Inhibition of the graft-versus-host response by BCGcw-induced suppressor cells or prostaglandin E1

Immunization of C57BL/6 mice with BCGcw stimulated a population of "suppressor cells" which had a decreased capacity to induce the graft-versus-host response. The graft-versus-host response was quantitated using the Simonsen splenomegaly assay. F1 mice (C57BL/6 X CBA) were inoculated intraperitoneally with 1 X 10(8) parental (C57BL/6) or (CBA) spleen cells. The F1 mice were sacrificed 13 days later and the resulting splenomegaly was 3-4 times the normal amount. F1 mice which were injected with parental BCGcw-primed C57BL/6 spleen cells had a 50% inhibition of splenomegaly, whereas BCGcw-primed CBA spleen cells (a strain which does not develop suppressor cells) did not show this inhibition. In vitro results also confirmed that only C57BL/6 mice and not CBA mice developed suppressor cells after BCGcw immunization. A second study showed that X-irradiated (1000 R) BCGcw-primed "suppressor cells" could inhibit splenomegaly caused by the inoculation of normal parental C57BL/6 cells into F1 mice. The mechanism by which BCGcw-primed "suppressor cells" caused this inhibition of splenomegaly was delineated and found to be dependent upon the secretion of prostaglandin (PGE-1). Indomethacin and aspirin, potent inhibitors of prostaglandin synthesis, blocked the activity of C57BL/6 BCGcw "suppressor cells" and splenomegaly resulted. Systemic administration of the prostaglandin (15S)-15-methyl PGE-1 reduced splenomegaly approximately 50% in F1 mice which were injected with C57BL/6 or CBA cells. These results indicated that immunization with BCGcw stimulated a population of "suppressor cells" which could cause a decrease in graft-versus-host response and that the secretion of prostaglandin was responsible for this inhibition.     

The effect soaking pea seeds with or without seedcoats has on seedling growth

Pea seeds (Pisum sativum L. `Alaska') with intact seedcoats (WC) and with seedcoats removed (WOC) were soaked in distilled water for 24 hours at 20°. The water, containing the pea diffusate, was decanted after the second, fourth, sixth, eighth, twelfth, and twenty-fourth hour and analyzed for total nitrogen, α-amino nitrogen, carbohydrate, and total solute dry weight. The seeds were germinated at 20° in a 16 hour photoperiod of 300 foot candles. Stem lengths and dry weights of roots, shoots and cotyledons were determined after 4, 11, and 18 days of growth. WOC seeds imbibed more water than WC seeds during the 24 hour imbibition period. Diffusates from WOC seeds always contained more solute than diffusates from WC seeds. Maltose, glucose, and fructose were not detected in the early diffusates from WOC seeds but were found in WC seed diffusates at all times. Seedlings from WC seeds had longer stems than those from WOC seeds. The dry weight of stems and roots of WC seedlings was greater than those from WOC seedlings. The dry weight of cotyledons from 18 day-old WC seedlings was less than from WOC seedlings. Water absorption by WC seeds was slower than by WOC seeds. Removal of the seedcoat allowed rapid imbibition resulting in seed injury presumably because of the loss of solutes which included monosaccharides, disaccharides, amino acids, and other nitrogen containing compounds. These results are consistent with the hypothesis that rapid imbibition disrupts membrane organization leading to reduction of seedling growth.     

FINE STRUCTURAL STUDIES OF ZEA MAYS POLLEN I: CELL MEMBRANES AND EXINE ONTOGENY

Electron microscopy was used to study pollen wall ontogeny in Zea mays. The initial stage of development consisted of compartmentalization of microspores within callose special walls. Microspore plasma membranes retracted and tubular elements of the endoplasmic reticulum became perpendicularly oriented to the plasma membranes. Evaginations of the endoplasmic reticulum into the microspore plasma membrane resulted in the establishment of a template or blueprint of the mature pollen wall. Sporopollenin deposition upon the template began immediately after dissolution of the callose special walls and release of the microspores into the anther locule. The columellae were the first pollen wall units to be formed; the tectum and foot layer became established shortly thereafter. The granular endexine was the last-formed unit. The relationships of membrane systems to the ontogeny of the pollen wall units and the mode of pollen wall growth are discussed.     

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Summary Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed. This study was supported by grants from the Swedish Medical Research Council (Project no. 4531 and 6816), the Swedish Association against Heart and Chest Diseases, the King Gustaf V and Queen Victoria Foundation, the National Institutes of Health, Bethesda, MD (grant HL 29873) and the Swedish National Board for Laboratory Animals.     

