is probably the isoform responsible for inhibition of histamine-stimulated adenylate cyclase in HGT-1 cells.     

Is recombinant human erythropoietin (rh-epo) more than just a treatment of anemia in cancer and chemotherapy?

Recombinant human eythropoietin (rh-epo) is a well established treatment for many kinds of anemia including the anemia of cancer with or without myelosuppressive chemotherapy.This review considers the effects of rh-epo in humans, tumour-bearing and healthy experimental animals treated with cisplatin with or without rh-epo, and proposes that the ability of rh-epo to improve the quality of life in cancer patients may also be due to interference with the prostaglandin pathways.     

Loss of Arnt (Hif1β) in mouse epidermis triggers dermal angiogenesis, blood vessel dilation and clotting defects

Targeted ablation of Aryl hydrocarbon receptor nuclear translocator (Arnt) in the mouse epidermis results in severe abnormalities in dermal vasculature reminiscent of petechia induced in human skin by anticoagulants or certain genetic disorders. Lack of Arnt leads to downregulation of Egln3/Phd3 hydroxylase and concomitant hypoxia-independent stabilization of hypoxia-induced factor 1α (Hif1α) along with compensatory induction of Arnt2. Ectopic induction of Arnt2 results in its heterodimerization with stabilized Hif1α and is associated with activation of genes coding for secreted proteins implicated in control of angiogenesis, coagulation, vasodilation and blood vessel permeability such as S100a8/S100a9, S100a10, Serpine1, Defb3, Socs3, Cxcl1 and Thbd. Since ARNT and ARNT2 heterodimers with HIF1α are known to have different (yet overlapping) downstream targets our findings suggest that loss of Arnt in the epidermis activates an aberrant paracrine regulatory pathway responsible for dermal vascular phenotype in K14-Arnt KO mice. This assumption is supported by a significant decline of von Willebrand factor in dermal vasculature of these mice where Arnt level remains normal. Given the essential role of ARNT in the adaptive response to environmental stress and striking similarity between skin vascular phenotype in K14-Arnt KO mice and specific vascular features of tumour stroma and psoriatic skin, we believe that further characterization of Arnt-dependent epidermal-dermal signalling may provide insight into the role of macro- and micro-environmental factors in control of skin vasculature and in pathogenesis of environmentally modulated skin disorders.     

Error-free replicative bypass of (6–4) photoproducts by DNA polymerase ζ in mouse and human cells

The ultraviolet (UV)-induced (6–4) pyrimidine–pyrimidone photoproduct [(6–4) PP] confers a large structural distortion in DNA. Here we examine in human cells the roles of translesion synthesis (TLS) DNA polymerases (Pols) in promoting replication through a (6–4) TT photoproduct carried on a duplex plasmid where bidirectional replication initiates from an origin of replication. We show that TLS contributes to a large fraction of lesion bypass and that it is mostly error-free. We find that, whereas Pol η and Pol ι provide alternate pathways for mutagenic TLS, surprisingly, Pol ζ functions independently of these Pols and in a predominantly error-free manner. We verify and extend these observations in mouse cells and conclude that, in human cells, TLS during replication can be markedly error-free even opposite a highly distorting DNA lesion.     

Inhibitory activity on matrix metalloproteinases and cell-proliferating activity of tissue inhibitor of metalloproteinases-1 (timp-1)—contrastive difference between human and bovine TIMP-1s on mouse cell proliferation

Either human or bovine TIMP-1 inhibited matrix metalloproteinase (MMP) activities, such as collagenolytic, gelatinolytic and caseinolytic, expressed by mouse cells as well as those expressed by human cells. Bovine TIMP-1 stimulated the proliferation of both human and mouse cells, but human TIMP-1 only proliferate human cells and not mouse cells indicating that the cell-proliferating activity has more strict species specificity than MMP inhibitory activity. All the results from [³H]thymidine incorporation, the phosphorylation of tyrosine-containing cellular proteins and extracellular-signal-regulated protein kinases supported the cell-proliferating properties of both human and bovine TIMP-1s. Human TIMP-1 seems not to bind to TIMP-1 receptors on mouse cells.     

