Comparative aspects and temperature dependence of [3H]1,4-dihydropyridine Ca2+ channel antagonist and activator binding to neuronal and muscle membranes

Binding of [3H]nitrendipine, [3H]nimodipine, and (+)[3H]PN 200-110 to microsomal preparations of guinea pig smooth and cardiac muscle and brain synaptosomes revealed high affinity interaction with KD values in the sequence, (+)PN 200-110 greater than nitrendipine greater than nimodipine. Bmax values for a particular tissue were independent of the 1,4-dihydropyridine employed in radioligand binding at 25 degrees C. The temperature dependence of [3H]nitrendipine binding in cardiac and smooth muscle microsomal preparations and brain synaptosomes was measured from 0 degrees to 37 degrees C and for skeletal muscle preparations from 0 degrees to 30 degrees C. Bmax values increased with temperature for cardiac membranes, but did not vary in other tissues. van't Hoff plots were nonlinear in all tissues, enthalpy and entropy changes becoming increasingly negative with increasing temperature. Competition binding of the activator-antagonist enantiomeric 1,4-dihydropyridine pairs of Bay k 8644 and PN 202-791 for [3H]nitrendipine in smooth muscle did not reveal significant thermodynamic differences between activator and antagonist molecules.     

A complete complementary DNA for the oncodevelopmental calcium-binding protein, oncomodulin

RNA from a rat liver tumor (Morris hepatoma 5123tc) was used to construct cDNAs together comprising the complete coding sequence of rat oncomodulin mRNA. Information obtained from these cDNAs as well as from primer extension analysis gave a deduced length for the complete oncomodulin mRNA of approximately 680 nucleotides (excluding the poly(A) tail) including a 5'-untranslated region of 97 +/- 2 nucleotides, a 324-nucleotide-coding sequence and a 259-nucleotide 3'-noncoding region. Comparison of the oncomodulin cDNA sequence with those coding for other members of the calcium-binding protein family shows little homology with the exception of a recently reported parvalbumin cDNA where the oncomodulin and parvalbumin nucleotide sequences are 59% identical in the protein-coding region. RNA blot analysis of poly(A+) RNA from normal adult rat liver gave no evidence of oncomodulin expression in this tissue. A single RNA species was detected, however, in RNA extracts from the hepatoma and from rat and human placentas. A probe prepared from one of the rat oncomodulin cDNAs hybridized with a single DNA species in restriction digests of hepatoma and normal DNA from rat and sequences in DNA of humans and other mammals. A 38-nucleotide sequence spanning the 5'-untranslated region and the first seven codons of the oncomodulin cDNA, was far less homologous than was the same region of a parvalbumin cDNA, to a chicken calmodulin cDNA sequence coding for the first calcium-binding domain. The oncomodulin gene appears to have diverged more from that of calmodulin than has the parvalbumin gene.     

Estimators of repeatability

Summary Analysis of variance and principal components methods have been suggested for estimating repeatability. In this study, six estimation procedures are compared: ANOVA, principal components based on the sample covariance matrix and also on the sample correlation matrix, a related multivariate method (structural analysis) based on the sample covariance matrix and also on the sample correlation matrix, and maximum likelihood estimation. A simulation study indicates that when the standard linear model assumptions are met, the estimators are quite similar except when the repeatability is small. Overall, maximum likelihood appears the preferred method. If the assumption of equal variance is relaxed, the methods based on the sample correlation matrix perform better although others are surprisingly robust. The structural analysis method (with sample correlation matrix) appears to be best.Paper number 776 from the Department of Meat and Animal Science, University of Wisconsin-Madison.     

Uptake and metabolism of [3H]norepinephrine in the cerebral hemispheres of chick embryos

Abstract— The uptake and metabolism of [³H]norepinephrine were studied in slices of cerebral hemispheres removed from chick embryos at 10, 15 and 20 days of embryonic age, as well as from 90-day-old hens. Brain tissue from all age groups concentrated [³H]norepinephrine to much greater levels at 37°C than at 0°C. There was a marked increase in the rate of accumulation of [³H]norepinephrine in tissues from 10 to 15 days of embryonic age, with no further increase in the rate observed from 15 to 20 days of embryonic age. Tissue slices were incubated for 20 min with [³H]norepinephrine, and the deaminated metabolites of norepinephrine were separated by paper chromatography. In tissues from all age groups, the neutral metabolites were produced in greater amounts than the acid metabolites. A significant increase in the amounts of deaminated metabolites formed was observed in the period from 10 to 15 days of embryonic age and a significant decrease in the amounts formed was observed in the period from 15 to 20 days of embryonic age. The deamination at 20 days was very similar to that observed in the adult. A significant decrease in the level of the deaminated metabolites was noted in all age groups in response to cocaine (an inhibitor of neuronal uptake mechanisms), an observation suggesting that mechanisms for neuronal uptake of NE are functional by 10 days of embryonic development in the chick. However, a significant increase in the level of deaminated metabolites in response to reserpine (an inhibitor of uptake of NE into storage granules) was observed only in slices taken from 20-day embryos and from the 90-day-old hen. The effect in the hen was more prominent than in the 20-day embryo. These results were interpreted to indicate that mechanisms for the uptake of NE develop at an earlier embryonic age than mechanisms for the storage of NE and that mechanisms for storage continue to develop after hatching.     

