Fibronectin initiates fibrin production by rabbit corneal endothelial cells in vitro

Confluent rabbit corneal endothelial cells incubated in the absence of serum do not produce fibrinogen. When exogenous fibronectin is added to these cultures, fibrinogen production is observed. Fibronectin concentrations stimulate fibrinogen synthesis by endothelial cells in a dose-response fashion. This direct interaction of fibronectin and fibrinogen may be important in both wound healing processes and pathological states.     

Collagen crosslinking and cartilage glycosaminoglycan composition in normal and scoliotic chickens

The amounts of lysine-derived crosslinks in collagens from tendon, cartilage, intervertebral disc, and bone and changes in the composition of sternal cartilage glycosaminoglycans were estimated in two lines of chickens, a control-isogenic line and a line that develops scoliosis. In the scoliotic line, scoliosis first appears at 3-4 weeks and progressively increases in severity and incidence so that 90% of the birds express the lesion by week 10. We have reported previously that cartilage, tendon, and bone collagens from scoliotic birds are more soluble than corresponding collagens from normal birds. Herein, collagen crosslinking and altered proteoglycan metabolism are examined as possible mechanisms for the differences in collagen solubility. At 1 week of age there were fewer reducible crosslinking amino acids (hydroxylysinonorleucine, dihydroxylysinonorleucine, and lysinonorleucine) in collagens from sternal cartilage and tendon in the scoliotic line than in the isogenic line. However, by week 3 and at weeks 5 or 7 values were similar in both groups. The amounts of hydroxypyridinium in vertebral bone and intervertebral disc collagen were also similar in both groups of birds. Consequently, differences in collagen crosslinking do not appear to be a persistent developmental defect underlying the expression of scoliosis in the model. However, differences were observed in cartilage proteoglycans and glycosaminoglycans from the scoliotic line that were not present in cartilage from the isogenic line. The average molecular weight of the uronide-containing glycosaminoglycans was 30% less in the scoliotic line than in the isogenic line, i.e., 12,000 compared to 18,000. The size distribution of cartilage proteoglycans from the scoliotic line also differed from that of proteoglycans from the isogenic line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of semen fractionation and dilution ratio on motility parameters of stallion spermatozoa. In Experiment 1, three ejaculates from each of three stallions were divided into sperm-rich (SR) and sperm-poor (SP) fractions to determine the difference in sperm concentration. Mean sperm concentration in SR fractions (349.5 x 10(6)/ml) was greater (P < 0.001) than that of SP fractions (96.9 x 10(6)/ml). In Experiment 2, three ejaculates from each of two stallions were divided into SR and SP fractions. Fifty percent of the original volume of SR fractions was combined with 50% of the original volume of SP fractions for each ejaculate to represent total ejaculates. SR and total ejaculates were diluted with skim milk-glucose semen extender as follows: 1) no dilution, or dilution to 2) 100 x 10(6)sperm/ml, 3) 50 x 10(6)sperm/ml, or 4) 25 x 10(6)sperm/ml. Semen samples were evaluated at 0.5, 3, 6, 12, and 24 h postejaculation (25 degrees C storage temperature) for percentages of total spermatozoal motility (TSM) and progressive spermatozoal motility (PSM). Mean TSM was greater (P < 0.05) in SR ejaculates than total ejaculates at 12 and 24 h postejaculation. Mean TSM of undiluted semen was lower (P < 0.05) than other dilution ratios over all periods. Mean TSM was greater (P < 0.05) at a 25 x 10(6)sperm/ml dilution ratio than a 50 x 10(6)sperm/ml dilution ratio at 12 and 24 h postejaculation, and greater (P < 0.05) than a 100 x 10(6)sperm/ml dilution ratio from 3 to 24 h postejaculation. Similar patterns were found for PSM. Collection of SR ejaculates and dilution to 25 x 10(6)sperm/ml improved longevity of spermatozoal motility.     

Bactericidal activity of peripheral blood neutrophils during the oestrous cycle and early pregnancy in the mare

The oestrous cycles of 20 mixed-breed mares were synchronized with daily injections of 10 mg oestradiol-17 beta and 150 mg progesterone given i.m. for 10 days. On the 10th day, 10-15 mg prostaglandin F-2 alpha was administered i.m. to induce oestrus. Neutrophils were isolated from jugular blood on the 2nd or 3rd day of oestrus, Days 5 and 7 after ovulation or during early pregnancy (Days 18-34 of pregnancy). Neutrophils were challenged with Staphylococcus aureus and their bactericidal activity examined after 30 and 120 min of incubation for a reduction of colony forming units. Bactericidal activity increased with the time of incubation (P less than 0.01) but did not differ for the oestrous cycle or pregnancy (P greater than 0.05).     

Characterization of murine monoclonal antibodies directed against basic fibroblast growth factor

Monoclonal antibodies (McAbs) were developed that identify the complete (1-146 aa) and the NH2-terminal truncated (des 1-15) form of bovine basic fibroblast growth factor (bFGF). Four McAbs, designated McAbs 6, 8, 38, and 42, bind the complete form of bFGF found in bovine pituitary, brain, and adrenal gland. One of these McAbs, McAbs 42, also binds to the des 1-15 aa form of bFGF found in bovine adrenal gland, kidney, and corpus luteum. None of the McAbs binds bovine-brain-derived acidic FGF (aFGF). McAbs 6, 8, and 38 recognized the same epitope located within the first ten residues of the NH2-terminal of complete bFGF. McAb 42 recognizes a "core" epitope found on both the complete and des 1-15 aa bFGFs. The McAbs are murine IgGs with affinity constants of 10(7)-10(8) liter/M for bovine-pituitary-derived bFGF. McAbs 8 and 42 have been used in a two-site ELISA to detect the complete form of bFGF. The ELISA is sensitive to 38.5 fmole/well of bFGF and is not affected by the presence of calf serum or bovine-brain-derived aFGF. These McAbs should be useful in distinguishing the native and des 1-15 aa forms of bFGF from each other, and from aFGF and other growth factors.     