Assignment of the gene encoding type 1γ protein phosphatase catalytic subunit (PPP1CC) on human,rat, and mouse chromosomes

Summary Using fluorescentin situ hybridization (FISH) method, a gene encoding the catalytic subunit of protein phosphatase type 1 (PPP1CC) was mapped to human 12q24.1-q24.2, rat 7q22, and mouse 10C. These results indicate that thePPP1CC is a member of conserved synteny group between rat chromosome 7, mouse chromosome 10 and human chromosome 12. These data and mapping data about other members of PP1 family show that in spite of the high identity of PP1 isoforms, each isoform is encoded by different genes which located on different chromosomes in human, rat, and mouse.     

Memantine treatment decreases levels of secreted Alzheimer's amyloid precursor protein (APP) and amyloid beta (Aβ) peptide in the human neuroblastoma cells

Memantine, an uncompetitive NMDA receptor antagonist, is a FDA-approved drug used for the treatment of moderate-to-severe Alzheimer's disease (AD). Several studies have documented protective roles of memantine against amyloid beta (Aβ) peptide-mediated damage to neurons in both in vitro and in vivo models. Memantine is also effective in reducing amyloid burden in the brain of APP transgenic mice. However, the exact mechanism by which memantine provides protection against Aβ-mediated neurodegenerative cascade, including APP metabolism, remains to be elucidated. Herein, we investigated the effect of memantine on levels of the secreted form of Aβ precursor protein (APP), secreted Aβ and cell viability markers under short/acute conditions. We treated neuronal SK-N-SH cells with 10 μM memantine and measured levels of secreted total APP (sAPP), APPα isoform and Aβ_(1–40) in a time dependent manner for up to 24 h. Memantine significantly decreased the levels of the secreted form of sAPP, sAPPα and Aβ_(1–40) compared to vehicle treated cells. This change started as early as 8 h and continued for up to 24 h of drug treatment. Unlike sAPP, a slight non-significant increase in total intracellular APP level was observed in 24-h treated memantine cells. Taken together, these results suggest a role for memantine in the transport or trafficking of APP molecules away from the site of their proteolytic cleavage by the secretase enzymes. Such a novel property of memantine warrants further study to define its therapeutic utility.     

Phagocytic activity of neutrophils and lymphocytes at various periods of storage in the state of cold anabiosis (-40°C)

We studied changes in phagocytic activity of neutrophils and lymphocytes subjected to cold anabiosis. The proposed effective method for introduction of human blood leukocytes into cold anabiosis at moderately low temperature (-40°C) in the presence of a cryopreserving agent does not require washout of the biological material after defrosting.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 10, pp. 400–402, October, 2004     

Voltage-gated calcium channel CACNB2 (β2.1) protein is required in the heart for control of cell proliferation and heart tube integrity

Background : L‐type calcium channels (LTCC) regulate calcium entry into cardiomyocytes. CACNB2 (β2) LTCC auxiliary subunits traffic the pore‐forming CACNA subunit to the membrane and modulate channel kinetics. β2 is a membrane associated guanylate kinase (MAGUK) protein. A major role of MAGUK proteins is to scaffold cellular junctions and multiprotein complexes. Results: To investigate developmental functions for β2.1, we depleted it in zebrafish using morpholinos. β2.1‐depleted embryos developed compromised cardiac function by 48 hr postfertilization, which was ultimately lethal. β2.1 contractility defects were mimicked by pharmacological depression of LTCC, and rescued by LTCC stimulation, suggesting β2.1 phenotypes are at least in part LTCC‐dependent. Morphological studies indicated that β2.1 contributes to heart size by regulating the rate of ventricle cell proliferation, and by modulating the transition of outer curvature cells to an elongated cell shape during chamber ballooning. In addition, β2.1‐depleted cardiomyocytes failed to accumulate N‐cadherin at the membrane, and dissociated easily from neighboring myocytes under stress. Conclusions : Hence, we propose that β2.1 may also function in the heart as a MAGUK scaffolding unit to maintain N‐cadherin‐based adherens junctions and heart tube integrity. Developmental Dynamics 241:648–662, 2012. © 2012 Wiley Periodicals Inc.     

