Accumulation and regulation of elastin in the rat uterus

The relative levels of elastin-specific mRNA were used as a measure of tropoelastin expression in uteri from pregnant Sprague-Dawley rats. The levels of elastin-specific mRNA were also correlated with values for net tropoelastin production and net deposition of mature, crosslinked elastin. The total content of uterine elastin increased throughout gestation, reaching maximal levels at Day 19 of gestation, which were three times those of nongravid tissue. Following involution, the elastin content decreased rapidly to near baseline values by 5 days postpartum. The content of soluble elastin, estimated using an enzyme-linked immunosorbent assay, paralleled in part the increase in elastin deposition and elastin mRNA levels. Uterine elastin metabolism appears to be unlike that in other elastic tissues, e.g., lung and large blood vessels. In most elastin containing tissues, the protein is synthesized during discrete developmental periods and is not readily degraded. However, uterine elastin is continuously expressed, and appears to be in a continual cycle of degradation and replacement.     

Ba2+ stimulation of luteinizing-hormone release demonstrates two mechanisms of Ca2+ entry in gonadotrope cells.

Kinetic studies on gonadotropin-releasing-hormone (gonadoliberin, GnRH)-stimulated luteinizing-hormone (lutropin, LH) release in the cultured rat gonadotrope demonstrated a biphasic pattern of LH release. The first rapid phase of release was unaffected by the voltage-gated Ca2+-channel blockers methoxyverapamil (D600) and nifedipine [a dihydropyridine (DHP)], whereas the later second phase was partially inhibited by both drugs. These results suggested that the initial phase of LH release is independent of Ca2+ entry through dihydropyridine (DHP)-sensitive Ca2+ channels and might depend on entry of extracellular Ca2+ by another mechanism. These mechanisms were further studied by utilizing Ba2+ as a Ca2+ substitute. Ba2+, which freely permeates DHP-sensitive Ca2+ channels in the absence of GnRH, induced LH release which was sensitive to blockade by D600 and nifedipine. However, in the presence of the channel blockers, Ba2+-induced LH release could be elicited when GnRH was added to the system. This indicates that GnRH stimulates LH release by initially activating a DHP-insensitive Ca2+-entry mechanism and then a DHP-sensitive mechanism. The DHP-sensitive mechanism freely allows Ba2+ entry in the absence of GnRH-receptor occupancy, whereas the DHP-insensitive mechanism requires GnRH-receptor activation for Ba2+ entry.     

Mutations conferring resistance to quinol oxidation (Qz) inhibitors of the cyt bc1 complex of Rhodobacter capsulatus.

Several spontaneous mutants of the photosynthetic bacterium Rhodobacter capsulatus resistant to myxothiazol, stigmatellin and mucidin--inhibitors of the ubiquinol: cytochrome c oxidoreductase (cyt bc1 complex)--were isolated. They were grouped into eight different classes based on their genetic location, growth properties and inhibitor cross-resistance. The petABC (fbcFBC) cluster that encodes the structural genes for the Rieske FeS protein, cyt b and cyt c1 subunits of the cyt bc1 complex was cloned out of the representative isolates and the molecular basis of inhibitor-resistance was determined by DNA sequencing. These data indicated that while one group of mutations was located outside the petABC(fbcFBC) cluster, the remainder were single base pair changes in codons corresponding to phylogenetically conserved amino acid residues of cyt b. Of these substitutions, F144S conferred resistance to myxothiazol, T163A and V333A to stigmatellin, L106P and G152S to myxothiazol + mucidin and M140I and F144L to myxothiazol + stigmatellin. In addition, a mutation (aer126) which specifically impairs the quinol oxidase (Qz) activity of the cyt bc1 complex of a non-photosynthetic mutant (R126) was identified to be a glycine to an aspartic acid replacement at position 158 of cyt b. Six of these mutations were found between amino acid residues 140 and 163, in a region linking the putative third and fourth transmembrane helices of cyt b. The non-random clustering of several inhibitor-resistance mutations around the non-functional aer126 mutation suggests that this region may be involved in the formation of the Qz inhibitor binding/quinol oxidation domain(s) of the cyt bc1 complex. Of the two remaining mutations, the V333A replacement conferred resistance to stigmatellin exclusively and was located in another region toward the C terminus of cyt b. The L106P substitution, on the other hand, was situated in the transmembrane helix II that carries two conserved histidine residues (positions 97 and 111 in R. capsulatus) considered to be the axial ligands for the heme groups of cyt b. The structural and functional roles of the amino acid residues involved in the acquisition of Qz inhibitor resistance are discussed in terms of the primary structure of cyt b and in relation to the natural inhibitor-resistance of various phylogenetically related cyt bc/bf complexes.     

The SV40 TC-II(kappa B) enhanson binds ubiquitous and cell type specifically inducible nuclear proteins from lymphoid and non-lymphoid cell lines.

