THE HORMONAL INTEGRATION OF SEXUAL REPRODUCTION IN OEDOGONIUM

Rawitscher -Kunkel , Erika , and L. Machlis . (U. California, Berkeley.) The hormonal integration of sexual reproduction in Oedogonium. Amer. Jour. Bot. 49 (2) : 177–183. Illus. 1962.—Sexual reproduction in a heterothallic, nannandrous species of Oedogonium was investigated cytologically and physiologically. Several new observations are reported. Oogonial mother cells release a substance which attracts androspores to them. The androspores, when attached to the oogonial mother cells, grow in well-defined directions apparently in response to a hormone originating in the oogonial mother cells. An oogonial mother cell divides into an oogonium and a suffultory cell only after the attached androspores complete their development into dwarf males, each bearing an antheridium. Presumably the developing dwarf males provide a chemical stimulus for the division of the oogonial mother cell. During development, the oogonia become enveloped in a massive gel which also encases the antheridia cut off at the apical ends of the dwarf male plants. The gel appears to function as a sperm trap, preventing the dissemination of the sperm into the surrounding liquid. The sperm are attracted to the protoplasmic papilla which briefly protrudes through the oogonial pore indicating the operation of a second chemotactic agent.     

Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L-forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform-methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform-methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic. The cytoplasmic membranes of the L-forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.     

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

Mice Lacking Platelet-Derived Growth Factor D Display a Mild Vascular Phenotype

Platelet-derived growth factor D (PDGF-D) is the most recently discovered member of the PDGF family. PDGF-D signals through PDGF receptor β, but its biological role remains largely unknown. In contrast to other members of the PDGF family of growth factors, which have been extensively investigated using different knockout approaches in mice, PDGF-D has until now not been characterized by gene inactivation in mice. Here, we present the phenotype of a constitutive Pdgfd knockout mouse model (Pdgfd^-/-), carrying a LacZ reporter used to visualize Pdgfd promoter activity. Inactivation of the Pdgfd gene resulted in a mild phenotype in C57BL/6 mice, and the offspring was viable, fertile and generally in good health. We show that Pdgfd reporter gene activity was consistently localized to vascular structures in both postnatal and adult tissues. The expression was predominantly arterial, often localizing to vascular bifurcations. Endothelial cells appeared to be the dominating source for Pdgfd, but reporter gene activity was occasionally also found in subpopulations of mural cells. Tissue-specific analyses of vascular structures revealed that NG2-expressing pericytes of the cardiac vasculature were disorganized in Pdgfd^-/- mice. Furthermore, Pdgfd^-/- mice also had a slightly elevated blood pressure. In summary, the vascular expression pattern together with morphological changes in NG2-expressing cells, and the increase in blood pressure, support a function for PDGF-D in regulating systemic arterial blood pressure, and suggests a role in maintaining vascular homeostasis.     

Preferential expression of cellular retinoic acid binding protein in a subpopulation of neural cells in the developing mouse embryo

The cellular retinoic acid binding protein is thought to be involved in the retinoic-acid-mediated signal transduction pathway. We have isolated the mouse cellular retinoic acid binding protein cDNA from an embryonal-carcinoma-derived cell line by using differential cDNA cloning strategies. In situ hybridization on sections of mouse embryos of various developmental stages indicated that the cellular retinoic acid binding protein gene, which we localized on mouse chromosome 9, is preferentially expressed in a subpopulation of neurectodermal cells. This restricted expression pattern suggests an important role for cellular retinoic acid binding protein in murine neurogenesis.     

N-ethylmaleimide inhibits Ca2+ influx induced by collagen or arachidonate on rabbit platelets

N-Ethylmaleimide dose dependently inhibited platelet aggregation induced by collagen or arachidonate but did not inhibit the aggregation by thrombin or ionophore A23187 within the concentrations tested. [3H]Arachidonate release from membrane phospholipids of the collagen-stimulated platelets was inhibited by N-ethylmaleimide in parallel with the inhibition of aggregation, but not in response to A23187. N-Ethylmaleimide prevented 45Ca2+ influx into platelet cells from outer medium induced by collagen, and also inhibited the increase in the concentration of cytoplasmic free Ca2+, which probably results from Ca2+ influx, as monitored by quin2 fluorescence, under stimulation with arachidonate. The concentration of N-ethylmaleimide giving a complete inhibition of Ca2+ influx was consistent with that required to inhibit collagen- or arachidonate-induced aggregation. Prostaglandin metabolism from arachidonate to thromboxane A2 was not disturbed by N-ethylmaleimide, while phosphatidate formation induced by arachidonate was slightly inhibited by it at concentrations at which aggregation was completely inhibited. These data suggest that N-ethylmaleimide preferentially suppresses increase in cytoplasmic free Ca2+ which is linked to thromboxane A2-receptor occupation in collagen- or arachidonate-stimulated platelets, probably due to blockage of Ca2+ influx through Ca2+-channel protein, thereby inhibiting aggregation induced by these agonists.     

Atrial granules in the dog heart after cardiac denervation

Summary Because the increase in sodium excretion during left atrial distension in conscious dogs is abolished after chronic cardiac denervation, we have investigated whether this is a result of the disappearance of specific atrial granules. Electron microscopy and light-microscopical and ultrastructural immunohistochemistry of canine atria show that atrial granules displaying immunoreactivity for cardiac hormones of the cardiodilatin/atrial natriuretic polypeptide (CDD/ANP) family are still present in denervated left and right atria, although reduced in quantity. It is concluded that the atrial-induced natriuresis is not only related to the existence of specific atrial granules. The functional link between atrial-induced natriuresis provoked by atrial distension and the release of atrial polypeptide hormones remains uncertain because the denervated heart can secrete CDD although the diuretic-natriuretic effect is altered.     

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding to the membrane receptor

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.     

Purification of cytochrome P-450 from polychlorinated biphenyl-treated crab-eating monkeys: high homology to a form of human cytochrome P-450

Cytochrome P-450, designated as P-450-MK2, was purified to an electrophoretic homogeneity from polychlorinated biphenyl (PCB)-treated female crab-eating monkeys. P-450-MK2 catalyzed nifedipine and nilvadipine oxidations, at a rate comparable to human P-450-HM1. The N-terminal amino acid sequence of P-450-MK2 was highly homologous to those of P-450-HM1 and NF 25. The antibodies to P-450-HM1 recognized P-450-MK2 and effectively inhibited the activity of testosterone 6 beta-hydroxylase in monkey liver microsomes. These results suggest that a form of cytochrome P-450 corresponding to human P-450-HM1 or P-450NF which belongs to the P450 III gene family is also present in liver microsomes of crab-eating monkeys.     

Purification and characterization of lymphocytic clonal growth factor (LCGF) derived from human-human hybridoma SH-76 cells

Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma.