Maintenance of motility of bull spermatozoa by a low molecular weight factor(s) released from cultured embryonic cells

This study was undertaken to investigate the factor(s) released from cultured embryonic cells that is responsible for the prolonged mortility of bull spermatozoa and to determine some of the properties of the factor(s). Spermatozoa were incubated at 37°C in a supernatant removed from a 4-day culture medium of embryonic chick skeletal muscle cells. Their motility was maintained for 25.2 h. In contrast, spermatozoa incubated in the fresh medium rapidly lost their motility. A filtrate of the 4-day culture medium, obtained by passing the fluid through an Amicon YC-05 ultrafiltration membrane, retained a favorable effect on prolonging motility of spermatozoa. Heating or freezethawing of the filtrate did not interfere with the ability of the spermatozoa to maintain their motility. Oxygen consumption of spermatozoa in the filtrate of the 4-day culture medium was similar to that of spermatozoa in the fresh medium. These results suggest that a low molecular weight factor(s) (Mol. Wt. < 500) supplied by the cultured cells effectively prolonged the survival of the spermatozoa.     

Maintenance of motility of fowl spermatozoa in vitro is prolonged by a low molecular weight factor derived from cultured chick embryo cells.

Gel filtration of a conditioned medium composed of the supernatant fluid removed from a 5-day culture of skeletal muscle cells from 9-day-old chick embryos with Bio-Gel P-2 revealed one peak of motility-prolonging activity (about 0.3 kDa), which was not present in fresh medium. Spermatozoa incubated in this fraction of the conditioned medium maintained their motility for at least 36 h at 37 degrees C. Both the formation of lipid peroxide and the leakage of lactic dehydrogenase of spermatozoa incubated in the conditioned medium fraction were lower than those incubated in the corresponding fresh medium. Initial rate of oxygen consumption of the spermatozoa incubated in the conditioned medium fraction increased compared with that of the fresh medium fraction. These results suggest that a low molecular weight factor(s) supplied by cultured cells effectively prolongs the motility of fowl spermatozoa, and that the effect could result from inhibition of the structural damage to the sperm membrane.     

Stimulation of sperm motility and oxygen consumption of fowl spermatozoa by a low molecular weight fraction of seminal plasma

Washed fowl spermatozoa were incubated in a phosphate buffer containing various concentrations of fowl seminal plasma at 41 degrees C, normal body temperature, and the motility and oxygen consumption of spermatozoa were determined. Immediately after the incubation, spermatozoa showed good motility in the various diluents. However, with concentrations of seminal plasma at or below 20%, spermatozoa quickly became immotile. In contrast, at concentrations higher than 40% seminal plasma, spermatozoa were motile even after 15 min. As the concentration of seminal plasma was increased, oxygen consumption of spermatozoa also increased. A filtrate of the seminal plasma, obtained by passing the fluid through an Amicon YM-2 ultra-filtration membrane (Mr less than 1000), also stimulated the motility and oxygen consumption of spermatozoa. These results suggest that some low molecular weight factor(s) in fowl seminal plasma stimulated motility and oxygen consumption of fowl spermatozoa at 41 degrees C. A physiological role of this factor(s) may be to assist passage of spermatozoa through the vagina after natural mating.     

The high molecular weight proteins released from cultured cells.

Studies have been performed with the serum-free culture medium taken from several fibroblast monolayer culture lines. A high molecular weight protein fraction was separated from the concentrated medium by sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis was used to assess the degree of purification obtained. In the electron microscope the negatively stained high molecular weight proteins were found to closely resemble the alpha2-macroglobulins. The suggestion that these proteins from cultured cells resemble the cylindrical protein complex isolated from mammalian erythrocyte ghosts is not supported by this study. The results are discussed in the light of the extensive literature now available on the electron microscopy of high molecular weight proteins.     

Association of a low molecular weight helper factor(s) with thymocyte proliferative activity.

