Manuel Krauskopf

Characterization of the major plasma apoliproteins of the high density lipoprotein in the carp (Cyprinus carpio)

Abstract 1. 1. Carp plasma DHL was isolated by a single chromatographic procedure using Affi-Gel Blue. Neither LDL nor VLDL were detected. 2. 2. Thus, HDL which comprises almost 40% of the total carp plasma proteins, also constitutes the major lipoprotein fraction in this fish, yielding two apolipoproteins: apo A-I and apo A-II with molecular weight of 29,500 and 12,000, respectively. These are present in a one to one molar ratio. 3. 3. Carp apo A-I and apo A-II lack cysteine and tryptophan. Although some similarities in the amino acid composition exist with respect to the same apoproteins from other organisms, antisera raised against carp apo-I cross-reacted only, and very slightly, with its rainbow trout counterpart. 4. 4. No reaction was detected with lamprey and the nothotenidae robalo fish. 5. 5. Four plasmatic apo A-I isoforms were identified. No differences were observed in carps acclimatized to winter and summer.

Behaviour of RNA and protein synthesis during the acclimatization of the Carp. Studies with isolated hepatocytes

1. Carp hepatocytes were isolated by dissociating the liver tissue with collagenase. The procedure yields viable cells with highly preserved ultrastructural and metabolic features. The isolated cells were able to self-aggregated and form tissue. 2. RNA and protein synthesis activity was significantly higher in the carp hepatocytes from summer acclimatized fish compared to the activity present in the cold adapted animals. 3. RNA synthesis assayed in carp hepatocytes suspensions obtained from summer and winter acclimatized fish exhibited a behaviour consistent with an inverse compensation to the cold acclimatization state, being apparently repressed, whereas protein synthesis did not show a compensatory activity.

Specific alanine-tRNA species associated with fibroin biosynthesis in the posterior silk-gland of Bombyx mori L.

During the course of our study on the role and mechanism of specific tRNA changes (tRNA adaptation) during the silk-fibroin secretion phase of the posterior silk-gland development in Bombyx mori L., we undertook fractionation and purification of the preponderant tRNAs, tRNAA”, tRNAGh/, tRNAMef, tRNASer and tRNATv’, by benzoylated DEAE-cellulose column chromatography, countercurrent distribution and polyacrylamide gel electrophoresis [2111. We observed major differences in compartmentalization of isoaccepting tRNAAk’ species in the two portions of the silk-gland, the posterior and middle silk-glands. Silk-fibroin (29% alanine, 46% glycine and 12% serine) is synthesized exclusively in the posterior part, whereas sericin (6% alanine, 17% glycine and 37% serine) is produced in the middle part of silk-gland. The Al! redominant tRNAAk’ species, tRNAe and tRNA,, , in the ratio of 2 : 1 (14% and 7% of the total tRNA content, respectively) are present in the posterior silk-gland during the fibroin secretion phase of gland development, whereas only one, tRNAg, species is the main tRNAk component of the middle silk-gland and of

APH-1, a POU homeobox gene expressed in the salt gland of the crustacean Artemia franciscana.

We characterized the first POU-homeoprotein in a crustacean (designated APH-1 for Artemia POU-Homeoprotein, EMBL Y15070). The amino acid sequence of the APH-1 POU-domain is identical, except for two residues, to that of the two class III POU proteins Cf1-a (Drosophila) and POU-M1 (Bombyx mori). Southern blot analysis suggests that crustaceans have only one class III POU gene. RT-PCR and whole-mount in situ hybridization show that APH-1 mRNA is present in larvae specifically in the salt gland, an organ which is involved in osmoregulation, and disappears in the adult.

A citationist perspective on science in Latin America and the Caribbean, 1981-1993

The publication productivity and citation record of the Latin American countries are analyzed and evaluated by using adequate databases.

An increased expression of nucleolin is associated with a physiological nucleolar segregation.

Nucleolar segregation is the most striking cellular phenotypic feature of cold-acclimatized carp and depicts the cyclical reprogramming that the physiology of the fish undergoes between summer and winter, where a clear differential expression of some nucleolar related genes occurs. We characterized carp nucleolin, a nucleolar protein involved in multiple steps of ribosome biogenesis, and evaluated its expression upon fish acclimatization. We show that the carp cDNA deduced amino acid sequence exhibits the same tripartite structural organization found in other species. Nevertheless, we observed that nucleolin mRNA expression was strongly induced in the cold-adapted carp as was the nuclear protein content, assessed by immunocytochemistry in liver sections. The physiological up-regulation of nucleolin in the cold-acclimatized carp, where rRNA transcription and processing are depressed concomitantly with the nucleolus segregation, is consistent with the notion that nucleolin plays a fundamental role in repressing rRNA synthesis.

