Polypeptides of the synaptic membrane antigens D1, D2, and D3

The rat brain synaptic membrane antigens D1, D2, and D3 were labelled by 125I and precipitated by antibodies in a crossed immunoelectrophoresis. The precipitates were stained, scraped off, reduced, and analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The D1 antigen was composed of two polypeptide chains, apparent molecular weights 50 300 and 116 000 D2 of only one polypeptide chain, apparent molecular weight 139 000, and D3 of three polypeptides, apparent molecular weights 14 100, 23 500, and 34 400. Higher apparent molecular weight polypeptides were present in variable amounts in the D3 precipitate, except when the synaptic membrane extracts had been pre-treated with phospholipase D.     

Unaltered D1, D2, D4, and D5 dopamine receptor mRNA expression and distribution in the spinal cord of the D3 receptor knockout mouse

Dopamine (DA) acts through five receptor subtypes (D1–D5). We compared expression levels and distribution patterns of all DA mRNA receptors in the spinal cord of wild-type (WT) and loss of function D3 receptor knockout (D3KO) animals. D3 mRNA expression was increased in D3KO, but no D3 receptor protein was associated with cell membranes, supporting the previously reported lack of function. In contrast, mRNA expression levels and distribution patterns of D1, D2, D4, and D5 receptors were similar between WT and D3KO animals. We conclude that D3KO spinal neurons do not compensate for the loss of function of the D3 receptor with changes in the other DA receptor subtypes. This supports use of D3KO animals as a model to provide insight into D3 receptor dysfunction in the spinal cord.     

Bovine serum transferrin phenotypes AA,D1D1, D2D2, EE: Their carbohydrate compositions and electrophoretic multiplicity

Samples of homozygous bovine serum transferrins have been prepared and their purity has been ascertained by immunological techniques and electrophoretic analysis in SDS. Measurements of carbohydrate composition show that no significant differences exist among the phenotypic variants AA, D₁D₁, D₂D₂, and EE. Chromatography of transferrin AA on DEAE-cellulose separated four subfractions, each of which corresponded well with one band obtained by polyacrylamide gel electrophoresis. Carbohydrate analyses of the individual subfractions did not show significant differences in sialic acid, hexose, or hexosamine contents. After desialylation with neuraminidase, each subfraction was converted to a major band and a minor band on gel electrophoresis. From the relative band positions of the desialylated transferrins, it was concluded that possession of sialyl residues by bovine transferrin is not the primary cause of electrophoretic multiplicity. Rather, sialic acid masks an underlying heterogeneity which most likely resides within the polypeptide chain. Further characterization of this heterogeneity will best be undertaken with the isolated asialotransferrin subfractions.This research was supported by Grants MT-4074 and MA-5554 from the Medical Research Council of Canada and a Senior Fellowship (to M. W. C. H.) from the Ontario Heart Foundation.     

Individual quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in human milk

Extraction, lipid-reduction, and chromatographic methods suitable for the resolution and subsequent quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxy-vitamin D3 from human milk are described. This procedure utilizes a methanol:methylene chloride extraction, precipitation of unwanted lipids with cold methanol and ether, backwash with alkaline buffer, silica Sep-Pak preparative chromatography, normal- and reverse-phase high-performance liquid chromatography with final quantitation of the antirachitic sterols by competitive protein binding assay. The described assay was used to determine these antirachitic sterols in milk from women receiving various supplements of vitamin D or undergoing ultraviolet phototherapy.     

Vitamin D,Vitamin D Binding Protein,and Longitudinal Outcomes in COPD

Background

Associations between Vitamin D₃ [25(OH)D], vitamin D binding protein (VDBP) and chronic obstructive pulmonary disease (COPD) are previously reported. We aimed to further investigate these associations on longitudinal outcomes.

Methods

426 COPD patients from western Norway, GOLD stage II-IV, aged 40–76, were followed every six-month from 2006 through 2009 with spirometry, bioelectrical impedance measurements and registration of exacerbation frequency. Serum 25(OH)D and VDBP levels were determined at study-entry by high-performance liquid chromatography coupled with mass spectrometry and enzyme immunoassays respectively. Yearly change in lung function and body composition was assessed by generalized estimating equations (GEE), yearly exacerbation rate by negative binomial regression models, and 5 years all-cause mortality by Cox proportional-hazard regression.