Immunohistological localisation of human FAT1 (hFAT) protein in 326 breast cancers. Does this adhesion molecule have a role in pathogenesis?

AIMS: To examine the immunohistological expression in human breast cancers of human FAT1 (hFAT) protein, a recently described member of the cadherin superfamily, and its correlation with histological type and grade. METHODS: A total of 326 cases of invasive and in situ breast cancer representing a broad spectrum of histological subtypes were immunostained with affinity-purified rabbit antibodies produced to the cytoplasmic region of hFAT using a standard avidin-biotin system. Staining intensity was arbitrarily graded on a scale of 0 to 3. RESULTS: All tumours showed diffuse staining for hFAT. Immunoexpression of the protein was generally strong in both lobular (LCIS, n = 2) and ductal in situ carcinoma (DCIS, n = 55). hFAT was also strongly immunoexpressed in all types of invasive carcinoma. Grade 3 DCIS displayed the highest hFAT intensity compared with lower grade tumours, with significant differences between grade 1 and 3 (p = 0.015) and grade 2 and 3 (p = 0.047). With invasive ductal carcinomas (n = 128) the difference was not as clear-cut, as most tumours showed moderate (n = 63) or strong staining (n = 49), although grade 3 IDC revealed significantly decreased immunoexpression compared with grade 1 IDC (p = 0.03). CONCLUSIONS: The results illustrate that hFAT1 does not display the pattern of expression seen with the E-cadherin-ss-catenin adhesion complex; however, its over-expression and diffuse expression in both in situ and invasive carcinoma strongly suggests a role in carcinogenesis. From the known functions of FAT1 it is suggested that the concurrent loss of classical cadherins from cell-cell junctions accompanied by increased FAT1 expression contributes to loss of duct formation, and increased cell migration and invasion.     

Different detectability of cyclooxygenase-2 (COX-2) protein in standard paraffin sections and tissue microarrays of human melanomas and naevi – Comparative study

Cyclooxygenase-2 (COX-2), overexpressed in many types of human cancer, may be a valuable marker for human melanoma. However, there are discrepancies between expression levels detected by different groups. The majority of the studies were carried out using standard paraffin sections. Tissue microarrays (TMAs) might enable analysis of COX-2 expression in numerous lesions. Our study assesses to what extent reprocessing of tissue samples used for preparing TMAs may influence reproducibility of data obtained for standard sections.     

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-β) expression

Parvoviridae is a family of small non-enveloped viruses and divided into two subfamilies. The family members infect a wide range of organisms from insects to humans and some of the members (e.g., nonpathogenic adeno-associated viruses) are effective gene therapy delivery vectors. We detailed the synonymous codon usage pattern of Parvoviridae family from the available 58 sequenced genomes through multivariate statistical methods. Our results revealed that nine viruses showed some degree of strong codon bias, and the others possessed a general weak trend of codon bias. ENc-plot and neutrality plot results showed that selective pressure dominated over mutation in shapes coding sequence’s composition. The overall GC content and GC content at the third synonymous codon position were the principal determinants behind the variations within the codon usage patterns, as they both significantly correlated with the first axis of correspondence analysis. In addition, gene length had no direct influence on the codon usage pattern. Densovirinae subfamily and Parvovirinae subfamily possessed nine identical preferred codons, though most of the two subfamilies codon usage frequencies were significantly different. The result of cluster analysis based on synonymous codon usage was discordant with that of taxonomic classification. Adeno-associated viruses formed a separated clade far from other Parvoviridae members in the dendrogram. Thus, we concluded that natural selection rather than mutation pressure accounts for the main factor that affects the codon bias in Parvoviridae family.     

