Probing the stabilizing role of C-terminal residues in trimethylamine dehydrogenase

In trimethylamine dehydrogenase, a homodimeric iron-sulfur flavoprotein, the C-terminal 17 residues of each subunit (residues 713- 729) embrace residues on the other subunit. The role of this unusual mode of interaction at the subunit interface was probed by isolating three mutant forms of trimethylamine dehydrogenase in which the C- terminus of the enzyme was deleted by five residues [delta(725-729], 10 residues [delta(720-729)] and 17 residues [delta(713-729)]. The solution properties and conformational states of the three mutant enzymes were investigated using optical, fluorescence and circular dichroism spectroscopies, ANS binding and a novel and conformationally sensitive hydrodynamic method. The data reveal that sequential deletion of the C-terminus of trimethylamine dehydrogenase does not affect significantly dimer stability or the overall structural integrity of the enzyme. However, deletion of the C-terminus severely compromises, but does not abolish, the ability of the enzyme to become covalently coupled with the redox cofactor FMN in the active site, located over 20 A from the C-terminus. Hydrodynamic studies reveal minor conformational changes in the deletion mutants that lead to a more compact enzyme structure. These conformational changes are probably transmitted to the active site via altering the interaction of the C-terminus with the second helix in the beta/alpha barrel of trimethylamine dehydrogenase, leading to poor flavinylation during the folding of the enzyme and assembly with FMN.      

Identification of a novel complement-dependent serum-elicited inward current in the Xenopus oocyte provoking Ca2+ influx and subsequent activation of Cl- channels

The membrane spanning complement channel is assumed to be a nonselective ion 'pore', although little evidence is available to support this hypothesis. In this paper we provide evidence that Ca2+ entry and Cl- exit occur rapidly after complement activation and precede the development of a long-lasting complement-dependent inward current. Addition of rabbit serum (a source of heterologous complement) and mouse anti-human insulin receptor antibody to a single Xenopus oocyte expressing human insulin receptor was shown to stimulate an initial hyperpolarising current followed by a sustained depolarising current. On voltage clamping the oocyte, a novel long-lasting inward current generated by serum addition was detected. Complement classical pathway-stimulated calcium influx into the oocyte was directly demonstrated using 45Ca influx measurements. In addition, we found that Ca2+ influx was required for the stimulation of the complement alternative pathway-dependent inward current. The novel conductance elicited by the classical pathway was outwardly rectifying, had a reversal potential of -35 +/- 8 mV (or -52 +/- 7 mV in the presence of chloride channel inhibitors), was inhibited by nifedipine, and was observed in the presence but not in the absence of the pore-forming complement component C9. As overactivation of complement does play a role in many inflammatory or autoimmune diseases, inhibition of early complement-mediated ion flux might restrict tissue damage and aid recovery from such diseases.     

The proteolytic maturation of prohormone convertase 2 (PC2) is a pH-driven process

Recombinant proPC2 purified from the medium of CHO cells overexpressing both the prohormone convertase (PC) precursor proPC2 and the 21-kDa amino terminal portion of the neuroendocrine protein 7B2 can spontaneously convert to an active species. In the present report, we have characterized the proPC2 zymogen conversion process. Sequencing of the mature 66 kDa enzyme revealed a single site of cleavage at the paired basic site amino terminal to the GYRDI sequence. In contrast to mature PC2 activity, proPC2 conversion was inhibited neither by the eukaryotic subtilisin inhibitor pCMS nor by the specific PC2 inhibitor, 7B2 CT peptide, suggesting significant differences between the proPC2 conversion reaction and the hydrolysis of synthetic substrates by mature PC2. In support of this idea, proPC2 conversion was not calcium dependent and was unaffected by 5 mM EDTA. The rate of conversion of proPC2 remained similar with a 10-fold difference in zymogen concentration, implicating an intramolecular rather than intermolecular mechanism of activation. Interestingly, the rate of proPC2 conversion was extremely pH dependent, occurring most extensively between pHs 4.0 and 4.9. Taken together, our results suggest that cellular proPC2 maturation occurs via an autocatalytic, intramolecular process controlled not by 7B2 inhibition nor by calcium levels, but by the decreasing pH gradient along the secretory pathway.     

Electron microscopy may reveal structure of docosahexaenoic acid-rich oil within Schizochytrium sp

Schizochytrium sp. is an algae-like microorganism utilized for commercial production of docosahexaenoic acid (DHA)-rich oil and dried microalgae for use as a source of DHA in foods, feeds, and nutritional supplements. Electron microscopic analysis of whole cells of Schizochytrium sp. employing sample preparation by high-pressure freeze substitution suggests the presence of secondary and tertiary semicrystalline structures of triacylglycerols within the oil bodies in Schizochytrium sp. A fine secondary structure consisting of alternating light-and darkstaining bands was observed inside the oil bodies. Dark bands were 29±1 Å in width, and light bands were 22±1 Å in width. The tertiary (three-dimensional) structure may be a multilayered ribbon-like structure which appears coiled and interlaced within the oil body. In freeze-fracture photomicrographs, Schizochytrium oil bodies exhibited fracture planes with terraces averaging 52±7 Å in height which could correspond to the combined width of two halves of two light bands and one dark band observed in the high-pressure freeze substitution photomicrographs. The results suggest that triacyglycerols within Schizochytrium sp. oil bodies may be organized in a triple chainlength structure. High-pressure freeze substitution electron micrographs of two other highly unsaturated oil-producing species of microalgae, Thraustochytrium sp. and Isochrysis galbana, also revealed this fine structure, whereas microalgae containing a higher proportion of saturated oil did not. The results suggest that the staining pattern is not an artifact of preparation and that the triple chain-length conformation of triacylglycerols in Schizochytrium sp. oil bodies may be caused by the unique fatty acid composition of the triacylglycerols.     

