Steroid-bovine serum albumin conjugates: molecular characterization and their interaction with androgen and estrogen receptors

Conjugates of testosterone-3-carboxymethyloxime (T-3-CMO), testosterone-17-hemisuccinate (T-17-HS), 17 beta-estradiol-6-carboxymethyloxime (E-6-CMO), or 17 beta-estradiol-17-hemisuccinate (E-17-HS) and bovine serum albumin (BSA) with varying steroid:protein ratios were prepared using the mixed anhydride method. Dialysis followed by molecular filtration yielded monomer steroid-BSA conjugates with a molecular weight of 70,000 dalton, and polymer conjugates with molecular weights of 140,000 dalton and higher. When conjugates were prepared with increasing initial steroid:BSA molar ratios the ratio of the obtained conjugates increased, in parallel with a decrease in the relative amount of monomers and an increase in the mean molecular size of polymers. The molecular properties of these conjugates were studied further by polyacrylamide gel electrophoresis (PAGE) in native and denaturing conditions. In native PAGE the monomer fractions showed one main band with a mobility slightly lower than BSA and a faint band corresponding with BSA-dimers. The polymer fractions consisted of a heterogeneous population of protein oligomers with molecular weights varying from 140,000 to over a million dalton. In the presence of sodium dodecylsulphate part of the polymers dissociated into monomers. In buffered aqueous solutions the bulk of the conjugate preparation retained its molecular size and composition, although the generated covalent bonds were found to be liable to spontaneous hydrolysis. Steroid-protein conjugates were shown to contain appreciable amounts of non protein-bound steroids. Binding of T-BSA to androgen receptors in rat ventral prostate cytosol was assayed using LH-20 chromatography and sucrose gradient centrifugation analysis. Binding of E-BSA to estrogen receptors was analysed with rat uterus cytosol using the dextran coated charcoal assay and the sucrose gradient centrifugation technique. Relative binding affinities (RBA) were analyzed in competition experiments using radiolabeled ligands. It was found that the molecular size of the conjugate does not influence its interaction with steroid receptors. Steroid coupled via the 17-position show a higher RBA to receptors than the T-3 or E-6 derivatives. The RBA of T-3-BSA, T-3-CMO, T-17-BSA and T-17-HS appeared to be very low, i.e. between 0.1 and 1.7% of the RBA of dihydrotestosterone. Consequently, high concentrations of conjugate are required to saturate androgen receptor binding sites. Under these conditions involvement of type II and eventually type III binding sites, which show less ligand specificity and lower affinity, may be anticipated preventing exclusive detection of androgen receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

Summary Synechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Ap^r gene of the E. coli plasmid pACYC177, yielding the Km^r hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Km^r shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm² gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cm^r Km^r shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.     

Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes

Stomatal conductances of normally oriented and inverted leaves were measured as light levels (photosynthetic photon flux densities) were increased to determine whether abaxial stomata of Vicia faba leaves were more sensitive to light than adaxial stomata. Light levels were increased over uniform populations of leaves of plants grown in an environmental chamber. Adaxial stomata of inverted leaves reached maximum water vapor conductances at a light level of 60 micromoles per square meter per second, the same light level at which abaxial stomata of normally oriented leaves reached maximum conductances. Abaxial stomata of inverted leaves reached maximum conductances at a light level of 500 micromoles per square meter per second, the same light level at which adaxial stomata of normally oriented leaves reached maximum conductances. Maximum conductances in both normally oriented and inverted leaves were about 200 millimoles per square meter per second for adaxial stomata and 330 millimoles per square meter per second for abaxial stomata. Regardless of whether leaves were normally oriented or inverted, when light levels were increased to values high enough that upper leaf surfaces reached maximum conductances (about 500 micromoles per square meter per second), light levels incident on lower, shaded leaf surfaces were just sufficient (about 60 micromoles per square meter per second) for stomata of those surfaces to reach maximum conductances. This `coordinated' stomatal opening on the separate epidermes resulted in total leaf conductances for normally oriented and inverted leaves that were the same at any given light level. We conclude that stomata in abaxial epidermes of intact Vicia leaves are not more sensitive to light than those in adaxial epidermes, and that stomata in leaves of this plant do not respond to light alone. Additional factors in bulk leaf tissue probably produce coordinated stomatal opening on upper and lower leaf epidermes to optimally meet photosynthetic requirements of the whole leaf for CO₂.     

Functional relationship among the gene clusters encoding F7(1), F7(2), F9, and F11 fimbriae of human uropathogenic Escherichia coli.

