Effect of testosterone and bovine follicular fluid on concentrations of luteinizing hormone and follicle-stimulating hormone in plasma of castrated rams that are homozygous carriers or non-carriers of the Booroola fecundity gene.

Castrated adult FecBFecB and Fec+Fec+ Booroola rams were injected with charcoal-treated bovine follicular fluid (bFF) (a source of inhibin-like activity) or given testosterone implants to examine whether the fecundity gene (FecB) influences sensitivity to negative feedback hormones in males. Mean concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) did not differ between genotypes before treatment. In Expt 1, injections of 5 ml bFF, but not of 1 ml (each given four times at intervals of 8 h), significantly (P < 0.05) depressed concentrations of LH and FSH, but there was no effect of genotype. After treatment, gonadotrophin concentrations returned to pretreatment values and for 2-2.5 days scaled (divided by pretreatment mean) LH values (235 +/- 49 for FecBFecB and 96 +/- 26% for Fec+Fec+ rams; P < 0.05) and scaled FSH values (106 +/- 5 for FecBFecB and 85 +/- 5% for Fec+Fec+ rams; P < 0.05) were significantly higher in FecBFecB than in Fec+Fec+ rams in the group that received 5 ml bFF. Irrespective of genotype, treatment with 5 ml bFF did not reduce mean FSH to concentrations observed in testis-intact rams. In Expt 2, Silastic envelopes were implanted subdermally to give physiological or supraphysiological circulating concentrations of testosterone. Both doses significantly reduced scaled LH values in a biphasic manner, such that there was an initial suppression followed by a short-lived increase. During the initial period of suppression in the lower dose group, mean scaled LH values were significantly higher in FecBFecB than in Fec+Fec+ rams (48.3 +/- 7.5 versus 23.1 +/- 5.5%; P < 0.05). Low doses of testosterone decreased LH pulse frequency in both genotypes but decreased (P < 0.05) pulse amplitude and mean concentrations in the Fec+Fec+ animals only. In nonimplanted control rams, mean LH concentrations (in samples taken every 10 min for 12 h) were significantly lower in FecBFecB than in Fec+Fec+ rams (0.6 +/- 0.2 versus 1.3 +/- 0.1 ng ml-1; P < 0.05). The mean FSH response to testosterone was not related to genotype. These data suggest that expression of the FecB gene results in an altered sensitivity of the pituitary gland to changes in negative feedback from testicular hormones and that, irrespective of genotype, neither testosterone nor inhibin-like activity alone can fully control FSH secretion in castrated rams.     

Changes in cellular dry mass during macrophage development.

Measurements of dry mass were made on developing human macrophages in suspension culture using a scanning and integrating microinterferometer. The mean dry mass (+/- SEM) of day 0 monocytes of 19 healthy individuals was 53.6 +/- 2.4 pg. There was a significant increase in cellular dry mass over the period of culture, mean values at 2, 4 and 6 days being 61.1, 79.8 and 110.4 pg, respectively. A comparison of the results with those obtained previously by ultrastructural morphometry inferred that there is a disproportionate increase in the water content of macrophages during development. The technique may have potential for investigating changes in mononuclear phagocytes in clinical states.     

Synthesis and characterization of a heterobifunctional mercurial cross-linking agent: incorporation into cobratoxin and interaction with the nicotinic acetylcholine receptor

The heterobifunctional organomercurial reagents 3-(acetoxymercurio)- and 3-(chloromercurio)-5-nitrosalicylaldehyde were prepared, characterized in model studies, and used to probe the interaction between cobratoxin, purified from the venom of the Thailand cobra (Naja naja siamensis), and the affinity-purified nicotinic acetylcholine receptor (AcChR) from Torpedo california electroplax. These reagents may also be useful in introducing chemically well-defined heavy metal atoms into proteins containing no reactive thiols. Model reagent adducts were prepared in situ by reductive amination with N-butylamine and N alpha-acetyllysine-N-methylamide. The nitrophenolic pKaS of the amine adducts were similar to those of the aldehyde reagents through reduced by 1.3-1.5 units when compared with the hydroxylmethyl reduction product. Reaction of either mercuriosalicylaldehyde with cobratoxin led to a single major modification product incorporating 1 mol of the reagent into cobratoxin at Lys 23. The Lys 23 modified toxin had a reduced binding affinity for the AcChR over that of the native toxin (Kd 2.75 nM cf. 0.3 nM). Reduction of the purified AcChR with 1 mM dithiothreitol (DTT) followed by removal of excess thiol led to cross-linking reactions with the Lys 23 modified cobratoxin to both the alpha and beta subunits of the AcChR complex. Reaction of DTT-treated AcChR with N-ethylmaleimide (NEM) blocked cross-linking, while treatment of the initially cross-linked toxin-AcChR complex with mercaptoethanol leads to reversal of cross-linking. These observations strongly support cross-linking mediated by the formation of a mercury-sulfur bond and further lend support the identity of the respective interacting sites in AcChR and toxin.     

