Incorporation of [14C] histidine into homocarnosine and carnosine of frog brain in vivo and in vitro.
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ABSTRACT:
SUBMITTER: Yockey WC
PROVIDER: S-EPMC1184931 | biostudies-other | 1969 Sep
REPOSITORIES: biostudies-other
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Project description:Purpose: Manganese (Mn) is a neurotoxic chemical which induces a wide range of complications in the brain tissue. Impaired locomotor activity and cognitive dysfunction are associated with high brain Mn content. At the cellular level, mitochondria are potential targets for Mn toxicity. Carnosine is a dipeptide abundantly found in human brain. Several pharmacological properties including mitochondrial protecting and antioxidative effects have been attributed to carnosine. The current study aimed to evaluate the effect of carnosine treatment on Mn-induced mitochondrial dysfunction in isolated brain mitochondria. Methods: Mice brain mitochondria were isolated based on the differential centrifugation method and exposed to increasing concentrations of Mn (10 µM-10 mM). Carnosine (1 mM) was added as the protective agent. Mitochondrial indices including mitochondrial depolarization, reactive oxygen species (ROS) formation, mitochondrial dehydrogenases activity, ATP content, and mitochondrial swelling and permeabilization were assessed. Results: Significant deterioration in mitochondrial indices were evident in Mn-exposed brain mitochondria. On the other hand, it was found that carnosine (1 mM) treatment efficiently prevented Mn-induced mitochondrial impairment. Conclusion: These data propose mitochondrial protection as a fundamental mechanism for the effects of carnosine against Mn toxicity. Hence, this peptide might be applicable against Mn neurotoxicity with different etiologies (e.g., in cirrhotic patients).
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Project description:Biologic scaffold materials are used for repair and reconstruction of injured or missing tissues. Such materials are often composed of allogeneic or xenogeneic extracellular matrix (ECM) manufactured by decellularization of source tissue, such as dermis. Dermal ECM (D-ECM) has been observed to degrade and remodel in vivo more slowly than other biologic scaffold materials, such as small intestinal submucosa (SIS-ECM). Histologic examination is a common method for evaluating material degradation, but it lacks sensitivity and is subject to observer bias. Utilization of (14)C-proline labeled ECM is a quantitative alternative for measuring degradation of ECM scaffolds. Using both methods, the amount of degradation of D-ECM and SIS-ECM was determined at 2, 4, and 24 weeks post-implantation in a rodent model. Results utilizing (14)C liquid scintillation counting (LSC) analysis showed distinct differences in degradation at the three time points. D-ECM material in situ stayed the same at 76% remaining from 2 to 4 weeks post-implantation, and then decreased to 44% remaining at 24 weeks. In the same time period, implanted SIS-ECM material decreased from 72% to 13% to 0%. Visual examination of device degradation by histology overestimated degradation at 2 weeks and underestimated device degradation at 24 weeks, compared to the (14)C method.
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Project description:1. Mycobacterium phlei (A.T.C.C. 356) cells were incubated with (14)C-labelled short-chain fatty acids and the 6-O-methylglucose-containing lipopolysaccharides that became esterified with radioactive acyl groups were isolated. The pattern of labelling of these lipopolysaccharides with the different acyl groups, the effects of different conditions on labelling patterns, and the kinetics of the turnover of (14)C-labelled acyl groups were studied. 2. The labelling patterns are summarized as follows. [1-(14)C]Acetate was incorporated into all of the acyl groups. [1-(14)C]Propionate led to labelling of propionate and succinate, while [1-(14)C]isobutyrate was incorporated mostly as such, along with a trace amount in iso-octanoate. 3. Under the conditions of the experiments, [1-(14)C]acetate was rapidly incorporated into succinyl (3-carboxypropionyl) and octanoyl groups, whereas the acetyl groups themselves were labelled more slowly. Radioactivity in propionyl and succinyl groups, originating from [1-(14)C]propionate, attained maximum values and then gradually decreased in both. Incorporation of [1-(14)C]isobutyrate proceeded slowly but reached a plateau and remained constant. While n-butyrate is not a normal constituent of methyl-glucose-containing lipopolysaccharides, it was incorporated as such when n-[1-(14)C]-butyrate was supplied in the medium. 4. [1-(14)C]Acetyl groups were readily displaced by unlabelled acetate. On the other hand, the specific radioactivity of the succinyl group continued to increase during a 3h incubation with unlabelled succinate. Propionyl and succinyl groups, labelled by [1-(14)C]propionate, were displaced slowly by unlabelled propionate or succcinate. The isobutyryl group of the lipopolysaccharides did not turn over, in contrast to the results obtained with the other acyl substituents.
