[Increase in tylosin production by a commercial strain of Streptomyces fradiae].

Conventional mutagenesis (UV irradiation and exposure to nitrosoguanidine) were used to produce and regenerate protoplasts, aiming at increasing the antibiotic activity of a Streptomycesfradiae strain producing tylosin. Variants exceeding the activity of the initial producer strain by 0.5-28.3% were obtained. The most active variants were produced by a combined exposure to UV and nitrosoguanidine, as well as upon regeneration of protoplasts formed from the cells of clones produced by UV irradiation. Unstable inheritance of the trait of increased tylosin production was demonstrated.

A family exhibiting arterial tortuosity syndrome displays homozygosity for markers in the arterial tortuosity locus at chromosome 20q13.

Arterial tortuosity associated with hyperextensible skin and hypermobility of joints, features that are characteristics of Ehlers-Danlos syndrome (EDS), has been described in several families. An arterial tortuosity locus has recently been mapped to chromosome 20q13. Here, we report a consanguineous Kurdish family in which an affected child manifested elongation and severe tortuosity of the aorta, carotid, and other arteries. Additional clinical symptoms include loose skin, hypermobile joints, hernias, and facial features that resemble EDS individuals. To examine whether the arterial tortuosity locus was involved in this child, homozygosity analysis was performed using microsatellite markers on 20q13. The affected child was found homozygous, whereas the unaffected parents and three siblings were heterozygous. Additional typing defined the genomic interval to a 37-cm region within which the arterial tortuosity locus is located. Three functional candidate genes (B4GALT5, KCNB1, and PTGIS) were sequenced. No mutations were discovered in the coding regions of these three genes and the promoter regions of B4GALT5 and KCNB1 genes. Moreover, the B4GALT5 mRNA expression was unaltered in patient-derived lymphoblastoid cells. In the PTGIS gene promoter, the affected child was homozygous for eight variable number of tandem repeats, while parents and unaffected siblings carried six repeats.

The restriction-modification system in Streptomyces flavopersicus.

To clone bifunctional vectors in streptomycetes, it was necessary to define the restriction-modification system of Streptomyces flavopersicus. Plasmid DNA from bifunctional vectors pIJ699 and pXED3-13, isolated from E. coli strains with different methylation systems: E. coli DH5 alpha (dam+ dcm+), E. coli MB5386 (dam dcm), E. coli CB51 (dam dcm+), E. coli NM544 (dam+ dcm), was used for transformation of protoplasts from strain S. flavopersicus. Restriction of dcm-methylated DNA from S. flavopersicus was established. As a host in the intermediate cloning strain E. coli NM544 (dam+ dcm) should be used, as the dcm-transmethylase-dependent strain S. flavopersicus does not process DNA from this strain.

Improved cholecalciferol nutrition in rats is noncalcemic, suppresses parathyroid hormone and increases responsiveness to 1, 25-dihydroxycholecalciferol.

We examined how cholecalciferol (vitamin D) nutrition affected serum 25-hydroxycholecalciferol (25(OH)D) and 1, 25-dihydroxycholecalciferol (1,25(OH)(2)D). Rats were fed conventional diet (vitamin D, 4.5 IU/g, or 7 nmol/d) or the same diet plus 18 nmol/d of extra vitamin D for 3 wk. The extra vitamin D resulted in greater serum 25(OH)D (51 +/- 3, vs. control of 21 +/- 2 nmol/L), and kidney mRNA for vitamin D receptor [VDR mRNA] (P = 0. 026) and lower serum 1,25(OH)(2)D (72 +/- 16 vs. control of 161 +/- 10 pmol/L, P = 0.001), and parathyroid hormone (PTH) (89 +/- 4 vs. control of 160 +/- 15 ng/L, P = 0.001). Kidney VDR mRNA relative to GAPDH mRNA correlated inversely with serum 1,25(OH)(2)D (r = -0.714, P = 0.006). There were no differences in serum calcium, phosphate, alkaline phosphatase, or weight gain. Experiment 2 compared groups supplemented with 0.2, 2 or 20 nmol/d of vitamin D orally, or 20 nmol/d dermally to see how vitamin D nutrition influenced the response of 1,25(OH)(2)D to changes in diet calcium. Vitamin D did not affect urinary calcium or pyridinoline excretion, serum calcium, phosphate, vitamin D binding protein or alkaline phosphatase. In groups given 20 nmol/d of vitamin D, renal mitochondrial 25(OH)D-1alpha-hydroxylase was lower (P < 0.01) and 25(OH)D-24-hydroxylase was higher (P < 0.05). Higher 25(OH)D concentration was related to proportionally lower 1,25(OH)(2)D at every calcium intake, indicating greater tissue sensitivity to 1, 25(OH)(2)D. We conclude suppression of 1,25(OH)(2)D and PTH, and higher renal VDR mRNA and 24-hydroxylase did not involve higher free 1,25(OH)(2)D concentration or a first pass effect at the gut. Thus, 25(OH)D or a metabolite other than 1,25(OH)(2)D is a physiological, transcriptionally and biochemically active, noncalcemic vitamin D metabolite.

