Radiation induced formation of giant cells inSaccharomyces uvarum III: Effect of X-rays on nuclear division

Summary Spindle formation and nuclear division of budding and irradiated yeast cells (Saccharomyces uvarum) was investigated by fluorescence microscopy of protoplasted cells. Protoplasts were treated with antitubulin antibodies and DAPI, a fluorescent dye staining DNA. In budding yeast cells, duplication of spindle pole bodies as well as formation of complete 1-µm spindles and elongated 8-µm spindles were documented. In X-irradiated cells, spindle pole bodies were duplicated as well, forming the complete 1-µm spindle. Nuclei of giant cells have lost the elongation ability and remain in a normal G₂-phase state, thus preventing nuclear as well as cellular division.     

Low concentrations of the stable prostaglandin endoperoxide U44069 stimulate shape change in quin2-loaded platelets without a measurable increase in [Ca2+]i

Dose-response relationships for raised cytoplasmic free calcium concentration, [Ca2+]i, and shape change were measured simultaneously in quin2-loaded human platelets. With the calcium ionophore ionomycin the threshold [Ca2+]i for shape change was 300 nM with a maximal response at 800 nM. With 1 mM external Ca2+ the U44069 concentrations required to stimulate half-maximal shape change and an increase in [Ca2+]i were 2 and 41 nM, respectively. For PAF these values were 8.7 and 164 pg/ml, respectively. Low concentrations of U44069 and PAF evoked substantial shape change without any rise in [Ca2+]i. In the absence of external Ca2+, U44069 stimulated half-maximal shape change at 2 nM, and half-maximal elevation of [Ca2+]i at 69 nM: here, increased [Ca2+]i never reached the threshold [Ca2+]i for shape-change derived with ionomycin. These results suggest that some transduction mechanism other than elevated [Ca2+]i, as yet unidentified, can cause shape change.     

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

Sweat gland density and response during high-intensity exercise in athletes with spinal cord injuries

Sweat production is crucial for thermoregulation. However, sweating can be problematic for individuals with spinal cord injuries (SCI), as they display a blunting of sudomotor and vasomotor responses below the level of the injury. Sweat gland density and eccrine gland metabolism in SCI are not well understood. Consequently, this study examined sweat lactate (S-LA) (reflective of sweat gland metabolism), active sweat gland density (SGD), and sweat output per gland (S/G) in 7 SCI athletes and 8 able-bodied (AB) controls matched for arm ergometry VO₂peak. A sweat collection device was positioned on the upper scapular and medial calf of each subject just prior to the beginning of the trial, with iodine sweat gland density patches positioned on the upper scapular and medial calf. Participants were tested on a ramp protocol (7 min per stage, 20 W increase per stage) in a common exercise environment (21±1°C, 45-65% relative humidity). An independent t-test revealed lower (p<0.05) SGD (upper scapular) for SCI (22.3 ±14.8 glands · cm⁻²) vs. AB. (41.0 ± 8.1 glands · cm⁻²). However, there was no significant difference for S/G between groups. S-LA was significantly greater (p<0.05) during the second exercise stage for SCI (11.5±10.9 mmol · l⁻¹) vs. AB (26.8±11.07 mmol · l⁻¹). These findings suggest that SCI athletes had less active sweat glands compared to the AB group, but the sweat response was similar (SLA, S/G) between AB and SCI athletes. The results suggest similar interglandular metabolic activity irrespective of overall sweat rate.     

Assembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted by Cell Wall Polysaccharides

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 μg ml⁻¹) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (β(1–3),β(1–6)-glucan) and β(1–3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment.Hydrophobins are a class of surface active proteins that play diverse roles in fungal growth and development. For instance, they allow fungi to escape an aqueous environment, confer hydrophobicity to fungal surfaces in contact with air, and mediate attachment of fungi to hydrophobic surfaces (1, 2). They also play a role in the architecture of the cell wall (3).Hydrophobins share eight conserved cysteine residues, but otherwise their sequences are diverse (4). Class I and II hydrophobins are distinguished on the basis of differences in hydropathy patterns and biophysical properties (5). SC3 of Schizophyllum commune is the best characterized class I hydrophobin. It self-assembles at interfaces between water and air, water and oil, and water and hydrophobic solids (6–8). The four disulfide bridges of SC3 prevent spontaneous self-assembly in solution and thus account for the controlled assembly at hydrophobic-hydrophilic interfaces (9).The water-soluble form of SC3 is oligomeric (10) and rich in β-sheet (11). Upon assembly at the water-air interface, SC3 proceeds via an intermediate form that has increased α-helical structure (α-helical state) to a stable end form that has increased β-sheet structure (β-sheet state) (11–13). SC3 in the β-sheet state initially has no clear ultrastructure (β-sheet I state) (12), but after prolonged incubation, the protein forms 10-nm wide amyloid-like fibrils (β-sheet II state) (12–14) that are called rodlets (6, 15). Like other amyloid fibrils (16), rodlets of the hydrophobins SC3 of S. commune and EAS of Neurospora crassa increase fluorescence of thioflavin T and bind Congo red (14, 17, 18). Moreover, x-ray diffraction of rodlets of EAS showed reflections at 4.8 Å (distance between strands in a β-sheet) and 10–12 Å (spacing between β-sheets stacked perpendicular to the fibril long axis) (19), which are indicative for amyloid fibrils.Notably, SC3 does not spontaneously self-assemble into amyloid fibrils at an interface between water and a hydrophobic solid. Instead, SC3 is arrested in the intermediate α-helical state. Transition to the β-sheet state is observed only by heating the sample in the presence of detergent (11, 12). These observations prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that amyloid formation of SC3 is promoted by increasing its concentration or by the presence of cell wall polysaccharides.     

