Biosurfactant production by Pseudomonas sp. and its role in aqueous phase partitioning and biodegradation of chlorpyrifos

Aim:  To study the effect of biosurfactant on aqueous phase solubility and biodegradation of chlorpyrifos.
Methods and Results:  A Pseudomonas sp. (ChlD), isolated from agricultural soil by enrichment culture technique in the presence of chlorpyrifos, was capable of producing biosurfactant (rhamnolipids) and degrading chlorpyrifos (0·01 g l⁻¹). The partially purified rhamnolipid biosurfactant preparation, having a CMC of 0·2 g l⁻¹, was evaluated for its ability to enhance aqueous phase partitioning and degradation of chlorpyrifos (0·01 g l⁻¹) by ChlD strain. The best degradation efficiency was observed at 0·1 g l⁻¹ supplement of biosurfactant, as validated by GC and HPLC studies.
Conclusion:  The addition of biosurfactant at 0·1 g l⁻¹ resulted in more than 98% degradation of chlorpyrifos when compared to 84% in the absence of biosurfactant after 120-h incubation.
Significance and Impact of the Study:  This first report, to the best of our knowledge, on enhanced degradation of chlorpyrifos in the presence of biosurfactant(s), would help in developing bioremediation protocols to counter accumulation of organophosphates to toxic/carcinogenic levels in environment.     

Identifying and Characterizing a Novel Protein Kinase STK35L1 and Deciphering Its Orthologs and Close-Homologs in Vertebrates

Background

The human kinome containing 478 eukaryotic protein kinases has over 100 uncharacterized kinases with unknown substrates and biological functions. The Ser/Thr kinase 35 (STK35, Clik1) is a member of the NKF 4 (New Kinase Family 4 ) in the kinome with unknown substrates and biological functions. Various high throughput studies indicate that STK35 could be involved in various human diseases such as colorectal cancer and malaria.

Methodology/Principal Findings

In this study, we found that the previously published coding sequence of the STK35 gene is incomplete. The newly identified sequence of the STK35 gene codes for a protein of 534 amino acids with a N-terminal elongation of 133 amino acids. It has been designated as STK35L (STK35 long). Since it is the first of further homologous kinases we termed it as STK35L1. The STK35L1 protein (58 kDa on SDS-PAGE), but not STK35 (44 kDa), was found to be expressed in all human cells studied (endothelial cells, HeLa, and HEK cells) and was down-regulated after silencing with specific siRNA. EGFP-STK35L1 was localized in the nucleus and the nucleolus. By combining syntenic and gene structure pattern data and homology searches, two further STK35L1 homologs, STK35L2 (previously known as PDIK1L) and STK35L3, were found. All these protein kinase homologs were conserved throughout the vertebrates. The STK35L3 gene was specifically lost during placental mammalian evolution. Using comparative genomics, we have identified orthologous sets of these three protein kinases genes and their possible ancestor gene in two sea squirt genomes.

Conclusions/Significance

We found the full-length coding sequence of the STK35 gene and termed it as STK35L1. We identified a new third STK35-like gene, STK35L3, in vertebrates and a possible ancestor gene in sea squirt genome. This study will provide a comprehensive platform to explore the role of STK35L kinases in cell functions and human diseases.     

Dual requirement for flexibility and specificity for binding of the coiled-coil tropomyosin to its target, actin

The coiled coil is a widespread motif involved in oligomerization and protein-protein interactions, but the structural requirements for binding to target proteins are poorly understood. To address this question, we measured binding of tropomyosin, the prototype coiled coil, to actin as a model system. Tropomyosin binds to the actin filament and cooperatively regulates its function. Our results support the hypothesis that coiled-coil domains that bind to other proteins are flexible. We made mutations that alter interface packing and stability as well as mutations in surface residues in a postulated actin binding site. Actin affinity, measured by cosedimentation, was correlated with coiled-coil stability and local instability and side chain flexibility, analyzed with circular dichroism and fluorescence spectroscopy. The flexibility from interruptions in the stable coiled-coil interface is essential for actin binding. The surface residues in a postulated actin binding site participate in actin binding when the coiled coil within it is poorly packed.     

Characteristics and programme-defined treatment outcomes among childhood tuberculosis (TB) patients under the national TB programme in Delhi

Background

Childhood tuberculosis (TB) patients under India''s Revised National TB Control Programme (RNTCP) are managed using diagnostic algorithms and directly observed treatment with intermittent thrice-weekly short-course treatment regimens for 6–8 months. The assignment into pre-treatment weight bands leads to drug doses (milligram per kilogram) that are lower than current World Health Organization (WHO) guidelines for some patients.

Objectives

The main aim of our study was to describe the baseline characteristics and treatment outcomes reported under RNTCP for registered childhood (age <15 years) TB patients in Delhi. Additionally, we compared the reported programmatic treatment completion rates between children treated as per WHO recommended anti-TB drug doses with those children treated with anti-TB drug doses below that recommended in WHO guidelines.

Methods

For this cross-sectional retrospective study, we reviewed programme records of all 1089 TB patients aged <15 years registered for TB treatment from January to June, 2008 in 6 randomly selected districts of Delhi. WHO disease classification and treatment outcome definitions are used by RNTCP, and these were extracted as reported in programme records.

Results and Conclusions

Among 1074 patients with records available, 651 (61%) were females, 122 (11%) were <5 years of age, 1000 (93%) were new cases, and 680 (63%) had extra-pulmonary TB (EP-TB)—most commonly peripheral lymph node disease [310 (46%)]. Among 394 pulmonary TB (PTB) cases, 165 (42%) were sputum smear-positive. The overall reported treatment completion rate was 95%. Similar reported treatment completion rates were found in all subgroups assessed, including those patients whose drug dosages were lower than that currently recommended by WHO. Further studies are needed to assess the reasons for the low proportion of under-5 years of age TB case notifications, address challenges in reaching all childhood TB patients by RNTCP, the accuracy of diagnosis, and the clinical validity of reported programme defined treatment completion.     