Queer theory,cyber-ethnographies and researching online sex environments

Both the act and the commission of the act of sex have been transformed by technology. This has in turn led to emerging research that seeks to consider online research methods and methodologies that take account of the new medium, with a number of studies examining specific groups and the behaviour of those groups from a socio-legal perspective. This paper will seek to consider the application of queer theory to researching so-called ‘virtual’ or online sex groups. It will examine how the virtual spaces, and the researchers who survey them, are constituted. The ethical and practical issues that emerge in surveying these groups from a queer theory perspective will also be explored.     

Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.     

Dopaminergic transmission between identified neurons from the mollusk, Lymnaea stagnalis

1. Dopaminergic transmission was investigated in the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis. 2. The giant pedal neuron, designated as right pedal dorsal one (RPeD1), makes chemical, monosynaptic connections with a number of identified follower cells in the CNS. Previous work has shown that RPeD1 is an interneuron and a important component of the Lymnaea respiratory central pattern generator. In this study, the hypothesis that RPeD1 uses dopamine as its neurotransmitter was tested by chromatographic, pharmacological, and electrophysiological methods. Characterization of RPeD1's transmitter pharmacology is essential to clearly understand its role in Lymnaea. 3. Earlier studies demonstrated that the soma of RPeD1 contains dopamine. This was quantitated in the present study by high-performance liquid chromatography (with electrochemical detection) of isolated RPeD1 somata and growth cones, which yielded 0.8 +/- 0.3 and 0.10 +/- 0.08 pmol of dopamine per soma and growth cone, respectively. 4. Bath or pressure application of dopamine to follower cells of RPeD1, in situ, mimicked the effects of RPeD1 stimulation. Dose-response curves were constructed for the excitatory effect of dopamine on follower cells, visceral dorsal two and three (VD2/3) (ED50 = 39 microM; Hill coefficient = 1.03), and the inhibitory effect of dopamine on follower cell, visceral dorsal four (ED50 = 33 microM; Hill coefficient = 0.92). 5. The following dopamine agonists (100 microM) were tested by bath application: 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN), apopmorphine, 2-bromo-alpha-ergocryptine, deoxyepinephrine (DE), mesulergine, (-) quinpirole, SKF 38393, and tyramine. Only the general dopamine agonists, ADTN and DE, mimicked RPeD1's effects on its follower cells. 6. When VD2/3 was isolated and plated in vitro, it maintained a depolarizing response to dopamine. This response was reduced by intracellular injection of the G-protein blocker, GDP-beta-S (2 mM in electrode). Similarly, incubation of VD2/3, in vitro for approximately 18 h, with pertussis toxin (PTX; 5 micrograms/ml), the G-protein inactivating exotoxin, also reduced the dopamine response. Injecting GDP or incubating in heat-inactivated PTX did not effect the response. 7. Several dopamine antagonists were used in an attempt to block RPeD1's synapses: chlorpromazine, ergonovine, fluphenazine, haloperidol, 6-hydroxydopamine, SCH 23390, (+/-) sulpiride, and tubocurarine. Only the D-2 dopamine receptor antagonist, (+/-) sulpiride, reversibly blocked synaptic transmission from RPeD1 to its follower cells. Both the (+) and the (-) enantiomer of sulpiride also antagonized synaptic transmission. A dose-inhibition curve for (+/-) sulpiride was constructed (IC50 = 47 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Lack of correlation of human fibroblast radiosensitivity in vitro with early skin reactions in patients undergoing radiotherapy

Fibroblasts from breast cancer patients were obtained as outgrowths in vitro from punch biopsies and their radiosensitivity tested in early passages. Skin erythema reactions in the same patients were also measured, as degree of redness using reflectance spectrophotometry. Measurements were taken before and during a 4-week radiotherapy treatment with electrons to the thoracic wall. Of 59 biopsies studied, radiosensitivity and erythema were concurrently studied in 32. In 24, evaluable data from both clinic and laboratory were obtained. A population growth assay in 96-well plates, using absorption of sulphur rhodamine B as the stain for cell numbers, showed good agreement with the colony-formation assay. Plating efficiencies and growth rates in the colony assay were higher using human serum in place of foetal calf serum. Cell survival curves with human serum were mostly exponential with little shoulder. The parameters of survival at 2 Gy (SF2) and the dose required to give 10% survival (D10) were used in the correlations with clinical data; these were 0.25 +/- 0.09 and 3.03 +/- 0.50 Gy, respectively. There was a strong correlation between these two survival curve parameters (r = 0.98). Skin redness was found to linearly increase with time during radiotherapy. The slope of the increase differed markedly from patient to patient, with a range of a factor approx. 10. No correlation was found between SF2 and erythema response in the 24 evaluable patients (r = 0.13, p > 0.5). A similar lack of correlation was found using D10 as the radiosensitivity parameter (r = 0.12, p > 0.5). These data indicate that fibroblast radiosensitivity measured in vitro cannot be used to predict erythema reactions to radiotherapy in breast cancer patients.