Geochronology of cave deposits at Liang Bua and of adjacent river terraces in the Wae Racang valley, western Flores, Indonesia: a synthesis of age estimates for the type locality of Homo floresiensis

A robust timeframe for the extant cave deposits at Liang Bua, and for the river terraces in the adjoining Wae Racang valley, is essential to constrain the period of existence and time of extinction of Homo floresiensis and other biota that have been excavated at this hominin type locality. Reliable age control is also required for the variety of artifacts excavated from these deposits, and to assist in environmental reconstructions for this river valley and for the region more broadly. In this paper, we summarize the available geochronological information for Liang Bua and its immediate environs, obtained using seven numerical-age methods: radiocarbon, thermoluminescence, optically- and infrared-stimulated luminescence (collectively known as optical dating), uranium-series, electron spin resonance, and coupled electron spin resonance/uranium-series. We synthesize the large number of numerical age determinations reported previously and present additional age estimates germane to questions of hominin evolution and extinction.     

Requirements of the engineered leader peptide of nisin for inducing modification, export, and cleavage

Nisin A is a pentacyclic peptide antibiotic produced by Lactococcus lactis. The leader peptide of prenisin keeps nisin inactive and has a role in inducing NisB- and NisC-catalyzed modifications of the propeptide and NisT-mediated export. The highly specific NisP cleaves off the leader peptide from fully modified and exported prenisin. We present here a detailed mutagenesis analysis of the nisin leader peptide. For alternative cleavage, we successfully introduced a putative NisP autocleavage site and sites for thrombin, enterokinase, Glu-C, and factor Xa in the C-terminal part of the leader peptide. Replacing residue F-18 with Trp or Thr strongly reduced production. On the other hand, D-19A, F-18H, F-18M, L-16D, L-16K, and L-16A enhanced production. Substitutions within and outside the FNLD box enhanced or reduced the transport efficiency. None of the above substitutions nor even an internal 6His tag from positions -13 to -8 had any effect on the capacity of the leader peptide to induce NisB and NisC modifications. Therefore, these data demonstrate a large mutational freedom. However, simultaneous replacement of the FNLD amino acids by four alanines strongly reduced export and even led to a complete loss of the capacity to induce modifications. Reducing the leader peptide to MSTKDFNLDLR led to 3- or 4-fold dehydration. Taken together, the FNLD box is crucial for inducing posttranslational modifications.     

Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Endpiece: The noble profession

ObjectivesTo assess whether bacterial vaginosis or chlamydial infection before 10 weeks'' gestation is associated with miscarriage before 16 weeks.DesignProspective cohort study.Setting32 general practices and five family planning clinics in south London.Participants1216 pregnant women, mean age 31, presenting before 10 weeks'' gestation.Results121 of 1214 women (10.0%, 95% confidence interval 8.3% to 11.7%) miscarried before 16 weeks. 174 of 1201 women (14.5%, 12.5% to 16.5%) had bacterial vaginosis. Compared with women who were negative for bacterial vaginosis those who were positive had a relative risk of miscarriage before 16 weeks'' gestation of 1.2 (0.7 to 1.9). Bacterial vaginosis was, however, associated with miscarriage in the second trimester at 13-15 weeks (3.5, 1.2 to 10.3). Only 29 women (2.4%, 1.5% to 3.3%) had chlamydial infection, of whom one miscarried (0.32, 0.04 to 2.30).ConclusionBacterial vaginosis is not strongly predictive of early miscarriage but may be a predictor after 13 weeks'' gestation. The prevalence of Chlamydia was too low to assess the risk, but it is unlikely to be a major risk factor in pregnant women.

What is already known on this topic

Miscarriages are common and associated with considerable morbidity and costsBacterial vaginosis is associated with miscarriage after 16 weeks'' gestation and preterm birth but the role of chlamydial infection is uncertain

What this study adds

Bacterial vaginosis is not a strong predictor of miscarriage before 16 weeks'' gestation but may be associated with miscarriage at 13-15 weeks'' gestationThe prevalence of chlamydial infection was too low for it to be a major risk factor for miscarriage in this population of healthy pregnant womenNon-invasive screening for bacterial vaginosis and chlamydial infection by using self administered vaginal swabs is feasible in pregnant women in the community