Drosophila basement membrane procollagen IV. I. Protein characterization and distribution

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.     

Ultrastructural changes in the cardiomyopathy of dystrophic hamsters and mice

Changes in the ultrastructure of the cardiac muscle cells have been followed in dystrophic mice and hamsters (22-40 weeks of age) and in both species a severe cardiomyopathy accompanies the cellular damage of the skeletal muscle. The degradative changes of the myofilament apparatus of the heart cells and the specific changes in mitochondrial ultrastructure (including swelling, septation and apparent division) are characteristic of the cellular damage of both the dystrophic skeletal muscle and of normal cardiac muscle in which [Ca]i has been experimentally raised, confirming the suggestions that (i) the same gene is responsible for the myopathy of skeletal and cardiac muscle in animal dystrophy and (ii) that changes in [Ca]i are implicated in the degradative changes of muscle cells.     

Failure to detect changes in sarcolemma permeability in amphibian muscle following severe cellular damage

1. Isolated amphibian hearts and pectoris cutaneous muscles were exposed either to DNP or to caffeine, thereby producing severe myofilament damage. 2. No accompanying change in sarcolemma permeability was detected by monitoring either CK or LDH release or Procion yellow entry in the heart, or by Procion entry in amphibian skeletal muscle. 3. The findings are in contrast with mammalian cardiac and skeletal muscles, and confirm that the pathways leading to myofilament degradation and to the breakdown in sarcolemma organization are separate.     

Effect of aldosterone on 86Rb fluxes in cultured kidney cells (A6)

This study was designed to evaluate the relative contributions of hormone induced changes in active and passive K+ transport in an epithelial cell line in continuous culture derived from toad kidney (A6) using 86Rb as a tracer for measuring unidirectional K+ fluxes. The effects of 24 h exposure to aldosterone (A) and aldosterone plus insulin (A+I) on unidirectional K+ fluxes were evaluated under short-circuited conditions and under open circuit conditions. In epithelia exposed to A, a small but significant amount of active K+ secretion was found, although it was not significantly greater than in control epithelia. The bidirectional fluxes in both A and A+I treated epithelia, under short-circuited conditions, increased by a similar amount over control values indicating an increase in apparent permeability of passive transepithelial K+ transport. Under open circuit conditions, A stimulated net K+ transport by about 5-fold over controls. The increase in K+ secretion produced by A under open circuit conditions could be explained by the combined effects of an increase in transepithelial K+ permeability and an increase in the transepithelial electrical potential difference (PD). The presence of I produced no additional effects to that of A on K+ transport under the conditions used in this study. It is concluded that the substantial increase in K+ secretion induced in A6 cells by 24 h exposure to A is primarily passive in nature. It is possible that the changes in both PD and transepithelial K+ permeability, which can account for the observed increase in K+ secretion, are secondary to the stimulation of active Na+ transport.     

Modulation of differentiation markers in human choriocarcinoma cells by extracellular matrix: on the role of a three-dimensional matrix structure

Abstract. During spontaneous or chemically induced differentiation human choriocarcinoma cells express typical characteristics of the normal differentiating trophoblast: 1) increased production of peptide and steroid hormones (chorionic gonadotropin, placental lactogen, estrogens, progesterone); 2) increased activity of cellular alkaline phosphatase; 3) morphological transition from cytotrophoblast to syncytiotrophoblast-like cells; and 4) arrested cell proliferation. Since the extracellular matrix is known to control gene expression we have examined the effects of different substrates composed of matrix macromolecules on the differentiation of BeWo choriocarcinoma cells. Matrices tested were; fibronectin, laminin, collagens type I and type IV, the basement membrane-like complex matrix Matrigel, and a complex matrix extracted from human term placenta. Irrespective of the type of molecule(s), it was consistently found that, whenever the matrix molecules were presented as threedimensional structures (as opposed to protein coatings on tissue culture plastic) the response of affected differentiation markers monitored was highly pronounced. Morphology was changed from monolayers to rounded colonies, cell proliferation was reduced, and the secretion of chorionic gonadotropin was increased up to tenfold. Heterogeneous effects were observed on progesterone secretion and on the activity of cellular alkaline phosphatase. Cell adhesion to matrix molecules, however, did not depend on the structure of the matrix. This study demonstrates that gene expression in these tumor cells can be modified by extracellular matrix and highlights that not only the presence of effector molecules in the matrix but also the three-dimensional structure of the matrix is important for the induction of differentiation.     