We have characterized the complexes resulting from the specific binding in vitro of proteins present in nuclear extracts of several lymphoid and non-lymphoid cell lines to the TC-I and TC-II sequences of the simian virus 40 (SV40) enhancer. No proteins could be detected, binding selectively to the TC-I sequence, but two proteins TC-IIA and TC-IIB were identified interacting specifically with both the TC-II/kappa B enhanson, 5'-GGAAAGTCCCC-3' (important for the activity of the SV40 enhancer in vivo), and with the related H-2Kb enhanson, 5'-TGGGGATTCCCCA-3'. The binding of these two proteins to mutated TC-II enhansons correlates with the effect of these mutations in vivo, suggesting that both proteins may be important for SV40 enhancer activity. The TC-IIA binding activity was present in nuclear extracts of mature lymphoid B cells and was increased in pre-B cell nuclear extracts by lipopolysaccharide (LPS) and cycloheximide treatment. Furthermore, complex formation between the TC-IIA protein and the TC-II enhanson was efficiently competed by the kappa B motif from the kappa chain enhancer, indicating that TC-IIA is the NF-kappa B factor or a closely related protein. However, in contrast to previous reports, a TC-IIA/NF-kappa B-like protein whose properties could not be distinguished from those of the TC-IIA protein present in lymphoid B cells, was found in nuclear extracts of several untreated non-lymphoid cell lines, notably of HeLa cells, but not of undifferentiated F9 embryonal carcinoma (EC) cells [F9(ND)]. The TC-IIA binding activity which was moderately increased in HeLa cell nuclear extracts by 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or cycloheximide treatment could be induced in nuclear extracts of F9(ND) cells by cycloheximide, but not by TPA. Moreover, the TC-IIA binding activity could be induced in cytosolic fractions from F9(ND) cells by treatment with deoxycholate, indicating that these cells contain an inhibitor protein similar to the previously described NF-kappa B inhibitor, I kappa B. The second TC-II enhanson binding protein, TC-IIB, which could be clearly distinguished from the TC-IIA/NF-kappa B-like protein, by a number of differential properties, resembles the previously described KBF1/H2TF1 protein as it binds with a higher affinity to the H-2Kb enhanson than to the TC-II/kappa B enhanson, and its pattern of methylation interference on the H-2Kb and TC-II/kappa B enhansons is identical to that reported for the KBF1/H2TF1 protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Steady-state kinetic analysis of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans.

A steady-state kinetic analysis was performed of the reaction of methylamine and phenazine ethosulphate (PES) with the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans. Experiments with methylamine and PES as varied-concentration substrates produced a series of parallel reciprocal plots, and when the concentrations of these substrates were varied in a constant ratio a linear reciprocal plot of initial velocity against PES concentration was obtained. Nearly identical values of V/Km of PES were obtained with four different n-alkylamines. These data suggest that this reaction proceeds by a ping-pong type of mechanism. The enzyme reacted with a variety of n-alkylamines but not with secondary, tertiary or aromatic amines or amino acids. The substrate specificity was dictated primarily by the Km value exhibited by the particular amine. A deuterium kinetic isotope effect was observed with deuterated methylamine as a substrate. The enzyme exhibited a pH optimum for V at pH 7.5. The absorbance spectrum of the pyrroloquinoline quinone prosthetic group of this enzyme was also effected by pH at values greater than 7.5. The enzyme was relatively insensitive to changes in ionic strength, and exhibited a linear Arrhenius plot over a range of temperatures from 10 degrees C to 50 degrees C with an energy of activation 46 kJ/mol (11 kcal/mol).     

Characterization of a cytoskeletal matrix associated with myelin from rat brain.

Extraction of rat brain myelin in a buffer containing Triton X-100 yielded a soluble fraction and an insoluble residue that was enriched in cytoskeletal elements. Immunoblot analysis of the detergent-soluble fraction and the insoluble cytoskeletal residue showed that all of the tubulin and more than half of the actin were found within the cytoskeletal fraction. The distribution of myelin-specific proteins was also examined, and revealed that 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) I and most of the myelin basic proteins (MBPs) were equally distributed between both fractions. By contrast, the large MBP (21.5 kDa) and CNPase II (50 kDa) were observed to partition almost entirely with the cytoskeletal fraction. Proteolipid protein was found predominantly in the detergent-soluble fraction, as was DM-20 protein. Analysis of the cytoskeletal fraction by sucrose-density-gradient centrifugation demonstrated that a distinct subset of lipids was tightly bound to the cytoskeletal protein residue. The cytoskeleton-associated lipid was considerably enriched in cerebroside and sphingomyelin by comparison with total myelin lipids. These results indicate that a cytoskeletal matrix is associated with multilamellar myelin, and suggest that this structure may play a fundamental role in myelinogenesis.     

Unusual redox properties of electron-transfer flavoprotein from Methylophilus methylotrophus

The most positive redox potential ever recorded for a flavin adenine dinucleotide (FAD) containing protein has been measured for an electron-transfer flavoprotein (ETF) synthesized by Methylophilus methylotrophus. This potential value, 0.196 V versus the standard hydrogen electrode (vs SHE), was measured at pH 7.0 for the one-electron reduction of fully oxidized ETF (ETFox) to the red anionic semiquinone form of ETF (ETF.-). Quantitative formation of ETF.- was observed. The first successful reduction of ETF from M. methylotrophus to its two-electron fully reduced form was also achieved. Although addition of the second electron to ETF.- was extremely slow, the potential value measured for this reduction was -0.197 V vs SHE, suggesting a kinetic rather than thermodynamic barrier to two-electron reduction. These data are believed to be consistent with the postulated catalytic function of ETF to accept one electron from the iron-sulfur cluster of trimethylamine dehydrogenase (TMADH). The second electron reduction appears to have no catalytic function. The very positive potential measured for this ETF and the wide separation of potentials for the two electron reduction steps show that this ETF is a unique and interesting flavoprotein. In addition, this work highlights that while ETFs exhibit similar structural and spectral properties, they display wide variations in redox properties.     

Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.     

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRTAnn Arbor)

Summary HPRT_{Ann Arbor} is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased K_m for both substrates, a normal V_max, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT_{Ann Arbor} and determined its complete nucleotide sequence. A single nucleotide change (TG) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.