Vascular supply of the anterior interventricular epicardial nerves and ventricular Purkinje fibers in the porcine hearts

The aim of this study was to perform a pilot histological and quantitative analysis of the blood vessels accompanying the epicardial nerves (vasa nervorum) in the porcine hearts. Twenty healthy porcine hearts were used in this study. The blood vessels were analyzed by light microscopy using four different staining techniques in transverse sections taken from the upper, middle, and lower segments of the anterior part of the interventricular region and the adjacent parts of the right and left ventricles containing epicardial nerves and the endocardial peripheral parts of the Purkinje fibers. In total, 317 epicardial nerves were detected. The vasa nervorum were present in 75.7% of these nerves. The vasa nervorum resembled arterioles and postcapillary and collecting venules. One hundred and forty nine epicardial nerves were perivascular, located in the adventitia of the anterior interventricular artery and vein. The remaining 168 nerves ran freely through the epicardial interstitium. The presence of the vasa nervorum was not related to topographical location or nerve diameter. Additionally, from a total of 33 analyzed ventricular complexes of Purkinje fibers small blood vessels located in their proximity were identified in only two cases. It can be concluded that the majority of the anterior epicardial nerves of porcine heart possess well-developed vasa nervorum. In contrast, similar blood vessels are rarely present in the vicinity of the Purkinje fibers. The data obtained contribute to a better understanding of the nutrition of the cardiac nerves.     

Salmonella pathogenicity islands encoding type III secretion systems.

Salmonella enterica harbours two Salmonella pathogenicity islands (SPIs) each encoding a type III secretion system for virulence proteins. SPI1 is required for invasion, while systemic infections and intracellular accumulation of Salmonella are dependent on SPI2 function. This review will describe and compare the genetic organisation, evolution, regulation and molecular functions of SPI1 and SPI2.     

Characterization of two D-glyceraldehyde-3-phosphate dehydrogenases from the extremely thermophilic archaebacterium Thermoproteus tenax

Thermoproteus tenax possesses two different glyceraldehyde-3-phosphate dehydrogenases, one specific for NADP+ and the other for NAD+. NADP(H) inhibits the NAD+-specific enzyme competetively with respect to NAD+ whereas NAD(H) virtually does not interact with the NADP+-specific enzyme. Both enzymes represent homomeric tetramers with subunit molecular masses of 39 kDa (NADP+-specific enzyme) and 49 kDa (NAD+-specific enzyme), respectively. The NADP+-specific enzyme shows significant homology to the known glyceraldehyde-3-phosphate dehydrogenases from eubacteria and eukaryotes as indicated by partial sequencing. The enzymes are thermostable, the NADP+-specific enzyme with a half-life of 35 min at 100 degrees C, the NAD+-specific enzyme with a half-line of greater than or equal to 20 min at 100 degrees C, depending on the protein concentration. Both enzymes show conformational and functional changes at 60-70 degrees C.     

Synthesis in vitro of a seven amino acid peptide encoded in the leader RNA of Rous sarcoma virus

Sequences of avian retroviral RNAs suggest that short open reading frames in the putatively untranslated leader sequences might direct the synthesis of small peptides. Previous analyses indicate that translation of Rous sarcoma virus (RSV) RNA in vitro faithfully reflects translation of the viral RNA in the chick cell. Accordingly, we sought to determine if the heptapeptide LP1, encoded in the open reading frame closest to the 5' end of RSV RNA, could be synthesized in vitro since this would strongly suggest that it might also be synthesized in vivo. Here we confirm that RSV RNA directs the synthesis of LP1 in rabbit reticulocyte lysates. LP1 is rapidly degraded in the lysate by an aminopeptidase activity. On the basis of the following observations, we propose that the open reading frame encoding LP1 plays a role in the life cycle of avian retroviruses. The LP1 open reading frame is ubiquitous with respect to position and length in 12 strains of avian retrovirus. In the amino acid sequences of the 12 strains, only three of the seven residues are invariant. On the basis of the conservation of the -3 and +4 nucleotides flanking the AUG codon, the strengths of initiation for translation of LP1 are approximately the same in the different viruses. The LP1 open reading frame is positioned in front of sites on retrovirus RNA that are required for initiation of cDNA synthesis and for packaging of the RNA into mature virus.     

