ED-71, a vitamin D analog, is a more potent inhibitor of bone resorption than alfacalcidol in an estrogen-deficient rat model of osteoporosis.

Although active vitamin D is used in certain countries for the treatment of osteoporosis, the risk of causing hypercalcemia/hypercalciuria means that there is only a narrow therapeutic window, and this has precluded worldwide approval. The results of our previous animal studies have suggested that the therapeutic effect of active vitamin D on bone loss after estrogen deficiency can be dissociated at least partly from its effect of enhancing intestinal calcium absorption and suppressing parathyroid hormone (PTH) secretion. To test this, we compared the effects of ED-71, a hydroxypropoxy derivative of 1alpha,25-dihydroxyvitamin D3, with orally administered alfacalcidol, on bone mineral density (BMD) and the bone remodeling process as a function of their effects on calcium metabolism and PTH, in a rat ovariectomy (ovx) model of osteoporosis. ED-71 increased bone mass at the lumbar vertebra to a greater extent than alfacalcidol, while enhancing calcium absorption (indicated by urinary calcium excretion) and decreasing serum PTH levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers. These results suggest that active vitamin D exerts an antiosteoporotic effect by inhibiting osteoclastic bone resorption while maintaining osteoblastic function, and that these anticatabolic/anabolic effects of active vitamin D take place independently of its effects on calcium absorption and PTH. The demonstration that ED-71 is more potent in these properties than alfacalcidol makes it an attractive candidate as an antiosteoporotic drug.

Effects of benidipine hydrochloride on cerebrovascular lesions in salt-loaded stroke-prone spontaneously hypertensive rats: evaluation by magnetic resonance imaging.

We determined possible protective effects of benidipine hydrochloride (benidipine), a dihydropyridine calcium antagonist, on cerebrovascular lesions in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). The animals were orally treated with benidipine at 1, 3 and 10 mg/kg daily for 7 weeks, and their neurological symptoms, body weight changes, systolic blood pressure and cerebrovascular lesions on magnetic resonance imaging (MRI) were determined at various time points of treatment. Moreover, the brains of the rats that showed cerebrovascular lesions on MRI in the course of treatment or completed 7-week treatment were examined histopathologically. Control rats presented such symptoms as sedation, ataxia and aggressiveness, while their MRI analysis revealed high signals over wide areas from the occipital to frontal cortex and from the corpus callosum to external capsule. These high signal areas corresponded in location to edematous or softening lesions revealed by the histopathological observation. Treatment with benidipine at 3 and 10 mg/kg ameliorated neurological symptoms, significantly suppressing cerebrovascular damages on MRI. Benidipine at 3 mg/kg significantly decreased blood pressure for the first four weeks but it did not thereafter. These findings demonstrate that benidipine can protect salt-loaded SHRSP from cerebrovascular injury as assessed by MRI.

Characterization of EngF from Clostridium cellulovorans and identification of a novel cellulose binding domain.

The physical and enzymatic properties of noncellulosomal endoglucanase F (EngF) from Clostridium cellulovorans were studied. Binding studies revealed that the Kd and the maximum amount of protein bound for acid-swollen cellulose were 1.8 microM and 7.1 mumol/g of cellulose, respectively. The presence of cellobiose but not glucose or maltose could dissociate EngF from cellulose. N- and C-terminally truncated enzymes showed that binding activity was located at some site between amino acid residues 356 and 557 and that enzyme activity was still present when 20 amino acids but not 45 amino acids were removed from the N terminus and when 32 amino acids were removed from the C terminus; when 57 amino acids were removed from the C terminus, all activity was lost. EngF showed low endoglucanase activity and could hydrolyze cellotetraose and cellopentaose but not cellotriose. Activity studies suggested that EngF plays a role as an endoglucanase during cellulose degradation. Comparative sequence analyses indicated strongly that the cellulose binding domain (CBD) is different from previously reported CBDs.

Characterization of engF, a gene for a non-cellulosomal Clostridium cellulovorans endoglucanase.

A new Clostridium cellulovorans (strain ATCC 35296) endoglucanase gene engF has been isolated and sequenced. The gene contains 1671 bp and codes for a protein containing 557 amino acids and a mass of 60.1 kDa. A putative signal peptide of 29 amino acids is present and the mature protein has a mass of 57.1 kDa. EngF does not have amino acid sequence homology to previously isolated EngB and EngD, but does show sequence homology to family 5 glycosyl hydrolases from Bacillus, Erwinia carotovora, and C. acetobutylicum species. EngF is not a component of the cellulosome and does not contain a duplicated sequence (DS) at its C-terminal region. EngF is capable of binding to cellulose and hydrolyzing carboxymethylcellulose but not xylan. The cellulose binding domain (CBD) differs from types I, II and III CBDs and no obvious homology has been found to other CBD types. The maximum activity of EngF occurs at pH 5.5 and at 47 degrees C. Its properties suggest that EngF plays an ancillary role in the degradation of cellulosic materials.

Cloning, nucleotide sequence, and expression of tef-1, the gene encoding translation elongation factor 1 alpha (EF-1 alpha) of Neurospora crassa.

The tef-1 gene encoding translation elongation factor 1 alpha was cloned from the ascomycete fungus Neurospora crassa. The sequences of genomic DNA and cDNA clones showed that the tef-1 gene contained one ORF of 1380 bp length that is interrupted by three short introns. The deduced polypeptide contained 460 amino acid residues, and the sequence had a high similarity with those of EF-1 alpha polypeptides from other species. The level of tef-1 mRNA was low in conidia but high in growing cells. When mycelia were transferred to poor nutrient media, the level of tef-1 gene mRNA decreased remarkably. The pattern of tef-1 expression was similar to the expression of genes for ribosomal proteins. The tef-1 gene was mapped between arg-3 and leu-4 loci on linkage group I by restriction fragment length polymorphism mapping. Southern blot analysis showed that Neurospora genomic DNA contained only one copy of the tef-1 gene in a genome.

Binding of aminoglycoside antibiotics to acidic mucopolysaccharides.

Binding of aminoglycoside antibiotics to acidic mucopolysaccharides has been studied by means of physicochemical methods. Reactivity was affected markedly by the ionic environment , e.g. pH and ionic strength of the medium, the concentrations and the molar ratios of the constituents. The ionic character of binding was further confirmed by gel chromatography. The reduction of metachromasis by an aminoglycoside was also observed. Their affinity is correlated with localization of the aminoglycosides in vivo. According to reactivity, the following descending order of affinity was obtained for each family: neomycin, gentamicin, sagamicin, kanamycin and streptomycin; heparin, chondroitin sulfate and hyaluronic acid. This sequence of aminoglycosides corresponds to the extent of oto-, nephro- and neuro-(acute)toxicity, suggesting that their affinity for acidic mucopolysaccharides contribute to their tissue toxicity.