Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Modification of a ribonuclease from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate

The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity. In the presence of 1 M cytidine, RNase Rh activity was protected from the CMC-modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C]CMC indicated that the RNase Rh lost its enzymatic activity with the incorporation of about one [14C]CMC. Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh. The binding of the CMC-modified RNase Rh with 2'-AMP was studied spectrophotometrically. The affinity of the modified RNase Rh towards 2'-AMP decreased markedly upon CMC modification.     

Elastase-1-secreting acinar cell carcinoma of the pancreas. A cytologic, electron microscopic and histochemical study

A case of acinar cell carcinoma of the pancreas was diagnosed by fine needle aspiration cytology during surgery. The cytologic characteristics of this neoplasm are described. Electron microscopy disclosed numerous zymogen granules in the tumor while histochemistry demonstrated the presence of elastase-1. Serum elastase-1 levels were markedly elevated. The cytologic differential diagnosis of pancreatic tumors is discussed.     

Ecotype-Specific Expression of a Flowering Mutant Phenotype in Arabidopsis thaliana

The majority of mutations that delay flowering in Arabidopsis thaliana have been identified in studies of the Landsberg erecta (Ler) ecotype. In this report we describe a gene (referred to as FLD) that, when mutated, delays flowering in the Columbia ecotype but has a minimal phenotype in the Ler genetic background. The late-flowering phenotype of fld mutants requires a non-Ler allele of another gene involved in the control of flowering time, Flowering Locus C. fld mutants retain a photoperiod response, and the flowering time of fld mutants can be reduced by cold treatment and low red/far-red light ratios.     

Simultaneous RP‐HPLC‐DAD Separation,and Determination of Flavonoids and Phenolic Acids in Plantago L. Species

A rapid reversed‐phase (RP) high‐performance liquid chromatography method was developed and applied for simultaneous separation, and determination of flavonoids and phenolic acids in eight Plantago L. taxa (P. altissima L., P. argentea Chaix , P. coronopus L., P. holosteum Scop . ssp. depauperata Pilger , P. holosteum ssp. holosteum, P. holosteum ssp. scopulorum (Degen) Horvati? , P. lagopus L., and P. maritima L.) growing in Croatia. Chromatographic separation was carried out on Zorbax Eclipse XDB‐C18 using gradient elution with a H₂O (pH 2.5, adjusted with CF₃COOH) and MeCN mixture at 30°. The contents of analyzed phenolic compounds (% of the dry weight of the leaves, dw) varied among examined species: rutin (max. 0.024%, P. argentea), hyperoside (max. 0.020%, P. lagopus), quercitrin (max. 0.013%, P. holosteum ssp. holosteum), quercetin (max. 0.028%, P. holosteum ssp. scopulorum), chlorogenic acid (max. 0.115%, P. lagopus), and caffeic acid (max. 0.046%, P. coronopus). Isoquercitrin was detected only in P. argentea (0.020%), while isochlorogenic acid content was below limit of quantification in all investigated species. Multivariate analyses (UPGMA and PCA) showed significant differences in contents of investigated polyphenolic compounds between different Plantago taxa. Accordingly, investigated substances might be employed as chemotaxonomic markers in the study of the complex genus Plantago.     

Peroxin Pex21p interacts with the C-terminal noncatalytic domain of yeast seryl-tRNA synthetase and forms a specific ternary complex with tRNA(Ser)

The seryl-tRNA synthetase from Saccharomyces cerevisiae interacts with the peroxisome biogenesis-related factor Pex21p. Several deletion mutants of seryl-tRNA synthetase were constructed and inspected for their ability to interact with Pex21p in a yeast two-hybrid assay, allowing mapping of the synthetase domain required for complex assembly. Deletion of the 13 C-terminal amino acids abolished Pex21p binding to seryl-tRNA synthetase. The catalytic parameters of purified truncated seryl-tRNA synthetase, determined in the serylation reaction, were found to be almost identical to those of the native enzyme. In vivo loss of interaction with Pex21p was confirmed in vitro by coaffinity purification. These data indicate that the C-terminally appended domain of yeast seryl-tRNA synthetase does not participate in substrate binding, but instead is required for association with Pex21p. We further determined that Pex21p does not directly bind tRNA, and nor does it possess a tRNA-binding motif, but it instead participates in the formation of a specific ternary complex with seryl-tRNA synthetase and tRNA(Ser), strengthening the interaction of seryl-tRNA synthetase with its cognate tRNA(Ser).     

Site-specific glycosylation of SARS-CoV-2: Big challenges in mass spectrometry analysis

Glycosylation of viral proteins is required for the progeny formation and infectivity of virtually all viruses. It is increasingly clear that distinct glycans also play pivotal roles in the virus's ability to shield and evade the host's immune system. Recently, there has been a great advancement in structural identification and quantitation of viral glycosylation, especially spike proteins. Given the ongoing pandemic and the high demand for structure analysis of SARS-CoV-2 densely glycosylated spike protein, mass spectrometry methodologies have been employed to accurately determine glycosylation patterns. There are still many challenges in the determination of site-specific glycosylation of SARS-CoV-2 viral spike protein. This is compounded by some conflicting results regarding glycan site occupancy and glycan structural characterization. These are probably due to differences in the expression systems, form of expressed spike glycoprotein, MS methodologies, and analysis software. In this review, we recap the glycosylation of spike protein and compare among various studies. Also, we describe the most recent advancements in glycosylation analysis in greater detail and we explain some misinterpretation of previously observed data in recent publications. Our study provides a comprehensive view of the spike protein glycosylation and highlights the importance of consistent glycosylation determination.     

Bystander effects of nucleoside analogs phosphorylated in the cytosol or mitochondria

The efficiency of nucleoside kinase suicide gene therapy for cancer is highly dependent on "bystander" cell killing, i.e., the transfer of cytotoxic phosphorylated nucleoside analogs to cells adjacent to those expressing the suicide enzyme. We have recently studied the possible use of mitochondrial nucleoside kinases as suicide genes. In the present study, we investigated if nucleoside analogs phosphorylated in the mitochondrial matrix cause bystander killing. We used deoxycytidine kinase-deficient Chinese hamster ovary cells reconstituted with deoxycytidine kinase targeted to either the cytosol or mitochondria matrix and determined the bystander cell killing when these cells were incubated with the nucleoside analogs 1-beta-D-arabinofuranosylcytosine and 2',2'-difluorodeoxycytidine. A bystander effect occurred when nucleoside analogs were phosphorylated in the cytosol, but not when these compounds were phosphorylated in the mitochondria. These findings suggest that nucleoside kinases targeted to the mitochondrial matrix have limited use in suicide gene therapy when efficient bystander cell killing is required.