A cysteine-activated protease isolated from Todarodes pacificus squid degrades collagen below its denaturation temperature

Collagen purified from the mantle muscle of the Japanese common squid, Todarodes pacificus, showed autodegradation during incubation under acidic conditions at 25 degrees C, without the addition of exogenous enzymes. This suggests that the collagenolytic proteases bind to collagen tightly through the steps of collagen preparation. Collagenolytic activity also was detected in a crude extract of mantle muscle, and leupeptin and E-64 were observed to inhibit collagenolytic activity within the collagen fraction and muscle extract. We purified these collagenolytic cysteine proteases by leupeptin column chromatography and cellulose acetate membrane electrophoresis. Optimal enzymatic activity was observed at pH 3.5, and collagenolytic activity was completely suppressed at neutral or alkaline pH. The purified enzymes were 28 kDa and 25 kDa in size, and both had gelatinolytic activity, as detected by gelatin zymography, and cut the specific site of denatured collagen alpha chain. The purified enzymes degraded squid collagen at 25 degrees C, which is 2.5 degrees lower than the temperature at which squid collagen normally denatures; however, the proteases were ineffective at 20 degrees C. Interestingly, the isolated proteases were capable of digesting both squid and bovine gelatin. In this article, we describe collagenolytic cysteine proteases that bind to the collagen of Todarodes pacificus, thereby digesting it by attacking microunfolding regions generated by incubation 2-3 degrees C below the denaturation temperature.     

Divergent expression and localization of aquaporin 5, an exocrine-type water channel,in the submandibular gland of Sprague-Dawley rats

By Western blot analysis, the expression level of aquaporin (AQP) 5 in the submandibular gland (SMG) was found to be different among individual rats of the Sprague-Dawley (SD) strain. Such differences were observed for AQP5 but not for AQP1 and consequently the SD strain was divided into two groups, one expressing a high level of AQP5 and the other a low one. The difference in average intensity of expression between the two groups was more than twofold. Immunohistochemical analysis of the SMG demonstrated that the AQP5 protein was localized in the basal and apical/lateral plasma membrane of acinar cells in rats expressing the high level of AQP5. In the rat expressing the low level, however, this channel protein was localized strongly in the apical/lateral plasma membrane, but only very weakly in the basal membrane of the acinar cells. Such a diverse localization of AQP5 was confirmed by Western blotting as well. Breeding between brother and sister was repeated for two times within high expressers and low expressers to obtain the third generation progenies (F2); the AQP5 level of the SMG in the third generation (F2 rats) from high expressers was significantly higher than the F2 from low expressers. Our present study suggests the existence of genetic variation in the expression of a water channel protein, AQP5, in rats.     

Activation of protease-activated receptor 2 stimulates proliferation and interleukin (IL)-6 and IL-8 secretion of endometriotic stromal cells

BACKGROUND: Inflammation has been proposed to play essential roles in the pathophysiology of endometriosis, in which neutrophils and mast cells have been suggested to be involved. We studied whether the protease-activated receptor 2 (PAR2), which is activated by enzymes from neutrophils and mast cells, in endometriotic stromal cells (ESC) has any implication in the development of the disease. METHODS: Cultured ESC were stimulated with various concentrations of a specific PAR2 agonist peptide. Proliferating activity of the cells was determined using immunostaining of proliferating cell nuclear antigen (a cell proliferation marker), 5-bromo-2'-deoxyuridine incorporation into DNA and cell count. The concentrations of interleukin (IL)-6 and IL-8 were measured using specific enzyme-linked immunosorbent assay kits. The phosphorylation of three mitogen-activated protein kinases (MAPK), i.e. p38 MAPK, p42/44 MAPK and stress-activated protein Kinase/c-jun N terminal Kinase, in ESC was examined with Western blot analysis. RESULTS: Activation of PAR2 stimulated the proliferation of ESC and the secretion of IL-6 and IL-8 from ESC in a dose-dependent manner. Activation of PAR2 stimulated the phosphorylation of all three MAPK, and inhibitors of each MAPK suppressed the PAR2 activation-induced proliferation of ESC. CONCLUSIONS: The activation of PAR2 in ESC may be involved in the pathophysiology of endometriosis by inducing the growth and inflammation of endometriotic lesions.     

Regulation of Fungal Infection by a Combination of Amphotericin B and Peptide 2, a Lactoferrin Peptide That Activates Neutrophils

To establish a novel strategy for the control of fungal infection, we examined the antifungal and neutrophil-activating activities of antimicrobial peptides. The duration of survival of 50% of mice injected with a lethal dose of Candida albicans (5 × 10⁸ cells) or Aspergillus fumigatus (1 × 10⁸ cells) was prolonged 3 to 5 days by the injection of 10 μg of peptide 2 (a lactoferrin peptide) and 10 μg of α-defensin 1 for five consecutive days and was prolonged 5 to 13 days by the injection of 0.1 μg of granulocyte-monocyte colony-stimulating factor (GM-CSF) and 0.5 μg of amphotericin B. When mice received a combined injection of peptide 2 (10 μg/day) with amphotericin B (0.5 μg/day) for 5 days after the lethal fungal inoculation, their survival was greatly prolonged and some mice continued to live for more than 5 weeks, although the effective doses of peptide 2 for 50 and 100% suppression of Candida or Aspergillus colony formation were about one-third and one-half those of amphotericin B, respectively. In vitro, peptide 2 as well as GM-CSF increased the Candida and Aspergillus killing activities of neutrophils, but peptides such as α-defensin 1, β-defensin 2, and histatin 5 did not upregulate the killing activity. GM-CSF together with peptide 2 but not other peptides enhanced the production of superoxide (O₂⁻) by neutrophils. The upregulation by peptide 2 was confirmed by the activation of the O₂⁻-generating pathway, i.e., activation of large-molecule guanine binding protein, phosphatidyl-inositol 3-kinase, protein kinase C, and p47^phox as well as p67^phox. In conclusion, different from natural antimicrobial peptides, peptide 2 has a potent neutrophil-activating effect which could be advantageous for its clinical use in combination with antifungal drugs.     

