Search for novel antifungal agents by monitoring fungal metabolites in presence of synthetically designed fluconazole derivatives using NMR spectroscopy

Resistance to currently available antifungal drugs necessitates development of new drugs using rapid, robust and automated methods to test a large number of newly synthesized drugs in less time. We have compared the effect of ketoconazole, fluconazole and its synthesized analogues on Candida albicans ATCC 10231. A metabolic profile of C.albicans ATCC 10231 in presence of drugs has been compared using ¹H NMR. Signals from metabolites have been monitored with time. MIC determined using conventional methods has been compared with Metabolic End Point (MEP) obtained from NMR spectroscopy. Results indicate that the activity of the fluconazole derivatives is in the order fluconazole p-methoxybenzoate > fluconazole = fluconazole benzoate > fluconazole toluate > fluconazole p-nitrobenzoate.     

Comparison of bacterial diversity from solar salterns and a simulated laboratory study

Bacterial diversity in solar salterns and a simulated solar saltern under laboratory conditions was studied. The two systems were compared at the pre-salt harvesting phase and salt harvesting phase using Denaturing Gradient Gel Electrophoresis (DGGE). Bacterial composition was dominated by Gammaproteobacteria and more specifically with members of Alteromonas, Vibrio, Pseudomonas, Tolumonas, Marinobacter, Pseudoalteromonas and novel uncultured bacteria. The Shannon–Weaver index (H), Simpson diversity index (D) and Equitability index (E) values showed that salterns can support a wide range of microbes during the pre-salt harvesting phase (3–4 % salinity) when compared to the salt harvesting phase (21–29 % salinity). Range-weighted richness (Rr) values indicated that solar salterns are habitable only by a limited group of microbes as they have medium richness, indicated by physico-chemical characteristics. Principal Coordinate Analysis (PCO) of the simulated study showed a variation in diversity at a large scale with increase in salinity. Solar salterns as well as the simulated tank showed more diversity during the pre-salt harvesting phase.     

Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma.     

Assembly and sorting of the tonoplast potassium channel AtTPK1 and its turnover by internalization into the vacuole

The assembly, sorting signals, and turnover of the tonoplast potassium channel AtTPK1 of Arabidopsis (Arabidopsis thaliana) were studied. We used transgenic Arabidopsis expressing a TPK1-green fluorescent protein (GFP) fusion or protoplasts transiently transformed with chimeric constructs based on domain exchange between TPK1 and TPK4, the only TPK family member not located at the tonoplast. The results show that TPK1-GFP is a dimer and that the newly synthesized polypeptides transiently interact with a thus-far unidentified 20-kD polypeptide. A subset of the TPK1-TPK4 chimeras were unable to assemble correctly and these remained located in the endoplasmic reticulum where they interacted with the binding protein chaperone. Therefore, TPK1 must assemble correctly to pass endoplasmic reticulum quality control. Substitution of the cytosolic C terminus of TPK4 with the corresponding domain of TPK1 was sufficient to allow tonoplast delivery, indicating that this domain contains tonoplast sorting information. Pulse-chase labeling indicated that TPK1-GFP has a half-life of at least 24 h. Turnover of the fusion protein involves internalization into the vacuole where the GFP domain is released. This indicates a possible mechanism for the turnover of tonoplast proteins.     

Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression

Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.     

Pregnancy after Treatment for Cervical Cancer Precursor Lesions in a Retrospective Matched Cohort

ObjectiveTo determine whether treatments for precancerous cervical lesions were associated with lower pregnancy rates compared to rates in unexposed women and women who had a diagnostic cervical biopsy or colposcopy.DesignMatched, retrospective cohort study.SettingKaiser Permanente Northwest (KPNW), an integrated healthcare delivery system in Oregon and Washington.PatientsWomen 14 to 53 years old with KPNW enrollment during the period 1998 through 2009.ResultsWe observed 570 pregnancies following cervical treatment in 4,137 women, 1,533 pregnancies following a diagnostic procedure in 13,767 women, and 7,436 pregnancies in a frequency-matched sample of 81,435 women unexposed to treatment or diagnostic procedures. After adjusting for age and contraceptive use, we observed a higher rate of pregnancies in the treatment group compared to unexposed women (hazard ratio (HR) = 1.42, 95% confidence interval (CI): 1.30–1.55), but no difference in pregnancy rates between the treatment and diagnostic procedure groups (HR = 1.03, 95% CI: 0.93–1.13).ConclusionsNo adverse effects of cervical procedures on subsequent rates of pregnancy were observed in this cohort with up to twelve years of follow-up time.     

Comparison of the activities of wild type and mutant enhancing factor/mouse secretory phospholipase A2 proteins

Enhancing factor (EF) protein, an isoform of secretory phospholipase A₂ (PLA₂), was purified as a modulator of epidermal growth factor from the small intestine of the Balb/c mouse. It was for the first time that a growth modulatory property of sPLA₂ was demonstrated. Deletion mutation analysis of EF cDNA carried out in our laboratory showed that enhancing activity and phospholipase activity are two separate activities that reside in the same molecule. In order to study the specific amino acids involved in each of these activities, two site-directed mutants of EF were made and expressedin vitro. Comparison of enhancing activity as well as phospholipase A₂ activity of these mutant proteins with that of wild type protein helped in identification of some of the residues important for both the activities.     

High-affinity binding of seminal plasma PSP94 to human immunoglobulin is through the Fab domain

Prostate secretory protein of 94 amino acids (PSP94) is one of the major proteins present in human seminal plasma. We had earlier reported that PSP94 has the ability to bind to human IgG. The aims of the present study were to further delineate the PSP94–IgG interaction and to understand whether this could have any significance in sperm function. Direct binding of IgG fragments to PSP94 showed maximal binding with F(ab′)₂ followed by Fab, while Fc displayed least binding in ELISA. Binding kinetics of PSP94–IgG interaction using surface plasmon resonance (SPR) revealed high-affinity binding of IgG to PSP94 with a dissociation constant (K_D) of 8.8 × 10⁻¹¹ M. PSP94–IgG interaction was found to be through the Fab domains of IgG. Real-time interaction kinetics revealed association constants for binding of IgG, Fab, and F(ab′)₂ towards PSP94 to be of the same order but with altered dissociation constants. IgG and its F(ab′)₂ fragment once complexed to PSP94 demonstrated negligible dissociation, while dissociation rate of Fab fragment was 6.6 × 10⁻⁴. In silico molecular modeling of PSP94–IgG complex identified N- and C-terminal β-strands of PSP94 to be the most plausible region involved in IgG interaction. Immunofluorescence studies revealed that IgG bound to human spermatozoa predominantly in the tail region, which could be prevented when IgG was preincubated with PSP94. This study reports for the first time that IgG forms a high-affinity complex with PSP94 through its F(ab′)₂ domain and reveals the ability of PSP94 to prevent binding of IgG to spermatozoa.