Modulation of the expression of GABA(A) receptors in rat cerebellar granule cells by protein tyrosine kinases and protein kinase C

The expression of GABA(A) receptors in rat cerebellar granules in culture has been studied by beta(2/3) subunit immunocytochemistry and fluorescence confocal microscopy. These cells show labeling all over the cell bodies' plasma membrane and dendrites. Treatment with the protein tyrosine kinase (PTK) inhibitor genistein results in a decrease of the labeling associated with the beta(2/3) subunit in both cell bodies and dendrites. No effect was found with an inactive genistein analogue, daidzein. A similar effect was found with a protein kinase C (PKC) activator, phorbol myristate acetate (PMA). The effects of genistein and PMA are additive.The interpretation of the results is that PTK inhibition blocks exocytotic deposit of newly synthesized GABA(A) receptors onto the neuronal plasma membrane. On the other hand, PKC activation speeds up endocytotic removal of GABA(A) receptors.     

Evaluation of the surface-averaged load exerted on a blood element by the Reynolds shear stress field provided by artificial cardiovascular devices

Implantable prosthetic devices can often affect the recipient's hemostasis, with possible hemolysis and thrombus formation. Since such devices can produce turbulent flow, it is important to characterize it as accurately as possible, by means of the Reynolds stress tensor. Some parameters related to the latter have been often used to provide a quantity related to the possible damage to blood constituents: the TSS_max, for instance, has been associated with hemolysis. It can be expressed as TSS_max=(σ₁−σ₃)/2, σ₁ and σ₃ being the highest and lowest principal normal stresses (PNSs) in each point of the flow.

In the present work, the average value of the shear stress over a spherical surface, representative of a blood component, is derived. All three PNSs (σ₁, σ₂ and σ₃) are found to have an equal role in the determination of this parameter, since the relative formula shows a marked symmetry with respect to the PNSs. The average shear stress level, for a given (σ₁, σ₃) pair (hence, for a given TSS_max), has a minimum and maximum value, depending on the particular σ₂ value yielded by the local structure of the turbulent flow field. A numerical investigation on more complex geometries shows similar results. The role of the intermediate PNS is thus shown for the first time to have a physical relevance. The presented results can be useful whenever a spatial averaging of the shear field is important to be assessed, such as in the case of platelet activation. A new parameter is thus proposed, which can be correlated with prosthetic devices complications.     

Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.     

Hyaluronan-CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9

Osteoclast (OC) precursors migrate to putative sites of bone resorption to form functionally active, multinucleated cells. The preOC FLG 29.1 cells, known to be capable of irreversibly differentiating into multinucleated OC-like cells, displayed several features of primary OCs, including expression of specific integrins and the hyaluronan (HA) receptor CD44. OC-like FLG 29.1 cells adhered to and extensively migrated through membranes coated with fibronectin, vitronectin, and laminins, but, although strongly binding to HA, totally failed to move on this substrate. Moreover, soluble HA strongly inhibited OC-like FLG 29.1 cell migration on the permissive matrix substrates, and this behavior was dependent on its engagement with CD44, as it was fully restored by function-blocking anti-CD44 antibodies. HA did not modulate the cell-substrate binding affinity/avidity nor the expression levels of the corresponding integrins. MMP-9 was the major secreted metalloproteinase used by OC-like FLG 29.1 cells for migration, because this process was strongly inhibited by both TIMP-1 and GM6001, as well as by MMP-9-specific antisense oligonucleotides. After HA binding to CD44, a strong down-regulation of MMP-9 mRNA and protein was detected. These findings highlight a novel role of the HA-CD44 interaction in the context of OC-like cell motility, suggesting that it may act as a stop signal for bone-resorbing cells.     

Atrial natriuretic peptide stimulates cat carotid body chemoreceptors in vivo

It is known that atrial natriuretic peptide (ANP) is released from cardiac myocyte and other stores during hypoxia and is involved in pulmonary-cardiovascular reflexes and in natriuresis and diuresis. Since the carotid body initiates hypoxic chemoreflexes, we hypothesized that ANP could potentiate the hypoxic stimulation of the carotid body chemoreceptor in vivo. We studied the effect of close intra-arterial injection of ANP on carotid chemoreceptor activity in anesthetized male cats which were paralyzed and artificially ventilated. Graded doses of ANP (0-10 nmoles) were administered by intra-arterial injections and they produced an excitatory response. Single dose of ANP (6.5 nmoles) at four steady-state levels of arterial PO(2), at constant PCO(2), produced increases of chemoreceptor activity. This increase of chemoreceptor activity with ANP in the presence of CO(2)-HCO(3)(-) in vitro could make a difference from those without CO(2)-HCO(3)(-) in vivo.     

