Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana)

Summary Vascular adrenergic sensitivity to exogenous catecholamines was examined in tadpoles of the American bullfrog (Rana catesbeiana), ranging from stage III to XIV. Central arterial blood pressure was measured in decerebrate bullfrog tadpoles to determine a reasonable initial infusion pressure. Solutions of epinephrine and phenylephrine were infused into the vasculature of pithed tadpoles, and the resulting changes in vascular resistance (R _v) were used to construct log dose-response relationships. Epinephrine infusion produced a dose-dependent increase in R _v (EC₅₀=5.3·10^-7 M), which could be reversed by sodium nitroprusside (a smooth muscle relaxant) and blocked by phenoxybenzamine (an -adrenergic antagonist). Larval R _v also increased with infusion of the -agonist phenylephrine (EC₅₀=7.4·10⁸ M). Infusion of 10^-6 M isoproterenol (a -agonist) largely reversed the phenylephrine-induced increase in R _v. These results indicate that the capacity exists for both -mediated vasoconstriction and -mediated vasodilation early in bullfrog ontogeny. Neither initial R _v nor the responses to infused epinephrine or phenylephrine were significantly correlated to development over the range of larval stages used in this study.Abbreviations ECG electrocardiogram - EPI epinephrine - ISO isoproterenol - PHE phenylephrine - POB phenoxybenzamine - R _v vascular resistance - SNP sodium nitroprusside     

Retention of the mitochondrial probe rhodamine 123 in normal lymphocytes and leukemic cells in relation to the cell cycle

The cationic fluorochrome rhodamine 123 (R123) is specifically taken up by mitochondria of live cells where it is retained due to the mitochondrial transmembrane potential. After pulse exposure of human normal quiescent or proliferating lymphocytes, human lymphocytic leukemic MOLT cells, and mice leukemic L1210 cells to 10 micrograms/ml of R123, the dye release was studied using flow cytometry. Two distinct phases of R123 release, each following first-order kinetics, were apparent; the half-time of retention for the rapidly and slowly released fractions of R123 was 0.8-1.1 and 2.8-4.2 h, respectively. Simultaneous supravital cell staining with R123 and Hoechst 33342 made it possible to correlate retention of R123 with cell position in the cell cycle. No significant differences were observed in the rate of R123 release from cells in G1 vs S or vs G2 + M phases of the cycle. The data rule out a possibility that the release of R123 is due to periodic depolarization of the mitochondria in the cell as may be postulated by cell cycle models that assume a transient passage of cells through resting phase following division. The observed similar rates of R123 release regardless of cell type or cell cycle phase suggest that the factors affecting the exchange are similar in normal lymphocytes vs leukemic cells and unrelated to cell proliferation rate or phase of the cell cycle. Two distinct rates of R123 release indicate the presence of two kinds of binding sites differing in affinity to the dye.     

Children's health risk assessment: incorporating a lifestage approach into the risk assessment process

This overview paper provides the historical context for the incorporation of lifestage‐specific concerns in human health risk assessment, briefly explains the process employed in a lifestage framework for risk assessment, and discusses the scientific rationale for how utilizing lifestage data will strengthen the overall risk assessment process. This risk assessment approach will add value by: (1) providing a more complete evaluation of the potential for vulnerability at different lifestages, including a focus on the underlying biological events and incorporation of mode of action information related to different critical developmental periods; (2) evaluating the potential for toxicity during all lifestages after early lifestage exposure; (3) reviewing the importance of integrating exposure information and adverse health effects across lifestages; and (4) serving as a basis to extend some aspects of the children's health risk assessment framework to all lifestages. Birth Defects Res (Part B), 2008. © 2008 Wiley‐Liss, Inc.     

