Isolation and properties of extracellular proteinases from Sporothrix schenckii.

Sporothrix schenckii, mainly in the yeast form of the organism, produced extracellular proteinases when cultivated in liquid media containing albumin or collagen as a nitrogen source, but did not do so in brain heart infusion medium. Isolation of two extracellular proteinases from albumin-containing medium was performed by chromatography on DEAE-Sepharose CL-6B and Sephacryl S-200. Proteinase I had a molecular weight of 36,500, an optimal pH at 6.0, and a pI at 4.8. Despite its activities in weakly acidic conditions, proteinase I demonstrated chymotrypsinlike characteristics, these being indicated by strong inhibitory activity by phenylmethylsulfonyl fluoride and chymostatin and good kinetic constants for a synthetic chymotrypsin substrate, Suc-Ala-Ala-Pro-Phe-MCA. Proteinase II had a molecular weight of 39,000, an optimal pH at 3.5, and a pI at 3.8. Proteinase II showed cathepsin D-like characteristics, these being indicated by strong inhibitory activity by pepstatin, an acidic optimal pH, and good kinetic constants for hemoglobin. These two enzymes hydrolyzed natural substrates such as stratum corneum, type I collagen, and elastin although not type IV collagen. Proteinase production and cell growth in collagen-containing medium and the enzymatic digestion of skin constituents by isolated proteinases suggested that these two proteinases cooperatively enable the organism to invade skin and to obtain peptides from insoluble proteins.     

Genetic polymorphisms of the CST2 locus coding for cystatin SA

A new genetic polymorphism of cystatin SA has been identified in human submandibular-sublingual saliva by means of basic gel electrophoresis and immunoblotting with anti-cystatin S. Two proteins, SA1 and SA2, are given by two alleles of CST2, viz., CST2^*1 and CST^*2. Inheritance is controlled by two codominant alleles at an autosomal locus. This hypothesis is supported by studies of 16 families 32 children. Gene frequencies for CST2^*1 and CST2^*2 are 0.935 and 0.065, respectively (n = 341). Eighteen amino acids determined among 20 N-terminal residues of cystatin SA2 are identical with the sequence encoded by CST2. Three forms of cystatin S (mono-phosphorylated cystatin S, di-phosphorylated cystatin S, and non-phosphorelated cystatin S) are present in the 341 saliva samples tested.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Changes in predominant energy substrate after hepatectomy

The changes in the energy substrate utilized by the remnant liver were studied in relation to the changes in the cellular energy status of 25 and 70% hepatectomized rabbits. In 25% hepatectomized rabbits, the energy charge $\text{(}\text{(}\text{ATP}\text{+0.5}\text{ADP}\text{)}\text{(}\text{ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}\text{)}$ level of the remnant liver remained unchanged, the energy substrate of which was predominantly glucose, rather than fatty acid. In contrast, in 70% hepatectomized rabbits, the energy production by the mitochondria was mainly dependent upon fatty acid oxidation at the early period after hepatectomy when the energy charge level decreased remarkably, and then upon glucose oxidation, concomitant with the restoration of the energy charge. It is suggested that the changes in the energy substrate utilized are closely related to those in the energy charge level and the mitochondrial phosphorylative activity of the remnant liver following hepatectomy.     

Whole-body vibration mediates mechanical hypersensitivity through Aβ-fiber and C-fiber thermal sensation in a chronic pain model

Whole-body vibration (WBV), which is widely used as a type of exercise, involves the use of vibratory stimuli and it is used for rehabilitation and sports performance programmes. This study aimed to investigate the effect of WBV treatment in a chronic pain model after 10 WBV sessions. An animal model (chronic pain) was applied in 60 male Wistar rats (±180 g, 12 weeks old) and the animals were treated with low intensity exercise (treadmill), WBV (vibrating platform), and a combined treatment involving both. The controls on the platform were set to a frequency of 42 Hz with 2 mm peak-to-peak displacement, g ≈ 7, in a spiral mode. Before and after the vibration exposure, sensitivity was determined. Aβ-fibers-mediated mechanical sensitivity thresholds (touch-pressure) were measured using a pressure meter. C-fibers-mediated thermal perception thresholds (hot pain) were measured with a hot plate. After each session, WBV influenced the discharge of skin touch-pressure receptors, reducing mechanical sensitivity in the WBV groups (P < 0.05). Comparing the conditions “before vs. after”, thermal perception thresholds (hot pain) started to decrease significantly after the third WBV session (P < 0.05). WBV decreases mechanical hyperalgesia after all sessions and thermal sensitivity after the third session with the use of WBV.     

Acid-Catalyzed Photosubstitution of 5-Fluoro-1,3-Dimethyluracil with Substituted Benzenes

Abstract

The substantial photosubstitution of 5-fluoro-1,3-dimethyluracil with substituted benzenes is first achieved by the addition of trifluoroacetic acid to the reaction mixture. The presence of protons is essential for the formation of 5-aryl-1,3-dimethyluracils.     

Synthesis of Iodoaminoimidazole Arabinoside (IAIA): A Potential Reductive Metabolite of the Spect Imaging Agent,Iodoazomycin Arabinoside (IAZA)

Abstract

The stereospecific synthesis of 1-(5-deoxy-5-iodo-α-D-arabino-furanosyl)-2-aminoimidazole (iodoaminoimidazole arabinoside: IAIA, 2) is described. The reaction of the protected sugar bromide (8) and trifluoroacetamidoimidazole (10B) gave the coupled product (11B), which gave the free nucleoside (4) on deblocking. It was identical with AIA obtained by reduction of AZA (azomycin arabinoside), whose anomeric configuration was found to be a by the X-ray crystallography.     

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans

Morbillivirus infection is a severe threat to marine mammals. Mass die‐offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain‐like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three‐dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.     

Mesenchymal stem cells contribute to endogenous FVIII:c production

Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative‐RT‐PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord‐derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.