Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.     

Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.     

Characterization of Actinobacillus actinomycetemcomitans Hemolysin

Twenty out of 33 Actinobacillus actinomycetemcomitans strains formed hemolytic colonies on horse blood agar plates under anaerobic conditions. The hemolytic activity found in A. actinomycetemcomitans strain 137HE was examined. This activity was detected in the late exponential to early stationary phases of growth. Human erythrocytes were the most susceptible, followed by rabbit, sheep, horse and swine red blood cells. The majority of activity was detected in the cell-associated vesicle fraction. Zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extract from whole cells was semipurified by ammonium sulfate precipitation, preparative isoelectric focusing (IEF) and gel-filtration chromatography to yield a major band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 12 kDa. Heating at 80 C for 30 min and treatment with proteinase K or trypsin resulted in complete disappearance of the hemolytic activity. Sulphydryl reagents enhanced activity and small amounts of cholesterol inhibited it. In summary, we demonstrated the presence of hemolysin in A. actinomycetemcomitans, and examined and characterized it.     

Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

Genetic control of amylose content in wheat endosperm starch and differential effects of three Wx genes

The endosperm starch of the wheat grain is composed of amylose and amylopectin. Genetic manipulation of the ratio of amylose to amylopectin or the amylose content could bring about improved texture and quality of wheat flour. The chromosomal locations of genes affecting amylose content were investigated using a monosomic series of Chinese Spring (CS) and a set of Cheyenne (CNN) chromosome substitution lines in the CS genetic background. Trials over three seasons revealed that a decrease in amylose content occurred in monosomic 4A and an increase in monosomic 7B. Allelic variation between CS and CNN was suggested for the genes on chromosomes 4A and 7B. To examine the effects of three Waxy (Wx) genes which encode a granule-bound starch synthase (Wx protein), the Wx proteins from CS monosomics of interest were analyzed using SDS-PAGE. The amount of the Wx protein coded by the Wx-B1 gene on chromosome arm 4AL was reduced in monosomic 4A, and thus accounted for its decreased amylose content. The amounts of two other Wx proteins coded by the Wx-A1 and Wx-D1 genes on chromosome arms 7AS and 7DS, respectively, showed low levels of protein in the monosomics but no effect on amylose content. The effect of chromosome 7B on the level of amylose suggested the presence of a regulator gene which suppresses the activities of the Wx genes.     

A Factor That Interrupts the Cell Cycle at G2 or Prophase is Present in Full-Grown Frog Oocytes

Full-grown amphibian oocytes that had been arrested at meiotic prophase I contained an activity that prevented the cell cycle from progressing beyond a G₂-like stage. Injection of the contents of germinal vesicles (GV-content) or cytoplasm obtained from oocytes of the frog Rana rugosa prevented fertilized eggs of Cynops pyrrhogaster or Bufo japonicus from cleaving. The nuclei in the arrested eggs consisted of thin chromosomes and nucleolus-like particles enclosed within clear nuclear membrane and their volume increased as a function of time after injection. Cycling of maturation-promoting factor (MPF) did not occur in the injected eggs, but DNA synthesis was not disturbed. The injection of exogenous MPF into the eggs induced the reinitiation of the cell cycle with progression to the M phase and subsequent cleavage. Furthermore, the injection into the full-grown oocytes of Bufo inhibited induction of the maturation of oocytes by progesterone. These results demonstrate that a factor that arrests the cell cycle either at a G₂-like stage of mitosis or at prophase in meiosis is present both in the GV and cytoplasm of frog oocytes. We refer to this factor as a G₂-specific cytostatic factor (G₂-CSF). G₂-CSF may play an important role not only in the physiological arrest at prophase I in meiosis, but also in regulation of the G₂/M transition in the cell cycle of early embryonic cells.     

Inactivation of erythropoietin receptor function by point mutations in a region having homology with other cytokine receptors.

The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a region, proximal to the transmembrane domain, that is essential for function and has homology with other members of the cytokine receptor family. To explore the functional significance of this region and to identify critical residues, we introduced several amino acid substitutions and examined their effects on erythropoietin-induced mitogenesis, tyrosine phosphorylation, and expression of immediate-early (c-fos, c-myc, and egr-1) and early (ornithine decarboxylase and T-cell receptor gamma) genes in interleukin-3-dependent cell lines. Amino acid substitution of W-282, which is strictly conserved at the middle portion of the homology region, completely abolished all the functions of the EpoR. Point mutation at L-306 or E-307, both of which are in a conserved LEVL motif, drastically impaired the function of the receptor in all assays. Other point mutations, introduced into less conserved amino acid residues, did not significantly impair the function of the receptor. These results demonstrate that conserved amino acid residues in this domain of the EpoR are required for mitogenesis, stimulation of tyrosine phosphorylation, and induction of immediate-early and early genes.