Prolactin gene expression and changes of prolactin pituitary level during the seasonal acclimatization of the carp

The effect of seasonal acclimatization on the extent of prolactin (PRL) gene expression and on the content of this was studied in summer- and winter-carp (Cyprinus carpio) hormone pituitary glands. PRL content in the rostral pars distalis (RPD) was evaluated by immunocytochemistry using antibodies against a cross-linked synthetic peptide comprising the sequence of 15 amino acids which conform to the primary structure of carp PRL. To assess the level of PRL gene transcription, a 24-mer synthetic oligonucleotide probe whose sequence included nucleotides 2041-2064 located in exon V of the carp PRL gene, was used. Employing in situ hybridization assays, a high expression of PRL mRNA was observed in the RPD of summer-acclimatized carp. A negligible level of transcription was observed in tissue sections of pituitary glands from winter-acclimatized carp. Concurrently, immunodetection of the PRL-producing cells in the RPD revealed that the pituitary hormone level was significantly higher in the warm season-adapted carp.

Environmental acclimatization of the carp modulates the transcription of β-actin

A cascade of mechanisms involving changes in gene expression are substantial to shape the adaptive responses that a eurythermal fish requires upon environmental changes in its habitat. We have previously shown that the cyclical reprogramming of rRNA transcription is a remarkable feature in carp under seasonal acclimatization. Using in situ hybridization and competitive RT‐PCR we found significant differences in β‐actin transcripts, generally accepted to be coded by a typical housekeeping gene, in tissues from summer‐ and winter‐acclimatized carp. The physiological differential β‐actin transcription herein reported places us on the alert for the reference genes estimated to be constitutive to quantitatively assess gene transcripts. J. Cell. Biochem. 80:223–228, 2000.

Transcription factor Pit-1 expression is modulated upon seasonal acclimatization of eurythermal ectotherms: Identification of two Pit-1 genes in the carp

A second Pit‐1 gene in carp (Cyprinus carpio), including the complete structural gene and 1.1 kb of promoter region, was identified and completely sequenced. The exon‐intron structure was determined, and reverse transcription‐polymerase chain reaction (RT‐PCR) experiments suggest that only one Pit‐1 splice variant is present in carp pituitary. The effect of seasonal acclimatization on the extent of Pit‐1 gene expression was studied in summer‐ and winter‐acclimatized carp. Quantitative RT‐PCR analysis revealed a clear increase of Pit‐1 mRNA in the pituitaries from summer‐acclimatized carp compared with the winter‐adapted fish. In situ hybridization of pituitary gland sections with riboprobes representing the complete 5′‐transactivating region of carp Pit‐1 depicted a significantly higher Pit‐1 mRNA level in the rostral pars distalis of the summer‐acclimatized fish where prolactin is expressed in a manner that resembles the seasonal increase observed in the proximal pars distalis and the pars intermedia. The cell‐ and temporal‐specific transcription of Pit‐1 supports its role in the molecular mechanisms that underly the acclimatization process undergone by eurythermal fish as a result of the physical effects of seasonal changes on their habitat. J. Cell. Biochem. 75:598–609, 1999.

Seasonal environmental changes regulate the expression of the histone variant macroH2A in an eurythermal fish

Adaptation to cold and warm conditions requires dramatic change in gene expression. The acclimatization process of the common carp Cyprinus carpio L. in its natural habitat has been used to study how organisms respond to natural environmental changes. At the cellular level, adaptation to cold condition is accompanied by a dramatic alteration in nucleolar structure and a down regulation of the expression of ribosomal genes. We show that the enrichment of condensed chromatin in winter adapted cells is not correlated with an increase of the heterochromatin marker trimethyl and monomethyl K20H4. However, the expression of the tri methyl K4 H3 and of the variant histone macroH2A is significantly increased during the winter season together with a hypermethylation of CpG residues. Taking into account the properties of macroH2A toward chromatin structure and dynamics and its role in gene repression our data suggest that the increased expression of macroH2A and the hypermethylation of DNA which occurs upon winter‐acclimatization plays a major role for the reorganization of chromatin structure and the regulation of gene expression during the physiological adaptation to a colder environment.