Use of an androgenized mare as an aid in detection of estrus in mares

A study was conducted over a 2-mo period to compare estrus detection results obtained using an androgenized teaser mare with those obtained with a stallion, using the same group of 10 normally cyclic mares. The teaser mare was androgenized by administration of boldenone undecylenate (500 mg i.m. every 1 to 2 wk), and allowed to run loose with the mare group. Estrus was determined by observation of the group for a 30-min period daily. In the second month of the experiment, a marking harness was used on the androgenized mare to help detect mares mounted when in estrus. Estrous periods detected by each teasing method were 1) first month: stallion, 18; androgenized mare, 5; 2) second month: stallion, 16; androgenized mare, 9. There were no estrous periods detected by the androgenized mare that were not also detected by the stallion. Under these conditions, the androgenized mare was not an adequate estrus detection aid. Also discussed are the successful results of an independent trial on a breeding farm using an androgenized mare as an estrus detection aid.     

Isolated cytoskeletons of human blood platelets: dark-field imaging of coiled and uncoiled microtubules

Detergent extraction of human blood platelets pre-treated with Taxol to stabilize microtubules allows isolation of marginal band (MB) cytoskeletons. We studied MB cytoskeleton structure using dark-field light microscopy and negative stain electron microscopy (EM). Dark-field illumination clearly demonstrated the "hoop" shape of MB cytoskeletons in unfixed suspensions where the microtubule coils had a mean diameter of 2.87 microns (+/- 0.18 micron, SD). Microtubules were uncoiled by brief exposure to trypsin (2 ng/micrograms protein) or by NaCl (154-600 mM) but not by DNase I, which removed approximately 40% of total actin, but had no effect on dark-field images of microtubule coils. As microtubules uncoiled, a single fiber emerged from the hoop and gradually lengthened as the brightness of the hoop diminished; these fibers correspond to the single microtubules seen by EM. Polypeptides of coiled and uncoiled MB cytoskeletons were analyzed by SDS-PAGE. When microtubules became uncoiled, no changes in the major components (alpha- and beta-tubulin, IEF-51K, or actin) were found. However, a number (greater than 10) of minor polypeptides, each less than 5% of total cytoskeletal protein and with an Mr ranging from 80,000- greater than 260,000, were decreased in "uncoiled" MB cytoskeletons. These results implicate one or more of these minor polypeptides in maintenance of hoop integrity. Dark-field light microscopy thus provides an approach toward investigating the mechanism(s) involved in maintaining the microtubule coil of the platelet marginal band.     

Methylcholine-activated eccrine sweat gland density and output as a function of age

The purpose of this investigation was to examine eccrine sweat gland responsiveness to intradermal injections of methylcholine (MCh) across three age groups of men [young (Y) = 22-24; middle (M) = 33-40; older (O) = 58-67 yr old, n = 5 per group]. Subjects were matched with respect to maximum O2 consumption, body size, and body composition, and were thoroughly heat acclimated before participation. Randomly ordered concentrations of acetyl-beta-methylcholine chloride ranging from 0% (saline) to 0.1% (5 x 10(-3) M) were injected into the skin of the dorsal thigh in a thermoneutral environment, and activated sweat glands were photographed at 30-s intervals for the next 8 min. Density of MCh-activated glands was independent of both age and [MCh] (e.g., 2 min after injection of 5 x 10(-3) M [MCh]: Y = 45 +/- 7, M = 46 +/- 12, O = 42 +/- 5 glands/cm2). However, sweat gland output (SGO) per active gland was significantly lower for the O group and failed to increase with increasing [MCh] above 5 x 10(-4) M. When MCh (5 x 10(-3) M) was injected after 1 h of exercise in the heat, higher SGO's were elicited in each group; however, the SGO of the O group was again significantly lower than that of the Y group (91 +/- 11 vs. 39 +/- 4 ng/gland, P less than 0.02) with the M group intermediate (69 +/- 11 nl/gland; 2 min postinjection data).(ABSTRACT TRUNCATED AT 250 WORDS)     

Densimetric determination of carbohydrate content in glycoproteins.

Carbohydrates play important roles in activity, stability and pharmacokinetics of glycoproteins and the degree of glycosylation varies with proteins. In this communication, a simple method of determining the carbohydrate content was developed, which consists of measuring the density increments of a glycoprotein and its non-glycosylated counterpart, and then dividing the difference between the two values by the density increment of carbohydrates. The density increment was relatively constant for various sugars except for sialic acid, and hence assumed to be 0.39. Thus, we obtained carbohydrate contents of 38, 28, 8 and 7% for Chinese hamster ovary cell-expressed erythropoietin (EPO), stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and platelet-derived growth factor (PDGF), respectively. These values are in close agreement with those determined by other methods.