An investigation of the ligand-binding properties of Pseudomonas aeruginosa nitrite reductase.

The low-temperature e.p.r. and m.c.d. (magnetic-circular-dichroism) spectra of Pseudomonas aeruginosa nitrite reductase, together with those of its partially and fully cyanide-bound derivatives, were investigated. The m.c.d. spectra in the range 600-2000 nm indicate that the native axial ligands to haem c are histidine and methionine, and furthermore that it is the methionine ligand that must be displaced before cyanide binding at this haem. The m.c.d. spectra in the range 1000-2000 nm contain no charge-transfer bands arising from low-spin ferric haem d1, a chlorin. New optical transitions in the region 700-850 nm were found for the cyanide adduct of haem d1. The g-values of haem d1 in the native enzyme are 2.51, 2.43 and 1.71, suggesting co-ordination by two histidine ligands in the oxidized state. There is clear evidence in the e.p.r. data of an interaction between the c and d1 haem groups. This is not apparent in the optical spectra. The results are interpreted in terms of haem groups that are remote from each other, their interaction being mediated through protein conformational changes. The possible implications of this in relation to reduction processes catalysed by the enzyme are considered.     

Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.     

An ultrastructural study of embryonic chick retinal neurons in culture

Summary The differentiation of cells and synapses in explants of 9-day-old chick embryo retina has been studied by light and electron microscopy over a period of 35 days in vitro, and samples of retina from the 9-day chick foetus were directly fixed and prepared for study.At the time of explantation the retinae were poorly differentiated and no lamination was apparent. From day 14 onwards, (i) outer and inner nuclear layers (ONL, INL) separated by a layer of neuropil corresponding to the outer plexiform layer (OPL) and (ii) a layer of scattered large ganglion cells separated from the INL by a zone of neuropil resembling the inner plexiform layer (IPL) were apparent, and (iii) a well-differentiated outer limiting membrane was established close to the surface of the explants. In the oldest cultures some development of photoreceptor outer segments occurred but a distinct optic nerve fibre layer did not form.Although cell identification presented problems even in the oldest cultures, the major retinal cell types described in vivo could be identified. Photoreceptor cells developed pedicles in the OPL which became filled with synaptic vesicles and synaptic ribbons and established ribbon synapses (including triads) with and were commonly invaginated by processes from horizontal and bipolar cells. Processes of bipolar cells in the IPL formed simple and dyad synapses. At least two types of presynaptic amacrine cells were also identified in the INL, one of which contained large numbers of dense-core vesicles. The ganglion cells, though sparse, were large and well differentiated.These findings show that all the major neuronal types of the retina are capable of developing and differentiating in vitro, lagging behind the time-table of development and differentiation in vivo by approximately 7 days, but resulting in a histotypically organised retina with synaptic neuropil showing many similarities to the corresponding neuropil in vivo.     

Preferential conservation of the globular domains of the beta A3/A1-crystallin polypeptide of the chicken eye lens

The primary structure of the beta 19/26-crystallin polypeptide of the chicken lens has been determined by cDNA sequencing and primer extension experiments. In addition, a primer extension experiment has corrected the sequence for the N-terminal arm of the murine beta 23 polypeptide, which is the homologue of the chicken beta 19/26 polypeptide. We also show that, in the chicken and mouse, the N-terminal arm of the polypeptide is encoded on two separate exons. For simplicity, we have changed the names of both chicken beta 19/26 and murine beta 23 to beta A3/A1, which is the name of the homologous bovine polypeptide. The deduced sequence of the chicken beta A3/A1 polypeptide fits the predicted three-dimensional structure involving two homologous domains, each folded into two 'Greek key' motifs, common to the beta gamma-crystallin superfamily of proteins. Comparison of the amino acid sequence of the chicken and mammalian beta A3/A1 polypeptides indicates that different regions of the protein, which are encoded on different exons, are diverging at different rates. The N-terminal extension is the fastest evolving region of the beta A3/A1 polypeptide. Hybrid-selected translation coupled with primer extension experiments suggest that a single chicken beta A3/A1 mRNA synthesizes two polypeptides, beta A3 (25 kDa) and beta A1 (23 kDa) by utilization of different translation initiation sites.     

Host receptor sites involved in the attachment of Bdellovibrio bacteriovorus and Bdellovibrio stolpii

Abstract The location of lipoteichoic acid and 3 cell wall-associated protein antigens in fresh isolates of Streptococcus mutans (serotype c ) and in variants derived from these parental strains by repeated subculture in vitro has been examined by electrophoretic and dot-immunobinding techniques. Following subculture there was a marked shift in the distribution of all four antigens from the cell wall to the extracellular culture supernatants. It is therefore apparent that that the decreased hydrophobicity and the impaired ability of the subcultured variants to colonise in vivo, as observed by others, cannot be correlated with changes in a single surface molecule.     