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Project description:1. The patterns of incorporation of (14)C into glycerolipid fatty acids of developing maize leaf lamina from supplied [1-(14)C]acetate and from (14)CO(2) during steady-state photosynthesis were similar. Oleate of phosphatidylcholine and palmitate of phosphatidylglycerol attained linear rates of labelling more rapidly than did other fatty acids, particularly the linoleate and linolenate of monogalactosyl diacylglycerol. 2. After the transfer of lamina from labelled to unlabelled acetate, there was a decrease in labelled oleate and linoleate of phosphatidylcholine and a concomitant increase in the amount of radioactivity in the linoleate and linolenate of monogalactosyl diacylglycerol. 3. The rapidly labelled phospholipids, phosphatidylcholine and phosphatidylglycerol, were shown by differential and sucrose-density-gradient centrifugation to be associated with different organelles, the former being mainly in a low-density membrane fraction, probably microsomal, and the latter mainly in chloroplasts. 4. During a 48h period after supplying spinach leaves with [(14)C]acetate, radioactivity was lost from the oleate of phosphatidylcholine present in fractions sedimented at 12000g and 105000g, and accumulated in the linolenate of monogalactosyl diacylglycerol of the chloroplast. 5. It is proposed that the phosphatidylcholine of some non-plastid membranes is intimately involved in the process of oleate desaturation and that this lipid serves as a donor of unsaturated C(18) fatty acids to other lipids, principally monogalactosyl diacylglycerol, of the chloroplasts.
| S-EPMC1172463 | biostudies-other
Project description:l-Carnosine (?-Ala-l-His) and several other histidine-containing peptides, including two N-methylated forms on the imidazole ring (l-anserine and l-balenine), two derivatives modified on the carboxyl function (carcinine and l-carnosinamide), two analogues differing in the length of the N-terminal residue (l-homocarnosine and Gly-l-His) and the N-acetyl derivatives, were investigated as activators of four isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The four human isoforms hCA I, II, VA and IX were activated in the low to high micromolar range, with a rather complex structure activity relationship. A performed computational study allowed us to rationalize these results and to propose a binding mode of these activators within the enzyme active site. Similarly to other CA activators, the here studied peptides could find relevant pharmacological applications such as in the management of CA deficiencies, for therapy memory and enhancing cognition or for artificial tissues engineering.
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Project description:1. Rat thyroid lobes were incubated for various periods of time in Krebs-Ringer bicarbonate containing [(3)H]leucine and either [1-(14)C]galactose or [1-(14)C]mannose. Radioactivity in soluble proteins was determined after their separation by sucrose-gradient centrifugation. 2. The time-course of incorporation of label from [(14)C]-mannose into soluble thyroid proteins was parallel to that observed for [(3)H]leucine. There was a lag of at least 30min. before either label appeared in non-iodinated thyroglobulin (protein 17-18s). During this time both labels were detected in two fractions known to contain subunit precursors of thyroglobulin (fractions 12s and 3-8s). Radioactivity from double-labelled fractions 12s and 3-8s was transferred to protein 17-18s during subsequent incubation in an unlabelled medium. 3. In contrast, most of the [(14)C]galactose was immediately incorporated into protein 17-18s. 4. During the first hour of incubation, puromycin almost completely inhibited the incorporation of label from [(3)H]leucine and [(14)C]mannose into all protein fractions, but had little effect on the incorporation of [(14)C]galactose into protein 17-18s. 5. These results indicate that mannose is incorporated into the carbohydrate groups of protein 17-18s at an earlier stage in its formation than galactose. It is suggested that the synthesis of the carbohydrate groups of ghyroglobulin begins soon after formation of the polypeptide components, more than 30min. before these are aggregated to protein 17-18s; carbohydrate synthesis then proceeds in a stepwise manner, galactose being incorporated at about the time of aggregation of subunits to protein 17-18s. Most, if not all, the carbohydrate is added to thyroglobulin before it is iodinated.
