A radioimmunoassay for the regulatory allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP)

The allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), found in gonadal and brain tissues by radiotracer and chemical methods, had been shown to play a role in gametogenesis, gonadotropin secretion and brain excitability. Since no simple assay was available, a radioimmunoassay for 3 alpha HP was developed using [3H]3 alpha HP and an antiserum raised against 3 alpha HP-20-CMO conjugated to bovine serum albumin. The specificity of the assay for the 3 alpha allylic configuration of 3 alpha HP was confirmed by examining 32 other steroids; cross-reaction with steroids containing different configurations (including metabolites of 3 alpha HP such as progesterone) was less than 0.9%. A Scatchard plot indicated a Ka of 1.56 X 10(9) M-1. Inter- and intra-assay coefficients of variation were 13.1 and 4.5%, respectively. The sensitivity of the assay was 6 pg and the 50% intercept of the standard curve was approx. 123 pg. The measurement by RIA of 3 alpha HP from standard solutions and HPLC purified tissue extracts was confirmed qualitatively and quantitatively by GC/MS methods. The RIA method was employed to determine 3 alpha HP levels in cultured Sertoli cells and in serum of intact and ovariectomized adult rats. Although for most uses, chromatography would not be necessary, two possible methods are presented to enable the separation of 3 alpha HP from other interfering steroids prior to RIA.     

High resolution 1H n.m.r. studies of vertebrate blood and plasma.

Spin echo Fourier transform proton n.m.r. spectra of whole blood contain resonances from both erythrocytes and plasma. A large number of well-resolved signals from mobile protons of low-molecular-weight metabolites in plasma and serum have been identified. Spectra from the plasmas of eight animal species and commercial, quality control sera are compared. CaEDTA2- and MgEDTA2- resonances can be used for the simultaneous determination of EDTA-chelatable calcium and magnesium concentrations in intact plasma and other biological fluids. Cholesterol is too immobile to contribute to the spectra of intact plasma, but is readily estimated by n.m.r. in both its free and esterified forms after extraction into methanol.     

The distribution of interspersed repetitive DNA sequences in the human genome

The distribution of interspersed repetitive DNA sequences in the human genome has been investigated, using a combination of biochemical, cytological, computational, and recombinant DNA approaches. "Low-resolution" biochemical experiments indicate that the general distribution of repetitive sequences in human DNA can be adequately described by models that assume a random spacing, with an average distance of 3 kb. A detailed "high-resolution" map of the repetitive sequence organization along 400 kb of cloned human DNA, including 150 kb of DNA fragments isolated for this study, is consistent with this general distribution pattern. However, a higher frequency of spacing distances greater than 9.5 kb was observed in this genomic DNA sample. While the overall repetitive sequence distribution is best described by models that assume a random distribution, an analysis of the distribution of Alu repetitive sequences appearing in the GenBank sequence database indicates that there are local domains with varying Alu placement densities. In situ hybridization to human metaphase chromosomes indicates that local density domains for Alu placement can be observed cytologically. Centric heterochromatin regions, in particular, are at least 50-fold underrepresented in Alu sequences. The observed distribution for repetitive sequences in human DNA is the expected result for sequences that transpose throughout the genome, with local regions of "preference" or "exclusion" for integration.     

The SF36 health survey questionnaire: an outcome measure suitable for routine use within the NHS?

OBJECTIVE--To assess the validity, reliability, and acceptability of the short form 36 (SF 36) health survey questionnaire (a shortened version of a battery of 149 health status questions) as a measure of patient outcome in a broad sample of patients suffering from four common clinical conditions. DESIGN--Postal questionnaire, followed up by two reminders at two week intervals. SETTING--Clinics and four training practices in north east Scotland. SUBJECTS--Over 1700 patients aged 16-86 with one of four conditions--low back pain, menorrhagia, suspected peptic ulcer, or varicose veins--and a comparison sample of 900 members of the general population. MAIN OUTCOME MEASURES--The eight scales within the SF36 health profile. RESULTS--The response rate exceeded 75% in the patient population (1310 respondents). The SF36 satisfied rigorous psychometric criteria for validity and internal consistency. Clinical validity was shown by the distinctive profiles generated for each condition, each of which differed from that in the general population in a predictable manner. Furthermore, SF36 scores were lower in referred patients than in patients not referred and were closely related to general practitioners'' perceptions of severity. CONCLUSIONS--These results provide support for the SF36 as a potential measure of patient outcome within the NHS. The SF36 seems acceptable to patients, internally consistent, and a valid measure of the health status of a wide range of patients. Before it can be used in the new health service, however, its sensitivity to changes in health status over time must also be tested.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.