人酸性成纤维细胞生长因子神经营养作用的初步研究

本实验研究了人酸性成纤维细胞生长因子(haFGF)的体外神经营养作用。结果表明,haFGF在体外能明显促进鸡胚(E-8)脊髓组织神经突起的生长,并能明显改变新生大鼠脑星形胶质细胞的形态,使扁平、多角形紧密联接的细胞转化为具有纤维样突起的胶质细胞,同时对胶质细胞DNA合成也有一定促进作用。实验还证明,haFGF可增加体外培养新生大鼠海马神经元的存活,且大大增加神经元胞体体积及突起长度。     

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and characterization of alloantisera specific for bovine class II major histocompatibility complex antigens

Ten alloantisera defining five major histocompatibility complex (MHC) class II specificities of the bovine lymphocyte antigen (BoLA) complex were produced and characterized. Eight antisera defining four of the specificities were generated by immunizing cattle with class I compatible-class II incompatible lymphocytes. The alloantiserum defining the fifth class II specificity was produced by skin implant immunization. A pregnancy serum specific for one of the class II specificities was also identified. The class II antigens recognized by these antisera were designated 'Dx' antigens to indicate that they are BoLA-D region antigens encoded by one or more undetermined class II loci. The molecules identified by the alloantisera are heterodimers composed of a 34-kd alpha and a 26- to 28-kd beta chain, and are expressed on B-lymphocytes but not on resting T-lymphocytes. In family studies the BoLA-Dx antigens segregated in linkage with the BoLA-A locus alleles. Most of the BoLA-A alleles present in the Cornell Holstein herd at a high frequency were found to exist in gametic association with two or more serologically defined class II haplotypes. On the basis of a population study it was determined that three pairs of class I and class II alleles (w10-Dx4, w31-Dx5, and c3-Dx2) were present in the Cornell herd at significantly increased frequencies.     

Segmental release of amino acid neurotransmitters from transcranial stimulation

The present study used microdialysis techniques in an intact rabbit model to measure the release of amino acids within the lumbar spinal cord in response to transcranial electrical stimulation. Dialysis samples from the extracellular space were obtained over a stimulation period of 90 minutes and were examined using high pressure liquid chromatography. Neuronal excitation was verified by recerding corticomotor evoked potentials (CMEPs) from the spinal cord. A significant increase in the release of glycine and taurine compared to sham animals was measured after 90 minutes of transcranial stimulation. Glutamate and aspartate release was not significantly elevated. GABA concentrations were consistently low. CMEP components repeatedly showed adequate activation of descending fiber pathways and segmental interneuron pools during dialysis sampling. Since glycine, and to a lesser extent taurine, have been shown to inhibit motor neuron activity and are closely associated with segmental interneuron pools, suprasegmental modulation of motor activity may be, in part, through these inhibitory amino acid neurotransmitters in the rabbit lumbar spinal cord.     

Management of Root-knot Nematodes by Phenamiphos Applied through an Irrigation Simulator with Various Amounts of Water

Phenamiphos (6.7 kg a.i./ha) was applied via an irrigation simulator to squash at planting (AP) and 2 weeks after planting (PP), and to corn AP and 1 week PP to manage root-knot nematodes (Meloidogyne incognita). The nematicide was applied with 0.25, 0.64, 1.27, and 1.91 cm surface water/ ha to a Lakeland sand in which the soil moisture was at or near field capacity. Based on efficacy and crop response, no additional benefits resulted when phenamiphos was applied in volumes of water greater than 0.25 crn/ha. The cost of applying each 0.25 cm of water over a hectare is approximately $1.08, or a 92% reduction in nematicide application cost over conventional methods ($13.50/ha). Low root-gall indices and high yields from squash and corn indicate more effective nematode management when phenamiphos was applied AP rather than PP. Results from this method of applying phenamiphos suggest that certain nematicides could be used as salvage alternatives when nematodes are detected in crops soon after planting. For multiple-pest management, nematicides, other compatible biocides, and fertilizers could be applied simultaneously with sprinkler irrigation.     

Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications

Cells subjected to the events occurring before, during, and after freezing and thawing are exposed to major changes in the osmotic pressure of the surrounding medium; i.e., the osmolalities can exceed 30. An important question in understanding the mechanisms of injury is whether cells respond as ideal osmometers to these strongly anisotonic solutions. Mouse and bovine embryos from eight-cell to blastocyst stage were used to investigate the question. They were found to behave as ideal osmometers at room temperature over a wide range of tonicities; i.e., from four times isotonic to almost 1/3 times isotonic, ideality being defined by a Boyle-van't Hoff equation. Embryo volumes increased from 40 to 200% of isotonic over this range and survivals of mouse embryos were unaffected. However, outside this range the membrane apparently becomes leaky and the survival of mouse embryos drops sharply. Osmolalities rise to high values during freezing and the paper develops the thermodynamic equations to show how computed cell volumes as a function of subzero temperature can be translated into the Boyle-van't Hoff format of cell volume as a function of the reciprocal of osmolality.     