Human mixed leukocyte supernatants contain thymocyte proliferative activity (TPA) and a low m.w. helper factor, designated HP-1, which is capable of partially restoring the antibody response of T-cell-deficient adherent murine spleen cells to the thymic-dependent antigen, SRC. TPA and HP-1 appear to have a comparable m.w. (14,000 to 14,500 daltons) by Sephadex gel filtration column chromatography. Furthermore, HP-1 and TPA exhibit similar patterns of heterogeneity on DEAE-cellulose chromatography, elute together on CM-cellulose chromatography, and manifest identical patterns of migration on polyacrylamide gel electrophoresis. These data suggest that the TPA and HP-1 activities reside in either the same molecule(s) or in different molecules with identical charge/mass ratios. Furthermore, the results support the hypothesis that the helper activity of HP-1 is derived from its capacity to activate T and/or pre-T cells.     

Inhibition of muscle cell development in culture by cells from spinal cord due to production of low molecular weight factor.

Relatively small numbers of cells cultured from chick embryo spinal cord had the property of inhibiting muscle cell growth and differentiation, as measured by protein synthesis, myoglobin synthesis, and myotube formation, when they had been in culture 4 days before the addition of dispersed muscles cells. Inhibition of pectoral white muscle and thigh red muscle development in culture was similar. Inhibition of this sort was not brought about by similar cocultivation with cells from liver, gizzard, intestine, lung, or skin, although skin cultures were slightly inhibitory. Simultaneous cocultivation of muscle and cord cells failed to result in inhibition of myogenesis. The inhibitory property was present in the medium, and inhibition was reduced by removal of conditioned medium and replenishment with fresh medium before introduction of myoblasts. Medium obtained from other tissues, similarly cultured, did not possess inhibitory properties. The inhibitiory properties of “cord-conditioned” medium were related to a factor or factors able to be concentrated by lyophilization and of relatively low molecular weight, as measured by membrane ultrafiltration and gel filtration chromatography. The nature of the cell type in spinal cord, e.g., neuronal glial, responsible for the production of this factor is not known.     

Long-term maintenance of in vitro cultured honeybee (Apis mellifera) embryonic cells

Background  

In vitro cultivation of cells allows novel investigation of in vivo- mechanisms and is a helpful tool in developmental biology, biochemistry and functional genomics. Numerous cell lines of insect species, e.g., silkworm and mosquito, have been reported. However, this is not the case for successful long-term cultivation of cells in honeybees.     

Suppression of the guinea-pig line-10 hepatocarcinoma by a factor(s) released from line-10 cells

Summary A factor or factors released from line-10 hepatocarcinoma cells during mixing in a fluid medium suppressed tumor growth when injected intradermally (ID) with viable line-10 cells to strain-2 guinea-pigs. No suppressive factor was released from other syngeneic normal adult, fetal, or tumor cells. The mechanism behind the tumor suppression was not determined, although antigens derived from line-10 cells were demonstrated in media containing the suppressive factor(s).     

低分子量神经丝蛋白（NF—L）的体外组装

Neurofilaments were isolated from bovine spinal cords by ultra-speed centrifugation and examined by negative staining. The neurofilament triplet proteins: NF-L, NF-M and NF-H were purified by DE-52 anion exchange chromatography in the presence of 6 mol/L urea. The reassembly of NF-L under controlled conditions was studied. NF-L can reassemble into 10 nm width filaments within 60 minutes at physiological condition of around 0.15 mol/L NaCl, 2 mmol/L MgCl2, neutral pH(pH 6.8) and 37 degrees C. In 6 mol/L urea, NF-L was examined as 12 nm-diameter particle by low angle rotary shadowing. When dialyzed against reassembly buffer for 20 minutes, some irregular filaments were formed. Further dialyzed for another 40 minutes, the long smooth filaments appeared. Some filaments were unraveled at the end regions, where existed 2-4 subfilaments. Four subfilaments were more often observed. That is to say, the 10 nm-width filament was composed of 4 subfilaments. While dialyzed against the alkaline buffer containing 0.15 mol/L NaCl, NF-L reconstituted into 45-180 nm-long, 10 nm-width filaments, which were not able to elongate into long filaments.     

