Legionella pneumophila NudA Is a Nudix hydrolase and virulence factor

We studied the identity and function of the 528-bp gene immediately upstream of Legionella pneumophila F2310 ptsP (enzyme I(Ntr)). This gene, nudA, encoded for a Nudix hydrolase based on the inferred protein sequence. NudA had hydrolytic activity typical of other Nudix hydrolases, such as Escherichia coli YgdP, in that Ap(n)A's, in particular diadenosine pentaphosphate (Ap(5)A), were the preferred substrates. NudA hydrolyzed Ap(5)A to ATP plus ADP. Both ptsP and nudA were cotranscribed. Bacterial two-hybrid analysis showed no PtsP-NudA interactions. Gene nudA was present in 19 of 20 different L. pneumophila strains tested and in 5 of 10 different Legionella spp. other than L. pneumophila. An in-frame nudA mutation was made in L. pneumophila F2310 to determine the phenotype. The nudA mutant was an auxotroph that grew slowly in liquid and on solid media and had a smaller colony size than its parent. In addition, the mutant was more salt resistant than its parent and grew very poorly at 25 degrees C; all of these characteristics, as well as auxotrophy and slow-growth rate, were reversed by transcomplementation with nudA. The nudA mutant was outcompeted by about fourfold by the parent in competition studies in macrophages; transcomplementation almost completely restored this defect. Competition studies in guinea pigs with L. pneumophila pneumonia showed that the nudA mutant was outcompeted by its parent in both lung and spleen. NudA is of major importance for resisting stress in L. pneumophila and is a virulence factor.     

Virulence Differences in Mice of Type A and B Histoplasma capsulatum Yeasts Grown in Continuous Light and Total Darkness

Type B yeasts were more virulent for mice than type A under most experimental conditions. Mice infected with type B yeasts grown in the light lived significantly longer than those with type B yeasts grown in the dark. Virulence differences of type A yeasts grown in continuous fluorescent light versus total darkness were not statistically significant.     

The variable influence of lifestyle and income on medical students' career specialty choices: data from two U.S. medical schools, 1998-2004.

PURPOSE: Recent reports on medical students' career choices suggest that lifestyle increasingly influences career decisions. The authors addressed the changing influence of lifestyle and income on career choice, how these influences differ by specialty, and the specific careers students identify as lifestyle friendly. METHOD: From 1998 to 2004, 1,334 (73%) fourth-year medical students from Brody School of Medicine at East Carolina University (no. = 485 graduates) and New York Medical College (no. = 1,348 graduates) completed a questionnaire that addressed career specialty preferences, as well as income and lifestyle concerns. Students were asked to rate career choice influences on a four-point scale (1 = no influence, 4 = major influence). Factor analysis of these influences identified seven factors including one each for lifestyle and income. RESULTS: A total of 1,327 students indicated a career preference. Lifestyle (p = .018) and income (p = .011) were found to increasingly influence medical students' career choices during the study period. Overall, the authors found significant differences between specialties in the relative contribution of these factors. Students' perceptions of specialties existed on a continuum of lifestyle friendly (e.g., radiology) to lifestyle unfriendly (e.g., obstetrics-gynecology). Contrary to previous reports, the students' responses indicate they perceived the primary care specialties as lifestyle intermediate compared to other specialties. CONCLUSIONS: Lifestyle and income have become more important to medical students in their career choice, and the relative influence of these factors varies considerably between specialties. This study suggests that previous efforts to dichotomize careers into those with controllable and uncontrollable lifestyles may mask important complexities.     

Community participation in primary health care (PHC) programmes: lessons from tuberculosis treatment delivery in South Africa

Background

Currently, there is renewed interest in the role community participation can play in Primary Health Care (PHC) programmes such as the delivery of effective anti-TB treatment to patients in high-burden settings.

Objectives

To explore the feasibility of community participation in a high-burden Tuberculosis Control Programme and to establish how supervision of treatment by lay volunteers compares with other methods of tuberculosis treatment delivery in the Northern Cape province of South Africa.

Methods

Prospective study involving 769 patients with confirmed pulmonary TB who were followed-up over a one-year period. Questionnaire interviews were also carried out with 135 lay volunteers participating in the TB programme.

Results

One-third of the TB patients in the study received their treatment from lay volunteers in the community. Treatment outcomes for new patients supervised from the community were found to be equivalent to those who received treatment through other modes of treatment delivery (RR=1.04[0.94–1.16], p=0.435). For the re-treatment patients, community-based treatment was found to be superior (RR=5.89[2.30–15.09], p<0.001), to self-administered therapy.

Conclusions

Health care planners should consider community participation as a viable way of ensuring accessibility and effectiveness in PHC programmes. There is need for more research into ways of achieving sustainability in resource-limited but high disease burden settings.     

Borrelia burgdorferi and Interleukin-1 Promote the Transendothelial Migration of Monocytes In Vitro by Different Mechanisms

A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) or Borrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to either B. burgdorferi or IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, or B. burgdorferi-stimulated HUVEC by 91% ± 3%, 65% ± 2%, or 25% ± 22%, respectively. Stimulation of HUVEC with B. burgdorferi also promoted a 6-fold ± 2-fold increase in the migration of human CD4⁺ T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes across B. burgdorferi-treated HUVEC, migration of CD4⁺ T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response to B. burgdorferi by approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium by B. burgdorferi may contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced by B. burgdorferi is significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates that B. burgdorferi and IL-1 employ distinct mechanisms to activate endothelial cells.     

