Molecular and biochemical mechanisms ofPasteurella haemolyticaleukotoxin-induced cell death

Pasteurella haemolyticaleukotoxin (LKT) is a member of the RTX family of pore-forming toxins that kill bovine immune cells. Several studies have suggested that RTX toxins kill target cells by the induction of apoptosis. In the present study, BL3 bovine leukaemia cells were exposed to LKT and assessed by molecular and flow cytometric techniques that measure different aspects of apoptotic cell death. The intoxicated cells demonstrated morphological, light scatter and Hoechst 33258 staining characteristics consistent with cells undergoing apoptosis. The cells also exhibited internucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, both indicators of apoptosis. LKT-treated cells bound annexin-V-FITC indicating that phosphatidylserine groups were translocated from the inner to the outer leaflet of the cell membrane. The effect of LKT on cells was dose dependent and inhibitable by incubation with anti-LKT monoclonal antibody. Finally, an early step for induction of apoptosis appears to be the binding of LKT to a β2 integrin since pre-incubating cells with anti-β2 integrin antibodies inhibited LKT-induced apoptosis. This study provides new insights into understanding the pathogenesis of bovine pasteurellosis and could lead to the development of both preventative and therapeutic strategies for disease management.     

A second group of hepatitis C viruses

cDNA clone 11–7 was isolated by immunoscreening a cDNA library that was prepared from a pooled plasma of non-A non-B hepatitis (NANBH) patients using expression vector gt 11. This cDNA corresponds to known nucleotide positions 3983–4745 of the genome of hepatitis C virus (HCV). This clone was used as a probe for screening the HCV-related cDNAs in a cDNA library similarly prepared by using gt 10. As a result, six more cDNA clones were isolated and analyzed for their nucleotide sequences. The results strongly suggested that there are at least two groups of HCV, group I and group II. According to our classification, the prototype HCV and clone 11–7 belong to group I HCV, and their nucleotide and deduced amino acid sequences were diverged from those of group II HCV. Genetic variation observed in the nucleotide and the amino acid sequences between the two groups resembles that in the NS3 region of the genome between Japanese encephalitis virus and West Nile fever virus. Polypeptides produced inEscherichia coli carrying a clone 11–7 or a group II cDNA clone E reacted with antibodies in the blood of 12 or 4 out of 14 individual chronic NANBH patients, respectively. Our data clearly indicate the existence of a second group of HCV.     

Pancreatic endocrine tumours associated with WDHA syndrome

Summary Nine pancreatic endocrine tumours of patients with watery diarrhoea hypokalaemia achlorhydria (WDHA) syndrome were examined by immunohistochemistry and electron microscopy. All cases revealed neoplastic proliferation of VIP (vasoactive intestinal peptide)-immunoreactive (IR) cells. Immunoreactivity to a novel peptide hormone PHM-27, which is processed from a common big precursor peptide of VIP (prepro VIP/PHM-27), was identified in VIP-IR cells of 8 tumours. VIP-PHM-IR cells had secretory granules measuring about 130 to 220 nm in diameter. Radioimmunoassay of tumour tissue extracts showed high VIP and PHM contents in proportional amounts in most cases. According to the results of immunostaining, the 8 tumours fell into two large groups; 5 with PP (pancreatic polypeptide)-IR cells and 3 with CT (calcitonin)-IR cells. The former group demonstrated VIP cells and PP cells intermingled in various proportions, including one tumour in which coexistence of PP-IR and VIP-IR in the same cells was demonstrated. Cell heterogeneity of the tumours and possible relationships of VIP, PP and CT cells were discussed.This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and from the Ministry of Education, Science and Culture, Japan     

Selective expression of L-serine synthetic enzyme 3PGDH in schwann cells, perineuronal glia, and endoneurial fibroblasts along rat sciatic nerves and its upregulation after crush injury

