Relative spectral response as a function of sequential ligand binding

A unique method is presented for the determination of the critical number of ligands that must bind to a macromolecule to elicit a spectroscopic response. This method is based on analysis of ligand binding data. For example, four Ca2+ and two Mg2+ ions are necessary for mirroring the relative decrease in the intrinsic fluorescence of bovine prothrombin fragment 1. For application of the method, ligand loading and relative spectroscopic response data must be measured over a full range of concentrations.     

Identification and characterization of mitochondria autoantigens in progressive systemic sclerosis: identity with the 72,000 dalton autoantigen in primary biliary cirrhosis

Sera from eight out of 62 (14.5%) patients with progressive systemic sclerosis (PSS) reacted by immunoblotting with a 72,000 dalton antigen and one, a patient with concomitant primary biliary cirrhosis (PBC), reacted with the 72,000 dalton and a 47,000 dalton antigen. Reactivity with these antigens was not seen with any of 111 control sera. The antigens with minor variations in m.w. were present in a variety of cultured cells and tissue homogenates from different species. Subcellular fractionation studies localized the antigens to the mitochondria. Of 19 sera from patients with other diseases selected for immunofluorescence staining for anti-mitochondria autoantibody, nine reacted with the 72,000 dalton antigen, seven reacted with both the 72,000 and 47,000 dalton antigens, and three reacted with the 47,000 dalton antigen. These results show that serum reactivity with the 72,000 dalton and 47,000 dalton mitochondria autoantigens is found with some patients with PSS. Because mitochondria autoantibodies that are reactive with the 72,000 dalton and 47,000 dalton polypeptides are also found in patients with PBC, the present finding provides additional support for the association of PSS with PBC. Prior absorption of rat liver homogenate with PBC sera removed PSS serum reactivity with a 63,000 dalton antigen, the equivalent 72,000 dalton antigen in rodents, and vice versa, showing that both PBC and PSS sera recognize the same antigen.     

Variations in concentration of oxytocin and vasopressin in the paraventricular nucleus of the hypothalamus during the estrous cycle in rats

Oxytocin (OT) and arginine-8-vasopressin (AVP) were measured by radioimmunoassay in micropunched hypothalamic neurosecretory nuclei of estrous cycling female Sprague-Dawley rats. In the paraventricular nucleus (PVN): the concentration (pg/microgram protein) of OT was significantly higher in rats in diestrus than during proestrus, estrus, or metestrus, while the concentration during metestrus was significantly greater than in proestrus and estrus; the concentration of AVP was significantly lower in animals in estrus than during the other three stages; because the paraventricular OT levels dropped before proestrus, the AVP/OT ratio was significantly greater in animals in proestrus than in diestrus, metestrus, and estrus. In the supraoptic nucleus (SON) a similar trend was noted: the concentration of OT was highest during diestrus, and AVP was lowest during estrus, though neither was significantly different from other stages. Because the OT and AVP cycles in the SON were asynchronous, the ratio of AVP to OT was significantly higher in proestrus than in metestrus or diestrus and significantly greater in estrus than during diestrus. In contrast to these two areas, peptide concentrations did not vary significantly across the estrous cycle in other sites of nonapeptide synthesis, i.e. the anterior commissural nucleus (ACN) and the suprachiasmatic nuclei (SCN).     

Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.     

Rosenhlattichthys nemotoi,a new species of scopelarchidae,from the south Indian Ocean Subtropical Convergence Zone

Rosenblattichthys nemotoi is described from a single larval specimen, 37 mm SL (standard length) taken from the southern Indian Ocean Subtropical Convergence Zone. This species differs from all otherRosenblattichthys in meristic characters (25 anal-fin rays, 26 or 27 pectoral-fin rays), configuration of accessory pigment areas (two areas present; a dorsal area (DA) and a midlateral peduncular area (PDA), and nonprecocity of pectoral-fin development. All other records ofRosenblattichthys are from tropical or subtropical waters.     

Do the frequencies of sister chromatid exchanges in endoreduplieated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplieated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.     

