Developmental gene expression in Bacillus subtilis crsA47 mutants reveals glucose-activated control of the gene for the minor sigma factor sigma(H).

The presence of excess glucose in growth media prevents normal sporulation of Bacillus subtilis. The crsA47 mutation, located in the gene for the vegetative phase sigma factor (sigma(A)) results in a glucose-resistant sporulation phenotype. As part of a study of the mechanisms whereby the mutation in sigma(A) overcomes glucose repression of sporulation, we examined the expression of genes involved in sporulation initiation in the crsA47 background. The crsA47 mutation had a significant impact on a variety of genes. Changes to stage II gene expression could be linked to alterations in the expression of the sinI and sinR genes. In addition, there was a dramatic increase in the expression of genes dependent on the minor sigma factor sigma(H). This latter change was paralleled by the pattern of spo0H gene transcription in cells with the crsA47 mutation. In vitro analysis of RNA polymerase containing sigma(A47) indicated that it did not have unusually high affinity for the spo0H gene promoter. The in vivo pattern of spo0H expression is not predicted by the known regulatory constraints on spo0H and suggests novel regulation mechanisms that are revealed in the crsA47 background.

An in vitro evaluation of condoms as barriers to a small virus.

BACKGROUND: Because of the possible presence of small holes, the effectiveness of condoms as barriers to virus transmission is controversial. GOALS: To determine the proportion of condoms that allow virus penetration and the amounts of virus that penetrate. STUDY DESIGN: A sensitive, static test was used to evaluate different condom types as barriers to a small virus, including brand with or without lubrication and ones of different materials. The test included some physiologic-based parameters and some parameters that exaggerated expected actual use conditions. RESULTS: Under test conditions, 2.6% (12 of 470) of the latex condoms allowed some virus penetration; the median level of penetration was 7 x 10(-4) ml. Lubricated condoms performed similarly to nonlubricated ones. Polyurethane condoms yielded results higher than but not statistically different from those for latex condoms. CONCLUSIONS: Few condoms allowed any virus penetration. The median amount of penetration for latex condoms when extrapolated to expected actual use conditions was 1 x 10(-5) ml (volume of semen). Thus, even for the few condoms that do allow virus penetration, the typical level of exposure to semen would be several orders of magnitude lower than for no condom at all.

PIP: Nine brands and 470 samples of latex condoms and two brands and 76 samples of polyurethane condoms bought from retail distributors were tested in vitro for their ability to block the penetration of virus. A sensitive, static test apparatus was designed for and used in the evaluation. The test included some physiological-based parameters as well as some which exaggerated the expected actual use conditions. Both lubricated and nonlubricated condoms were tested. Before testing, however, most of the lubrication was removed from the lubricated condoms through rinsing with Dulbecco's phosphate-buffered saline and blotting with sterile paper towels. The 0X174 bacteriophage of 27 nm particle diameter, 32 nm including its bulky spikes, was used as the proxy challenge virus. Under test conditions, 12 of the latex condoms (2.6%) allowed some virus penetration of median quantity 0.0007 ml. Just two of the latex condoms were responsible for 99.8% of the total penetration among latex condoms overall. The performance of lubricated condoms was similar to that of nonlubricated ones. Four of the polyurethane condoms allowed penetration, but only one condom was responsible for 98.6% of total penetration. The difference in performance between latex and polyurethane condoms is not statistically significant. The median amount of penetration for latex condoms when extrapolated to expected actual use conditions was 0.00001 ml of semen. Therefore, even for the few condoms which allow virus penetration, the typical level of exposure to semen is several orders of magnitude lower than the amount of exposure expected when not using a condom.

An analysis of the complete nucleotide sequence of the Haemophilus ducreyi broad-host-range plasmid pLS88.

We present an analysis of the complete nucleotide sequence of pLS88, a naturally occurring, 4.8-kb broad-host-range plasmid isolated from Haemophilus ducreyi and encoding resistance to sulfonamides, streptomycin, and kanamycin. Sequence analysis of the genes encoding sulfonamide and streptomycin resistance revealed homology to the RSF1010 sulII and strA genes. The sulII-strA intergenic region of pLS88 has a 38-bp deletion identical to that of the RSF1010-like plasmid pHD8.1, isolated from Actinobacillus pleuropneumoniae. The kanamycin resistance gene shows strong homology to Tn903, but lacks the inverted repeats of the transposon. No other genes have been identified. The region downstream of the kanamycin resistance gene shows homology to the RSF1010 oriV region; however, this region is not essential to plasmid replication. The ori of pLS88 is contained within a 1060-bp region and does not appear to contain structures typical of plasmid origins. This region is flanked by DNA showing strong homology to regions both upstream and downstream of the Haemophilus influenzae ROB-1 beta-lactamase gene. Because of the small size of the origin, pLS88 appears to resemble the structure of narrow-host-range plasmids, but replicates, via an as yet unidentified mechanism, as a broad-host-range plasmid.