The P fimbrial gene clusters encoding the serologically different F7(1), F7(2), F9, and F11 fimbriae were compared functionally. The results show that these gene clusters are closely related.     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

A Method for Visualizing the Acrosome by Light Microscopy

A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol: acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.     

Triangularis sterni: a primary muscle of breathing in the dog

The isolated action, pattern of neural activation, and mechanical contribution to eupnea of the triangularis sterni (transversus thoracis) muscle were studied in supine anesthetized dogs. Linear displacement transducers were used to measure the axial displacements of the ribs and sternum. Tetanic stimulation of the triangularis sterni in the apneic animal caused a marked caudal displacement of the ribs, a moderate cranial displacement of the sternum, and a decrease in lung volume. During quiet breathing, there was invariably a rhythmic activation of the muscle in phase with expiration that was independent of the presence or absence of activity in the abdominal and internal interosseous intercostal muscles. This phasic expiratory activity in the triangularis sterni was of large amplitude and caused the ribs to be more caudal and the sternum to be more cranial during the spontaneous expiratory pause than during relaxation. Additional studies on awake animals showed that rhythmic activation of the triangularis sterni occurs in all body positions and is not caused by anesthesia. These findings indicate that expiration in the dog is not a passive process and that the end-expiratory volume of the rib cage is not determined by an equilibrium of static forces alone. Rather, it is actively determined and maintained below its relaxation volume by contraction of the triangularis sterni throughout expiration. The use of this muscle is likely to facilitate inspiration by increasing the length of the parasternal intercostals and taking on a portion of their work.     

Synthesis of glycoconjugates in mouse primordial germ cells

The synthesis of protein-bound carbohydrates has been studied in primordial germ cells (PGCs) and in somatic cells of 12.5 to 13.5-days-postcoitum (dpc) fetal mouse gonads. Both cell types were shown to synthesize asparagine-linked glycopeptides and glycosaminoglycans (GAGs). In addition, PGCs also synthesize lactosaminoglycans (LAGs) although in different proportions in female and male germ cells. Female PGCs, which at 13.5 dpc are entering meiosis, synthesize mainly LAGs, and minor amounts of hyaluronic acid (HA) and chondroitin sulfate (CS). Male germ cells, on the other hand, synthesize mainly CS. Furthermore, somatic cells of fetal gonads synthesize HA as the major class of GAGs. It is suggested that the activation of LAG synthesis in developing germ cells might be related to the beginning of meiosis. Moreover, we propose that HA synthesis might be developmentally regulated in somatic cells of the gonad, in order to regulate the establishment of specific interactions with germ cells.     

Inhibition by verapamil of neutrophil responses to formylmethionylleucylphenylalanine and phorbol myristate acetate. Mechanisms involving Ca2+ changes, cyclic AMP and protein kinase C

Verapamil inhibits in human neutrophils the respiratory burst, the secretion and the change of transmembrane potential induced by formylmethionylleucylphenylalanine, a Ca2+-dependent stimulus, and by phorbol myristate acetate, a Ca2+-independent stimulus. Besides the blocking of Ca2+ channels, many mechanisms are responsible for the inhibition of neutrophil responses. In fact, verapamil (i) increases the intracellular cAMP concentration, potentiates the cAMP response induced by the chemotactic peptide and induces the appearance of a cAMP response also when the stimulant is phorbol myristate acetate; (ii) causes a decrease of Ca2+ association to cell membranes, so depleting the pools of exchangeable Ca2+ and depressing the 'Ca2+ response' in terms of rise in [Ca2+]i monitored with Quin 2 and of rapid mobilization from cell membranes monitored by chlorotetracycline fluorescence change; (iii) inhibits the Ca2+-activated phospholipid-dependent protein kinase C. The data, discussed in relation to the biochemical mechanisms of the stimulus-response coupling, are compatible with the hypothesis of an involvement of the activation of protein kinase C as key step in the sequence of transduction events for the induction of many neutrophil functions.     

Studies on enzyme-substrate interactions of cholinephosphotransferase from rat liver

In order to elucidate the reaction mechanism and the substrate-binding sites, CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), prepared from rat liver microsomal fraction, has been subjected to kinetic analysis and substrate specificity studies. Kinetic evidence supports the hypothesis of a Bi-Bi sequential mechanism, involving a direct nucleophilic attack of diacylglycerol on CDPcholine during the reaction. To investigate the substrate requirements for recognition and catalysis, several CDPcholine analogs, modified in the nitrogen base or in the sugar or in the pyrophosphate bridge, have been synthesized, characterized and assayed as substrates and/or inhibitors of the reaction. The amino group on the pyrimidine ring, the 2'-alcoholic function of the ribose moiety as well as the pyrophosphate bridge have been identified as critical sites for enzyme-substrates interactions.