Regulation of hepatic malic enzyme by perfluorodecanoic acid

Perfluorodecanoic acid (PFDA) administration to adult male rats increased both the activity of hepatic malic enzyme and liver weight in a dose-dependent manner. Hepatomegaly and augmented activity of malic enzyme in liver were apparent within one day following PFDA administration and reached a plateau by three days posttreatment. Malic enzyme quantity per liver in PFDA-treated rats was elevated within one day following dosing and increased continually throughout five days posttreatment. Administration of PFDA to rats in the fed state also led to an increase in the specific activity of hepatic malic enzyme that peaked at three days following dosing. When compared to the fed condition, rats fasted for 48 hours had a decrease in both relative liver weight and the quantity of supernatant protein per liver. The total activity (U/liver) and specific activity of malic enzyme in the liver were also reduced in the fasted state. During the 24 hours after treatment in rats fasted for 48 hours, the body weight as well as the absolute and relative liver weight of animals receiving vehicle declined continuously in the absence of feed. Following the administration of PFDA to fasted rats, body weight was maintained until eight hours posttreatment but then declined at a rate similar to that found with the vehicle-treated group. Absolute and relative liver weight in PFDA-treated rats were increased significantly at eight hours posttreatment when compared to those receiving vehicle, and this increment was maintained throughout the rest of the 24 hours following dosing. While the activity and enzyme content of hepatic malic enzyme decreased in the vehicle-treated group, administration of PFDA to rats fasted for 48 hours prevented their decline. The specific activity of hepatic malic enzyme in 48 hours fasted rats receiving PFDA was also elevated significantly at 16 hours posttreatment. Thus, the administration of PFDA to the adult male rat in both the fed and fasted nutritional states was found to regulate hepatic malic enzyme by not only increasing enzyme quantity but also by augmenting the specific activity, (ie, catalytic state) of the enzyme.     

Karyotype of Saguinus labiatus labiatus (red-bellied marmosets)

The karyotype of Saguinus labiatus labiatus was determined by the Giemsa-banding technique on leukocytes cultured from 10 marmosets. The diploid chromosome number (2n = 46) was the same and the chromosome complement similar to other marmosets of genus Saguinus. Small karyotypic differences were found between S. l. labiatus and white-lipped marmosets (Saguinus fuscicollis) in the size of the X chromosome and in the banding pattern of one pair of metacentric chromosomes. A karyotypic variant was detected in 1 S. l. labiatus, characterized by a diploid chromosome number of 45 with balanced autosomal translocation involving two pairs of acrocentric chromosomes (T 16/19).     

Electrophoretic map of boar sperm plasma membrane polypeptides and localization and fractionation of specific polypeptide subclasses

A high resolution, two-dimensional (2-D) electrophoretic map of the plasma membrane (PM) polypeptides from the ejaculated boar spermatozoon is described. 2-D silver-stained polyacrylamide gel electrophoresis (PAGE) gels revealed over 250 polypeptides; Coomassie blue staining revealed more than 100. Fifty Coomassie-staining polypeptides were catalogued and biochemically characterized, with twenty of these designated major sperm PM polypeptides. Cavitation pressures ranging from 50 PSI to 1000 PSI were used to enrich 2-D maps either in head PM (50 PSI) or in flagellar PM (1000 PSI) and provided tentative localization of certain PM polypeptides. Immunoabsorption chromatography showed that most major polypeptides seen in the 2-D map were antigenic. Major polypeptide bands from single dimensional (1-D) gels were excised, antibodies against them were produced in rabbits, and the polypeptides were localized via indirect fluorescein isothiocyanate (FITC) technique. Cross-reacting antigenic determinants in the PAGE PM polypeptides were determined by transblotting and staining the transblots by an indirect peroxidase technique. Cross-reactivity was extensive with several polypeptide groups, but specific enough with others to allow tentative localization. Lectin affinity chromatography using Con A, WGA, RCA-1, PNA, and DBA indicated the lectin specificity of PM polypeptides. These data together with periodic acid-Schiff (PAS) and carbohydrate-specific silver staining permitted identification of glycoproteins in the 2-D maps. FITC coupled to specific lectins showed the regional distribution of these lectins on the sperm surface. The 2-D polypeptide map and the catalogue of properties of major Coomassie-stained PM polypeptides provides a reference for future studies in the boar and other species.