Low intracellular magnesium in patients with acute pancreatitis and hypocalcemia.

To determine the role of magnesium deficiency in the pathogenesis of hypocalcemia in acute pancreatitis, we measured magnesium levels in serum and in peripheral blood mononuclear cells in 29 patients with acute pancreatitis, 14 of whom had hypocalcemia and 15 of whom had normal calcium levels. Only six patients had overt hypomagnesemia (serum magnesium less than 0.70 mmol per liter [1.7 mg per dl]). The mean serum magnesium concentration in hypocalcemic patients was not significantly lower than in normocalcemic patients, but the mononuclear cell magnesium content in hypocalcemic patients with pancreatitis was significantly lower than in normocalcemic patients with pancreatitis (P less than .01). The serum magnesium level did not correlate with that of serum calcium or the mononuclear cell magnesium content, but the latter did significantly correlate with the serum calcium concentration (r = .81, P less than .001). Most patients with hypocalcemia had a low intracellular magnesium content. Three normomagnesemic, hypocalcemic patients with alcoholic pancreatitis also underwent low-dose parenteral magnesium tolerance testing and showed increased retention of the magnesium load. We conclude that patients with acute pancreatitis and hypocalcemia commonly have magnesium deficiency despite normal serum magnesium concentrations. Magnesium deficiency may play a significant role in the pathogenesis of hypocalcemia in patients with acute pancreatitis.     

Centromere-Linkage Analysis and Consolidation of the Zebrafish Genetic Map

The ease of isolating mutations in zebrafish will contribute to an understanding of a variety of processes common to all vertebrates. To facilitate genetic analysis of such mutations, we have identified DNA polymorphisms closely linked to each of the 25 centromeres of zebrafish, placed centromeres on the linkage map, increased the number of mapped PCR-based markers to 652, and consolidated the number of linkage groups to the number of chromosomes. This work makes possible centromere-linkage analysis, a novel, rapid method to assign mutations to a specific linkage group using half-tetrads.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA.

A transformation scheme for Cryptococcus neoformans to yield high-frequency, integrative events was developed. Adenine auxotrophs from a clinical isolate of C. neoformans serotype A were complemented by the cryptococcal phosphoribosylaminoimidazole carboxylase gene (ade2) with a biolistic DNA delivery system. Comparison of two DNA delivery systems (electroporation versus a biolistic system) showed notable differences. The biolistic system did not require linear vectors and transformed each auxotrophic strain at similar frequencies. Examination of randomly selected transformants by biolistics showed that 15 to 40% were stable, depending on the recipient auxotroph, with integrative events identified in all stable transformants by DNA analysis. Although the ade2 cDNA copy transformed at a low frequency, DNA analysis found homologous recombination in each of these transformants. DNA analysis of stable transformants receiving genomic ade2 revealed ectopic integration in a majority of cases, but approximately a quarter of the transformants showed homologous recombination with vector integration or gene replacement. This system has the potential for targeted gene disruption, and its efficiency will also allow for screening of DNA libraries within C. neoformans. Further molecular strategies to study the pathobiology of this pathogenic yeast are now possible with this transformation system.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Usefulness of the stomacher in a microbiological regulatory laboratory.

The relative efficiency of the Waring blender, the Stomacher 400, and the Stomacher 3500 for preparing food samples for microbiological analysis was studied. Comparative aerobic plate count (APC) values were determined on 671 samples, representing 30 categories of foods. Of the 26 categories of nonfatty foods, the blender gave significantly higher geometric mean APC values than those given by the Stomacher 400 and the Stomacher 3500 in 65 and 69 percent of the categories, respectively. In a comparison of the two Stomacher models, the Stomacher 400 gave significantly higher geometric mean APC values than these given by the Stomacher 3500 in 73 percent of the food categories. Addition of Tween 80 to four categories of fatty foods at concentrations of 0.5, 1.0, and 2.0 percent did not raise the APC values given by either model of stomacher to the levels given by the Waring blender. Overall, the efficiency of both models of Stomacher, relative to the blender and to each other, was specific and depended upon the particular food being analyzed.     

Studies on pulmonary aryl hydrocarbon hydroxylase activity in inbred strains of mice.

Pulmonary and hepatic levels of aryl hydrocarbon hydroxylase (AHH) were studied in inbred strains of mice following intratracheal (i.t.) instillation of 3-methylcholanthrene (MCA). I.t. instillation of 188 mug MCA in sterile 0.2% gelatin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dose of MCA, the pulmonary AHH levels of strains C57BL/6Cum, C57BL/6J, BALB/cMai, C3H/fMai, and C57L/J were observed to be induced within 24 h after treatment. Strains DBA/2Cum, AKR/J, SJL/J, DBA/2J and RF/J expressed no such increase. At a dose of 500 mug MCA, the pulmonary tissue of DBA/2 mice did express a 4-fold increase. This increase in AHH was determined to be quite different from the increase observed in C57BL/6 mice by: (1) specific activity of the enzymes, (2) genetic regulation, (3) susceptibility to inhibition by 7,8-benzoflavone, and (4) spectral properties of the associated cytochromes. It was of major importance that induction of pulmonary AHH was observed to be regulated by a single dominant gene in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice. Results were discussed with the view in mind that these genetically regulated levels of AHH may play a role in susceptibility to cancers induced by polycyclic aromatic hydrocarbon carcinogens.     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.