Properties of heart fibroblasts of adult rats in culture

Summary In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cell used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate. Direct observation of type IV collagen is consistent with the previous finding of type IV mRNA in cardiac fibroblasts in situ and in freshly isolated populations of these cells.     

Decrease in insulin-containing secretory granules and mitochondrial gene expression in mouse pancreatic islets maintained in culture following streptozotocin exposure

We have previously described a preferential reduction in the secretory response to nutrient secretagogues in pancreatic mouse islets maintained in culture after in vitro exposure to streptozotocin (SZ). This reduction was associated with an impaired substrate metabolism at the mitochondrial level. To further clarify this issue, mouse pancreatic islets were exposed in vitro to 2.2 mM SZ for 30 min. At 4 h after SZ treatment ultrastructural changes were apparent in the endoplasmic reticulum and Golgi areas of the B-cells. However, 2 and 6 days following SZ exposure the B-cells appeared well preserved, except for a marked decrease in the number of insulin-containing secretory granules. A morphometric analysis of the B-cells 6 days after SZ exposure showed a normal B-cell size and a normal volume fraction of B-cell mitochondria. However, there was a decrease in total islet size and a 13% decrease in the volume fraction of B-cells in the islets. These mouse islets exhibited a decreased content of the mitochondrial DNA-encoded cytochrome b mRNA, as evaluated by dot-blot analysis. As a whole, the data obtained indicate that SZ treatment does not induce a decrease in the number of mitochondria or long-lasting ultrastructural damage to this organelle. However, there is a clear decrease in the cytochrome b mRNA, suggesting that SZ can induce damage to the mitochondrial DNA.     

The codon 408 mutation associated with haplotype 2 is predominant in Polish families with phenylketonuria

Summary The incidence of phenylketonuria (PKU) in the western part of Poland is 1 in 5000 live births. Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase locus have been analysed in 46 Polish families with PKU. Among 43 fully-informative families 16 RFLP haplotypes were identified. Haplotype 2 is the most frequently (62%) associated with Polish PKU alleles, and the codon 408 mutation is in complete linkage disequilibrium with this haplotype in Poland. This finding is in agreement with observations in other eastern European countries (German Democratic Republic, Czechoslovakia, and Hungary) and in contrast to the genotype distribution observed in western European countries. The present observation suggests the spread of classical PKU, due to the codon 408 mutation associated with haplotype 2, from east to west in European populations. Perhaps more important for genetic counselling, 62% of all PKU chromosomes in the Polish population can now be detected using only one mutantspecific oligonucleotide probe.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.     

Brain injury: New insights into neurotransmitter and receptor mechanisms

The studies reviewed here represent a continuing search for mechanisms which play a role in neurological disturbances resulting from brain injury. Focal cortical freezing lesions in rats were shown to cause a widespread decrease in local cerebral glucose utilization (LCGU) in cortical areas of the lesioned hemisphere and this was interpreted as reflecting a depression of cortical activity. Such an interpretation was supported by the finding that in lesioned brain reduction of cerebral metabolism by pentobarbital and isoflurane was limited by the metabolic depression that has already occurred as a result of injury and by the demonstration that the energy status and substrate (glucose) supply in the cortical areas in the injured brain have not been compromised at the time when LCGU was decreased. Both the serotonergic and the noradrenergic neurotransmitter systems were implicated in functional alterations associated with injury. Cortical serotonin (5-HT) metabolism was increased throughout the lesioned hemisphere and complete inhibition of 5-HT synthesis withp-chlorophenylalanine ameliorated the decrease in cortical LCGU, interpreted as reflecting cortical functional depression. Cortical norepinephrine metabolism was bilaterally increased in focally injured brain, while prazosin, a selective ₁-noradrenergic receptor blocker, normalized cortical LCGU in the lesioned hemisphere. Low-affinity in vivo binding of [¹²⁵I]HEAT, another selective ₁-receptor ligand, was specifically increased in cortical areas of the lesioned hemisphere at the time of the greatest depression in LCGU, suggesting that ₁-adrenoreceptors may be of functional importance in injured brain. The general conclusion from this series of studies on mechanisms underlying functional disturbances in injured brain is that both the serotonergic and the noradrenergic neurotransmitter systems are involved in the widespread cortical depression which develops with time as a consequence of a focal lesion. The data are compatible with the inhibitory effects of NE and 5-HT in the cortex and with the hypothesis that these two transmitter systems affect cortical information processing.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

Habitat and population size of the coelacanth Latimeria chalumnae at Grand Comoro

Synopsis In 1987 and 1989 coelacanths were observed for the first time in their natural habitat with the help of submersibles. Coelacanths were found between 150–253 m depth, their preferential depth seems to be around 200 m; the water temperature ranged between 16.5–22.8° C. During the day coelacanths aggregate in small non-aggressive groups in sheltered lava-caves. Caves might be a limiting factor for distribution. At night they leave the caves for hunting by drifting singly along the steep lava slopes. They migrate between different caves located within a large home range covering more than 8 km coastline. Coelacanths are site-attached, some for a period of at least 2 years. Our own observations and earlier catch records show that only the west coast of Grand Comoro is a suitable coelacanth habitat with more structural complexity and prey fish abundance than other coastlines of the island. From our survey we estimated a total coelacanth population off Grand Comoro to be 150–210 individuals; a saturated population would be 370–510 individuals. This small relict population seems to be stable. International protection of coelacanths against commercial interests is needed     

Divalent cation involvement in the action of Clostridium perfringens type A enterotoxin. Early events in enterotoxin action are divalent cation-independent

Clostridium perfringens type A enterotoxin (CPE) is a membrane-active cytotoxin. There are a number of recognized early steps in CPE cytotoxicity including binding of CPE to a protein receptor, insertion of CPE into membranes, and CPE-mediated induction of changes in membrane permeability for small molecules such as ions and amino acids. Further support for the existence of these early steps and further characterization of these events are presented in this report. We now report that these early steps in CPE action are largely independent of extracellular divalent cations. It is also shown that 3H-nucleotide release, known to be a later CPE effect, is Ca2+-dependent. A model for CPE cytotoxicity is suggested involving CPE action as a two-step process with Ca2+-independent early steps and Ca2+-dependent late steps.