The conformations and basal conformational dynamics of translocation factor SecDF vary with translocon SecYEG interaction.

The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered. Atomic force microscopy (AFM) is a powerful technique for studying the dynamics of single-molecule systems including membrane proteins in near-native conditions. The sharp tip of the AFM provides direct access to membrane-external protein conformations. Here, we acquired AFM images and kymographs (∼100 ms resolution) to visualize SecDF protrusions in near-native supported lipid bilayers and compared the experimental data to simulated AFM images based on static structures. When studied in isolation, SecDF exhibited a stable and compact conformation close to the lipid bilayer surface, indicative of a resting state. Interestingly, upon SecYEG introduction, we observed changes in both SecDF conformation and conformational dynamics. The population of periplasmic protrusions corresponding to an intermediate form of SecDF, which is thought to be active in precursor protein handling, increased more than ninefold. In conjunction, our dynamics measurements revealed an enhancement in the transition rate between distinct SecDF conformations when the translocon was present. Together, this work provides a novel vista of basal-level SecDF conformational dynamics in near-native conditions.

Differential invasion of Trypanosoma brucei brucei and lymphocytes into the brain of C57BL/6 and 129Sv/Ev mice.

Trypanosoma brucei subspecies invade the brain parenchyma at late stages of human and experimental rodent infections. In this study, we compared the outcome of infection with T. b. brucei in MHC-matched (H-2b) C57BL/6 (B6) and 129Sv/Ev (Sv-129). Sv-129 showed higher parasitaemia and lower specific IgM (but not IgG) antibody levels than B6 mice. The number of trypanosomes, CD4+ and CD8+ T cells in the brain parenchyma was higher in B6 mice. B6 mice lost weight and showed higher cumulative mortality when compared with Sv-129 mice. Higher levels of IL-1beta, IL-6, IL-10, TNF-alpha, IFN-gamma, ICAM-1 and E-selectin, but low levels of TGF-beta mRNA were present in brains of B6 when compared with Sv-129-infected mice. Thus, host genetics differentially determine the invasion of T. b. brucei into the brain parenchyma, which is paralleled by the severity of inflammation in the brain and course of the disease, but not by parasitaemia nor by antibody titres.

Positive Kveim test in patients with coexisting sarcoidosis and human immunodeficiency virus infection.

We present two cases of sarcoidosis complicated by HIV infection. Each case had a different level of sarcoidosis activity and coexisted with either an AIDS-related infection or a HIV-positive state. Manifestations of sarcoidosis were not apparent in the patient with the AIDS-defining opportunistic infection, but were active in the patient with asymptomatic HIV infection. Both patients had granulomatous reactions to Kveim antigen, and one had such a reaction following an AIDS-defining infection. These findings suggest that non-T-cell mechanisms may be involved in granuloma formation in sarcoidosis.

Influence of major cardiopulmonary surgery on serum levels of procalcitonin and other inflammatory markers.

Procalcitonin is a marker of significant bacterial infection. With Food and Drug Administration approval of a new high-sensitive procalcitonin assay in the United States, we felt it would be important to assess its normal elevation and time characteristics, as compared to other inflammatory markers in patients undergoing routine cardiac surgery. This is a prospective observational study including consecutive patients after routine cardiac surgery. Blood was sampled preoperatively, immediately postoperatively and daily until discharge or to postoperative day five for measurement of blood markers of infection. Patients were classified into different groups based on the type of surgery (on-pump and off-pump) and progression of recovery (complicated and uncomplicated). Patients after coronary artery bypass grafting off-pump (n=61) had significantly lower mean (0.90 vs 1.13 µg/l, P=0.006) and peak (2.09 vs 2.35 µg/l, P=0.002) procalcitonin levels in the postoperative course compared to patients with either on-pump valve surgery alone, on-pump coronary artery bypass grafting alone (n=28) or valve surgery with coronary artery bypass grafting (n=16). In addition, mean and peak procalcitonin levels were significantly higher (P=0.004 and P=0.002 respectively) in the 60 patients with a complicated course. This study provides insights into 'normal' kinetics of a new high-sensitive procalcitonin assay after different types of cardiac surgery, and in patients with and without a complicated postoperative course. Our results suggest that using a single procalcitonin level to guide antibiotic therapy decisions during the early period after major cardiac surgery may not be useful and that monitoring its kinetic may be the preferred strategy.

Functional antagonism between hormone receptor systems: modulation of glycoprotein secretion in secretory epithelial cells.

A physiologic response such as mucin secretion from epithelial cells in vivo may be under the control of several endogenous substances such as acetylcholine, norepinephrine, and vasoactive intestinal peptide (VIP). These substances may simultaneously activate distinct membrane receptors that exist on the same epithelial cells, and this activation may result in reciprocal physiologic responses or functional antagonism. To test whether simultaneous activation of the VIP and muscarinic receptors or of beta-adrenoreceptors and muscarinic receptors affect mucin secretion in a reciprocal manner, we studied some characteristics of the resultant physiologic response in human epithelial cells secreting radiolabeled mucin-like glycoprotein (MLGP). Both basal and methacholine (M.chol)-induced MLGP secretion could be blocked by VIP (1 pM to 1 microM) and by isoproterenol (ISO) (0.1 nM to 10 nM) in a concentration-dependent and reversible manner. In a membrane preparation from the same cells, VIP (1 to 1,000 nM) and ISO (0.1 to 10 microM) stimulated adenylyl cyclase activity in a concentration-dependent and nonadditive manner. In the same membrane preparation, no effect of M.chol was observed on this response to VIP or to ISO. It is proposed that functional antagonism at the cellular level between basal or cholinergic-stimulated mucin secretion and either activated beta-adrenergic or VIP receptors may play a crucial role in modulation of mucin secretion from epithelial cells.