Polymerization mechanism of N-carboxy-α-amino acid anhydride by organometallic compounds. II.. Reaction with organozinc compounds

Reactions of N-carboxy-α-amino acid anhydride (NCA) with dialkylzinc or related organozinc compounds were studied to elucidate the polymerization mechanism of NCA by dialkylzinc as initiator. The first stage of initiation reaction is a hydrogen abstraction reaction of dialkylzinc from NH group of α-amino acid NCA resulting in the formation of an activated NCA. The second stage of initiation is a reaction between two molecules of the activated NCA forming a zinc carbamate group. Propagation reaction is a carbonyl addition of the zinc carbamate group to the activated NCA to form a mixed anhydride which changes into an amide group releasing carbon dioxide. Regeneration of the activated NCA is supposed to be done by the reaction of free α-amino acid NCA with the zinc atom bonded to nitrogen atom at the growing chain end.     

Dominant-Negative Rho, Rac, and Cdc42 Facilitate the Invasion Process of Vibrio parahaemolyticus into Caco-2 Cells

To clarify the invasive process of Vibrio parahaemolyticus, an invasion assay was performed using cells expressing dominant negative small GTPases of the Rho family. This assay showed that the dominant negative host phenotype facilitates bacterial invasion, suggesting that the mechanism of V. parahaemolyticus invasion differs from that reported for other invasive bacteria.     

Signal transduction in human B cells initiated via Ig{beta} ligation

Ig and Igß heterodimers are non-covalently associatedwith Ig to compose the antigen receptor complexes on B cells.The demonstration that different sets of tyrosine kinases bindto the cytoplasmic tails of Ig and Igß suggests thatIg and Igß may activate distinct second messengerpathways. In this study, we examined the effects of mAbs againstan exposed epitope of human Igß on pre-B and B celltriggering. Cross-linkage of Igß on B cells leadsto activation of tyrosine kinases, hydrolysis of phosphatidylinositides,and elevation of intracellular Ca²⁺, effects qualitatively identicalto those of anti-_µ mAbs. Our observations thus indicatethat cross-linking of Igß does not segregate signaltransduction pathways connected with the cytoplasmic talls ofIg and Igß. Ig ligation has been reported to be moreeffective in triggering pre-B than B cells, whereas our resultsindicated that Igß ligation is more efficient in triggeringB than pre-B cells. In addition to their activation properties,the anti-Igß mAbs effectively modulated B cell receptorcomplexes and blocked terminal differentiation of all plasmacell isotypes. The findings support the idea that anti-Igßcould serve as a universal B cell immunosuppressant.     

Synphilin-1 transgenic mice exhibit mild motor impairments

Synphilin-1 represents a cytoplasmic protein that interacts with α-synuclein and localizes close to synaptic vesicles. The interaction of synphilin-1 with several proteins involved in Parkinson's disease suggests that it might be involved in the pathogenesis of the disease. Nonetheless, the function of synphilin-1 remains unclear. In the present study, we generated transgenic mice expressing human synphilin-1 under the prion protein promoter. Synphilin-1 was widely expressed in neurons in the brain including the substantia nigra, where massive loss of dopamine neurons was not observed. In the transgenic mouse brain, synphilin-1 protein was polyubiquitinated, and partially insoluble. Although modified-SHIRPA revealed no significant difference in behavior and morphology, the reduced rotarod performance and step length were observed in transgenic mice as compared with non-transgenic littermates. Synphilin-1 might be involved in motor function, and its accumulation in the central nervous system can cause motor impairments.     

Neurotoxicity of Clostridium perfringens Epsilon-Toxin for the Rat Hippocampus via the Glutamatergic System

The neurotoxicity of epsilon-toxin, one of the major lethal toxins produced by Clostridium perfringens type B, was studied by histological examination of the rat brain. When the toxin was injected intravenously at a lethal dose (100 ng/kg), neuronal damage was observed in many areas of the brain. Injection of the toxin at a sublethal dose (50 ng/kg) caused neuronal damage predominantly in the hippocampus: pyramidal cells in the hippocampus showed marked shrinkage and karyopyknosis, or so-called dark cells. The dark cells lost the immunoreactivity to microtubule-associated protein-2, a postsynaptic somal and dendric marker, while acetylcholinesterase-positive fibers were not affected. Timm’s zinc staining revealed that zinc ions were depleted in the mossy layers of the CA3 subfield containing glutamate as a synaptic transmitter. The cerebral blood flow in the hippocampus was not altered significantly before or after administration of the toxin, as measured by laser-Doppler flowmetry, excluding the possibility that the observed histological change was due to a secondary effect of ischemia in the hippocampus. Prior injection of either a glutamate release inhibitor or a glutamate receptor antagonist protected the hippocampus from the neuronal damage caused by epsilon-toxin. These results suggest that epsilon-toxin acts on the glutamatergic system and evokes excessive release of glutamate, leading to neuronal damage.