Isolation and reconstitution of the n-butylmalonate-sensitive dicarboxylate transporter from rat liver mitochondria

The mitochondrial dicarboxylate carrier has been substantially purified from rat liver mitoplasts by extraction with Triton X-114 in the presence of cardiolipin followed by chromatography on hydroxylapatite. Upon incorporation of the hydroxylapatite eluate into phospholipid vesicles, an n-butylmalonate-sensitive malonate/malate exchange has been demonstrated. This exchange activity is enhanced 226-fold relative to the starting material (i.e. detergent-extracted mitoplasts). Silver-stained sodium dodecyl sulfate-polyacrylamide gradient gels verify the high purity of this fraction relative to the starting material. Nonetheless, the banding pattern indicates that several protein species are still present. As isolated, the dicarboxylate transporter is rather unstable but can be stabilized either by the addition of 10% ethylene glycol and subsequent storage at -20 degrees C or by incorporation into phospholipid vesicles in the presence of malate followed by freezing in liquid nitrogen. Such proteoliposomes catalyze a [14C]malonate uptake which is characterized by a first order rate constant of 1.02 min-1 and a t 1/2 of 41 s. This uptake can be inhibited by dicarboxylates (e.g. succinate, malate, unlabeled malonate) but not by either alpha-ketoglutarate or by tricarboxylates (e.g. citrate, threo-Ds-isocitrate). Furthermore, the reconstituted malonate transport is dependent on internal malate and can be inhibited by n-butylmalonate, mersalyl, p-chloromercuribenzoate, and Pi, but not by N-ethylmaleimide. It is concluded that this highly purified fraction contains a reconstitutively active dicarboxylate transporter which, based on its substrate specificity and inhibitor sensitivity, appears to be identical to the native dicarboxylate transport system found in intact rat liver mitochondria.     

The value of provoked expectoration in obtaining sputum samples for cytologic investigation. A prospective, consecutive and controlled investigation of 134 patients

A prospective controlled investigation in 134 consecutive outpatients compared the cytologic adequacy of sputum samples obtained by spontaneous and provoked expectoration. Inhalation of nebulized 10% sodium chloride was used for provoked expectoration. A significantly higher number of adequate samples was produced after provocation, as judged by the presence of alveolar macrophages (X2 = 5.63; p less than 0.02). The improvement in sample adequacy was limited to the nonsmokers and ex-smokers in the study. This result, together with the relatively high cost of cytologic sputum examinations, indicates that provoked expectoration should at least be applied to the collection of sputum samples from nonsmokers and ex-smokers.     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.     

Influence of Deep Ocean Sewage Outfalls on the Microbial Activity of the Surrounding Sediment

The microbial activity near two deep ocean sewage outfalls off the coast of the island of Oahu, Hawaii, was characterized. Water samples and sediment samples to a depth of 4.5 cm were analyzed from an area of approximately 4.5 × 10⁴ m² surrounding the outfalls. Although the effluent water at both sites exhibited heterotrophic activity that was 2 orders of magnitude greater than water from a control site, ambient water samples taken within 1 m of the discharge ports exhibited activity only twice that of the control water. The heterotrophic activity of the outfall sediment was only elevated above that of the control site for surface samples collected within 10 m of the outfall. Likewise, the rates of microbial nucleic acid synthesis and carbon production in the sediment were only elevated immediately adjacent to the outfalls. Total microbial biomass, as determined by the ATP content of the sediment, varied spatially but was generally elevated at the outfall sites. The specific growth rates calculated for the sediment microbial populations, however, were not greater at the outfall sites. At one site the rocks surrounding the diffuser pipe were covered with copious amounts of slime that appeared to be composed entirely of microbial cells and filaments. This microbial mat was extremely active with respect to heterotrophic activity and biomass production. Overall, it appears that the impact of the sewage discharge on the ambient seawater microbiota is slight and that the effect on the sediment microbiota is confined to an area immediately adjacent to the diffuser ports. In the sand itself, the effect is limited to the upper 2 cm at most.