Results

1/3 of the patients had vitamin D deficiency (<20ng/mL) and a greater decline in both FEV1 and FVC, compared to patients with normal levels; for FEV1 this difference only reached statistical significance in the 28 patients with the lowest levels (<10ng/mL, p = 0.01). Neither 25(OH)D nor VDBP levels predicted exacerbation rate, change in fat free mass index or risk of death.

Conclusion

Severe vitamin D deficiency may affect decline in lung function parameters in COPD. Neither 25(OH)D nor VDBP levels did otherwise predict markers of disease progression.     

Electrophoretic mobility of N- and C-terminal monoferric fragments of bovine transferrin phenotypes AA,D1D1, D2D2, and EE,and N-terminal amino acid sequences

Iron-saturated bovine transferrins A, D1, D2, and E were cleaved by trypsin yielding monoferric fragments. The N-terminal fragments (F) of transferrins A and D2 had identical mobility in cellulose acetate electrophoresis, that of transferrin D1 a slower mobility, and that of E a still slower mobility. The C-terminal fragments (S) gave multiple bands which were essentially identical in the case of transferrins A, D1, and E, but of slower mobility in the case of transferrin D2. All four variants had identical N-terminal amino acid sequences. The electrophoretic mobility of the C-terminal fragments was reduced by neuraminidase treatment, but the N-terminal fragments were unaffected. The four transferrin variants therefore appear to be made up from three electrophoretically distinguishable N-terminal halves and two C-terminal halves. The feature responsible for the electrophoretic double banding of homozygous bovine asialotransferrins is consistently associated with the C-terminal half of the molecule.     

Relationships among the O-Antigen Gene Clusters of Salmonella enterica Groups B, D1, D2, and D3

The O antigen is an important cell wall antigen of gram-negative bacteria, and the genes responsible for its biosynthesis are located in a gene cluster. We have cloned and sequenced the DNA segment unique to the O-antigen gene cluster of Salmonella enterica group D3. This segment includes a novel O-antigen polymerase gene (wzy_D3). The polymerase gives α(1→6) linkages but has no detectable sequence similarity to that of group D2, which confers the same linkage. We find the remnant of a D3-like wzy gene in the O-antigen gene clusters of groups D1 and B and suggest that this is the original wzy gene of these O-antigen gene clusters.     

西北汉族人群D10S1248、D2S441、D1S1677、D9S1122、D10S1435等5个miniSTR基因座的遗传多态性及其法医学应用

为了调查D10S1248、D2S441、D1S1677、D9S1122、D10S1435等5个miniSTR(mini short tandem repeats)基因座在西北汉族人群中的遗传多态性、遗传稳定性及其在陈旧降解检材中的法医学应用价值, 文章采用荧光PCR和基因分型技术对西北汉族154份无关个体血样、10个家系血液样本及10份陈旧降解检材进行片段长度分析。在西北汉族人群中, 5个miniSTR基因座分别检测出了8、7、7、6、7个等位基因, 等位基因频率分布符合Hardy-Weinberg平衡定律, 杂合度(Heterozygosity, H)为0.662~0.792, 个人识别率(Power of discrimination, PD)为0.869~0.915, 非父排除率(Power of exclusion, PE)为0.382~0.585, 多态信息含量(Polymorphism informa-tion content, PIC)为0.650~0.750。家系和陈旧降解检材的研究表明, 5个miniSTR基因座具有高度的遗传稳定性, 可对陈旧降解检材DNA进行有效的分型。5个miniSTR基因座适合作为西北汉族人群的遗传标记, 用于陈旧降解检材的法医学个人识别和亲权鉴定案件中。     

Serum Vitamin D,Vitamin D Binding Protein,and Risk of Colorectal Cancer

Background

We previously reported a positive association between serum 25-hydroxyvitamin D (25(OH)D) and colorectal cancer risk. To further elucidate this association, we examined the molar ratio of 25(OH)D to vitamin D binding protein (DBP), the primary 25(OH)D transport protein, and whether DBP modified the association between 25(OH)D and colorectal cancer risk.

Methods

In a nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study, controls were 1∶1 matched to 416 colorectal cancer cases based on age and date of blood collection. Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI) for quartiles of 25(OH)D, DBP, and the molar ratio of 25(OH)D:DBP, a proxy for free, unbound circulating 25(OH)D.