Secretion of the sweet-tasting plant protein thaumatin byStreptomyces lividans

Summary To produce and direct the export inStreptomyces lividans of the sweet plant protein thaumatin, thaumatin II cDNA was fused in the correct reading frame to the -galactosidase leader peptide, under the control of the -galactosidase promoter and ribosome binding site. The export of the recombinant thaumatin may allow the correct formation of the thaumatin disulfide bonds. The recombinant thaumatin was purified from the medium on an S-Sepharose column and detected with western blots by sheep -thaumatin antibodies. The recombinant thaumatin was the same size as authentic thaumatin and changed position on an acrylamide gel in response to reduction by 2-mercaptoethanol in the same manner.     

Syncro-mate B induces estrus in ovariectomized cows and heifers

Eleven ovariectomized Hereford x Simmental cows and 10 ovariectomized crossbred heifers (primarily Angus and Hereford) were given the Syncro-Mate B (SMB) estrous synchronization treatment. The SMB treatment consisted of a 2 ml i.m. injection containing 5 mg of estradiol valerate and 3 mg of norgestomet plus a hydron ear implant containing 6 mg of norgestomet. The ear implant was removed 9 d later. Cows and heifers were considered in estrus only if they stood for mounting by a herdmate or a bull. Observations for estrus were made four or six times each day for 3 d after implant removal. The 21 animals were used in eight trials. Each trial involved 9 or 11 cows or 5 or 10 heifers. Four days to three weeks elapsed between implant removal and implant insertion for the next trial. No ovariectomized cow or heifer was observed in estrus for 21 d before treatment with SMB. In the eight trials, 3 of 9, 7 of 9 and 6 of 11 cows exhibited estrus, whereas 5 of 10, 1 of 5, 3 of 5, 3 of 5 and 5 of 5 heifers exhibited estrus after treatment. When data were pooled, 16 of 29 (55.2%) cows and 17 of 30 (56.7%) heifers exhibited estrus after treatment. Our data indicate that the SMB treatment can induce estrus in cows and heifers, independently of the ovaries.     

Induction of rabies virus-specific T-helper cells by synthetic peptides that carry dominant T-helper cell epitopes of the viral ribonucleoprotein.

The T-helper cell response to the internal proteins of rabies virus was investigated. The rabies virus nucleoprotein was shown to be a major target antigen for T-helper cells that cross-react between rabies and rabies-related viruses. T-helper cells were assayed in vitro by testing virus-induced lymphocytes for lymphokine secretion in response to antigen. Immunodominant T-helper cell epitopes of the viral nucleoprotein were identified in vitro by using synthetic peptides delineated from the amino acid sequence of the nucleoprotein. The response to synthetic peptides were under Ir gene control. Antigenic peptides were tested in vivo for stimulation of rabies virus-specific T-helper cells. Inoculation of mice with peptides bearing immunodominant T-helper cell epitopes resulted in an accelerated and enhanced neutralizing antibody response upon booster immunization with inactivated rabies virus.     

Substance P modulation of DAMGO binding in the brain of CXBK and Swiss-Webster mice.

The effects of substance P (SP) on the binding of the selective mu opioid agonist [3H]DAMGO to brain membranes of CXBK and Swiss-Webster (SW) mice were compared. We have previously shown that subnanomolar concentrations of SP and N-terminal fragments of SP modulate DAMGO binding in SW brain membranes and hypothesized that modulation occurs via SP interaction with mu 1 sites. In the present study, binding assays using CXBK mice, a strain deficient in mu receptors including mu 1 sites, were performed to assess the effect of mu receptor deficiency on SP-induced modulation of DAMGO binding. Whereas the addition of 0.1 nM SP to the binding mixtures produced up to 30% increase in the values of Kd and maximum binding capacity (R) for the SW strain, SP produced little or no change in the case of CXBK strain. Maximum binding capacity for DAMGO was 43% less in the brain of CXBK mice than in SW mice. No difference was observed in the estimated binding parameters of the spinal cord for the two strains. Whereas pretreatment of brain membranes of SW mice using beta-funaltrexamine (beta-FNA) increased from 2- to 10-fold the modulatory effect of SP, CXBK brain membranes pretreated with beta-FNA remained nearly insensitive to modulation by SP. The effect of SP on the affinity of DAMGO binding in SW mice, but not in CXBK mice, was reversed by the addition of GTP. It is concluded that mu receptor deficiency can markedly influence SP-induced modulation of DAMGO binding.