Breed influences on in vitro development of abattoir-derived bovine oocytes

ABSTRACT: BACKGROUND: There is a discrepancy in the reproductive performance between different cattle breeds. Using abattoir-derived ovaries and data base information we studied the effects of breed on in vitro fertilization and early embryo development. METHODS: The in vitro developmental competence of oocytes from cattle (n = 202) of Swedish Red (SR), Swedish Holstein (SH) and mixed beef breeds was compared, retrospectively tracing donors of abattoir-derived ovaries using a combination of the national animal databases and abattoir information. Age was significantly lower and carcass conformation score was higher in the beef breeds than in the dairy breeds. Cumulus oocyte complexes (n = 1351) were aspirated from abattoir-derived ovaries from animals of known breed (visual inspection confirmed through databases), age (databases), and abattoir information. Oocytes were matured, fertilized (frozen semen from two dairy bulls) and cultured according to conventional protocols. On day 8, blastocysts were graded and the number of nuclei determined. RESULTS: Cleavage rate was not different between the breeds but was significantly different between bulls. The percentage of blastocysts on day 8 was significantly higher when the oocyte donor's breed was beef or SR than SH. There was no significant difference in blastocyst grades or stages between the breeds, but the number of nuclei in day 8 blastocysts was significantly lower in SH compared to the beef. CONCLUSIONS: The use of abattoir-derived ovaries from animals whose background is traceable can be a valuable tool for research. Using this approach in the present study, oocyte donor breed was seen to affect early embryo development during in vitro embryo production, which may be a contributing factor to the declining fertility in some dairy breeds seen today.     

Organization of the 5S RNA genes in macro- and micronuclei of Tetrahymena pyriformis

The organization of the 5S genes in macro- and micronuclei of Tetrahymena pyriformis was studied using restriction endonucleases. After complete digestion of macronuclear DNA with BamH-I or Hpa I, 5S RNA hybridized to a DNA fragment of approximately 280 base pairs (bp). When macronuclear DNA was only partially digested with these enzymes, hybridization with ³²P-5S RNA demonstrated an oligomeric series with a spacing of 280 bp. These results indicate that the 5S genes are tandemly repeated in macronuclei and that the repeating unit is 280 bp (or 180,000 daltons). Since 5S RNA is 120 nucleotides, we conclude that the 5S repeat units contain a 120 bp transcribed region and a 160 bp spacer region. When macronuclear DNA was digested with Eco RI, Bgl I, or Eco RI + Bgl I, 5S RNA hybridized to DNA of molecular weight 3–4×10⁶, suggesting that these enzymes do not cleave within a 5S repeat. These 3–4×10⁶ dalton fragments define the maximum size of an average cluster of 5S repeated units. Assuming the size of the 5S repeat to be 0.18×10⁶ daltons, there are about 15–20 5S repeats per average tandem cluster, and since there are 350 5S-genes per haploid genome, there must be approximately 15–20 tandem arrays. Results obtained using micronuclear DNA suggest that organization of the 5S-genes is very similar in macro- and micronuclei. Macronuclear rRNA genes are extracnromosomal palindromic dimers. In contrast, 5S genes in Tetrahymena were found to be integrated within the genomes of both macro- and micronuclei and not linked to the rRNA genes. Moreover, it is unlikely that they are palindromes; rather they appear to be tandemly repeated in head-to-tail linkages. Thus, the organization of the 5S genes in Tetrahymena is similar to that of higher eukaryotes.     

Rate and directionality of mutations and effects of allele size constraints at anonymous, gene-associated, and disease-causing trinucleotide loci.

We studied the patterns of within- and between-population variation at 29 trinucleotide loci in a random sample of 200 healthy individuals from four diverse populations: Germans, Nigerians, Chinese, and New Guinea highlanders. The loci were grouped as disease-causing (seven loci with CAG repeats), gene-associated (seven loci with CAG/CCG repeats and eight loci with AAT repeats), or anonymous (seven loci with AAT repeats). We used heterozygosity and variance of allele size (expressed in units of repeat counts) as measures of within-population variability and GST (based on heterozygosity as well as on allele size variance) as the measure of genetic differentiation between populations. Our observations are: (1) locus type is the major significant factor for differences in within-population genetic variability; (2) the disease-causing CAG repeats (in the nondisease range of repeat counts) have the highest within-population variation, followed by the AAT-repeat anonymous loci, the AAT-repeat gene-associated loci, and the CAG/CTG-repeat gene-associated loci; (3) an imbalance index beta, the ratio of the estimates of the product of effective population size and mutation rate based on allele size variance and heterozygosity, is the largest for disease-causing loci, followed by AAT- and CAG/CCG-repeat gene-associated loci and AAT-repeat anonymous loci; (4) mean allele size correlates positively with allele size variance for AAT- and CAG/CCG-repeat gene-associated loci and negatively for anonymous loci; and (5) GST is highest for the disease-causing loci. These observations are explained by specific differences of rates and patterns of mutations in these four groups of trinucleotide loci, taking into consideration the effects of the past demographic history of the modern human population.     

