Differences in ovarian activity between booroola X merino ewes which were homozygous, heterozygous and non-carriers of a major gene influencing their ovulation rate

Differences in the function and composition of individual ovarian follicles were noted in Booroola Merino ewes which had previously been segregated on at least one ovulation rate record of greater than 5 (FF ewes, N = 15), 3-4 (F+ ewes, N = 18) or less than 3 (++ ewes, N = 18). Follicles in FF and F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In FF (N = 3), F+ (N = 3) and ++ (N = 3) ewes, the respective mean +/- s.e.m. diameters for the presumptive preovulatory follicles were 3.4 +/- 0.3, 4.1 +/- 0.2 and 6.8 +/- 0.3 mm and in each of these follicles the respective mean +/- s.e.m. numbers of granulosa cells (X 10(6)) were 1.8 +/- 0.3, 2.2 +/- 0.3 and 6.6 +/- 0.3. During a cloprostenol-induced follicular phase, the oestradiol secretion rates from FF ewes with 4.8 +/- 0.4 'oestrogenic' follicles, F+ ewes with 3.2 +/- 0.2 'oestrogenic' follicles and ++ ewes with 1.5 +/- 0.02 'oestrogenic' follicles were not significantly different from one another. Moreover, the mean total numbers of granulosa cells from the 'oestrogenic' follicles from each genotype were identical, namely 5.4 X 10(6) cells. Irrespective of genotype the mean weight of each corpus luteum was inversely correlated to the ovulation rate (R = 0.91, P less than 0.001). Collectively, these findings support the notion that the maturation of greater than or equal to 5 follicles in FF ewes and 3-4 follicles in F+ ewes may each be necessary to provide a follicular-cell mass capable of producing the same quantity of oestradiol as that from 1-2 preovulatory follicles in ++ ewes.     

The subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

Incubation of subcellular fractions isolated from rat cerebral cortex with [-³²P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes.A preliminary account of part of this work has been published (1).     

Biosynthesis and processing of rat alpha 1-antitrypsin

Various biosynthetic forms of rat alpha 1-antitrypsin (alpha 1AT) have been isolated by immunoprecipitation of in vitro and in vivo synthesized products. Rat alpha 1AT is synthesized in a rabbit reticulocyte system as a 45,000-Da preprotein with a 23-amino acid signal sequence. The majority of the amino acids in the signal sequence have been identified and resemble the signal peptides of other secretory proteins with respect to the abundance and positions of hydrophobic amino acids. Evidence from the translation of rat liver RNA in the presence of dog pancreas microsomes, from the translation of rat liver polysomes, and from tunicamycin-treated rat hepatocytes established that cleavage of the signal peptide of pre-alpha 1AT results in the formation of a 42,000-Da protein, the polypeptide backbone of mature alpha 1AT. A 50,000-Da glycoprotein is immunoprecipitated from translations programmed with rat liver microsomes or with rat liver mRNA and dog pancreas microsomes. Cotranslational glycosylation of alpha 1AT appears to occur in a stepwise fashion since three glycosylated forms of alpha 1AT (approximately 45,000, 47,000, and 50,000 Da) can be detected in polysome translations. These proteins are susceptible to cleavage by endo-beta-N-acetylglucosaminidase H and are digested to the same product, indicating that they have identical polypeptide chains. Two intracellular forms of alpha 1AT were detected in cultured rat hepatocytes, a 50,000- and a 52,000-Da protein; only the larger protein was immunoprecipitated from the medium of these cells. Digestion with endo-beta-N-acetylglucosaminidase H indicated that the 50,000-Da protein is a core glycosylated processing intermediate, whereas the 52,000-Da protein, which comigrated with purified serum alpha 1AT, appears to contain complex carbohydrate sidechains. When glycosylation was inhibited by incubation of hepatocytes with tunicamycin, a nonglycosylated 42,000-Da protein was immunoprecipitated from the cells and the culture medium, indicating that glycosylation of alpha 1AT is not essential for its secretion.     

Compartmentation between glycolysis and gluconeogenesis in rat liver

1. The specific radioactivity-time relationships of glucose, glucose 6-phosphate, glycerol 1-phosphate and UDP-glucose were determined in rat liver after the intravenous injection of [U-(14)C]fructose, and a kinetic analysis was carried out. The glucose 6-phosphate pool was found to be compartmented into gluconeogenic and glycolytic components, and evidence was obtained that the triose phosphates were similarly compartmented. The glycolytic pathway was fed by glycogenolysis and glucose phosphorylation. There was no direct evidence that glycogenolysis fed only the glycolytic pathway, but this interpretation would make the liver resemble other organs in this respect. 2. UDP-glucose was not formed solely from gluconeogenic glucose 6-phosphate, as there was some dilution of label in the intervening glucose 1-phosphate pool, probably from glycogenolysis, though other pathways cannot be excluded. 3. The data cannot be explained by isotopic exchange.     

Ca transport in membrane vesicles from pinto bean leaves and its alteration after ozone exposure

The influence of ozone on Ca²⁺ transport in plant membranes from pinto bean (Phaseolus vulgaris L. var Pinto) leaves was investigated in vitro by means of a filtration method using purified vesicles. Two transport mechanisms located at the plasma membrane are involved in a response to ozone: (a) passive Ca²⁺ influx into the cell and (b) active Ca²⁺ efflux driven by an ATP-dependent system, which has two components: a primary Ca²⁺ transport directly linked to ATP which is partially activated by calmodulin and a H⁺/Ca²⁺ antiport coupled to activity of a H⁺-ATPase. The passive Ca²⁺ permeability is increased by ozone. A triangular pulse of ozone stimulates a higher influx of Ca²⁺ than does a square wave, even though the total dose was the same (0.6 microliter per liter × hour). Leaves exposed to a square wave did not exhibit visible injury and were still able to recover from oxidant stress by activation of calmodulin-dependent Ca²⁺ extrusion mechanisms. On the other hand, leaves exposed to a triangular wave of ozone, exhibit visible injury and lost the ability of extruding Ca²⁺ out of the cell.     

Investigating causation in cancer clusters

Questions concerning clusters of cancer cases frequently arise in public health practice. The process of investigating any such cluster requires awareness that such case groupings can easily occur by chance and that any search for biologically meaningful causes will be severely constrained by various methodologic difficulties. These include (1) the long and probably variable latent periods between causative events and cancer diagnosis, (2) the limited numbers of cases available for study in any given cluster situation, and (3) the clinical non-specificity of cancer cases whereby no readily available means are at hand to identify the specific causes for any particular case. Evaluation of any given cluster should involve careful consideration of such limitations, together with a preliminary assessment of the specific cases involved and their community or workplace setting, before more intensive study is undertaken. Received: 22 February 1996 / Accepted in revised form: 13 June 1996