Transformation of some hydroxy amino acids to other amino acids.

It has been observed that beta-hydroxy-alpha-amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehyde reactions, glycine also reacts with acetone to give beta-hydroxyvaline under prebiologically feasible conditions. It is suggested, therefore, that the formation of beta-hydroxy-alpha-amino acids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions.     

Androgen receptor acetylation site mutations cause trafficking defects, misfolding, and aggregation similar to expanded glutamine tracts

Kennedy's disease is a degenerative disorder of motor neurons caused by the expansion of a glutamine tract near the amino terminus of the androgen receptor (AR). Ligand binding to the receptor is associated with several post-translational modifications, but it is poorly understood whether these affect the toxicity of the mutant protein. Our studies now demonstrate that mutation of lysine residues in wild-type AR that are normally acetylated in a ligand-dependent manner mimics the effects of the expanded glutamine tract on receptor trafficking, misfolding, and aggregation. Mutation of lysines 630 or 632 and 633 to alanine markedly delays ligand-dependent nuclear translocation. The K632A/K633A mutant also undergoes ligand-dependent misfolding and aggregation similar to the expanded glutamine tract AR. This acetylation site mutant exhibits ligand-dependent 1C2 immunoreactivity, forms aggregates that co-localize with Hsp40, Hsp70, and the ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP), and inhibits proteasome function. Ligand-dependent nuclear translocation of the wild-type receptor and misfolding and aggregation of the K632A/K633A mutant are blocked by radicicol, an Hsp90 inhibitor. These data identify a novel role for the acetylation site as a regulator of androgen receptor subcellular distribution and folding and indicate that ligand-dependent aggregation is dependent upon intact Hsp90 function.     

Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis. Like its Salmonella homologues SopE and SopE2, BopE is a guanine nucleotide exchange factor for Rho GTPases. It is thought that, in order to be secreted by the type III system, proteins must be unfolded or only partially folded. As part of a study of B. pseudomallei virulence proteins, we have expressed, purified and characterized the catalytic domain of BopE (amino acids 78-261). Analytical ultracentrifugation experiments in conjunction with analytical size exclusion chromatography show that BopE(78-261) is monomeric in aqueous solution. CD spectroscopy indicates that the protein is predominantly alpha-helical, with predicted secondary structure composition of 59% alpha-helix and 7% beta-strand. NMR spectroscopy confirms that BopE(78-261) adopts a single, stable conformation. In differential scanning calorimetry experiments, thermal denaturation of BopE(78-261) (T(m) 52 degrees C) is reversible. Also, the secondary structure composition of BopE(78-261) changes little over a range of pH values from 3.5 to 10.5. BopE may therefore fold spontaneously to a functional form upon secretion into the host cell cytoplasm, and retains a native or native-like fold in varied environments. These properties are likely to be advantageous for a secreted bacterial effector protein.     

Pseudomonas cepacia lipase--mediated transesterification reactions of hydrocinnamates

Use of lipase from Pseudomonas cepacia in transesterifcation reactions of ethyl hydrocinnamate with different alcohols has been examined. Among the alcohols tested, viz., n-butanol, iso-amyl alcohol, benzyl alcohol, n-octanol and 1-phenylethanol, only n-butanol yielded the transesterified product. Among the solvents tested, viz., n-heptane, n-hexane, cyclohexane, toluene, diisopropylether and n-butanol, the initial rate of transesterification proceeded in the order cyclohexane > n-heptane > n-hexane > diisopropylether > n-butanol > toluene. Using hexane as the solvent and a substrate to enzyme ratio of 1:5, the substrate to alcohol ratio was varied to maximize the yield. n-Butyl hydrocinnamate was obtained in 92% yield in 48 hr by employing a 1:1 (wt/wt) ratio of ethyl hydrocinnamate to lipase and a 1:5 (vol/vol) ratio of ethyl hydrocinnamate to n-butanol in hexane.     

Super sural neurofasciocutaneous flaps in acute traumatic heel reconstructions

Conventional reversed sural flaps have been used to cover heel defects; however, the experience of the authors indicates that the reach of these flaps falls just short of the critical area to be covered. With the limitation being the location of the flap (the middle third of the leg), the authors thought that if the flap territory were extended proximally, they would have a super flap with immense potential. Nevertheless, the critical question remained, "How far?" The massive earthquake in January of 2001 in Gujarat, India, made medical personnel pressed for time, manpower, resources, and other ancillary supports. The authors were forced to make some innovations in their management of extensive heel defects. On the basis of preexisting anatomic studies, they developed the possibility of using distally based neuroskin flaps of huge dimensions that extend well beyond the conventional confines. The versatility of this extended, reversed, neuro-fasciocutaneous flap in regard to its reliability and safety, despite its huge dimensions, is commendable. The hallmarks of this successful extended sural flap, which the authors used to cover large heel defects, were basically accurate understanding of the anatomy and the use of Doppler to map the perforators and the lesser saphenous vein for inclusion in the lie of the pedicle. The authors share their experience of five cases of difficult heel reconstructions salvaged with this flap, which made them attempt to define maximum flap dimensions that can be harvested. The authors learned that the flap can be extended proximally to include the entire upper one-third of the leg posteriorly, drastically improving its reach and size, without compromising safety. The largest flap used measured 17 x 16 cm, far more than ever reported in the literature. The flaps truly deserve the classification of "super," which is usually reserved for Ponten's flaps.