Developmental regulation of 16S acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line

We have studied the appearance, distribution and regulation of acetylcholinesterase (AChE) and acetylcholine receptors (AChRs) in a mouse skeletal muscle cell line (C2), that was originally isolated and described by Yaffe & Saxel [54]. In culture, cells from this line form spontaneously contracting myotubes, with overshooting action potentials that are TTX-sensitive. After fusion of myoblasts into myotubes, there was a dramatic increase in the amount of both AChE and AChR. Three forms of AChE, distinguished by their sedimentation on sucrose gradients, were synthesized: 4-6S, 10S, and 16S. The 4-6S and 10S forms appeared 1 day after the cells began to fuse, whereas the 16S form appeared only 2 days after fusion began. Maximal levels of the 16S AChE form (25-30% of the total) were obtained by reducing the concentration of horse serum in the fusion medium. Prevention of myoblast fusion by reducing the calcium levels in the medium decreased the total AChE by 70%, and only the 4-6S form was synthesized. Blocking spontaneous contractile activity of the myotubes by tetrodotoxin (TTX) led to a 50% reduction in all three esterase forms. Thus, the 16S, or endplate form of AChE is not specifically regulated by electrical or contractile activity in the C2 cell line. After fusion the number of AChRs increased rapidly for 3-4 days and then stabilized. Receptor clusters, ranging from 10-30 micron in length, appeared 1 day after myoblast fusion began. When cells were grown in medium containing reduced Ca2+, the total number of AChRs was decreased by 20-50%. Reduction of Ca2+ after myotubes and AChR clusters had formed resulted in dispersal of AChR clusters. Inhibition of muscle contractions with TTX did not affect the number of AChRs or their distribution.     

Correlative changes in sucrose uptake, ATPase activity and membrane fluidity in carnation petals during senescence

Apparent sucrose uptake. ATPase activity and membrane fluidity changes were studied during the development and senescence of carnation flowers ( Dianthus caryophyllus L., cv. Cerise Royallette). Typical changes associated with senescence of a cut flower, such as respiration, ethylene production and fresh weight, were measured. Concomitant with a rise in respiration and ethylene production and a decline in fresh weight, a sharp decrease in apparent sucrose uptake was observed. Sucrose uptake was pH dependent (pH optimum, 5.5) and influenced by membrane integrity. Apparently, the activity of ATPase is related to sucrose uptake, because similar changes occurred during flower development. In addition, the activity of ATPase was well correlated with membrane fluidity.
It is suggested that sucrose uptake is controlled by ATPase activity, which in turn is modulated by membrane lipid fluidity. The decline in membrane fluidity associated with senescence leads to a corresponding reduction in ATPase activity and sucrose uptake. Further evidence supporting this view comes from experiments in which senescence was enhanced by 1-aminocyclopropane-l-carboxylic acid. It shortened the time to decline in fresh weight, rise in respiration and ethylene production. In parallel, reduction in membrane fluidity, ATPase activity and sucrose uptake were observed.     

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes.

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.     

Transcription-Independent Activation of Ornithine Decarboxylase Activity by Heparin in Cloned Cerebral Endothelial Cells

Abstract: Heparin, a highly sulfated glycosaminoglycan, is known to be obligatory for long-term endothelial cell cultures; it potentiates the mitogenic activities of endothelial cell growth factors and prolongs the replicative life span of the cells. Here we have shown that besides its growth factor-supportive role, heparin exerts a specific action on cerebral capillary endothelial cells (cECs), unrelated to serum or growth factors, by increasing activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in these cells. For our experiments we have used two different types of cloned cECs: type I cECs, grown in the presence of endothelial cell growth factor and heparin, and type II cECs, usually cultivated without growth factors. Heparin action on ODC activity was shown to be dose dependent within the range of 1–100 μg/ml. Increasing concentrations of or depletion of endothelial cell growth factor from type I cultures had no effect on ODC activity. The increase in enzyme activity was highest after 30 min to 1 h of heparin treatment. As evidenced by northern analysis, the heparin-mediated enhancement of ODC activity was not accompanied by changes of ODC mRNA levels. Studies of DNA replication revealed that in the absence of heparin-binding growth factors, heparin did not affect the proliferative activity of cloned cECs.