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.     

Siblings with renal tubular acidosis and nerve deafness. The first family in Japan

Summary Two siblings with renal tubular acidosis (RTA) and nerve deafness were examined. It was found by ammonium chloride and bicarbonate loading tests that the 6-year-old brother had a hybrid type of RTA and his 4-year-old sister, a distal type of RTA. Enzyme activity and amount of enzyme protein of carbonic anhydrase isoenzyme I and II in red blood cells, measured using an immunoadsorbent method, were normal in both cases. Although this indicated that the RTAs of these patients are not generated by the carbonic anhydrase deficiency, an investigation with renal tissue is necessary to arrive at a final conclusion.     

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.     

Nitrogen and phosphorus fertilization negatively affects strigolactone production and exudation in sorghum

Strigolactones (SLs) are essential host recognition signals for both root parasitic plants and arbuscular mycorrhizal fungi, and SLs or their metabolites function as a novel class of plant hormones regulating shoot and root architecture. Our previous study indicated that nitrogen (N) deficiency as well as phosphorus (P) deficiency in sorghum enhanced root content and exudation of 5-deoxystrigol, one of the major SLs produced by sorghum. In the present study, we examined how N and P fertilization affects SL production and exudation in sorghum plants subjected to short- (5 days) or long-term (10 days) N or P deficiency and demonstrated their common and distinct features. The root contents and exudation of SLs in the N- or P-deficient sorghum plants grown for 6, 12 or 24 h with or without N or P fertilization were quantified by LC–MS/MS. In general, without fertilization, root contents and exudation of SLs stayed at similar levels at 6 and 12 h and then significantly increased at 24 h. The production of SLs responded more quickly to P fertilization than the secretion of SLs, while regulation of SL secretion began earlier after N fertilization. It is suggested that sorghum plants regulate SL production and exudation when they are subjected to nutrient deficiencies depending on the type of nutrient and degree of deficiency.     

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.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.