Results

Comparing highest to lowest quartiles, DBP was not associated with colorectal cancer risk (OR = 0.91; 95% CI: 0.58, 1.42, p for trend  = 0.58); however, a positive risk association was observed for the molar ratio of 25(OH)D:DBP (OR = 1.44; 95% CI: 0.92, 2.26, p for trend  = 0.04). In stratified analyses, the positive association between 25(OH)D and colorectal cancer was stronger among men with DBP levels above the median (OR = 1.89; 95% CI: 1.07, 3.36, p for trend  = 0.01) than below the median (OR = 1.20; 95% CI: 0.68, 2.12, p for trend  = 0.87), although the interaction was not statistically significant (p for interaction  = 0.24).

Conclusion

Circulating DBP may influence the association between 25(OH)D and colorectal cancer in male smokers, with the suggestion of a stronger positive association in men with higher DBP concentrations. This finding should be examined in other populations, especially those that include women and non-smokers.     

Staphylococcal Enterotoxins A,D, and E

Abstract

Staphylococcal enterotoxins (SEs) are a family of structurally related exotoxin molecules produced by certain Gram-positive Staphylococcus aureus bacterial strains. SEs are a major cause of food poisoning and are involved in bacterial Gram-positive shock in humans. SEs bind to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) and subsequently activate a large fraction, 5–20%, of T lymphocytes (1). This property has led to their classification of superantigens (SAg). The T cells are activated by SAg to proliferate and produce cytokines such as interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor-α and β (TNF-α and β) (2,3). Depending on origin, superantigens can be divided in two groups, viral and bacterial. For reviews see Refs. 4–8.     

Goldilocks,vitamin D and sarcoidosis

While low levels of vitamin D can increase the risk for osteoporosis, excessive amounts of vitamin D may also be problematic. Hypercalcemia and hypercalcuria due to increased vitamin D activity occur in a significant proportion of sarcoidosis patients. Saidenberg-Kermanac’h and colleagues compared vitamin D levels with bone fragility fractures in their sarcoidosis clinic. They found that a 25-(OH) vitamin D level between 10 and 20 ng/ml was associated with the lowest risk of bone fractures and paradoxically higher levels increased the risk of bone fractures. Using less vitamin D supplementation may simultaneously lower the risk for bone fracture and hypercalcemia in sarcoidosis.In the previous issue, Saidenberg-Kermanac’h and colleagues provide more information regarding the complexity of vitamin D activity in sarcoidosis [1]. A few years ago, vitamin D was declared the nutrient of the decade. This was heady stuff for a sterol that was originally felt important only in preventing rickets. Studies have demonstrated its key role in calcium absorption and bone growth. Beyond that, vitamin D has been considered an important sterol in various aspects of health. Low levels of vitamin D have been associated with increased risk for cancer, type 2 diabetes, and heart disease.Most of these observations have been based on measurements of 25-(OH)-vitamin D3 (ergocalcitrol). This sterol is converted by 1-alpha hydroxylase to 1,25-(OH)2-vitamin D3 (calcitrol), the active form of vitamin D. This conversion occurs in the kidney and patients with chronic renal failure require calcitrol replacement.In tuberculosis, vitamin D supplementation has been recommended in patients because vitamin D is crucial in the granulomatous reaction against the organism. However, what may be good for tuberculosis eradication may not be good for sarcoidosis. It has been noted that excessive amounts of vitamin D are associated with a worse clinical outcome in sarcoidosis [2]. In granulomas, there may be increased activity of 1-alpha hydroxylase. As part of the Th-1 immune response, calcitrol has a paracrine effect within the granuloma. In some cases, this leads to excessive calcitrol, resulting in hypercalcuria or hypercalcemia [3]. At least 10% of sarcoidosis patients have hypercalcemia, half of whom can develop associated renal dysfunction [3,4]. In some cases, hypercalcemic renal failure can be reversed by simply withdrawing vitamin D supplementation [3]. There are case reports of excessive vitamin D replacement leading to hypercalcemia in patients with mycobacterial infections [5].The sarcoidosis patient may be treated with glucocorticoids, sometimes for years. Obviously, long-term glucocorticoid administration places the patient at risk for developing osteoporosis [6,7]. In rheumatoid arthritis, patients undergoing prolonged glucocorticoid treatment are recommended to receive calcium and vitamin D replacement [8]. While this is the cornerstone of prevention of osteoporosis, the role of calcium and vitamin D replacement in sarcoidosis remains unclear [9].Into this quandary comes the study by Saidenberg-Kermanac’h and colleagues reported in the previous issue of Arthritis Research & Therapy[1]. After studying a large cohort of sarcoidosis patients from their clinic, the authors found that fragility fractures occurred in nearly a quarter of them. The fracture risk was increased for those treated with corticosteroids. Although low levels of ergocalcitrol was an independent risk for osteoporosis, ironically high levels of ergocalcitrol were also associated with an increased risk for osteoporosis. They found that ergocalcitrol levels of 10 to 20 ng/ml was associated with the lowest fracture risk for patients. This J shaped risk for bone fragility has been noted in non-sarcoidosis patients, although the proposed target levels are higher for these patients [10]. For the clinician treating sarcoidosis, one has to balance not only the risk for osteoporosis, but also the risk for hypercalcemia and renal failure (Figure 1).Open in a separate windowFigure 1Schematic depicting vitamin D metabolism in the body from ergocalcitrol to calcitrol. The conversion is enhanced by increased activity of 1-alpha hydroxylase activity in the granuloma of sarcoidosis patients. The untoward consequences of low or high vitamin D activity are summarized at the bottom of the figure.One possible explanation for the lower ideal ergocalcitrol level in sarcoidosis is the enhanced activity of 1-alpha hydroxylase in sarcoidosis granulomas. The authors did not provide information regarding calcitrol levels in their patients. The proportion of calcitrol to ergocalcitrol appears to be higher in sarcoidosis compared to non-sarcoidosis conditions. In one study of 270 sarcoidosis patients, 80% had low ergocalcitrol levels, but less than 1% had low calcitrol levels. In fact, that study found that 10% of patients had elevated calcitrol levels [3]. Those with elevated calcitrol were more likely to have a history of hypercalcemia or hypercalcuria. Higher levels of calcitrol have been associated with more advanced pulmonary sarcoidosis [2].The other potential benefits of vitamin D replacement in sarcoidosis are unclear. Should sarcoidosis patients with low ergocalcitrol but normal calcitrol levels be prescribed vitamin D supplementation to reduce their risk for cancer and type 2 diabetes? If so, do they increase their risk for hypercalcemia or hypercalcuria? Could this increased vitamin D intake raise the functional level of vitamin D even higher and therefore increase the risk for osteoporosis?To paraphrase Goldilocks, one does not want too little or too much vitamin D. You want just the right amount.     