A low molecular weight factor from dividing cells activates phospholipase D in caveolin-enriched membrane microdomains

Phospholipase D (PLD) activity is elevated in Ras-transformed NIH 3T3 cells. This difference in PLD activity between Ras-transformed and nontransformed parental cells disappeared in isolated membranes from these cells. In reconstitution experiments, heat-denatured cytosolic fractions from Ras-transformed, but not parental, NIH 3T3 cells elevated PLD activity in isolated membranes. This heat-resistant PLD-stimulating activity from the Ras-transformed cells was sensitive to proteases and passed through a 1-kDa MW cutoff membrane, suggesting that the factor is a peptide of less than 10 amino acids. The ability of this PLD-stimulating factor, designated PLD-SF, to elevate PLD activity in isolated membranes was restricted to the caveolin-enriched light membranes, where many signaling molecules are localized. PLD-SF was also elevated in v-Src- and v-Raf-transformed cells and in serum-stimulated NIH 3T3 cells. PLD-SF was detected in a variety of rat tissues but was highest in testes, where a large percentage of cells are dividing. A similar low molecular weight PLD-stimulating activity was found in actively dividing, but not stationary yeast, cells. The data here provide evidence for a highly conserved PLD-stimulating peptide that is elevated in response to mitogenic stimuli.     

Low molecular weight RNAs hydrogen-bonded to nuclear and cytoplasmic poly(a)-terminated RNA from cultured chinese hamster ovary cells

A group of RNAs 90–100 nucleotides long were isolated by melting them from poly(A)-terminated nuclear or cytoplasmic RNA from cultured Chinese hamster ovary cells. Conditions that favor hydrogen bond formation allowed the reassociation of these low molecular weight RNAs with poly(A)-terminated RNA. The nuclear poly(A)-terminated molecules contained 1.3 moles of the low molecular weight RNAs per mole of poly(A), while the cytoplasmic poly(A)-terminated RNA contained only one seventh as much. These low molecular weight RNAs were also isolated from the total 4S RNA of either the nucleus or cytoplasm by polyacrylamide gel electrophoresis. They formed a prominantly labeled band of RNA in the gels after cells had been labeled with H₃³²PO₄ for 4 hr. The low molecular weight RNAs melted from the nuclear poly(A)-terminated RNA were slightly different (although not necessarily in primary nucleotide sequence) from those melted from the cytoplasmic poly(A)-terminated RNA.     

Isolation and characterization of a low molecular weight growth-promoting factor from human plasma

A low molecular weight growth factor (LMW-GF) enriched preparation was purified from human plasma after ultrafiltration or gel filtration by means of molecular sieving chromatography low pressure reversed phase chromatography (LP-RPLC) and electrophoresis. Purification was monitored by a biological assay testing the capacity of the fractions to enhance the sulfation activity of the somatomedins/insulin-like growth factors on chick embryo cartilage. Analysis of its chemical nature show that it is hydrophilic, stable to heat, resistant to most of the proteases but that it is degraded by acid hydrolysis or carboxypeptidase Y action. UV absorption spectrum and ion-exchange chromatographic retention behavior support the hypothesis that the most purified active preparation includes a peptide structure. The presence of sugar is suggested by concanavalin A binding experiments. The fact that the purification fractions also enhance thymidine uptake by other cell lines (fibroblasts, activated lymphocytes) widens the role of such small plasma molecules in the field of growth factor activities.     

Conversion of high molecular weight CRF (corticotropin-releasing factor) or PRF (prolactin-releasing factor) into lower molecular weight forms by boiling at low pH.