A branching process model of gene amplification following chromosome breakage.

We have devised a mathematical model of gene amplification utilizing recent experimental observations concerning dihydrofolate reductase (DHFR) gene amplification in CHO cells. The mathematical model, based on a biological model which proposes that acentric elements are the initial intermediates in gene amplification, includes the following features: (1) initiation of amplification by chromosomal breakage to produce an acentric structure; (2) replication of acentric DNA, once per cell cycle; (3) dissociation of replicated acentric DNA; (4) unequal segregation of acentric DNA fragments to daughter cells at mitosis; (5) subsequent reintegration of acentric fragments into chromosomes. These processes are assumed to be independent for each element present in a cell at a given time. Thus, processes of unequal segregation and integration may occur in parallel, not necessarily in a unique sequence, and may be reiterated in one or multiple cell cycles. These events are described mathematically as a Galton-Watson branching process with denumerable infinity of object types. This mathematical model qualitatively and quantitatively reproduces the major elements of the dynamical behavior of DHFR genes observed experimentally. The agreement between the mathematical model and the experimental data lends credence to the biological model proposed by Windle et al. (1991), including the importance of chromosome breakage and subsequent gene deletion resulting from resection of the broken chromosome ends as initial events in gene amplification.     

Developmental uncoupling between blastoderm and yolk cell in the embryo of the teleost Fundulus

During cleavage and blastula stages of embryos of the teleost Fundulus heteroclitus all of the cells are both electotonically coupled and dye coupled to one another, as determined by microelectrode impalements and spread of Lucifer Yellow. At about the time that gastrulation begins we observed a specific loss of junctional coupling between the yolk cell and cells of the blastoderm. Passage of Lucifer Yellow between the yolk cell and blastoderm was reduced at stage 12 (late blastula), and not detected at stage 13 and thereafter, although cells of the blastoderm remain dye coupled to one another through gastrula stages. Also, junctional electrical coupling between the yolk cell and blastoderm became substantially reduced at stage 13 and thereafter. The loss of coupling at this specific cell apposition and time and the large size of the yolk cell may prove useful in analyzing the underlying cellular mechanisms.     

Origin and organization of the zebrafish fate map

We have analyzed lineages of cells labeled by intracellular injection of tracer dye during early zebrafish development to learn when cells become allocated to particular fates during development, and how the fate map is organized. The earliest lineage restriction was described previously, and segregates the yolk cell from the blastoderm in the midblastula. After one or two more cell divisions, the lineages of epithelial enveloping layer (EVL) cells become restricted to generate exclusively periderm. Following an additional division in the late blastula, deep layer (DEL) cells generate clones that are restricted to single deep embryonic tissues. The appearance of both the EVL and DEL restrictions could be causally linked to blastoderm morphogenesis during epiboly. A fate map emerges as the DEL cell lineages become restricted in the late blastula. It is similar in organization to that of an amphibian embryo. DEL cells located near the animal pole of the early gastrula give rise to ectodermal fates (including the definitive epidermis). Cells located near the blastoderm margin give rise to mesodermal and endodermal fates. Dorsal cells in the gastrula form dorsal and anterior structures in the embryo, and ventral cells in the gastrula form dorsal, ventral and posterior structures. The exact locations of progenitors of single cell types and of local regions of the embryo cannot be mapped at the stages we examined, because of variable cell rearrangements during gastrulation.