Effect of laminin on structural karyotype variability of kangaroo rat kidney cell lines

The structural karyotypic variability has been investigated in the "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, and in cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, there is a significant increase in the frequency of chromosomal aberrations, both chromosomal breaks and dicentrics (telomeric associations). Different sensitivity of individual chromosomes to inducing chromosomal breaks was observed in addition to a preferential involvement of some chromosomes in dicentric formation. Structural instability of chromosomes at cultivation on laminin demonstrates nonspecific reaction of the "markerless" cell lines to unfavourable factors of the environment. We discuss possible reasons of differences in the character of karyotypic variability between a cell line of the Indian muntjac skin fibroblasts and epithelial-like Rat kangaroo kidney cell lines cultivated on laminin.     

Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species

The nucleotide sequences corresponding to bovine alpha S2- and beta- casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.      

Changes in alpha-actinin localization and myofibrillogenesis in rat cardiomyocytes under cultivation

By indirect immunofluorescence with monoclonal anti-alpha-actinin antibodies the localization of this contractile protein was studied in ventricular cardiac myocytes from newborn 2-4-day old rats in the course of their cultivation. In freshly isolated heart muscle cells a predominant longitudinal orientation of myofibrils was observed; in some cells on the periphery of cytoplasm the contours of Z-lines are indistinct. During cell spreading, in the areas of intercalated discs, growing processes were observed mostly containing no contractile structures at earlier stages of cultivation. On days 3 to 14, the cytoplasmic processes and ruffles are filled with developing myofibrils. The cultures are heterogeneous in the morphology of contractile apparatus of individual cells. In most cardiomyocytes mature myofibrils are well-developed in the central part of the cytoplasm, whereas in its peripheral areas non-myofibrillar stress-fiber-like structures and bundles with continuous distribution of alpha-actinin frequently connected to myofibrils are more typical. In the areas of active myofibrillogenesis, located mainly on the cell periphery, numerous alpha-actinin dots are observed; most of them are arranged linearly and periodically at a distance of 0.3-1.5 microns and seem to be structural precursors of Z-lines. The data obtained show that the cultures of mammalian cardiac cells may be a convenient object for studying myofibrillogenesis in the course of cardiomyogenic differentiation.     

Hydrophobic surface properties of myosin in solution as studied by partition in aqueous two-phase systems: effects of ionic strength,pH and temperature

Summary The effect of gonadectomy (at the 10th day of life) and treatment with sexual steroids (during the first month) upon development of alpha-amylase activity in rat parotid gland has been studied.Alpha-amylase specific activity of parotid glands from 20-day-old orchidectomized rats and from 25-day-old ovariectomized animals was significantly higher than that of intact male and female rats of the same age respectively. Spayed males treated with testosterone (10 g/day on the 13th, 15th, and 17th day) and ovariectomized rats treated with oestradiol (2.5 g/day from the 16th to the 22nd day) showed values of enzymic activity similar to those of normal animals.Results indicate that oestradiol and testosterone have an inhibitory effect upon the increase of alpha-amylase activity in parotid gland during a very defined period of development.Career investigators of Consejo Nacional de Investigaciones Cientificas y Técnicas.     

Effect of cibachron blue F3GA on the polymerization of actin

Summary The effect of the dye Cibachron Blue F3GA on the G-F transformation of rabbit muscle actin has been studied with viscosimetry. The presence of the dye which is known to bind to nucleotide binding sites, decreased both the initial rate of polymerization of actin as well as the final viscosity of actin. Both these effects can be ascribed to an increase in the critical concentration of actin. The inhibitory effect of Cibachron Blue F3GA was counteracted by ATP, suggesting a competition between Cibachron Blue F3GA and ATP for the binding site/sites on actin.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

The Role of Polygalacturonase (PG) in the Ripening of Apple Fruits

Polygalacturonase (PG) activity and changes in respiratory intensity of apple fruits were investigated. The respiratory rate was decreased to a preclimacteric minimum from 30 Aug. to 20 Sept., Then increased to a climacteric peak (20–30 Sept.) and again drop down gradually with approaching the senescence stage. The PG activity was undetectable in a developing fruit until the onset climacteric phase. It rose rapidly after harvest, and reaching its highest level on 27 Oct. Just a month after the climacteric peak. The PG activity fell gradually. The amount of the fractions of pectic acid in fruits changed with the modifications of PG activity. With the ripening of fruits, the content of alcohol-soluble small molecules of pectic acids was increased from 12 to 13 5 mg/100 g of tissue, while the amount of alcohol- insoluble large molecules of pectic acids reduced from 530 to 280/100 g of tissue. PG activity would indicate the destruction of cell walls and the separation of cells. The onset of softening of fruits occurred 20 days after the rise of PG activity. It is supposed that the process of softening is directly controlled by PG activity.     

Fractionation of chromosomes : II. Use of hydrophobic affinity partition in aqueous two-phase system

A simple method for separation of large quantities of isolated metaphase chromosomes in Single-Tube Partition (STP), using hydrophobic ligand in an aqueous two-phase system is presented. The two-phase system is composed of an aqueous solution of Dextran 500 and poly(ethylene) glycol 6000 (PEG). The concentration of chromosomes to be separated has no influence on the distribution behaviour in the partition system and up to 10(7) chromosomes can be used in a phase system as small as 3-5 g (5 ml tube). Different groups of chromosomes differ in their distribution in the two phases and the introduction of PEG with covalently attached hydrophobic ligand provides a means of controlling the distribution of chromosomes. A combination of positively charged trimethylaminomethane PEG (TMA-PEG) together with palmitat PEG (P-PEG) gives a fairly good condition for separating chromosomes on the basis of their net surface charge differences.