The interactive effects of temperature,food level and maternal phenotype on offspring size in Daphnia magna

Invertebrate offspring are usually larger in colder environments. To test for possible effects of covariates (e.g. maternal phenotype and feeding conditions) on this pattern, we performed a laboratory experiment to look at the effect of temperature on newborn weight in the planktonic crustacean Daphnia magna. Three tempèratures (12°C, 16°C and 22°C) and two food levels (10,000 cells ml^–1 and 150,000 cells ml^–1) were used, and offspring were examined from the first five clutches of mothers that had been maintained under the constant experimental conditions for three generations. Preliminary analysis suggested that newborn weight was significantly affected by temperature although patterns in the data were not clear cut. In addition, the covariates mother weight and clutch size were positively and negatively correlated with newborn weight, respectively; and later clutches tended to contain heavier offspring. Therefore, in an effort to control for the effects of the covariates, repeated-measures analysis of covariance was performed using ratio values of newborn weight/mother weight (relative newborn weight) as the dependent variable, clutch size as the covariate and clutch number as the repeated measures term. Now, temperature as a main effect in an ANCOVA model did not significantly influence relative newborn weight. The repeatedmeasure term clutch number also became nonsignificant, indicating that when differences in mother weight due to age were accounted for there were no overall differences in relative newborn weight between clutches from a particular mother. Temperature effects on relative newborn weight were only significant as part of interaction terms with food concentration and with clutch number. Thus there were different weight responses to temperature within food levels, and between clutch numbers within food levels. Under the low-food conditions newborn were heaviest at 16°C, lightest at 12°C and intermediate at 22°C. Conversely, under the high-food condition newborn were lightest at 16°C, heaviest at 12°C and again intermediate at 22°C. However, newborn tended to be heavier under the low food condition, and food concentration was highly significant as a main effect. Mother growth rate showed no significant relationship with newborn weight. It is concluded that direct temperature effects on relative newborn weight are marginal and nonsignificant. Temperature effects through interactions with food concentration and clutch number are important determinants of newborn weight, but relatively speaking account for only a small proportion of observed variance in newborn weight (25%), compared with the direct effect of food concentration (67%).     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Butyrate-induced cell differentiation of cell-cycle mutants and ''wild-type'' mastocytoma cells: Histamine, 5-hydroxytryptamine and metachromatic granules as independently regulated differentiation markers

In cultures of heat-sensitive (hs; arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (cs; arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants that had been isolated from the same subclone (K21) of the murine P-815-X2 mastocytoma line, the degree of cell differentiation was assessed by determining the cellular histamine and 5-hydroxytryptamine (5-HT) content as well as the number of metachromatic granules per cell. The findings were compared with those obtained for 'wild-type' K21 and P-815-X2 cells. The addition of butyrate to 'wild-type' cells or to mutant cells maintained at the respective permissive temperature resulted in a relative increase in the level of all three differentiation markers. In cs mutant cells, essentially the same pronounced increase in granule numbers was observed during butyrate treatment at 39.5 degrees C and during incubation at 33 degrees C without butyrate, thereby suggesting that butyrate induces morphological cell differentiation in cs mutants via the same mechanisms as exposure to the nonpermissive temperature. In contrast, the histamine and 5-HT levels reached in hs and cs mutant cells in the presence of butyrate were higher than those observed during incubation at the nonpermissive temperature. Large quantitative differences were detected with respect to the potential of individual cell lines to express the three differentiation parameters. High levels of histamine were characteristic of 'wild-type' P-815-X2 cells treated at 33 degrees C with butyrate, while low amine levels and small numbers of granules were observed in K21 cells (i.e., the parent line of hs and cs mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo

The organization of the actin cytoskeleton was studied in unfertilized porcine oocytes and preimplantation stage embryos from Day 1 through Day 8 of development. Fixed and detergent-extracted oocytes and embryos were analyzed by fluorescence microscopy after staining with either rhodamine-phalloidin to localize filamentous actin or with affinity-purified anti-actin antibodies to localize the total immunodetectable actin. Whereas unfertilized oocytes contain immunoreactive cytoplasmic actin, rhodamine-phalloidin binding is not detected until fertilization when a prominent cortical staining pattern becomes apparent. In early cleavage stage embryos, filamentous actin is concentrated in the cell cortex of blastomeres especially at sites of cell-cell contact. Compacting morulae exhibit a marked accumulation of actin at the margins of blastomeres where numerous interdigitating cell processes are located. The predominantly pericellular distribution of actin becomes a distinguishing feature of trophectodermal cells in the expanding blastocyst at Day 6 of development; these cells form a prominent actin-limited zone circumscribing the inner cell mass. In Day 8 blastocysts, three cell types are present that are readily distinguishable based upon their actin displays among other cytological features. Trophectodermal cells exhibit continuous actin-rich lateral borders and stress fibers along their basal surface. Inner cell mass cells contain a discontinuous actin boundary and prominent foci of actin along their blastocoelic surface. Lining the blastocoel are patches of endodermal cells in which the actin is exclusively cortical. The data are discussed with respect to differences between species and the chronology of actin rearrangements during preimplantation development of the porcine embryo.     