A review and update of animal toxicoses associated with fumonisin-contaminated feeds and production of fumonisins by Fusarium isolates

During the 1989 corn harvest season, numerous reports of equine leukoencephalomalacia (ELEM) outbreaks and a pulmonary edema (PPE) syndrome in swine from several regions of the United States were received by the National Veterinary Services Laboratories (NVSL), Ames, Iowa. Previous and concurrent research linked Fusarium moniliforme and fumonisin-contaminated feeds to both diseases. Chemical and mycological investigations revealed fumonisin B₁ (FB₁) concentrations of 20 to 360 ppm in suspect swine feeds and 8 to 117 ppm in suspect equine feeds. Nonproblem feeds contained concentrations below 8 ppm. Fusarium moniliforme and Fusarium proliferatum were isolated from both problem and nonproblem equine and swine feeds. When cultured on autoclaved corn, the F. moniliforme and F. proliferatum isolates produced respective FB₁ and fumonisin B₂ (FB₂) that range from less than 5 to more than 2450 ppm and less than 5 to more than 1000 ppm, respectively. Isolates from both problem and nonproblem feeds produced high levels (greater than 500 ppm) in culture. Reported here is a review of chemical and mycological data resulting from the study of several cases of PPE and ELEM.     

Modeling problems in conservation genetics using captive Drosophila populations: Consequences of equalizing founder representation

Equalizing founder representation is a recommended practice for maintaining captive populations. However, this procedure has not been subject to controlled experimental evaluation. The effects on inbreeding, genetic variation, and reproductive fitness of maintaining small captive populations by equalizing founder representation (EFR) versus randomly choosing parents (RC) were compared. Ten replicate lines were created with unequal founder representations, split into EFR and RC lines, and maintained for a further eight generations. Founder representations computed from pedigrees were closer to equality in the EFR lines than in the RC lines or the base population, most of the changes being evident after one generation. Significant benefits of EFR were found in lowered inbreeding (mean inbreeding coefficients of 0.35 and 0.41, respectively, for EFR and RC lines) and average heterozygosity (0.141 for EFR, 0.084 for RC, compared with 0.216 in the base population). However, EFR was not significantly better than RC in moving allele frequencies towards equalized founder representation. No significant difference was found in reproductive fitness between EFR and RC (relative fitnesses compared to the base population were 0.179 for EFR and 0.182 for RC). The use of equalization of founder representation for a few generations can be recommended in the genetic management of captive populations derived from a small number of founders that contribute unequally. © 1992 Wiley-Liss, Inc.     

Production of hydroxyl radicals and their disassociation from myocardial cell injury during calcium paradox.

The production of hydroxyl radicals during calcium paradox injury was investigated by measuring the production of 2,5-dihydroxybenzoic acid (2,5-DHBA) from salicylate. Four groups of rats were analyzed. In the first group, isolated hearts were perfused with calcium-free medium for 10 minutes followed by perfusion with medium containing Ca++ for 10 minutes. In the other groups, 0.25 microM N,N'-diphenyl-1,3-phenylenediamine (DPPD), 80 microM cytochrome c, or 450 U/ml catalase was added. Coronary effluent was analyzed for the presence of 2,5-DHBA, and tissue sections were examined using light microscopy. In the first group, 2,5-DHBA production began during the calcium-free period, peaked tenfold 60-90 sec. into the Ca repletion period, and declined thereafter. The increase in 2,5-DHBA was accompanied by severe cell damage. Cytochrome c reduced 2,5-DHBA production, and catalase almost completely inhibited 2,5-DHBA production, while DPPD had no effect on 2,5-DHBA production. None of the three additives provided any complete morphological protection. The data provide evidence for the production of hydroxyl radicals during calcium-paradox injury, that their production is dependent upon the presence of hydrogen peroxide, and that cell damage in the calcium paradox is not primarily mediated by the extracellular hydroxyl radicals.     

Trade-offs inDaphnia vertical migration strategies

Summary Planktonic animals performing diel vertical migration (DVM) experience a tradeoff between reduced mortality and reduced reproductive output due to lower food availability in their refuge. Models of DVM as an evolutionarily stable strategy predict that, under certain conditions, strategies of both migration and non-migration can coexist. Vertical profiles of animal abundances during day and night, however, do not allow any discrimination between the behaviour of individuals or subpopulations. We used length-body protein regressions as a measure of the nutritional state ofDaphnia to distinguish possible sub-populations differing in their migration strategy. An overwhelming part of the population migrated downwards during the day. However, the few daphnids in the epilimnion during the day had significantly higher protein content than the animals in the deep water, indicating that these daphnids did not migrate randomly but remained in the surface food-rich water all day. This shows that migrating animals gain no metabolic advantage over non-migrating ones.Supported by a F.P.U. grant (Spanish Goverment)