Characterization of FMRF Amide-Like Immunoreactivity in Rat Spinal Cord by Region-Specific Antibodies in Radioimmunoassay and HPLC

Material in rat spinal cord extracts that reacts with antibodies to the molluscan tetrapeptide FMRF amide (Phe-Met-Arg-Phe-NH2) has been characterized by HPLC and radioimmunoassay using region specific antibodies. An antibody to the N-terminally extended analogue, Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH2 (YGGFMRF amide), did not react with the rat material. Two antibodies to FMRF amide were characterized that differed markedly in their affinities for analogues with substitutions in the second and third positions from the C-terminus; both required the C-terminal amide, and neither showed appreciable sensitivity to substitutions in the fourth position from the C-terminus. With both antibodies the relative potency of the avian brain peptide, LPLRF amide, was about 0.1. Both antibodies revealed similar concentrations of immunoreactive material in rat spinal cord extracts. On reversed-phase HPLC using Techsil C18 and Spherisorb-phenyl columns, two peaks were separated that could be distinguished in retention times from FMRF amide, Leu-Pro-Leu-Arg-Phe-NH2 (LPLRF amide), and YGGFMRF amide. The results suggest that the rat spinal cord peptides are structurally related to the C-terminal tripeptide of FMRF amide and are probably extended at the N-terminus by sequences immunochemically distinct from other known peptides.     

Electrostatic effect of trypsin binding on the hydrogen exchange rate of bovine pancreatic trypsin inhibitor beta-sheet NH's

The changes of H-D exchange rates upon protein-protein interactions are generally interpreted as a result of the changes of the dynamic properties of the proteins. The effect of trypsin binding on the H-D exchange kinetics of some trypsin inhibitor amide H's was reported (Simon et al., 1984). In this paper the electrostatic potential originating from the trypsin molecule is calculated at the positions of the studied amide H's in the trypsin-trypsin inhibitor complex. We conclude that the observed decrease of the exchange rates is mainly due to the electrostatic field of the trypsin molecule.     

Cardiovascular responses to intrathecal administration of arginine vasopressin in rats

Arginine vasopressin (AVP) has been localized in numerous extrahypothalamic brain regions and in the spinal cord. The results of intracerebroventricular AVP injections and microinjection of AVP into the brain stem suggest that this peptide, acting centrally at higher levels, may influence cardiovascular function. No function for the AVP occurring at spinal levels has been reported. In this study we report that AVP, in picomole quantities, increased arterial blood pressure and integrated heart rate in a dose-dependent manner following intrathecal application to the thoracic region in the rat. This response was not blocked by intravenous administration of the AVP antagonist d(CH₂)₅-d-Tyr-VAVP. These results suggest that AVP, acting within the spinal cord, may alter neural outflow regulating blood pressure and heart rate.     

Radioimmunoassay for fibroblast growth factor (FGF): release by the bovine anterior pituitary in vitro

A radioimmunoassay (RIA) was developed to measure fibroblast growth factor (FGF) using antiserum generated against a synthetic replicate of [Tyr¹⁰]FGF(1–10). The antisera, previously shown to be capable of inhibiting the biological action of FGF on bovine aortic arch endothelial cells in vitro [1], are highly specific for the amino-terminus of FGF. In the RIA, the antisera recognize the decapeptide antigen [Tyr¹⁰]FGF(1–10) and the intact mitogen on an equimolar basis and show less than 0.01% cross-reactivity with N-acetyl-[Tyr¹⁰]FGF(1–10).

Bovine adenohypophysial cells maintained in primary monolayer culture release and ir-FGF which is indistinguishable from the intact mitogen in as much as it is retained on heparin-Sepharose affinity columns and shows a dose-dependent and parallel displacement in RIA. The release of ir-FGF by the bovine adenohypophysis can be increased with forskolin (10⁻⁵ M) or KCl (50 mM). Preincubation of pituitary cells with 17β-estradiol has no measurable effects on basal ir-FGF, but increases the release after KCl treatment 2–3-fold. These results show that ir-FGF can be released by the bovine adenohypophysis in vitro and lend credence to the hypothesis that FGF plays a physiological role in the homeostatic mechanisms regulating mesoderm-derived cell growth.