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.     

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.     

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.     

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.     

Fertilization potential of follicular oocytes classified by stage of cycle and size of follicle

Bovine follicular oocytes, collected from two sizes of vesicular follicles and from donor animals from three stages of the estrous cycle, were matured and fertilized in vitro . Frequency of fertilization and ability to form male pronuclei after in vitro maturation were found to be independent of both estrual stage and follicular size.     

The effects of superovulation on Brucella abortus infection in the bovine uterus

Uterine flushings were collected three times at predetermined intervals from 11 mixed-breed beef cows and cultured for Brucella abortus . Prior to sampling, all cows had aborted fetuses from which brucellae had been isolated. Initial collections were made between 21 and 34 days following abortion. The second flushing was conducted at the onset of injections used for inducing superovulation and the third flushing was conducted 6 to 8 days after the ensuing estrus. The latter two flushes were conducted between 60 and 120 days following abortion. Brucellae were isolated from uterine flushings collected from 6 of the 11 cows on the initial round of sampling. Cultures of all subsequent uterine flushings collected before and after injections for superovulation were negative. It was concluded that the superovulatory treatment is not likely to reactivate the release of brucellae into the uterine lumen during the period when embryos are normally collected.     

Successful transfer of embryos recovered from a cow with chronic cervicitis of mixed aerobic and anaerobic bacterial aetiology a case report

An imported 13-year-old Simmentaler cow was presented with severe purulent cervicitis and endometritis from which Corynebacterium pyogenes , Fusobacterium necrophorum , Bacteroides melaninogenicus and an untyped Clostridium perfringens were isolated. The endometritis responded to treatment but the cervix did not and remained fibrous, continuously patent and purulent. Natural pregnancy was considered unlikely and the cow was prepared as an embryo transfer donor. Eight embryos were recovered and six transferred, resulting in five confirmed pregnancies and four live births.     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.     

Regulation of Na/K/Cl cotransport in vascular smooth muscle cells

The regulation of Na/K/Cl cotransport was investigated in vascular smooth muscle cells. That a Na/K/Cl cotransport system exists was established by the finding that the ouabain insensitive K influx was sensitive to the "loop" diuretic bumetanide. Furthermore, bumetanide sensitive K influx was dependent upon the presence of both Na and Cl in the extracellular milieu. Bumetanide sensitive K influx was inhibited by agents which elevate cellular cyclic AMP levels, and to a lesser extent by agents which elevate cellular cyclic GMP levels. When serum, EGF or TPA was added, bumetanide sensitive K influx was enhanced. These results suggest that vascular smooth muscle cells have a ouabain insensitive, bumetanide sensitive Na/K/Cl cotransport system which is stimulated by serum, EGF or TPA and inhibited by cAMP or cGMP.