Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator

In the cyanobacteria Synechococcus elongatus and Thermosynechococcus elongatus, the KaiA, KaiB and KaiC proteins in the presence of ATP generate a post-translational oscillator (PTO) that can be reconstituted in vitro. KaiC is the result of a gene duplication and resembles a double doughnut with N-terminal CI and C-terminal CII hexameric rings. Six ATPs are bound between subunits in both the CI and CII ring. CI harbors ATPase activity, and CII catalyzes phosphorylation and dephosphorylation at T432 and S431 with a ca. 24-h period. KaiA stimulates KaiC phosphorylation, and KaiB promotes KaiC subunit exchange and sequesters KaiA on the KaiB-KaiC interface in the final stage of the clock cycle. Studies of the PTO protein-protein interactions are convergent in terms of KaiA binding to CII but have led to two opposing models of the KaiB-KaiC interaction. Electron microscopy (EM) and small angle X-ray scattering (SAXS), together with native PAGE using full-length proteins and separate CI and CII rings, are consistent with binding of KaiB to CII. Conversely, NMR together with gel filtration chromatography and denatured PAGE using monomeric CI and CII domains support KaiB binding to CI. To resolve the existing controversy, we studied complexes between KaiB and gold-labeled, full-length KaiC with negative stain EM. The EM data clearly demonstrate that KaiB contacts the CII ring. Together with the outcomes of previous analyses, our work establishes that only CII participates in interactions with KaiA and KaiB as well as with the His kinase SasA involved in the clock output pathway.     

Variation of hamstrings lengths and velocities with walking speed

Crouch gait, one of the most prevalent movement abnormalities among children with cerebral palsy, is frequently treated with surgical lengthening of the hamstrings. To assist in surgical planning many clinical centers use musculoskeletal modeling to help determine if a patient’s hamstrings are shorter or lengthen more slowly than during unimpaired gait. However, some subjects with crouch gait walk slowly, and gait speed may affect peak hamstring lengths and lengthening velocities. The purpose of this study was to evaluate the effects of walking speed on hamstrings lengths and velocities in a group of unimpaired subjects over a large range of speeds and to determine if evaluating subjects with crouch gait using speed matched controls alters subjects’ characterization as having “short” or “slow” hamstrings. We examined 39 unimpaired subjects who walked at five different speeds. These subjects served as speed-matched controls for comparison to 74 subjects with cerebral palsy who walked in crouch gait. Our analysis revealed that peak hamstrings length and peak lengthening velocity in unimpaired subjects increased significantly with increasing walking speed. Fewer subjects with cerebral palsy were categorized as having hamstrings that were “short” (31/74) or “slow” (38/74) using a speed-matched control protocol compared to a non-speed-matched protocol (35/74 “short”, 47/74 “slow”). Evaluation of patients with cerebral palsy using speed-matched controls alters and may improve selection of patients for hamstrings lengthening procedures.     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 Å crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G–ribosome complex.     

Thermodynamic effects of replacements of Pro residues in helix interiors of maltose-binding protein

Introduction of Pro residues into helix interiors results in protein destabilization. It is currently unclear if the converse substitution (i.e., replacement of Pro residues that naturally occur in helix interiors would be stabilizing). Maltose-binding protein is a large 370-amino acid protein that contains 21 Pro residues. Of these, three nonconserved residues (P48, P133, and P159) occur at helix interiors. Each of the residues was replaced with Ala and Ser. Stabilities were characterized by differential scanning calorimetry (DSC) as a function of pH and by isothermal urea denaturation studies as a function of temperature. The P48S and P48A mutants were found to be marginally more stable than the wild-type protein. In the pH range of 5-9, there is an average increase in T(m) values of P48A and P48S of 0.4 degrees C and 0.2 degrees C, respectively, relative to the wild-type protein. The other mutants are less stable than the wild type. Analysis of the effects of such Pro substitutions in MBP and in three other proteins studied to date suggests that substitutions are more likely to be stabilizing if the carbonyl group i-3 or i-4 to the mutation site is not hydrogen bonded in the wild-type protein.     

