Conformational chemistry of surface-attached calmodulin detected by acoustic shear wave propagation

A thickness shear-mode acoustic wave device, operated in a flow-through format, was used to detect the binding of ions or peptides to surface-attached calmodulin. On-line surface attachment of the protein was achieved by immobilisation of the biotinylated molecule via a neutravidin-biotin linkage onto the surface of the gold electrode of the detector. The interaction between calmodulin, and calcium and magnesium ions induced an increase in resonant frequency and a decrease in motional resistance, which were reversible on washing with buffer. Interestingly, the changes in resonant frequency and motional resistance induced by the binding were opposite to the normal operation of the detector. The response was interpreted as a decrease in surface coupling (partial slip at the liquid/solid interface) instigated by exposure of hydrophobic domains on the protein, and an increase in the thickness, and hence effective wavelength, of the acoustic device, corresponding to an increase in the length of calmodulin by 1.5 A. This result is consistent with the literature value of 4 A. In addition, the interaction of the protein with peptide together with calcium ions was detected successfully, despite the relatively low molecular mass of the 2-kDa peptide. These results confirm the potential of acoustic wave physics for the detection of changes in the conformational chemistry of monolayer of biochemical macromolecules at the solid/liquid interface.     

Intraoperative cytology (squash smear) in neurosurgical practice – pitfalls in diagnosis experience based on 3057 samples from a single institution

OBJECTIVES: The smear technique is challenging for a neuropathologist where rapid and accurate diagnosis is to be given on small biopsies. The present study, a large retrospective analysis of squash smears in neurosurgical practice, was conducted to assess the usefulness, accuracy and the diagnostic pitfalls of smear diagnosis. METHODS: The authors analysed 3057 central nervous system (CNS) lesions sent for intraoperative cytology (IC) during the years 1988-2005. The stain used was 1% alcoholic toluidine blue. The smear diagnosis was compared with the histological diagnosis to evaluate the diagnostic accuracy. RESULTS: Diagnostic accuracy irrespective of lesion and site ranged from 83.0% to 86.0% per year (mean=85%). The highest rate of correlation among common brain tumours was noted in schwannoma (96.6%) and pituitary adenoma (92.2%), followed by meningiomas (88.9%), astrocytomas (88.4%), chordomas (86.4%) and neurocytomas (86.9%). Infections as a whole contributed 380 cases. The most common infection was tuberculosis. CONCLUSION: This is the largest series reported from India to the best of our knowledge. Squash smear technique is a very reliable and rapid method of intraoperative diagnosis. Knowledge of clinical and neuroimaging details helps the experienced neuropathologist to improve the diagnostic accuracy.     

Caenorhabditis elegans lin-35/Rb, efl-1/E2F and other synthetic multivulva genes negatively regulate the anaphase-promoting complex gene mat-3/APC8

Retinoblastoma (Rb)/E2F complexes repress expression of many genes important for G(1)-to-S transition, but also appear to regulate gene expression at other stages of the cell cycle. In C. elegans, lin-35/Rb and other synthetic Multivulva (SynMuv) group B genes function redundantly with other sets of genes to regulate G(1)/S progression, vulval and pharyngeal differentiation, and other unknown processes required for viability. Here we show that lin-35/Rb, efl-1/E2F, and other SynMuv B genes negatively regulate a component of the anaphase-promoting complex or cyclosome (APC/C). The APC/C is a multisubunit complex that promotes metaphase-to-anaphase progression and G(1) arrest by targeting different substrates for ubiquitination and proteasome-mediated destruction. The C. elegans APC/C gene mat-3/APC8 has been defined by temperature-sensitive embryonic lethal alleles that strongly affect germline meiosis and mitosis but only weakly affect somatic development. We describe severe nonconditional mat-3 alleles and a hypomorphic viable allele (ku233), all of which affect postembryonic cell divisions including those of the vulval lineage. The ku233 lesion is located outside of the mat-3 coding region and reduces mat-3 mRNA expression. Loss-of-function alleles of lin-35/Rb and other SynMuv B genes suppress mat-3(ku233) defects by restoring mat-3 mRNA to wild-type levels. Therefore, Rb/E2F complexes appear to repress mat-3 expression.     

The Lec23 Chinese hamster ovary mutant is a sensitive host for detecting mutations in alpha-glucosidase I that give rise to congenital disorder of glycosylation IIb (CDG IIb)

