Hypervitaminosis D and Metastatic Calcification in a Colony of Inbred Strain 13 Guinea Pigs, Cavia porcellus.

A commercial diet fed to a colony of inbred strain 13 guinea pigs for approximately 6 weeks was subsequently recalled for excessive levels of vitamin D. Twenty-one of 62 animals exhibited clinical signs, including anorexia, lethargy, and poor body condition. Nine affected and 4 clinically normal animals were euthanized for further evaluation, including serum chemistry, urinalysis, and gross and/or histopathology. Macroscopic findings included white discoloration in multiple organs in 8 animals, and microscopic evaluation confirmed multiorgan mineralization in tissues from 7 animals. Serum 25-hydroxyvitamin D levels were elevated in 10 animals. Serum inorganic phosphorus and alkaline phosphatase levels were increased in all exposed animals; however, total calcium and ionized calcium levels were not significantly higher in exposed animals than in control strain 13 guinea pigs from a different institution. The data support a diagnosis of hypervitaminosis D with metastatic calcification. Following the diet recall, the remaining guinea pigs increased their food intake and regained body condition. Diagnostic testing of 8 animals euthanized approximately 3 months after returning to a normal diet demonstrated that serum parathyroid hormone remained significantly lower, and ionized calcium and ionized magnesium were significantly higher, in recovered animals compared to controls and exposed animals. These results indicate that diagnostic tests other than serum calcium are necessary for a diagnosis of hypervitaminosis D in guinea pigs.

Salmonella enterica causes more severe inflammatory disease in C57/BL6 Nramp1G169 mice than Sv129S6 mice.

Salmonella enterica serovar Typhimurium (S. Typhimurium) causes systemic inflammatory disease in mice by colonizing cells of the mononuclear leukocyte lineage. Mouse strains resistant to S. Typhimurium, including Sv129S6, have an intact Nramp1 (Slc11a1) allele and survive acute infection, whereas C57/BL6 mice, homozygous for a mutant Nramp1 allele, Nramp1(G169D) , develop lethal infections. Restoration of Nramp1 (C57/BL6 Nramp1(G169) ) reestablishes resistance to S. Typhimurium; mice survive at least 3 to 4 weeks postinfection. Since many transgenic mouse strains are on a C57/BL6 genetic background, C57/BL6 Nramp1(G169) mice provide a model to examine host genetic determinants of resistance to infection. To further evaluate host immune response to S. Typhimurium, we performed comparative analyses of Sv129S6 and C57/BL6 Nramp1(G169) mice 3 weeks following oral S. Typhimurium infection. C57/BL6 Nramp1(G169) mice developed more severe inflammatory disease with splenic bacterial counts 1000-fold higher than Sv129S6 mice and relatively greater splenomegaly and blood neutrophil and monocyte counts. Infected C57/BL6 Nramp1(G169) mice developed higher proinflammatory serum cytokine and chemokine responses (interferon-γ, tumor necrosis factor-α, interleukin [IL]-1ß, and IL-2 and monocyte chemotactic protein-1 and chemokine [C-X-C motif] ligand 1, respectively) and marked decreases in anti-inflammatory serum cytokine concentrations (IL-10, IL-4) compared with Sv129S6 mice postinfection. Splenic dendritic cells and macrophages in infected compared with control mice increased to a greater extent in C57/BL6 Nramp1(G169) mice than in Sv129S6 mice. Overall, data show that despite the Nramp1 gene present in both strains, C57/BL6 Nramp1(G169) mice develop more severe, Th1-skewed, acute inflammatory responses to S. Typhimurium infection compared with Sv129S6 mice. Both strains are suitable model systems for studying inflammation in the context of adaptive immunity.

Hot topic: association of telomere length with age, herd, and culling in lactating Holsteins.

Telomere length variation may provide a quantitative measure of the effects of dairy management and selection practices on animal stress and welfare. The objective of this study was to evaluate the association between telomere length in Holstein cattle with age, herd, and survival. A multiplex quantitative PCR (qPCR) procedure was utilized to estimate telomere length for 201 Holstein cows from 10 herds following DNA extraction from blood. Primers were designed to amplify a 79-bp telomere product and a 144-bp product of a standard reference gene (ß-globin). Both primer sets were included in the same reaction well to enable the analysis of relative quantity (qT) of telomere product compared with ß-globin product. Triplicate samples were run for each cow, and mixed models were used to analyze the qPCR results. Younger cows were significantly associated with higher qT, and significant variation was observed among herds for qT. Cows with short telomeres were more likely to be culled in the subsequent year than cows with above-average telomere lengths. Multiplex qPCR provides a cost-effective method of assessing telomere length. Variation in telomere length might provide insights into how management practices and genetic selection influence cow stress and physiological responses to stress.

BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor alpha chimeric protein (PMLRARalpha) to block neutrophil differentiation and initiate acute leukemia.

