The role of transmembrane domain III in the lactose permease of Escherichia coli.

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.     

Recovery of a cell surface fetal antigen from circulating immune complexes of melanoma patients

Summary A well-characterized 69.5×10³ dalton glycoprotein fetal antigen (FA), isolated from the spent culture medium of a melanoma cell line, UCLA-SO-14 (M14), was utilized to characterize the antigen component of circulating immune complexes (CIC) from melanoma patients. Ten serum samples from five patients with stage II melanoma at 1 and 4 months prior to the clinical detection of recurrent disease were selected for study. The CIC were dissociated with low pH and ultrafiltered through a 100¢10³ dalton exclusion limit membrane. The low pH treatment resulted in an increase in antibody titer in eight of ten serum samples. The antibody activity in membrane immunofluorescence was quantitatively inhibited by the filtered antigen fraction and purified FA, suggesting the presence of anti-FA antibodies in the treated serum, which possibly were complexed with FA in the untreated sample. As determined by competitive inhibition in an enzyme-linked immunosorbent assay, the filtrate (antigen fraction) contained an antigen that was immunologically similar to FA. These results clearly demonstrate that FA, expressed on the cell surface of melanoma cells, is present in CIC of selected melanoma patients.Supported in part by NIH grants CA 30019, CA 12582, CA 09010, CA 29605 awarded by DHSS     

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

Candida albicans, the most prevalent human fungal pathogen, is generally diploid. However, 50% of isolates that are resistant to fluconazole (FLC), the most widely used antifungal, are aneuploid and some aneuploidies can confer FLC resistance. To ask if FLC exposure causes or only selects for aneuploidy, we analyzed diploid strains during exposure to FLC using flow cytometry and epifluorescence microscopy. FLC exposure caused a consistent deviation from normal cell cycle regulation: nuclear and spindle cycles initiated prior to bud emergence, leading to “trimeras,” three connected cells composed of a mother, daughter, and granddaughter bud. Initially binucleate, trimeras underwent coordinated nuclear division yielding four daughter nuclei, two of which underwent mitotic collapse to form a tetraploid cell with extra spindle components. In subsequent cell cycles, the abnormal number of spindles resulted in unequal DNA segregation and viable aneuploid progeny. The process of aneuploid formation in C. albicans is highly reminiscent of early stages in human tumorigenesis in that aneuploidy arises through a tetraploid intermediate and subsequent unequal DNA segregation driven by multiple spindles coupled with a subsequent selective advantage conferred by at least some aneuploidies during growth under stress. Finally, trimera formation was detected in response to other azole antifungals, in related Candida species, and in an in vivo model for Candida infection, suggesting that aneuploids arise due to azole treatment of several pathogenic yeasts and that this can occur during the infection process.     

Ligand Binding Modulates the Structural Dynamics and Compactness of the Major Birch Pollen Allergen

Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.     

Promiscuity in multidrug recognition and transport: the bacterial MFS Mdr transporters

Multidrug (Mdr) transport is an obstacle to the successful treatment of cancer and infectious diseases, and it is mediated by Mdr transporters that recognize and export an unusually broad spectrum of chemically dissimilar toxic compounds. Therefore, in addition to its clinical significance, the Mdr transport phenomenon presents intriguing and challenging mechanistic queries. Recent studies of secondary Mdr transporters of the major facilitator superfamily (MFS) have revealed that they are promiscuous not only regarding their substrate recognition profile, but also with respect to matters of energy utilization, electrical and chemical flexibility in the Mdr recognition pocket, and surprisingly, also in their physiological functions.     

Membrane Protein Biogenesis in Ffh- or FtsY-Depleted Escherichia coli

Background

The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically.

Methodology/Principal Findings

We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization.

Conclusions

Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane.     

Analysis of bacterial communities in sponges and coral inhabiting Red Sea,using barcoded 454 pyrosequencing

Microbial communities are linked with marine sponge are diverse in their structure and function. Our understanding of the sponge-associated microbial diversity is limited especially from Red Sea in Saudi Arabia where few species of sponges have been studied. Here we used pyrosequencing to study two marine sponges and coral species sampled from Obhur region from Red sea in Jeddah. A total of 168 operational taxonomic units (OTUs) were identified from Haliclona caerulea, Stylissa carteri and Rhytisma fulvum. Taxonomic identification of tag sequences of 16S ribosomal RNA revealed 6 different bacterial phyla and 9 different classes. A proportion of unclassified reads were was also observed in sponges and coral sample. We found diverse bacterial communities associated with two sponges and a coral sample. Diversity and richness estimates based on OUTs revealed that sponge H. caerulea had significantly high bacterial diversity. The identified OTUs showed unique clustering in three sponge samples as revealed by Principal coordinate analysis (PCoA). Proteobacteria (88–95%) was dominant phyla alonwith Bacteroidetes, Planctomycetes, Cyanobacteria, Firmicutes and Nitrospirae. Seventeen different genera were identified where genus Pseudoalteromonas was dominant in all three samples. This is first study to assess bacterial communities of sponge and coral sample that have never been studied before to unravel their microbial communities using 454-pyrosequencing method.     