Lec23 Chinese hamster ovary cells are defective in alpha-glucosidase I activity, which removes the distal alpha(1,2)-linked glucose residue from Glc(3)Man(9)GlcNAc(2) moieties attached to glycoproteins in the endoplasmic reticulum. Mutations in the human GCS1 gene give rise to the congenital disorder of glycosylation termed CDG IIb. Lec23 mutant cells have been shown to alter lectin binding and to synthesize predominantly oligomannosyl N-glycans on endogenous glycoproteins. A single point mutation (TCC to TTC; Ser to Phe) was identified in Lec23 Gcs1 cDNA and genomic DNA. Serine at the analogous position is highly conserved in all GCS1 gene homologues. A human GCS1 cDNA reverted the Lec23 phenotype, whereas GCS1 cDNA carrying the lec23 mutation (S440F in human) did not. By contrast, GCS1 cDNA with an R486T or F652L CDG IIb mutation gave substantial rescue of the Lec23 phenotype. Nevertheless, in vitro assays of each enzyme gave no detectable alpha-glucosidase I activity. Clearly the R486T and F652L GCS1 mutations are only mildly debilitating in an intact cell, whereas the S440F mutation largely inactivates alpha-glucosidase I both in vitro and in vivo. However, the S440F alpha-glucosidase I may have a small amount of alpha-glucosidase I activity in vivo based on the low levels of complex N-glycans in Lec23. A sensitive test for complex N-glycans showed the presence of polysialic acid on the neural cell adhesion molecule. The Lec23 Chinese hamster ovary mutant represents a sensitive host for detecting a wide range of mutations in human GCS1 that give rise to CDG IIb.     

Novel water soluble 2,6-dimethoxyphenyl ester derivatives with intravenous anaesthetic activity

A number of water soluble bis-amino-2,6-dimethoxyphenyl ester derivatives were found to exhibit improved anaesthetic activity in mice relative to propofol 1. Of the analogues disclosed, 44 was further profiled in rodents and found to be a superior agent to propofol for the induction and maintenance of anaesthesia.     

Functional and molecular characterization of nucleobase transport by recombinant human and rat equilibrative nucleoside transporters 1 and 2. Chimeric constructs reveal a role for the ENT2 helix 5-6 region in nucleobase translocation

The human (h) and rat (r) equilibrative (Na(+)-independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine and coronary vasoactive drugs. Structurally, the proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, hENT1, rENT1, hENT2, and rENT2 were produced in Xenopus laevis oocytes and investigated for their ability to transport pyrimidine and purine nucleobases. hENT2 and rENT2 efficiently transported radiolabeled hypoxanthine, adenine, guanine, uracil, and thymine (apparent K(m) values 0.7-2.6 mm), and hENT2, but not rENT2, also transported cytosine. These findings were independently confirmed by hypoxanthine transport experiments with recombinant hENT2 produced in purine-cytosine permease (FCY2)-deficient Saccharomyces cerevisiae and provide the first direct demonstration that the ENT2 isoform is a dual mechanism for the cellular uptake of nucleosides and nucleobases, both of which are physiologically important salvage metabolites. In contrast, recombinant hENT1 and rENT1 mediated negligible oocyte fluxes of hypoxanthine relative to hENT2 and rENT2. Chimeric experiments between rENT1 and rENT2 using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) identified TMs 5-6 of rENT2 (amino acid residues 172-231) as a determinant of nucleobase transport activity, suggesting that this domain forms part(s) of the ENT2 substrate translocation channel.     

C. elegans ksr-1 and ksr-2 have both unique and redundant functions and are required for MPK-1 ERK phosphorylation

Kinase Suppressor of Ras (KSR) is a conserved protein that positively regulates Ras signaling and may function as a scaffold for Raf, MEK, and ERK. However, the precise role of KSR is not well understood, and some observations have suggested that KSR might act in a parallel pathway. In C. elegans, ksr-1 is only required for a specific Ras-mediated process (sex myoblast migration) and is a nonessential positive regulator of other Ras-mediated developmental events. We report the existence of a second C. elegans ksr gene, ksr-2, which is required for Ras-mediated signaling during germline meiotic progression and functions redundantly with ksr-1 during development of the excretory system, hermaphrodite vulva, and male spicules. Thus, while the ksr-1 and ksr-2 genes are individually required only for specific Ras-dependent processes, together these two genes appear necessary for most aspects of Ras-mediated signaling in C. elegans. The finding that ksr-2; ksr-1 double mutants have strong ras-like phenotypes and severely reduced or absent levels of diphosphorylated MPK-1 ERK strongly supports models where KSR acts to promote the activation or maintenance of the Raf/MEK/ERK kinase cascade.     

Structural basis of the thermostability of monomeric malate synthase from a thermophilic Bacillus.

Malate synthases from a thermophilic Bacillus and Escherichia coli have been isolated in a high state of purity. Molecular weights of these two proteins determined in the native state and after denaturation in sodium dodecyl sulfate-mercaptoethanol show that the enzymes are monomeric. This conclusion is supported, for the thermophile enzyme, by the result of an electrophoretic analysis of that protein after treatment with dimethylsuberimidate and denaturation. The thermophilic Bacillus malate synthase is considerably more thermostable than its mesophilic counterparts from E. coli, Bacillus licheniformis, and Pseudomonas indigofera. It is, however, markedly labilized by an increase in the ionic strength of the medium brought about by the addition of 0.2 M potassium chloride or in pH above 9. Increased ionic strength has little effect on the thermostability of the mesophilic bacterial malate synthases. These observations provide strong support for the idea that monomeric proteins in thermophiles owe their unusual heat stability to the presence of salt bridges in their tertiary structure.