Constraints on proper folding of the amino terminal domains of group III metabotropic glutamate receptors.

The glutamate binding site of the G-protein coupled metabotropic glutamate receptors (mGluRs) is contained within the large extracellular amino terminal domain (ATD) of the receptor. In this study, we examined the ligand binding properties and cellular dispositions of the membrane-bound mGluR4 and mGluR8 subtypes of mGluRs, and a series of truncated versions of these receptors. Truncation of the ATDs of mGluR4 and mGluR8 40 amino acids upstream of the first transmembrane domain produced soluble proteins that were secreted into the cell culture media of transfected human embryonic kidney cells. The soluble receptors retained ligand binding capabilities. Additional constructs of the ATDs of mGluR4 and mGluR8 were assessed for their ability to bind the agonist [(3)H]L-AP4 and for secretion from cells. A shorter mGluR4 construct truncated 98 amino acids upstream from the first transmembrane domain failed to bind [(3)H]L-AP4, while the analogous mGluR8 construct displayed a low level of binding. Unlike the full-length receptors, which were expressed on the cell surface, or the soluble constructs which were secreted, the shorter constructs were primarily associated with intracellular membranes. These observations suggest that the cysteine-rich region may be important for efficient secretion, but not absolutely obligatory for ligand binding. Surprisingly, longer constructs encoding the entire ATDs of mGluR4 and mGluR8 failed to bind ligand and were localized intracellularly. Together, these findings demonstrate that there are strict limitations on the proper folding of truncated versions of the ATDs of mGluR4 and mGluR8. Specifically, all of the leucine-isoleucine-valine binding protein homology region, and part of the cysteine-rich region is required for optimal secretion in a soluble form that retains ligand binding activity.

Vitamin D-receptor genotypes as independent genetic predictors of decreased bone mineral density in primary biliary cirrhosis.

BACKGROUND & AIMS: Hepatic osteodystrophy is a complication of primary biliary cirrhosis (PBC). Allelic polymorphisms of the vitamin D receptor (VDR) gene are related to bone mineral density (BMD) in normal cohorts and those with primary osteoporosis. We sought to establish the prevalence of reduced bone mass in PBC, correlate BMD with VDR gene polymorphisms, and identify risk factors for the development of hepatic osteodystrophy. METHODS: Seventy-two female patients with PBC were evaluated prospectively. Clinical information, BMD assessment, disease severity, and osteoporosis risk factors were documented, and multivariate regression modeling was performed. RESULTS: Twenty-four percent of the patients were osteoporotic at the lumbar spine and 32% at the femur. Severe bone loss (z score <-2.0) occurs 4 times more frequently in patients with PBC compared with controls. Body weight (P = 0.003) and postmenopausal status (P = 0.012) correlated independently with BMD. VDR genotype (P = 0.01) correlated with lower BMD at the spine only. CONCLUSIONS: Osteoporosis is a common complication of PBC. VDR genotype predicts lower BMD in patients with PBC. Studies are warranted to investigate the mechanism(s) by which VDR as well as other candidate genes may contribute to the development of hepatic osteodystrophy in PBC.

Probing the ligand-binding domain of the mGluR4 subtype of metabotropic glutamate receptor.

Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool. Although the conversion of Arg(78), Ser(159), or Thr(182) to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist L-[(3)H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser(157) and Ser(160)) or a relatively modest effect (Ser(181)) on ligand affinity compared with the Arg(78), Ser(159), and Thr(182) mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog L-serine O-phosphate (L-SOP) was "docked" into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser(159) and Thr(182) of mGluR4 form hydrogen bonds with the alpha-carboxyl and alpha-amino groups on L-SOP, respectively, whereas Arg(78) forms an electrostatic interaction with the acidic side chains of L-SOP or glutamate. The conservation of Arg(78), Ser(159), and Thr(182) in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors.

Determinants of peak bone mass: clinical and genetic analyses in a young female Canadian cohort.