Validation of the Candida albicans delayed-type hypersensitivity (DTH) model in the female B₆C₃F₁ mouse for use in immunotoxicological investigations

Although numerous models are used to evaluate the immunotoxic effects of xenobiotics on cell-mediated immunity (CMI), no holistic model for evaluating such effects on the delayed-type hypersensitivity (DTH) response has gained widespread acceptance. Due to a lack of interference from antigen-specific antibody production, the Candida albicans DTH model has recently been demonstrated to be a more appropriate model for assessing effects on CMI than other DTH models that utilize different sensitizing antigens, such as sheep erythrocytes (SRBC) or keyhole limpet hemocyanin (KLH). The present studies were conducted to validate the C. albicans DTH model for its ability to detect suppression (or the lack thereof) of CMI following exposure for 28 days to well-characterized immunosuppressive drugs, each having a different mechanism of action. The compounds evaluated included azathioprine (AZA), cyclophosphamide (CPS), cyclosporin A (CSA), dexamethasone (DEX), and the non-immunotoxic compound, benzo[e]pyrene (B[e]P). Exposure to each of the four known immunotoxicants resulted in statistically significant decreases in the DTH response to C. albicans. Footpad swelling was decreased following exposure to AZA at ≥ 20?mg/kg but not at 10?mg/kg, CPS at ≥ 10?mg/kg but not at 5?mg/kg, CSA at ≥ 3?mg/kg but not at 1?mg/kg, or DEX at ≥ 0.3?mg/kg (intermittently at 0.1?mg/kg) but not at 0.03?mg/kg. As expected, exposure to B[e]P for 28 days at doses up to 40?mg/kg had no effect on the DTH response. These results demonstrated that the C. albicans DTH assay in the B?C?F? mouse was capable of appropriately classifying each test article as to its immunotoxic effects on CMI. Furthermore, comparisons of these results with previous reports of effects on ex vivo CMI end points suggest that this DTH assay may be more sensitive than standard ex vivo assays at detecting immunosuppressive effects.     

Novel first-dose adverse drug reactions during a phase I trial of olipudase alfa (recombinant human acid sphingomyelinase) in adults with Niemann–Pick disease type B (acid sphingomyelinase deficiency)

PurposeEnzyme replacement therapy with olipudase alfa (recombinant human acid sphingomyelinase) is being developed for Niemann–Pick disease type B (NPD B).MethodsA single-center, open-label, nonrandomized, single-ascending-dose trial evaluated the safety of intravenous olipudase alfa (0.03–1.0 mg/kg) in 11 adults with NPD B. Patients were monitored in the hospital for 72 h after infusion and had follow-up visits on days 14 and 28.ResultsPlasma ceramide, a product of sphingomyelin catabolism by olipudase alfa, showed dose-dependent elevations by 6 h postdose, or postinfusion. No serious adverse drug reactions (ADRs) occurred during the study. Acute phase reaction-type ADRs, as evidenced by elevated inflammatory biomarkers (high-sensitivity C-reactive protein, interleukin-8, and calcitonin) and constitutional symptoms (fever, pain, nausea, and/or vomiting) emerged 12–24 h following doses ≥0.3 mg/kg olipudase alfa. Three patients experienced hyperbilirubinemia. The study was terminated after a patient dosed at 1 mg/kg exhibited severe hyperbilirubinemia; he was subsequently diagnosed with Gilbert syndrome.ConclusionThe maximum tolerated dose of olipudase alfa in adults with NPD B was 0.6 mg/kg. First-dose ADRs were likely induced by elevated concentrations of ceramide (or its downstream derivatives) generated by the catabolism of accumulated sphingomyelin. Within-patient dose escalation to slowly catabolize sphingomyelin stores may be a strategy to mitigate first-dose ADRs in patients with NPD B.