Explant organ culture: A review

Organ explant culture models offer several significant advantages for studies of patho-physiologic mechanisms like cell injury, secretion, differentiation and structure development. Organs or small explants/slices can be removed in vivo and maintained in vitro for extended periods of time if careful attention is paid to the media composition, substrate selection, and atmosphere. In the case of human tissues obtained from autopsy or surgery, additional attention must be paid to the postmortem interval, temperature, hydration, and cause of death. Explant organ culture has been effectively utilized to establish outgrowth cell cultures and characterize the histiotypic relationships between the various cell types within an organ or tissue.J. Resau is a visiting scientist at the NCI-LMO-DCE in Frederick, MD 21702, U.S.A.K. Sakamoto is a visiting scientist from the Department of Surgery, Gunma University School of Medicine, Maebashi, Japan     

Chin marking behavior, sexual receptivity, and pheromone emission in steroid-treated, ovariectomized rabbits

The effect of daily injections of estradiol benzoate (1 or 10 micrograms) and of progesterone (10 mg) on chin marking activity, sexual receptivity, and emission of nipple-search pheromone in ovariectomized rabbits was investigated. Both estradiol treatments resulted in a significant increase in all three measures over baseline and control group levels within 1-3 days, and withdrawal in a return to pretreatment levels within 2 weeks (Experiment I). In contrast, the administration of progesterone to such estradiol-primed does resulted in an almost immediate suppression of chin marking and lordosis, but in marked enhancement of pheromone emission and aggressive behavior (Experiment II). However, progesterone given alone to nonprimed does had no effect on any of these measure (Experiment III). The response profiles resulting from these treatments correspond well to patterns reported for intact does during estrus (= estradiol alone), pregnancy (= estradiol plus progesterone), and at parturition (= progesterone withdrawal).     

A merozoite receptor protein from Plasmodium knowlesi is highly conserved and distributed throughout Plasmodium

The 66-kDa merozoite surface antigen (PK66) of Plasmodium knowlesi, a simian malaria, possesses vaccine-related properties that are thought to originate from a receptor-like role in parasite invasion of erythrocytes. We report the complete sequence of PK66 which allowed the demonstration that highly conserved analogues exist throughout Plasmodium including a recently reported gene from P. falciparum (Peterson, M. G., Marshall, V. M., Smythe, J. A., Crewther, P. E., Lew, A., Silva, A., Anders, R. F., and Kemp, D. J. (1989) Mol. Cell. Biol. 9, 3151-3155). These analogues are highly promising vaccination candidates. The distribution of PK66 changes after schizont rupture in a coordinate manner associated with merozoite invasion. The protein is concentrated at the apical end prior to rupture, following which it can distribute itself entirely across the surface of the free merozoite. During invasion, immunofluorescence studies suggest that, PK66 is excluded from the erythrocyte at, and behind, the invasion interface.     

Seasonal variations in RNA–DNA ratios and in indicators of feeding, reproduction, energy storage, and condition in a population of bluegill, Lepomis macrochirus Rafinesque

Liver and epaxial muscle RNA concentrations and RNA-DNA ratios (RNA/DNA) of bluegill Lepomis macrochirus from a central Tennessee lake were maximum in the spring, low during the summer, high again in the fall, and low during the winter. Liver-somatic indexes and DNA concentrations indicated that liver cell volume and energy storage varied seasonally and were lowest during late spring and summer. Seasonal variations in gonosomatic indexes were typical of this species, and RNA/DNA decreased as gonads matured during the spring. RNA/DNA and energy storage in the liver decreased during the major spawning season. Seasonal variations in food consumption may have effected seasonal growth and energy storage. However, a summer depression in RNA/DNA may have been more closely associated with the effects of thermal stratification and dissolved oxygen stress.     

Cloning of metallothionein cDNA from neonatal rat liver

A metallothionein cDNA clone was isolated from a cDNA bank prepared from neonatal r a t liver poly(A)-containing RNA by a colony screening procedure using [³²P]cDNA probes prepared from mRNA of either metal-induced or uninduced rat livers. Nucleotide sequence analysis of this clone showed that it contained the entire 3' untranslated region and 30% of the coding sequence for a rat metallothionein. The sequence is remarkably homologous with the mouse metallothionein-I gene.     

Human serum albumin. Spectroscopic studies of binding and proximity relationships for fatty acids and bilirubin.

Binding and proximity relationships of hydrophobic ligands on human serum albumin have been studied using absorption, fluorescence, circular dichroism, and electron paramagnetic resonance spectroscopy. The ligands studied were bilirubin, two conjugated linear polyene fatty acids, cis-parinaric acid and cis-eleostearic acid, and three nitroxide derivatives of stearic acid with doxyl groups at positions 5, 10, and 12, respectively. Binding of polyene fatty acids was monitored by absorption peak shifts, induced circular dichroism, enhancement of fluorescence, and energy transfer between albumin's single tryptophanyl residue and the polyene chromophore. Induced circular dichroism studies indicate excitonic ligand-ligand interaction between bound fatty acids. Fluorescence enhancement of cis-parinaric acid was analyzed using a stepwise multiple equilibrium model, and six binding constants in the range 10(8) to 10(6) M-1 were obtained, in agreement with previous measurements for other fatty acids. The temperature dependence of the equilibrium constants indicates that the binding enthalpy is nearly zero. Fluorescence energy transfer was similarly used to quantitate bilirubin binding to albumin. Energy transfer, nitroxide quenching of fluorescence, and electron paramagnetic resonance spectroscopy were used to elucidate binding geometries which support and extend proposed structural models for albumin. It is suggested that the first two fatty acids bind side-by-side in an antiparallel fashion in domain III of human serum albumin.