The amino acid sequence of the double-headed protein proteinase inhibitor from dog submandibular glands, I. Structural homology to the pancreatic secretory trypsin inhibitors (author's transl)]

The primary structure of the broad specificity proteinase inhibitor from dog submandibular glands was elucidated. The inhibitor consists of a single polypeptide chain of 117 amino acids which is folded into two domains (heads) connected by a peptide of three amino acid residues. Both domains I and II show a clear structural homology to each other as well as to the single-headed pancreatic secretory trypsin inhibitors (Kazal type). The trypsin reactive site (-Cys-Pro-Arg-Leu-His-Glx-Pro-Ile-Cys-) is located in domain I and the chymotrypsin reactive center (-Cys-Thr-Met-Asp-Tyr-Asx-Arg-Pro-Leu-Tyr-Cys-) in domain II, cf. the Figure. The inhibitor is thus double-headed with two independent reactive sites. Whereas head I is responsible for the inhibition of trypsin and plasmin, head II is responsible for the inhibition of chymotrypsin, subtilisin, elastase and probably also Aspergillus oryzae protease and pronase. Remarkably, the structural homology exists also to the single-headed acrosin-trypsin inhibitors from seminal plasma[12] and the Japanese quail inhibitor composed of three domains[13].     

Isolation and characterization of a virulent bacteriophage from Staphylococcus carnosus

Abstract A virulent bacteriophage, øSK311, was isolated from Staphylococcus carnosus , an organism used as a starter culture for the production of dry sausage. Electron microscopic studies revealed that this bacteriophage showed some morphological similarities with the Escherichia coli phages T4 and λ. The host range of øSK311 extends over 9 different staphylococcal species. A phage-resistant mutant of S. carnosus could be isolated. Cells of this mutant exhibited a capsule-like structure. The DNA of øSK311 possesses a G + C content of 31.4 mol% and appears to be highly modified.     

Sodium periodate stimulation of human lymphocytes : A comparison between normal and chronic lymphocytic leukemia lymphocytes

Lymphocytes from healthy donors and from patients with chronic lymphocytic leukemia (CLL) were stimulated to divide with sodium periodate. The time of maximal response of normal lymphocytes to sodium periodate (NaIO₄) was earlier than that observed to phytohemagglutinin (PHA), but the magnitude was lower. In comparison, CLL lymphocytes responded to NaIO₄ more extensively and earlier than to PHA.     

Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium

Alternative splicing of ryanodine receptor subtype 3 (RYR3) may generate a short isoform (RYR3S) without channel function and a functional full-length isoform (RYR3L). The RYR3S isoform has been shown to negatively regulate the native RYR2 subtype in smooth muscle cells as well as the RYR3L isoform when both isoforms were coexpressed in HEK-293 cells. Mouse myometrium expresses only the RYR3 subtype, but the role of RYR3 isoforms obtained by alternative splicing and their activation by cADP-ribose during pregnancy have never been investigated. Here, we show that both RYR3S and RYR3L isoforms are differentially expressed in nonpregnant and pregnant mouse myometrium. The use of antisense oligonucleotides directed against each isoform indicated that only RYR3L was activated by caffeine and cADP-ribose in nonpregnant myometrium. These RYR3L-mediated Ca²⁺ releases were negatively regulated by RYR3S expression. At the end of pregnancy, the relative expression of RYR3L versus RYR3S and its ability to respond to cADP-ribose were increased. Therefore, our results suggest that physiological regulation of RYR3 alternative splicing may play an essential role at the end of pregnancy. ryanodine receptor; smooth muscle; alternative splicing     

Localization of kallikrein in porcine pancreas and submandibular gland as revealed by the indirect immunofluorescence technique.

The glandular kininogenase kallikrein is known to occur in many mammalian organs and glands but direct histochemical localization has been achieved in only a few cases. We have now been able to localize porcine kallikrein in the acinar cells of the pancreas and in the striated and collecting duct cells of the submandibular gland. Incubation of frozen and fixed sections with one of the crossreacting antibodies, anti-pancreatic, anti-submandibular or anti-urinary kallikrein IgG resulted in the same immunofluorescence pattern. There was evidence of a specific fluorescence neither in the acinar cells, nor in the interstitial tissue or blood cells of the submandibular gland nor in the islets of Langerhans, the interlobular ducts or blood vessels of the pancreas. From all data now available about glandular kallikreins, it seems that the kallikreins in these organs are very similar.     

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H_I and H_IV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca²⁺ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca²⁺ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca²⁺ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca²⁺. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.