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Project description:Incorporation of selenocysteine (Sec) in bacteria requires a UGA codon that is reassigned to Sec by the Sec-specific elongation factor SelB and a conserved mRNA motif (SECIS element). These requirements severely restrict the engineering of selenoproteins. Earlier, a synthetic tRNASec was reported that allowed canonical Sec incorporation by EF-Tu; however, serine misincorporation limited its scope. We report a superior tRNASec variant (tRNAUTuX) that facilitates EF-Tu dependent stoichiometric Sec insertion in response to UAG both in vivo in Escherichia coli and in vitro in a cellfree protein synthesis system. We also demonstrate recoding of several sense codons in a SelB supplemented cell-free system. These advances in Sec incorporation will aid rational design and directed evolution of selenoproteins.
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Project description:We have recently shown that the histidine-rich calcium binding protein (HRC) promotes the invasion and metastasis of hepatocellular carcinoma (HCC). In the current study, we evaluated whether HRC may also affect the growth of HCC. We found that ectopic expression of HRC obviously enhanced proliferation and colony formation, while suppression of HRC exhibited inhibitory effects. Furthermore, we demonstrated that HRC promoted tumor growth in nude mice. These effects may result from the ability of HRC to upregulate cyclinD1 and cyclin-dependent kinase 2 (CDK2) expressions and promote G1/S transition. Further study showed that MEK/ERK signaling pathway was involved in HRC-induced cell proliferation. Interestingly, overexpression or depletion of HRC revealed its regulation on endoplasmic reticulum stress (ERS) and apoptosis, which was partially dependent on PERK/ATF4/CHOP signaling pathway. In addition, blocking ERS using 4-phenylbutyric acid (4-PBA) not only downregulated the expression of PERK, ATF4 and CHOP, but also significantly decreased apoptosis induced by HRC silence, whereas ERS inducer thapsigargin (TG) exerted the opposite effects. Our study thus demonstrates a role of HRC in promoting HCC growth, besides its role in inducing HCC metastasis, and highlights HRC as a promising intervention target for HCC.
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Project description:A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogues in both Escherichia coli and mammalian cells. In combination with tRNACUA(Pyl), a pyrrolysyl-tRNA synthetase mutant was able to site-specifically incorporate 3-methyl-histidine, 3-pyridyl-alanine, 2-furyl-alanine, and 3-(2-thienyl)-alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogues created mutant proteins with distinct spectral properties. This work further expands the structural and chemical diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.
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Project description:In excitable tissues, the endogenous dipeptide carnosine (CAR, β-Ala-l-His) sustains homeostatic responses to various challenges. By eliciting hypoglycemic effects via actions on the autonomic nervous system and protection of pancreatic beta-cells, CAR is also relevant in diabetes. We investigated the expression of genes involved in CAR biosynthesis, degradation, and membrane transport pathways, in the pancreas and brains of mice treated with streptozotocin (STZ) and then exposed to dietary CAR. We induced hyperglycemia by STZ intraperitoneal injections; then, STZ-treated mice received drinking water with or without CAR for two weeks. We report that CAR administration elicits beneficial effects on blood glucose levels and weight loss in STZ-treated mice and, remarkably, on the insulin gene products in the pancreas, preserving gene expression from STZ challenge. Also, we describe mRNA downregulation of the Slc15a2/Pept2 (dipeptide transporter) and Cndp2 (intracellular dipeptidase) genes in the pancreas of hyperglycemic mice, and dysregulation of Carns1 (CAR synthase), Pept2 and Cndp2 in brains; interestingly, dietary CAR elicits counteracting effects. These expression patterns associate with variations of CAR content in tissues of mice. Overall, our report suggests a direct role of CAR in the diabetes-affected pancreas and in the diabetes-targeted CNS, proposing (dys)regulation of CAR’s homeostasis as a marker condition.
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