Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs

Presence of the dihydrouridine (D) stem in the mitochondrial cysteine tRNA is unusually variable among lepidosaurian reptiles. Phylogenetic and comparative analyses of cysteine tRNA gene sequences identify eight parallel losses of the D-stem, resulting in D-arm replacement loops. Sampling within the monophyletic Acrodonta provides no evidence for reversal. Slipped-strand mispairing of noncontiguous repeated sequences during replication or direct replication slippage can explain repeats observed within cysteine tRNAs that contain a D-arm replacement loop. These two mechanisms involving replication slippage can account for the loss of the cysteine tRNA D-stem in several lepidosaurian lineages, and may represent general mechanisms by which the secondary structures of mitochondrial tRNAs are altered.      

cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family

The complete cDNA sequence and protein reading frame of a developmentally regulated hemocyanin subunit in the Dungeness crab (Cancer magister) is presented. The protein sequence is aligned with 18 potentially homologous hemocyanin-type proteins displaying apparent sequence similarities. Functional domains are identified, and a comparison of predicted hydrophilicities, surface probabilities, and regional backbone flexibilities provides evidence for a remarkable degree of structural conservation among the proteins surveyed. Parsimony analysis of the protein sequence alignment identifies four monophyletic groups on the arthropodan branch of the hemocyanin gene tree: crustacean hemocyanins, insect hexamerins, chelicerate hemocyanins, and arthropodan prophenoloxidases. They form a monophyletic group relative to molluscan hemocyanins and nonarthropodan tyrosinases. Arthropodan prophenoloxidases, although functionally similar to tyrosinases, appear to belong to the arthropodan hexamer- type hemolymph proteins as opposed to molluscan hemocyanins and tyrosinases.      

Microsatellite allele frequencies in humans and chimpanzees, with implications for constraints on allele size

The distributions of allele sizes at eight simple-sequence repeat (SSR) or microsatellite loci in chimpanzees are found and compared with the distributions previously obtained from several human populations. At several loci, the differences in average allele size between chimpanzees and humans are sufficiently small that there might be a constraint on the evolution of average allele size. Furthermore, a model that allows for a bias in the mutation process shows that for some loci a weak bias can account for the observations. Several alleles at one of the loci (Mfd 59) were sequenced. Differences between alleles of different lengths were found to be more complex than previously assumed. An 8-base-pair deletion was present in the nonvariable region of the chimpanzee locus. This locus contains a previously unrecognized repeated region, which is imperfect in humans and perfect in chimpanzees. The apparently greater opportunity for mutation conferred by the two perfect repeat regions in chimpanzees is reflected in the higher variance in repeat number at Mfd 59 in chimpanzees than in humans. These data indicate that interspecific differences in allele length are not always attributable to simple changes in the number of repeats.      

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.     

Bombesin-like peptides in rat spinal cord: Biochemical characterization,localization and mechanism of release

Bombesin-like peptides were characterized in rat spinal cord by radio-immunoassay. The density of bombesin-like peptides was eight-fold greater in the dorsal than ventral horn or white matter of the spinal cord. Using high pressure liquid chromatography fractionation techniques, the main peak of immunoreactivity coeluted with synthetic bombesin. Also, the mechanism of release spinal cord peptides was determined. K⁺ and veratridine stimulated release of immunoreactive bombesin in a Ca⁺⁺-dependent mechanism. These data indicate that bombesin-like peptides may function as a unique class of neuroregulatory agents in mammalian spinal cord.     

Bombesin-like peptides in rat brain: Localization in synaptosomes and release from hypothalamic slices

The subcellular distribution of bombesin-like immunoreactivity in rat brain was investigated. The number of receptors was significantly greater in the synaptosomal than mitochondrial fraction and quantity of bombesin-like immunoreactivity was greatest in the synaptosomal fraction. Also, the release of bombesin-like peptides from rat hypothalamic slices was investigate K⁺ and veratridine stimulated release of immunoreactivity in a Ca⁺⁺-dependent manner.     

Acute effects of heroin and morphine on newly synthesized serotonin in rat brain.

Heroin and morphine, in acute intraperitoneal doses of 2 and 10 mg/kg respectively, produced significant increments in the formation of newly formed brain serotonin from tritiated (³H)-L-tryptophan to ³H-serotonin. Opiate analgesia, Straub tail sign and catatonia, were observed during the increase in the synthesis of serotonin. The transport of radio-labelled tryptophan into the rat brain was not increased by the acute injection of the opiates, but brain levels of ³H-serotonin and of its main metabolite, 5-hydroxyindoleacetic acid, were significantly elevated. These opiates do not interfere with the accumulation of serotonin or with the transport of its metabolite in serotonergic neurons after inhibition of monoamine oxidases with Pargyline. An increase in the activity of tryptophan hydroxylases was more pronounced in the forebrain than in the brain stem. Stimulation of newly synthesized serotonin is probably mediated by an increase in tryptophan hydroxylase activity and not by an increase in the transport of tryptophan into the brain.     

Mechanism of release of norepinephrine from peripheral adrenergic neurones by the calcium ionophores X 537A and A 23187

The effects of the calcium ionophores, X 537A and A 23187, in causing release of norepinephrine (NE) were examined in isolated guinea-pig vas deferens and rat atrial segments. Examination of calcium dependence, dopamine-β-hydroxylase (DBH) release and release of deaminated metabolites of NE suggest that while A 23187 releases by both an exocytotic and non-exocytotic mechanism, X 537A releases NE predominantly through a non-exocytotic mechanism. The relative impotence of A 23187 in effecting release of NE may explain its inactivity in alteration of cardiac responses.     

Norcocaine: a pharmacologically active metabolite of cocaine found in brain

N-Demethylation of cocaine results in the production of norcocaine, which has been identified by gas chromatography-mass spectrometry in the brains of monkeys given repeated doses of cocaine. This metabolite is about as active as cocaine in inhibiting uptake of ³H-norepinephrine by synaptosomes prepared from rat brain. Other cocaine derivatives have been found to be relatively inactive in inhibiting uptake of this amine.