Distinct firing properties of higher order thalamic relay neurons

It has been proposed that the thalamus is composed of at least two types of nuclei. First-order relay nuclei transmit signals from the periphery to the cortex while higher order nuclei may route information from one cortical area to another. Although much is known about the functional properties of relay neurons in first-order nuclei, little is known about relay neurons belonging to higher-order nuclei. We investigated the electrophysiological properties of relay cells in a higher-order thalamic nucleus using in vitro intracellular recordings from thalamic slices of the rat's lateral posterior nucleus (LPN). We found neurons of the LPN possess many of the same membrane properties as first-order relay neurons. These included low-threshold calcium spikes (IT) and burst firing, a mixed cation conductance (IH) that prevented membrane hyperpolarization, and a transient K+ conductance that delayed spike firing (IA). The repetitive firing characteristics of LPN neurons were more distinct. One group of cells, located in the more caudal regions of the LPN responded to depolarizing current pulses with a train of action potentials or in a regular spiking (RS) mode. This form of firing showed a steep but highly linear increase in firing frequency with increasing levels of membrane depolarization. Another group of cells, located in the more rostral regions of the LPN, responded to depolarizing current pulses with clusters of high-frequency bursts or in a clustered spiking (CS) mode. The overall firing frequency rose nonlinearly with membrane depolarization, but the frequency of a given burst remained relatively constant. The caudal LPN receives input from the superior colliculus, whereas the rostral LPN receives input from layers V and VI of the visual cortex. Thus the RS and CS cells may be driven by subcortical and cortical inputs respectively, and the distinct temporal properties of their response modes may be a necessary component of the LPN circuitry.     

Molecular mechanism of temperature sensing by the circadian clock of Neurospora crassa

Expression levels and ratios of the long (l) and short (s) isoforms of the Neurospora circadian clock protein FREQUENCY (FRQ) are crucial for temperature compensation of circadian rhythms. We show that the ratio of l-FRQ versus s-FRQ is regulated by thermosensitive splicing of intron 6 of frq, a process removing the translation initiation site of l-FRQ. Thermosensitivity is due to inefficient recognition of nonconsensus splice sites at elevated temperature. The temperature-dependent accumulation of FRQ relative to bulk protein is controlled at the level of translation. The 5'-UTR of frq RNA contains six upstream open reading frames (uORFs) that are in nonconsensus context for translation initiation. Thermosensitive trapping of scanning ribosomes at the uORFs leads to reduced translation of the main ORF and allows adjustment of FRQ levels according to ambient temperature.     

The degradation of serum amyloid A protein by activated polymorphonuclear leucocytes: participation of granulocytic elastase

To determine the role of inflammation in amyloidogenesis, we have studied the degradation of human serum amyloid A (SAA) protein by purified preparations of human blood polymorphonuclear leucocytes (PMN) and monocytes. When both PMN and monocytes were incubated in SAA-containing medium, the concentration of SAA as measured by a competitive anti-AA radioimmunoassay decreased over time. The rate of decrease of SAA was similar for both monocytes and PMN and there were no differences between four patients with amyloidosis and three normal controls. Resting PMN from normal volunteers were able to degrade SAA to smaller acid-soluble peptides within 16 hr while zymosan-activated PMN produced significant degradation within 1 hr (31%–50%). The supernatants from zymosan-treated PMN also caused marked SAA degradation within 1 hr.

The following enzyme inhibitors were able to prevent degradation of SAA by PMN supernatants; phenylmethylsulphonyl fluoride, a serine esterase inhibitor; α₁ anti-trypsin and soybean trypsin inhibitor; and acetyl-ala-ala-pro-val-chloromethyl ketone, an elastase inhibitor. The ability of a neutral lysosomal enzyme to degrade SAA was further confirmed by showing that purified PMN elastase significantly degraded ¹²⁵I-SAA.

We conclude that PMN contain one or more lysosomal enzymes capable of degrading SAA, an apoprotein of HDL₃ serum lipoproteins. Alteration in SAA proteolysis by activated PMN may contribute to the deposition of amyloid fibrils in the tissues of patients with chronic inflammatory disease.

     

Growth hormone co-treatment for ovulation induction may enhance conception in the co-treatment and succeeding cycles, in clonidine negative but not clonidine positive patients

To investigate the effect of co-treatment with growth hormone(GH) for ovulation induction with human menopausal gonadotrophins(HMG) on conception, we compared the pregnancy rate and responseto co-treatment with GH versus HMG/human chorionic gonadotrophin(HCG) alone in a prospective, randomized, cross-over protocolof volation induction for either in-vivo or in-vitro fertilization(IVF). The main outcome measures were the amount of gonadotrophinused and conception. Co-treatment with GH was associated witha reduction of 30% in gonadotrophin requirement. In 24 clonidinenegative patients 14 pregnancies were achieved (58.3%) eitherin the GH/HMG/HCG cycle or in the succeeding one. GH co-treatmentdid not generate any pregnancy in eight clonidine positive patients.We conclude that growth hormone may increase the pregnancy ratewhen combined with HMG/HCG for ovulation induction, not onlyin the co-treatment cycle but also in the succeeding one. Thebeneficial, synergistic effect of GH co-treatment was detectedin clonidine negative but not in clonidine positive infertilepatients.