Human herpesvirus 8 (HHV-8/KSHV) and hematologic malignancies

Human herpesvirus 8 (HHV-8), also defined Kaposi's sarcoma (KS)-associated herpesvirus, was identified by Chang and colleagues in 1994 using purely molecular techniques, before any serological evidence or virus isolation in cell culture could be achieved. HHV-8 is unique among herpesviruses because its prevalence in the general population is low and because it possesses the richest weaponry of viral oncogenes and tumor-promoting factors ever described. Eleven HHV-8-specific genes are homologs of cellular genes, which were hijacked from the host during a long parallel evolution, and at least five of such genes show both in vitro and in vivo transforming ability. HHV-8 is the causative agent of KS, but it has also been associated with different hematologic malignancies, including primary effusion lymphoma (PEL), multicentric Castelman's disease (MCD), MCD-related immunoblastic/plasmablastic lymphoma and various atypical lymphoproliferative disorders. Although low-level silent infection was detected in bone marrow stromal cells from patients with multiple myeloma, a role of HHV-8 in this disease is unlikely. As seen with KS, the incidence of HHV-8-associated lymphoproliferative disorders is increased in the setting of human immunodeficiency virus infection.     

Glycosylation enhances functional stability of the chemotactic cytokine CCL2

The human chemokine CCL2 gene was expressed in the yeast P.pastoris and gave rise to a mixture of differently glycosylated recombinant proteins. In comparison to non-glycosylated E.coli-derived CCL2, glycosylated yeast CCL2L was 4-20 times less active in a chemotactic assay in vitro. However, CCL2L could maintain full activity upon prolonged incubation at 37 degrees C, whereas the non-glycosylated chemokine readily lost activity. It could be hypothesized that glycosylation is a mechanism used by the organism to modulate CCL2 stability. The partial loss of specific activity due to glycosylation is balanced by the advantage of prolonging the effectiveness of chemokine. Thus, differential glycosylation allows one to obtain highly effective short-lived CCL2 or less-effective long-lived CCL2 and may thus represent a novel mechanism of adaptation to pathological versus physiological conditions.     

Primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin

The proteinase inhibitor WSCI, active in inhibiting bacterial subtilisin and a number of animal chymotrypsins, was purified from endosperm of exaploid wheat (Triticum aestivum, c.v. San Pastore) by ion exchange chromatography and its complete amino acid sequence was established by automated Edman degradation. WSCI consists of a single polypeptide chain of 72 amino acid residues, has a molecular mass of 8126.3 Da and a pl of 5.8. The inhibition constants (Ki) for Bacillus licheniformis subtilisin and bovine pancreatic alpha-chymotrypsin are 3.92 x 10(-9) M and 7.24 x 10(-9) M, respectively. The inhibitor contains one methionine and of tryptophan residue and has a high content of essential amino acids (41 over a total of 72 residues), but no cysteines. The primary structure of WSCI shows high similarity with barley subtilisin-chymotrypsin isoinhibitors of the Cl-2 type and with maize subtilisinchymotrypsin inhibitor MPI. Significant degrees of similarity were also found between sequences of WSCI and of other members of the potato inhibitor I family of the serine proteinase inhibitors. The wheat inhibitor WSCI has a single reactive site (the peptide bond between methionyl-48 and glutamyl-49 residues) as identified by affinity chromatography and sequence analysis.     

Is a 3-month course of treatment with Peg-interferon effective in acute HCV hepatitis?

From February 2002, all the consecutive patients referring to the Department of Infectious Diseases, University of Turin, who were diagnosed as having acute HCV hepatitis were included in a prospective cohort study to evaluate if a 3-month course of Peg-Interferon alpha-2b (1.5 microg/kg once weekly) is effective to decrease the risk of progression to chronic disease. ALT and HCV-RNA measurements were scheduled at week 4 and 12 during treatment, and 24 weeks after the end of therapy. As of April 2003, ten patients were enrolled in the study. As to HCV genotype, seven patients had type 1 and 3 type non-1. At entry, median HCV-RNA level was 129500 (range: 3000-3100000 copies/mL) and six patients were symptomatic. Treatment was given within 20 days (range: 8-30) of the ALT peak. All patients completing 4 weeks (n = 9) and 12 weeks of treatment (n = 7) had undetectable HCV-RNA. Five patients who completed the 24-week follow-up after the end of treatment had a sustained viral response with ALT levels within normal range. Therapy was well tolerated in all patients. Even if our data are not definitive, our results show that once-weekly administration of Peg-interferon alfa-2b in patients with acute HCV infection may be an effective and convenient regimen.