Peak bone mass has been shown to be a significant predictor of risk for osteoporosis. Previous studies have demonstrated that skeletal mass accumulation is under strong genetic control, and efforts have been made to identify candidate loci. Determinants of peak bone mass also include diet, physical activity, hormonal status, and other clinical factors. The overall contribution of these factors, genetic and nongenetic, and their interaction in determining peak bone density status have not been delineated. Six hundred and seventy-seven healthy unrelated Caucasian women ages 18-35 years were assessed. A detailed, standardized interview was conducted to evaluate lifestyle factors, menstrual and reproductive history, medical conditions, medication use, and family history of osteoporosis. Bone mineral density (BMD) was measured at the lumbar spine (L2-L4) and the femoral neck (hip) using dual-energy X-ray absorptiometry. Genotyping of the vitamin D receptor (VDR) locus at three polymorphic sites (BsmI, ApaI, and TaqI) was performed. In bivariate analyses, BMD at the lumbar spine and hip was positively correlated with weight, height, body mass index (BMI), and level of physical activity, both now and during adolescence, but negatively correlated with a family history of osteoporosis. Hip, but not spine BMD, correlated positively with dietary intake of calcium, and negatively with amenorrhea of more than 3 months, with caffeine intake, and with age. Spine, but not hip BMD, correlated positively with age and with number of pregnancies. VDR haplotype demonstrated significant associations with BMD at the hip, level of physical activity currently, and BMI. In multivariate analysis, independent predictors of greater BMD (at the hip or spine) were: age (younger for the hip, older for the spine), greater body weight, greater height (hip only), higher level of physical activity now and during adolescence, no family history of osteoporosis, and VDR genotype (hip only). Weight, age, level of physical activity, and family history are independent predictors of peak BMD. Of these factors, weight accounts for over half the explained variability in BMD. VDR alleles are significant independent predictors of peak femoral neck, but not lumbar spine BMD, even after adjusting for family history of osteoporosis, weight, age, and exercise. However, the overall contribution of this genetic determinant is modest. Taken together, these factors explained approximately 17% and 21% of the variability in peak spine and hip BMD, respectively, in our cohort. Future research should be aimed at further evaluation of genetic determinants of BMD. Most importantly, understanding the critical interactive nature between genes and the environment will facilitate development of targeted strategies directed at modifying lifestyle factors as well as earlier intervention in the most susceptible individuals.

A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations.

BACKGROUND: The regulation of extracellular calcium concentration by parathyroid hormone is mediated by a calcium-sensing, G-protein-coupled cell-surface receptor (CASR). Mutations of the CASR gene alter the set-point for extracellular ionised calcium [Ca2+]o and cause familial hypercalcaemia or hypocalcaemia. The CASR missense polymorphism, A986S, is common in the general population and is, therefore, a prime candidate as a genetic determinant of extracellular calcium concentration. METHODS: We genotyped the CASR A986S variant (S allele frequency of 16.3%) in 163 healthy adult women and tested samples of their serum for total calcium, albumin, total protein, creatinine, phosphate, pH, and parathyroid hormone. A prospectively generated, random subset of 84 of these women provided a whole blood sample for assay of [Ca2+]o. FINDINGS: The A986S genotype showed no association with total serum concentration of calcium, until corrected for albumin. In a multivariate regression model, biochemical and genetic variables accounted for 74% of the total variation in calcium. The significant predictors of serum calcium were: albumin (p<0.001), phosphate (p=0.02), parathyroid hormone (p=0.007), pH (p=0.001), and A986S genotype (p=0.009). Fasting whole-blood [Ca2+]o also showed an independent positive association with the 986S variant (p=0.013). INTERPRETATION: The CASR A986S variant has a significant effect on extracellular calcium. The CASR A986S polymorphism is a likely candidate locus for genetic predisposition to various bone and mineral disorders in which extracellular calcium concentrations have a prominent part.

Calcium sensing receptor gene: analysis of polymorphism frequency.

The role that the Calcium Sensing Receptor (CASR) plays in extracellular calcium regulation had been ascertained through studies of inactivating as well as activating mutations of CASR gene in a number of multiplex families. We have extended these observations to a polymorphism analysis of the intercellular domain of CASR in a cohort of healthy young women. The results demonstrate significant allelic polymorphism as a result of nonconservative changes at two specific sites. Further studies will be required to determine what, if any, relationship this may have to CASR phenotype.

In vitro ruminal degradation and synthesis of protein on fractions extracted from alfalfa hay and silage.

Net release of degraded N as NH3 and total AA plus microbial protein synthesis, quantified from incorporation of 15NH3 into microbial protein, was used to estimate the rate and extent of in vitro degradation of protein fractions isolated from alfalfa hay and silage. Seven proteins (casein, alfalfa hay, alfalfa silage, extracts from alfalfa hay and silage, and residues from alfalfa hay and silage) were studied. Results from (NH4)2SO4 and SDS-PAGE fractionations suggested that soluble proteins in alfalfa hay and silage differed in susceptibility to proteolytic attack. Although the net release of NH3 plus total AA N from alfalfa silage and alfalfa silage extract was twofold greater than that from alfalfa hay and alfalfa hay extract, net microbial protein synthesis on alfalfa hay and alfalfa hay extract was 33 and 43% greater. Despite greater NPN content in alfalfa silage, protein degradation rate and estimated escape were similar for intact alfalfa hay (0.103/h and 43%) and silage (0.067/h and 43%). This result might be explained by the less efficient microbial utilization of silage NPN, greater protozoal numbers on hay, greater soluble true protein in hay, or differences in molecular mass and stability of soluble proteins in hay versus silage. Use of a two-compartment model, based on water-soluble and insoluble CP fractions assumed to pass with the liquid and solid phases, respectively, yielded RUP estimates for alfalfa hay and silage that were similar to NRC estimates.