Targeted Routine Antenatal Anti-D Prophylaxis in the Prevention of RhD Immunisation - Outcome of a New Antenatal Screening and Prevention Program

Objective

To estimate the incidence of RhD immunisation after implementation of first trimester non-invasive fetal RHD screening to select only RhD negative women carrying RHD positive fetuses for routine antenatal anti-D prophylaxis (RAADP).

Materials and Methods

We present a population-based prospective observational cohort study with historic controls including all maternity care centres and delivery hospitals in the Stockholm region, Sweden. All RhD negative pregnant women were screened for fetal RHD genotype in the first trimester of pregnancy. Anti-D immunoglobulin (250–300 µg) was administered intramuscularly in gestational week 28–30 to participants with RHD positive fetuses. Main outcome measure was the incidence of RhD immunisation developing during or after pregnancy.

Results

During the study period 9380 RhD negative women gave birth in Stockholm. Non-invasive fetal RHD genotyping using cell-free fetal DNA in maternal plasma was performed in 8374 pregnancies of which 5104 (61%) were RHD positive and 3270 (39%) RHD negative. In 4590 pregnancies with an RHD positive test the women received antenatal anti-D prophylaxis. The incidence of RhD immunisation in the study cohort was 0.26 percent (24/9380) (95% CI 0.15–0.36%) compared to 0.46 percent (86/18546) (95% CI 0.37 to 0.56%) in the reference cohort. The risk ratio (RR) for sensitisation was 0.55 (95% CI 0.35 to 0.87) and the risk reduction was statistically significant (p = 0.009). The absolute risk difference was 0.20 percent, corresponding to a number needed to treat (NNT) of 500.

Conclusions

Using first trimester non-invasive antenatal screening for fetal RHD to target routine antenatal anti-D prophylaxis selectively to RhD negative women with RHD positive fetuses significantly reduces the incidence of new RhD immunisation. The risk reduction is comparable to that reported in studies evaluating the outcome of non selective RAADP to all RhD negative women. The cost-effectiveness of this targeted approach remains to be studied.     

Comparative Molecular Docking Analysis of Cytoplasmic Dynein Light Chain DYNLL1 with Pilin to Explore the Molecular Mechanism of Pathogenesis Caused by Pseudomonas aeruginosa PAO

Cytoplasmic dynein light chain 1 (DYNLL1) is a component of large protein complex, which is implicated in cargo transport processes, and is known to interact with many cellular and viral proteins through its short consensus motif (K/R)XTQT. Still, it remains to be explored that bacterial proteins also exhibit similar recognition sequences to make them vulnerable to host defense mechanism. We employed multiple docking protocols including AUTODOCK, PatchDock, ZDOCK, DOCK/PIERR and CLUSPRO to explore the DYNLL1 and Pilin interaction followed by molecular dynamics simulation assays. Subsequent structural comparison of the predicted binding site for DYNLL1-Pilin complex against the experimentally verified DYNLL1 binding partners was performed to cross check the residual contributions and to determine the binding mode. On the basis of in silico analysis, here we describe a novel interaction of DYNLL1 and receptor binding domain of Pilin (the main protein constituent of bacterial type IV Pili) of gram negative bacteria Pseudomonas aeruginosa (PAO), which is the third most common nosocomial pathogen associated with the life-threatening infections. Evidently, our results underscore that Pilin specific motif (KSTQD) exhibits a close structural similarity to that of Vaccinia virus polymerase, P protein Rabies and P protein Mokola viruses. We speculate that binding of DYNLL1 to Pilin may trigger an uncontrolled inflammatory response of the host immune system during P. aeruginosa chronic infections thereby opening a new pioneering area to investigate the role of DYNLL1 in gram negative bacterial infections other than viral infections. Moreover, by manifesting a strict correspondence between sequence and function, our study anticipates a novel drug target site to control the complications caused by P. aeruginosa infections.     

A non-cyclic baboon θ-defensin derivative exhibiting antimicrobial activity against the phytopathogen Verticillium dahliae

θ-Defensins are the only natural cyclic proteins found in primates. They have strong antimicrobial activity related to their trisulfide ladders and macrocyclic conformation. A non-cyclic baboon θ-defensin (BTD) was synthesized by substituting valine with phenylalanine at position 17, at the C-terminal end of the BTD; this was termed “BTD-S.” The antimicrobial activities of this synthetic peptide were investigated against Escherichia coli and two cotton phytopathogens: Verticillium dahliae and Fusarium oxysporum. The minimum inhibitory concentration (MIC) of BTD-S for E. coli was 10 μg/mL and for V. dahliae was 5 μg/mL, significantly lower than that for F. oxysporum (40.0 μg/mL). A time course analysis of fungal cultures indicated that the growth of V. dahliae was completely inhibited after 96 h of BTD-S treatment. Furthermore, hemolysis assays revealed that BTD-S was not toxic to mammalian cells as it could not induce lysis of sheep red blood cells even at ten times the MIC (50 μg/mL). Scanning electron microscopy and double-stained (calcofluor white and propidium iodide binding) fluorescence microscopy showed that exposure of spores of V. dahliae to BTD-S either disabled normal germination or disintegrated the spores. The size of cells exposed to BTD-S was significantly reduced compared with controls, and their number increased in a dose-dependent curve when measured by flow cytometry. These findings suggest that BTD-S has great potential to inhibit the growth of V. dahliae and can be utilized as an effective remedy to control economic losses caused by Verticillium wilt in the development of wilt-resistant cotton.