Extracts (0.1 N HCl) of bovine hypophyseal stalk had 2 CRF peaks, one (CRF-A) in the void volume with Sephadex G-100 chromatography, the other more retarded (CRF-B). PRF activity of the same extracts eluted in 2 peaks from G-100, one in the void volume (PRF-A) and the other (PRF-B) between A and B CRF peaks. On rechromatography, isolated CRF-A and PRF-A remained in the void volume. However, heating to 100 C at pH 1–2 for 15 min converted CRF-A to CRF-B and PRF-A to PRF-C, which eluted after PRF-B on G-100. We conclude that CRF or PRF can be converted from high to low molecular weight forms with full retention of biological activity.     

GH-dependency of lymphocyte multiplication factor with a low molecular weight

Liquid chromatography (FPLC) separation at pH 6.85 shows that the fractions in human serum which stimulate the multiplication of human lymphocyte are mainly in the 28-42 kd and the 1.3-1.9 kd fractions. The low molecular weight fraction is absent or very low in both GH-deficient and GH-resistant children and T lymphocyte-deficient children, and appears after appropriate treatment, i.e. GH or marrow transplantation. The data suggest the GH-dependency of 1.3-1.9 kd factor(s) produced by immuno-competent lymphocytes.     

The absorption by boar spermatozoa of zinc bound to low molecular weight ligands.

Most of the zinc accumulated by boar spermatozoa at 4 degrees C from seminal plasma appears to arise from the low molecular weight zinc ligands. Zinc added to semen in low concentrations (0-1 to 0-6 mM) is preferentially absorbed by the spermatozoa, particularly at 4 degrees C.     

In vitro synthesis of thyroxine in a low molecular weight polypeptide fragment from human thyroglobulin

A peptide fragment of Mr 16 K was purified from the cyanogen bromide digest of human thyroglobulin either normally iodinated in vivo (0.21 % I) or highly iodinated in vitro (1.40 % I). This peptide segment represents in the native molecule a zone in which tyrosine residues are not or poorly accessible to iodination and consequently do not produce thyroxine. In contrast, after isolation from thyroglobulin and iodination in vitro, the peptide is capable of synthesizing thyroxine with a high efficiency. It is concluded that the peptide described which probably represents a potential hormone forming site in the whole thyroglobulin molecule should constitute a valuable model to study the mechanism of thyroxine formation in vitro.     

Effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa in vitro

The effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa were investigated in vitro. The addition of taurine, within the range of 0-5 mmol l(-1), did not appreciably affect the motility of intact fowl spermatozoa. Motility remained almost negligible at 40 degrees C, while vigorous movement was observed at 25 degrees C. Even with the addition of Ca2+ before the addition of taurine, neither stimulation nor inhibition of motility was observed compared with the control (no addition of taurine). Similar results were obtained by the addition of taurine and calyculin A, a specific inhibitor of protein phosphatases. There were no changes in intracellular free Ca2+ concentrations, measured by a fluorescent Ca2+ indicator, fura-2, in taurine-treated spermatozoa. These results suggest that taurine is not involved in the regulation of fowl sperm motility and metabolism by intracellular Ca2+ mobilization in vitro.     

Production of a low molecular weight growth inhibitory factor by adenovirus 12-transformed cells.

Malignant rodent cells transformed by human adenovirus 12 produce a potent cell growth inhibitory factor. The cell growth inhibitory factor inhibits the growth of and DNA synthesis in normal fibroblasts in vitro. Extent of the production of the cell growth inhibitory factor appears to be proportional to that of the malignancy of the transformed cells. C57AT1-AB cells, an adenovirus 12-transformant of C57BL/6 mouse origin, are highly tumorigenic in the syngeneic and allogeneic mice. The cell growth inhibitory factor produced by these cells was characterized for the physicochemical properties; the cell growth-inhibitory activity was quantitatively recovered in the filtrates of YM-2 membrane (M(r) less than 1,000), resistant to the heat treatments at 56 degrees C for 30 min and 100 degrees C for 5 min, and extractable by ethyl acetate under acid-condition. These results suggest that the cell growth inhibitory factor may be lipid or oligopeptides.