Exploiting Phosphate-Starved cells of <Emphasis Type="Italic">Scenedesmus</Emphasis> sp. for the Treatment of Raw Sewage

Phosphate depletion is one of the favorable ways to enhance the sewage water treatment with the algae, however, detailed information is essential with respect to internal phosphate concentration and physiology of the algae. The growth rate of the phosphate-starved Scenedesmus cells was reduced drastically after 48 h. Indicating cells entered in the stationary phase of the growth cycle. Fourier Transform Infrared analysis of phosphate-starved Scenedesmus cells showed the reduction in internal phosphate concentration and an increase in carbohydrate/phosphate and carbohydrate/lipid ratio. The phosphate-starved Scenedesmus cells, with an initial cell density of, 1 × 10⁶ cells mL^?1 shows 87% phosphate and 100 % nitrogen removal in 24 h. The normal Scenedesmus cells need approximately 48 h to trim down the nutrients from wastewater up to this extent. Other microalgae, Ankistrodesmus, growth pattern was not affected due to phosphate starvation. The cells of Ankistrodesmus was able to reduce 71% phosphate and 73% nitrogen within 24 h, with an initial cell density of, 1 × 10⁶ cells mL^?1.     

Reactive Oxygen Species Activity and Antioxidant Properties of Fusarium Infected Bananas

Fusarium infection of bananas is a global problem that threatens the production of bananas. This study looks at the effects of the infection upon the reactive oxygen species (ROS) system, as well as the induced antioxidant properties in the roots, stems, leaves and fruits. Results show that there is a greater amount of damage in infected tissue samples as opposed to non‐infected. The damage was observed to be higher in the root samples. ROS assays were divided into two classes: ROS assays and ROS‐scavenging assays. Of the ROS assays, lipoxygenase was observed to be higher in the infected samples, while peroxidase (POD) and polyphenol oxidase (PPO) were significantly higher in infected stem, leaf and fruit samples. Among root samples, there was no significant difference in POD activity and PPO was lower in infected samples. Induction of ROS is important for the hypersensitive response (HR) to function properly. The ROS‐scavenging enzymes, namely ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase, exhibited higher levels in the infected tissue. This is most likely to counter the build‐up of the ROS enzymes and to prevent further cell death. The increase in ROS‐scavenging assays also correlates with higher antioxidant properties as antioxidants play a critical role in regulating the HR free radicals.     

RasG protein accumulation occurs just prior to amoebae emergence during spore germination in Dictyostelium discoideum

Abstract RasG protein levels in dormant and germinating spores of Dictyostelium discoideum strains JC1 and SG1 were estimated by Western blotting. Ras Glevels were very low in dormant spores and remained low during the lag period, regardless of whether spores were heat activated or treated with autoactivator during the early stages of spore germination. RasG levels increased late during spore swelling just prior to the emergence stage of germination. These data are consistent with a requirement for RasG during vegetative growth.     

Significant role of estrogen and progesterone receptor sequence variants in gallbladder cancer predisposition: a multi-analytical strategy

Background

Carcinoma of gallbladder (GBC) is an aggressive malignancy. The higher incidence of gallbladder cancer in women has been partly attributed to hormonal factors. Therefore the present study was designed to explore the role of genetic variants in estrogen (ESR1, ESR2) and progesterone (PGR) receptors in conferring risk of gallbladder cancer.

Materials and Methods

The present case-control study recruited total of 860 subjects, including 410 GBC patients, 230 gallstone patients and 220 controls. We examined the associations of 6 selected polymorphisms in three genes: ESR1 (rs2234693, rs9340799, rs1801132), ESR2 (rs1271572, rs1256049) and PGR (rs1042838) with GBC risk. Genotyping for all the polymorphisms was done using PCR-RFLP. Multifactor dimensionality reduction and classification and regression tree approaches were combined with logistic regression to discover high-order gene-gene interactions in hormonal pathway.

Results

On comparing the genotype frequency distribution in gallstone and GBC patients with that of healthy subjects, the homozygous variant genotypes of ESR1-397TT (rs2234693) polymorphism showed significant risk for developing gallstone [odds ratio: OR = 2.9] and GBC [OR = 1.8] respectively. Detailed haplotypes analysis suggested that ESR1 T _rs2234693G _rs9340799C _rs1801132 have significant association in conferring risk for both gallstones [OR = 2.2] and GBC [OR = 3.0]. However, the variant-containing genotypes (DI+II) of PGR (rs1042838) showed low risk in both GBC [OR = 0.4] and gallstone patients [OR = 0.4].On performing the MDR analysis, ESR1 IVS1-397C>T, ESR1 IVS1-351A>G, and ESR2-789 A>C yielded the highest testing accuracy of 0.634. These results were further supported by the CART analysis which revealed that individuals with the combined genotypes of ESR1-397 CT or TT, ESR1-351 AG or GG and ESR2 -789 AA had the highest risk for GBC [OR  = 3.9].

Conclusion

Using multi-analytical approaches, our study showed important role of ESR1 IVS1-397C>T, ESR1 IVS1-351A>G, and ESR2-789 A>C variants in GBC susceptibility and the risk appears to be mediated through gallstone dependent pathway.