Segregation of random amplified DNA markers in F1 progeny of conifers

Summary The recently developed approach to deriving genetic markers via amplification of random DNA segments with single primers of arbitrary nucleotide sequence was tested for its utility in genetic linkage mapping studies with conifers. Reaction conditions were optimized to reproducibly yield clean and specific amplification products. Template DNA from several genotypes of Douglas-fir (Pseudotsuga menziesii) and white spruce (Picea glauca) were tested against eight ten-base oligonucleotide primers. Most of the tested primer/parent tree combinations yielded polymorphic PCR products (RAPD markers). Selected primers were then used in PCR reactions with template DNA isolated from offspring in Douglas-fir and black spruce diallel crosses among the same parental lines. The diallel study confirmed the appropriate inheritance of RAPD markers in the F₁ generation. The value of these dominant RAPD markers for genetic linkage mapping in trees was established from both theoretical and applied perspectives.     

Lower sodium lactate in Whitten's medium improves in vitro developmental capacity of one-cell mouse embryos

A modification of Whitten's medium, involving a reduced content of Na-lactate syrup (0.2 ml/100 ml; 11.65 mM) and osmolarity (251 mOsm), was compared with normal Whitten's medium (0.37 ml/100 ml; 21.6 mM) for ability to support mouse embryonic development in vitro from one-cell to the blastocyst stage. In a pilot study utilizing 10 ICR donor female mice, in vitro developmental capacity (IVDC; percentage of fertilized one-cell embryos developing to blastocysts in vitro per female donor) was significantly enhanced by the modified medium (68.0 versus 24.0%; P<0.001). In the main study, utilizing 134 ICR and 17 ICR x C57BL/6J F(1) donor females, the modified medium supported increased IVDC for both ICR (67.9 versus 51.1%; P<0.001) and F(1) females (98.5 versus 89.4%; P<0.05). A large degree of among donor-female variation in IVDC was observed for both media in the ICR stock (SD = 30.0). The beneficial role of the reduction of Na-lactate in Whitten's medium may be related to an improved provision of energy requirements for first cleavage and/or a more suitable osmolarity for development.     

Influences of prenatal and postnatal fraternity size on ovarian development in the mouse

An experiment was conducted to test effects of prenatal and postnatal fraternity size (size of litter in which an individual develops prenatally or is reared postnatally) on ovarian development in mice. Fraternity size treatments were created by standardizing sizes of prenatal and postnatal fraternities in which mice were gestated and reared. Prenatal fraternity size was standardized by surgery on Day 9 of gestation to 6, 10, and 14 fetuses. Postnatal fraternity size was standardized by randomly assigning pups to litters of 5, 10, or 15 pups within 24 h of birth. Female pups were killed at either 3 or 20 wk of age and right ovaries were prepared for histology. Follicles were classified by size and morphology, and numbers of follicles in each class were tabulated. Interaction of postnatal fraternity size and age was observed for number of antral follicles (p less than 0.05). Mice reared in small postnatal fraternities had more antral follicles at weaning (3 wk) and fewer antral follicles at maturity (20 wk of age) than mice reared in large postnatal fraternities. No effect of either prenatal or postnatal fraternity size on other follicle populations was observed (p greater than 0.20). Numbers of Type 2 (primordial), Type 3a, and Type 3b follicles changed with age (p less than 0.01); numbers of primordial follicles declined with age, but numbers of Type 3a and 3b follicles increased. A hypothesis of a negative association between postnatal fraternity size and number of antral follicles at 3 wk of age was supported, but a hypothesis of a positive association between fraternity size and number of primordial follicles was not supported.     

