Transgenic animals as a tool for studying the effect of the c-myc proto-oncogene on cardiac development

Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Nascent muscle fiber appearance in overloaded chicken slow-tonic muscle

The application of a weight overload to the humerus of chickens induces a hypertrophy of anterior latissimus dorsi (ALD) muscle fibers. This growth is accompanied by a rapid and almost complete replacement of one slow-tonic myosin isoform, SM-1, by another slow-tonic isoform, SM-2. In addition, a population of small fibers appears mainly in extrafascicular spaces and, concurrently, three additional myosin bands are detected by gel electrophoresis. Five antibodies against myosin heavy chain (MHC) isoforms were selected as immunocytochemical probes to determine the cellular location and nature of these myosins. The antibodies react with ventricular, fast skeletal muscle and either SM-1 or SM-2, or both the slow-tonic MHCs. The antifast and antiventricular antibodies react with myosin present in the 10-day embryonic ALD muscle but do not react with myosin in posthatch ALD muscle. The small fibers in overloaded muscle contain a myosin isoform characteristically expressed during the embryonic stage of ALD muscle development and therefore are named nascent myofibers. Some of the nascent myofibers do not react with the antibody to both slow-tonic MHCs, indicating the lack of the normal adult slow-tonic myosins which are expressed in 10-day embryos. In order to explore the origin of the nascent fibers, an electron microscopic study was performed. Stereological analysis of the existing fibers shows a stimulation of numbers and sizes of satellite cells. In addition, the volume occupied by nonmuscle and undifferentiated cells increases dramatically. Myotube formation with incipient myofibrils is seen in extrafascicular spaces. These data suggest that new muscle fiber formation accompanies hypertrophy in overloaded chicken ALD muscle and the process may involve satellite cell migration.     

Derivation of the sequence of the signal peptide in human C4b-binding protein and interspecies cross-hybridisation of the C4bp cDNA sequence

A 5' cDNA clone coding for human C4b-binding protein (C4bp) was isolated, characterised and sequenced to complete the cDNA sequence coding for residues 1-32 thus confirming the protein sequence data of Chung et al. [(1985) Biochem. J. 230, 133-141]. The sequence extended to allow derivation of the putative leader peptide sequence which was 32 residues in length and showed a high of hydrophobicity typical of other documented leader sequences. Cross hybridisation was detected between the human C4bp cDNA probes and genomic DNA isolated from various species on Southern blots suggesting that genomic sequence homologous to that coding for C4bp has been conserved during evolution.     

Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Movement of ions and small solutes across endothelium and epithelium of perfused rabbit lungs

In situ and isolated fluid-filled rabbit lungs were used to study the transport of indicators between the air space and vascular compartments. These indicators were placed in either the perfusate or air spaces and samples were collected from the perfusate at intervals during a 1-h perfusion period. At the end of the hour, fluid was pumped out of the air space compartment into serial tubes and indicator concentrations were determined in both the air space and perfusion fluids. One hour after introducing the indicators into the air space, the relative decreases in solute concentration were (arranged from the greatest to the least decline): [14C]urea greater than 36Cl- = 125I- greater than 22Na+ greater than [3H]mannitol. The relative rates at which the indicators appeared in the perfusate were similar. When the indicators were placed in the perfusate, a similar relationship was observed in the increase in air space concentrations, but the loss of 22Na+ from the perfusate was similar to those of 36Cl- and 125I-. Losses of all indicators from the perfusate were two or more times those from the air spaces, and although the loss of [3H]mannitol from the perfusate was similar to that of 22Na+ for about 30 min, subsequent loss was much slower. Very little 125I-albumin traversed the tissue barrier, and the small changes in the concentrations of 125I-albumin in the air spaces suggested that little fluid movement had occurred. These studies suggest that the epithelium is less permeable to solutes than the endothelium and permits passage of anions at a faster rate than 22Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Initial filtration rate and initial clogging in the Hemorheometre

The role of different factors contributing to red cell filterability in the Hemorheometre has been investigated. Although the original method uses a small volume of suspension to determine red cell filterability, the present experiments showed that the results obtained are still significantly affected by filter clogging. Consequently a change in filterability could be due to a change in filter clogging possibly by residual leucocytes. An adaptation of filter chamber and filling method is described, resulting in a simpler and faster measuring procedure. The inaccuracy in measuring low haematocrits contributes significantly to experimental errors. Therefore a definition of red cell filterability based on the red cell count (instead of haematocrit) of the suspension is suggested.     

1H NMR studies of lambda cro repressor. 1. Selective optimization of two-dimensional relayed coherence transfer spectroscopy

Two-dimensional relayed coherence transfer NMR spectroscopy (RELAY) has been used to corroborate side chain spin system identities in crowded regions of the 1H NMR spectrum of the lambda cro repressor protein. The mixing time in the RELAY experiments was optimized for specific preselected spin systems by using recently developed methods [Bax, A., & Drobny, G. (1985) J. Magn. Reson, 61, 306-320], which utilize the transverse relaxation time (T2) of the molecule and relevant J couplings for the defined spin system. We demonstrate that a mixing time of 26 ms gives rise to strong C alpha H-C gamma H3 RELAY cross peaks for all valine, threonine, and isoleucine residues, while RELAY cross peaks for other spin systems are weak or are not observed. This allows for rapid and unambiguous identification of the side chain resonances for valine, isoleucine, threonine, and alanine (by elimination). The use of optimized RELAY for analyzing and identifying spin systems in complex spectra is discussed.     

1H NMR studies of lambda cro repressor. 2. Sequential resonance assignments of the 1H NMR spectrum

The cro repressor protein from bacteriophage lambda has been studied in solution by two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Following the approach of Wüthrich and co-workers [Wüthrich, K., Wider, G., Wagner, G., & Braun, W. (1982) J. Mol. Biol. 155, 311-319], individual spin systems were identified by J-correlated spectroscopy (COSY) supplemented, where necessary, by relayed coherence transfer spectroscopy (RELAY). Nuclear Overhauser effect spectroscopy (NOESY) was used to obtain sequence-specific assignments. From the two-dimensional spectra, the peptide backbone resonances (NH and C alpha H) for 65 of the 66 amino acids were assigned, as well as most of the side chain resonances. The chemical shifts for the assigned protons are reported at 35 degrees C in 10 mM potassium phosphate, pH 6.8, and in 10 mM potassium phosphate, pH 4.6, 0.2 M KCl, and 0.1 mM EDTA. Small shifts were observed for some resonances upon addition of salt, but no major changes in the spectrum were seen, indicating that no global structural change occurs between these ionic strengths. NOE patterns characteristic of alpha-helices, beta-strands, and turns are seen in various regions of the primary sequence. From the location of these regions the secondary structure of cro in solution appears to be virtually identical with the crystal structure [Anderson, W. F., Ohlendorf, D. H., Takeda, Y., & Matthews, B. W. (1981) Nature (London) 290, 754-758]. Missing assignments include the Pro-59 resonances and the peripheral protons of the eight lysine, the three arginine, and three of the five isoleucine residues.