Eccrine sweat gland anatomy in cockayne syndrome: a possible diagnostic aid.

Cockayne syndrome is an autosomal recessive disease, which includes as major features motor and mental retardation (beginning in the second year), microcephaly, ataxia, retinal degeneration and pigmentation, cataracts, progeroid features, intracranial calcification, hypogonadism, and growth retardation. Many other diseases have some of these features, so that diagnosis of Cockayne syndrome can be difficult, especially in younger children. Eccrine sweat glands were microdissected from autopsy or biopsy specimens from patients with Cockayne syndrome, and mean values for duct length, secretory coil volume, ratio of coil volume to duct length, and axis ratio of the secretory coil were determined. In comparison with values for eccrine glands of patients with no known genetic or chromosomal disease, eccrine glands in Cockayne syndrome are abnormally small for age. Whether other diseases with various similarities to Cockayne syndrome produce similar growth abnormality of eccrine sweat glands is not known, but determination of sweat gland size may provide data suggesting or supporting the diagnosis of Cockayne syndrome.

Myopathic skeletal muscle fiber abnormalities in cardiomyopathies of childhood.

Skeletal muscle fibers isolated from 50 muscle specimens from 10 children with cardiomyopathy of unknown cause are compared to those from 18 specimens from 5 patients with skeletal muscle myopathies, 45 specimens from 18 patients with congenital heart disease, and 15 specimens from 7 patients with no genetic, chromosomal, or cardiac disease. Muscle fibers from the myopathy specimens show increased nuclei/mm of fiber and increased nuclei/mm/micron of diameter (R value), as well as reduced surface area and volume of cytoplasm per nucleus, compared to control values. The values for cardiomyopathy deviate from normal in the same way as, but to a lesser degree than, those for myopathy--namely, in this material, diseases with cardiomyopathy tend also to produce mild myopathy. Since cardiac and skeletal muscle pathologic findings have not been adequately studied for the majority of the approximately 50 genetic disorders causing cardiomyopathy or otherwise affecting cardiac function described to date, the data indicate primarily that skeletal muscle biopsy will undoubtedly be more useful in cardiomyopathic disorders when the appropriate correlative studies of cardiac and skeletal muscle in such diseases have been done. Because larger biopsy specimens can be obtained, skeletal muscle merits further exploitation in biochemical research on basic mechanisms of disorders causing cardiomyopathy.

Papillary pore counts: a method of studying developmental aberrations in diseased juvenile kidneys.

Counts of the number of pores of primary collecting tubules (ducts of Bellini) on renal papillae, and values calculated by adjusting the counts of compound papillae to those of "virtual" single papillae, were determined for kidneys of patients with end-stage renal failure. Values for chronic glomerulonephritis, Alport's disease, infantile polycystic disease, trisomy 18, and trisomy 13 were not abnormal. Kidneys of patients with CUTO showed significantly low pore counts, indicating that this process in some cases is a true hypoplasia, with mean reduction of number of ducts of Bellini of 26%. FGS showed a high proportion of single papillae (80% vs. normal 60%) with high virtual pore counts, suggesting that a developmental abnormality underlies this disorder (or this outcome of nephrotic syndrome). Cystinosis showed a high proportion of compound papillae (80% vs. 40%) but low virtual pore counts, implying that this genetic disorder causes both maldevelopment and postnatal functional abnormality of the kidneys. A Jeune syndrome kidney produced very low pore counts (mean 8 vs. 16.6 for virtual pore counts), and Down's syndrome also showed low pore counts (mean VPC 15.1 vs. normal 16.6), indicating that the low kidney weights demonstrated by others with Down's syndrome reflect a true hypoplasia.

Histochemical methods for dissociated muscle fibers.

Skeletal or cardiac muscle fibers can be separated by brief (3--5 second) dissociation of formalin-fixed pieces with a Willems Polytron (Brinkmann Instrument Co.). Such separated fibers are useful for demonstration of abnormal accumulations of lipids, carbohydrates, proteins and minerals in metabolic diseases. Staining techniques for demonstration of various stored materials include: 1) toluidine blue at pH 2.8 for acid mucopolysaccharide in skeletal muscle fibers in Pompe's glycogenesis 2, 2) one-step trichrome stain for nemaline myopathy and for abnormal mitochondria in X-linked infantile cardiomyopathy, 3) periodic acid-methenamine silver stain for glycolipid-containing lysosomes in I-cell disease (mucolipidosis 2), 4) Sudan black B stain for lipid in skeletal muscle fibers in Reye's syndrome, infantile lactic acidosis, Leigh's infantile subacute necrotizing encephalopathy and Jansky-Bielschowsky late infantile ceroid lipofuscinosis, 5) iron stain for iron in cardiac and skeletal muscle fibers in thalassemia with advanced hemosiderosis, and 6) autofluorescence for "ceroid" in skeletal muscle fibers in Jansky-Bielschowsky disease.