The effects of dissolved oxygen and aquatic surface respiration on the critical thermal maxima of three intermittent-stream fishes

Synopsis Critical thermal maxima (CTM) were determined for three species of fish in hypoxic (1.2 mg l^-1), normoxic (7 mg l^-1) and hyperoxic (12 mg l^-1) oxygen tensions. All fish were acclimated to 30°C under a regulated photoperiod of 12:12. During each CTM determination, one-half of the test fish were allowed free access to the surface (to permit aquatic surface respiration) while the remaining one-half were denied access to the surface. Under hypoxic conditions, CTMs of Fundulus notatus, Notropis lutrensis and Pimephales vigilax given access to the surface were significantly higher than those of conspecifics without access to the surface. With access to the surface, all species had significantly lower CTMs under hypoxic conditions than under normoxic and hyperoxic conditions. CTMs measured under normoxic and hyperoxic conditions were not different for any of the test species. Under all comparable oxygen tensions with surface access, F. notatus had the highest CTM, N. lutrensis intermediate and P. vigilax had the lowest CTM. The critical oxygen concentration for N. lutrensis during CTM determinations occurred between 1.2 and 2 mg l^-1.     

Failure of therapy with 2,3-dihydroxybenzoic acid to modify the course of sepsis-induced lung injury

Oxidant-induced injury of the pulmonary microvasculature reportedly contributes to an increase in microvascular permeability and pulmonary hypertension, both of which are principal features of acute lung injury (ALI). We tested the hypothesis that antioxidant therapy with 2,3-dihydroxybenzoic acid (DHB), initiated in awake sheep after the development of sepsis-induced ALI, would ameliorate the progression of these lesions. DHB has many actions that suggested to us the potential for demonstrating benefit in ALI complicating sepsis; it is a nontoxic hydroxyl-radical scavenger that also inhibits the cyclooxygenase pathway and acts as a weak iron chelator. In preliminary experiments, we demonstrated that pretreatment with DHB prevented an increase in mean pulmonary arterial pressure, plasma thromboxane A2, measured as its metabolite thromboxane B2, and lymph total protein clearance that otherwise followed an infusion of zymosan-activated plasma (ZAP) in sheep. In subsequent experiments, 12 additional sheep were rendered septic by cecal ligation and perforation. Twenty-four to 36 h after cecal ligation and perforation, an increase in lung microvascular permeability was confirmed, because pulmonary lymph flow had increased by 82% while the mean lymph-to-plasma total protein ratio was unchanged from baseline. At this point, six sheep were then treated with parenteral DHB and six with DHB vehicle for the subsequent 24 h. In contrast to the demonstrated benefit of DHB pretreatment in preventing ALI secondary to an infusion of ZAP, the progressive increase in lymph total protein clearance that complicated septic lung injury in the DHB vehicle group throughout this 24-h study period was not ameliorated in the DHB treatment group. However, DHB did prevent a modest increase in mean pulmonary arterial pressures that was demonstrated in the DHB vehicle group throughout this 24-h treatment period. Although pretreatment prevented ALI after a ZAP infusion, we conclude that DHB only incompletely modified disease progression when administered after the onset of sepsis-induced ALI because it ameliorated the pulmonary hypertensive response without concurrently modifying an increase in lung microvascular fluid flux.     

A novel c.592-4_c.592-3delTT mutation in DGUOK gene causes exon skipping

Deoxyguanosine kinase (DGUOK) catalyzes the first step of the mitochondrial deoxypurine salvage pathway, the phosphorylation of purine deoxyribonucleosides. Mutations in the DGUOK gene have been linked to inherited mtDNA depletion syndromes, neonatal liver failure, nystagmus, and hypotonia. Previously, we reported the first case of a heterozygous unclassified c.592-4_c.592-3delTT alteration in a patient with DGUOK deficiency without the demonstration of its pathogenicity (Dimmock et al., 2008). This alteration was predicted to cause aberrant splicing based upon two computer algorithms. We now report a homozygous c.592-4_c.592-3delTT mutation found in two affected siblings of asymptomatic consanguineous parents. The proband presented with symptoms of idiopathic hepatitis, liver dysfunction, nystagmus, and retinal blindness. This individual died at 6 months of age due to liver failure. This individual’s affected sibling presented similarly and has remarkable elevations of tyrosine, methionine, and alanine. Many organic acids were elevated in urine, including lactic acid, Krebs cycle intermediates, and para-hydroxy compounds; ketone bodies were also present. RNA studies support aberrant splicing. Sequencing of cDNA detected exon 5 skipping in the two affected siblings, but not in the normal control. These results indicate that the homozygous c.592-4_c.592-3delTT is deleterious and responsible for the DGUOK deficiency. The parents were subsequently confirmed to be carriers of this mutation. In summary, we have demonstrated that c.592-4_c.592-3delTT is a pathogenic splice acceptor site mutation leading to DGUOK deficiency.