Allelic variation in HLA-B and HLA-C sequences and the evolution of the HLA-B alleles

Several new HLA-B (B8, B51, Bw62)- and HLA-C (Cw6, Cw7)-specific genes were isolated either as genomic cosmid or cDNA clones to study the diversity of HLA antigens. The allele specificities were identified by sequence analysis in comparison with published HLA-B and -C sequences, by transfection experiments, and Southern and northern blot analysis using oligonucleotide probes. Comparison of the classical HLA-A, -B, and -C sequences reveals that allele-specific substitutions seem to be rare events. HLA-B51 codes only for one allelespecific residue: arginine at position 81 located on the 1 helix, pointing toward the antigen binding site. HLA-B8 contains an acidic substitution in amino acid position 9 on the first central sheet which might affect antigen binding capacity, perhaps in combination with the rare replacement at position 67 (F) on the ul helix. HLA-B8 shows greatest homology to HLA-Bw42, -Bw41, -B7, and-Bw60 antigens, all of which lack the conserved restriction sites Pst I at position 180 and Sac I at position 131. Both sites associated with amino acid replacements seem to be genetic markers of an evolutionary split of the HLA-B alleles, which is also observed in the leader sequences. HLA-Cw7 shows 98% sequence identity to the JY328 gene. In general, the HLA-C alleles display lower levels of variability in the highly polymorphic regions of the 1 and 2 domains, and have more distinct patterns of locus-specific residues in the transmembrane and cytoplasmic domains. Thus we propose a more recent origin for the HLA-C locus.     

Role of phospholipases in myocardial ischemia: effect of cardioprotective agents on the phospholipases A of heart cytosol and sarcoplasmic reticulum in vitro

Increased breakdown of myocardial phospholipids to fatty acids and lysophosphoglycerides is an early feature of myocardial ischemic injury and many investigators believe that enhanced phospholipase action is an important factor in the process. Several recent reports indicate that inhibitors of phospholipase A, such as mepacrine, chloroquine and chlorpromazine, can prevent heart phosphoglyceride breakdown in vivo. We isolated the phospholipases A from rat heart cytosol and sarcoplasmic reticulum and examined the effects of various cardioprotective substances on their activity. Most of the cardioprotective agents studied inhibited the heart phospholipases in vitro, providing further evidence that phospholipid degradation in ischemic myocardial injury may be modulated by pharmacologic agents.     

Ontogeny of hormone-secreting cells of the rat pituitary gland: An immunocytochemical study on dissociated cells

Summary An immunocytochemical study was undertaken in foetal, prepubertal and mature rats to determine the time of differentiation of various types of adenohypophyseal cells during development. Freshly dissociated pituitary cells from foetal (18–21 days postconception), neonatal (from birth up to 30 days) and adult rats (more than 8 weeks) were characterized using immunocytochemical methods. All types of hormone-producing cells were present at day 18 postconception, although only 20% of the cells were immunolabelled. Adrenocorticotropin (ACTH)-secreting cells accounted for the highest number of hormone-positive cells. Growth hormone-secreting cells increased remarkably from day 18 postconception onwards. Prolactin-secreting cells were not seen in the foetal adenohypophysis and did not start to increase until 10 days after birth, whereas by that time the number of ACTH, thyrotropin, follicle-stimulating and luteinizing hormone-secreting cells had stopped increasing. By day 30 after birth, 80–95% of the cells were immunoreactive.     

δ and κ Opiate receptors in primary astroglial cultures from rat cerebral cortex

The effects of , , and receptor-agonists on forskolin stimulated cyclic adenosine-3, 5-monophosphate (cAMP) formation were examined in astroglial enriched primary cultures from the cerebral cortex of newborn rats. Intracellular cAMP accumulation was quantified by radioimmunoassay. Morphine was used as a -receptor agonist, D-Ala-D-Leu-Enkephalin (DADLE) as a -receptor agonist and dynorphine 1–13 (Dyn) as a -receptor agonist. Basal cAMP levels were unaffected by either the opiate agonists or the antagonists used. In the presence of the cAMP stimulator forskolin, morphine had no significant effect on the cytoplasmic cAMP levels. DADLE caused a dose related inhibition of the forskolin stimulated cAMP accumulation. The effects of this receptor stimulation was blocked with the selective antagonist ICI 174.864. In the presence of Dyn, the forskolin stimulated cAMP accumulation was inhibited in a dose related manner. This receptor stimulation was blocked with the selective antagonist MR 2266. Co-administration of DADLE and Dyn resulted in a non additive inhibition of the forskolin stimulated accumulation of cAMP. These findings indicate that astroglial enriched cultures from the cerebral cortex of rats express and -receptors co-localized ont he same population of cells, and that these receptors are inhibitory coupled to adenylate cyclase.     

Plant regeneration by organogenesis in Vitis rootstock species

A procedure for the regeneration of Vitis rootstocks plantlets by organogenesis from foliar tissues is described. Leaves from mature plants grown in growth chambers or from plantlets grown in tubes were wounded with a scalpel and cultured on a modified Murashige and Skoog liquid medium containing different concentrations of benzyl-aminopurine. The presence of benzyl-aminopurine is required for shoot formation. The age of the source explant, the composition of the culture medium and the culture temperature are important parameters of the regeneration process.Abbreviations BA 6-benzyl-aminopurine - MS Murashige and Skoog medium - MM modified Murashige and Skoog medium     

Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.