Non-essential amino acid L-serine functions as a highly potent, glia-derived neurotrophic factor, because it is a precursor for syntheses of proteins, other amino acids, membrane lipids, and nucleotides, and also because its biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH) is preferentially expressed in particular glial cells within the brain. Here we pursued 3PGDH expression in peripheral nerves and its change after crush injury. In the pathway of rat sciatic nerves, 3PGDH was selectively expressed in non-neuronal elements: Schwann sheaths and endoneurial fibroblasts in sciatic nerves, satellite cells in dorsal root ganglia, and astrocytes and oligodendrocytes in the spinal ventral horn. In contrast, 3PGDH was immunonegative in axons, somata of spinal motoneurons and ganglion cells, and endoneurial macrophages. One week after crush injury, 3PGDH was upregulated in the distal segment of injured nerves, where 3PGDH was intensified in activated Schwann cells and fibroblasts. 3PGDH was still negative in activated macrophages, which were instead associated or surrounded by activated Schwann cells with intensified 3PGDH. These results suggest that in the peripheral nervous system, these non-neuronal cells synthesize and may supply L-serine to satisfy metabolic demands for maintenance and regeneration of peripheral nerves and for proliferation and activation of macrophages upon nerve injury.     

Morphometric comparison of human nerve cells: pyramidal motor system

We compared morphological and morphometric data on various motor neurons in the human pyramidal system using the modified Klüver-Barrera staining method with extremely minimized shrinkage ratio and an image-analyzer. We classified motor neurons in the human pyramidal system into three groups according to the measurement data. This report may be of interest to better understand the process of nerve conduction in the human pyramidal system.     

The plasminogen activation system reduces fibrosis in the lung by a hepatocyte growth factor-dependent mechanism

Mice deficient in the plasminogen activator inhibitor-1 gene (PAI-1-/- mice) are relatively protected from developing pulmonary fibrosis from bleomycin administration. We hypothesized that one of the protective mechanisms may be the ability of the plasminogen system to enhance hepatocyte growth factor (HGF) effects, which have been reported to be anti-fibrotic in the lung. HGF is known to be sequestered in tissues by binding to extracellular matrix components. Following bleomycin administration, we found that HGF protein levels were higher in bronchoalveolar lavage fluid from PAI-1-/- mice compared to wild-type (PAI-1+/+) mice. This increase could be suppressed by administering tranexamic acid, which inhibits plasmin activity. Conversely, intratracheal instillation of urokinase into bleomycin-injured PAI-1+/+ mice to activate plasminogen caused a significant increase in HGF within bronchoalveolar lavage and caused less collagen accumulation in the lungs. Administration of an anti-HGF neutralizing antibody markedly increased collagen accumulation in the lungs of bleomycin-injured PAI-1-/- mice. These results support the hypothesis that increasing the availability of HGF, possibly by enhancing its release from extracellular matrix by a plasmin-dependent mechanism, is an important means by which activation of the plasminogen system can limit pulmonary fibrosis.     

REACTIVE LYMPHOID HYPERPLASIA OF THE STOMACH

The surgically excised stomachs were re-examined histopathologically, and eighteen cases were placed in the category of reactive lymphoid hyperplasia (RLH). The distribution and the classes of infiltrating immunoglobulin (Ig) bearing cells were examined on lesions of RLH cases together with ten histopathologically determined malignant lymphomas (ML) of the stomach and the control stomachs. It was found that in eleven cases of RLH and one case of ML, many lymphoid cells bearing different classes of Ig were present in those lesions in an intermingled way (polyspecific group). Meanwhile, lymphoid cells in three RLH cases and two ML cases bore only a single mono-specific Ig (monospecific group). In other cases, the number of Ig bearing cells were not sufficient to reach any clear conclusions (undetermined group). It was speculated that regardless of the histopathological diagnosis, the mono-specific group might belong to the category of neoplasm of B cell type and the polyspecific group in the category of true reactive process. The possible histopathological criteria for differentiation of the reactive process and lymphoid neoplasm of the stomach were re-checked, and the importance of immunohistochemical study on these cases were stressed.