Inversion of proton translocation in bacteriorhodopsin mutants D85N, D85T, and D85,96N.

Proton translocation activity of bacteriorhodopsin mutants lacking the proton acceptor Asp-85 was investigated using the black lipid membrane technique. Mutants D85N, D85T, and D85,96N were constructed and homologously expressed in Halobacterium salinarium to yield a membrane fraction with a buoyant density of 1.18 g/cm3, i.e., identical to that of wild-type purple membrane. In all mutants, the absorbance maximum was red-shifted between 27 and 49 nm compared with wild type, and the pKa values of the respective Schiff bases were reduced to between 8.3 and 8.9 compared with the value of > 13 in wild type. Therefore, a mixture of chromophores absorbing at 410 nm (deprotonated form) and around 600 nm (protonated form) exists at physiological pH. In continuous blue light, the deprotonated form generates stationary photocurrents. The currents are enhanced by a factor of up to 50 upon addition of azide in D85N and D85,96N mutants, whereas D85T shows no azide effect. The direction of these currents is the same as in wild type in yellow light. Yellow light alone is not sufficient to generate stationary currents in the mutants, but increasing yellow light intensity in the presence of blue light leads to an inversion of the current. Because all currents are carried by protons, this two-photon process demonstrates an inverted proton translocation by BR mutants.     

23,24,25-Trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3, and 23-dehydro-25-hydroxyvitamin D3: new in vivo metabolites of vitamin D3

Four new in vivo metabolites of vitamin D3 were isolated from the blood plasma of chicks given large doses of vitamin D3. The metabolites were isolated by methanol-chloroform extraction and a series of chromatographic procedures. By use of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions, the metabolites were identified as 23,24,25-trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3 and 23-dehydro-25-hydroxyvitamin D3.