The promyelocytic leukemia retinoic acid receptor alpha (PMLRARalpha) chimeric protein is associated with acute promyelocytic leukemia (APL). PMLRARalpha transgenic mice develop leukemia only after several months, suggesting that PMLRARalpha does not by itself confer a fully malignant phenotype. Suppression of apoptosis can have a central role in tumorigenesis; therefore, we assessed whether BCL-2 influenced the ability of PMLRARalpha to initiate leukemia. Evaluation of preleukemic animals showed that whereas PMLRARalpha alone modestly altered neutrophil maturation, the combination of PMLRARalpha and BCL-2 caused a marked accumulation of immature myeloid cells in bone marrow. Leukemias developed more rapidly in mice coexpressing PMLRARalpha and BCL-2 than in mice expressing PMLRARalpha alone, and all mice expressing both transgenes succumbed to leukemia by 7 mo. Although both preleukemic, doubly transgenic mice and leukemic animals had abundant promyelocytes in the bone marrow, only leukemic mice exhibited thrombocytopenia and dissemination of immature cells. Recurrent gain of chromosomes 7, 8, 10, and 15 and recurrent loss of chromosome 2 were identified in the leukemias. These chromosomal changes may be responsible for the suppression of normal hematopoiesis and dissemination characteristic of the acute leukemias. Our results indicate that genetic changes that inhibit apoptosis can cooperate with PMLRARalpha to initiate APL.

A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains.

A previously unknown redox cofactor has been identified in the active site of lysyl oxidase from the bovine aorta. Edman sequencing, mass spectrometry, ultraviolet-visible spectra, and resonance Raman studies showed that this cofactor is a quinone. Its structure is derived from the crosslinking of the epsilon-amino group of a peptidyl lysine with the modified side chain of a tyrosyl residue, and it has been designated lysine tyrosylquinone. This quinone appears to be the only example of a mammalian cofactor formed from the crosslinking of two amino acid side chains. This discovery expands the range of known quino-cofactor structures and has implications for the mechanism of their biogenesis.

Ganglioside modulation of lipopolysaccharide-initiated complement activation.

Highly purified preparations of mouse gangliosides have been demonstrated to bind to purified preparations of lipopolysaccharide (LPS). In some instances, the binding has been demonstrated to be dependent upon the presence of sialic acid in the ganglioside preparation. The binding of gangliosides to LPS from the deep rough Salmonella minnesota Re mutant has suggested that the interaction involves the lipid A-2-keto-3-deoxyoctulosanate region of the LPS macromolecule. The interaction between gangliosides and LPS has been demonstrated to result in an abrogation of lipid A dependent activation of the classical pathway of serum complement by Re LPS. Surprisingly, however, the presence of sialic acid containing glycolipids has been shown to enhance significantly the capacity of LPS to initiate activation of the alternative pathway of complement. These data suggest that sialic acid can enhance as well as inhibit the formation of a stable alternative-pathway C3 convertase.

Functional and structural diversity of the human Dickkopf gene family.

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.

Identification of the quinone cofactor in a lysyl oxidase from Pichia pastoris.

A copper amine oxidase from Pichia pastoris is the only known non-mammalian lysyl oxidase [Tur, S.S. and Lerch, K. (1988) FEBS Lett. 238, 74-76]. Recently, the cofactor in mammalian lysyl oxidase has been identified as a novel lysine tyrosylquinone moiety [Wang, S.X., Mure, M., Medzihradszky, K.F., Burlingame, A.L., Brown, D.E., Dooley, D.M., Smith, A.J., Kagan, H.M. and Klinman, J.P. (1996) Science 273, 1078-1084]. In order to identify the cofactor in P. pastoris lysyl oxidase, we have isolated the phenylhydrazone-derivative of the active-site peptide. This peptide has the active-site sequence conserved among topa quinone containing amine oxidases. The resonance Raman spectra of the phenylhydrazone derivatives of the enzyme, active-site peptide, and a topa quinone model compound are essentially identical. Collectively, these results establish that P. pastoris lysyl oxidase is a topa quinone enzyme.

A novel approach to the assessment of variations in the human platelet count.

This is the first report of a method to assess the significance of numerical changes in the platelet count based upon a result exceeding the normal intra-individual variation in platelet numbers. Serial platelet counts from 3,789 subjects were analysed to determine the intra-individual variation in platelet numbers. A platelet count difference of 98 x 10(9)/L in males was found to represent a change that would occur by chance in less than 1 in 1,000 platelet count determinations. Tables to determine the significance of platelet number variations, given N previous observations, are provided at two probability levels. The repeatability of the platelet count was calculated as 0.871 (males) and 0.849 (females) indicating that the heritability of platelet count is high and that the platelet count is predominantly genetically determined. A seasonal variation in platelet count was found with a 'winter' versus 'summer' difference of 5.10 X 10(9)/L (males) and 5.82 x 10(9)/L (females).

Fed-batch culture technology.

The use of fed-batch procedures offers distinct advantages over other modes of operation of bioreactors, and is a widely researched technique. These advantages are discussed; some uses of fed-batch procedures and the associated methods of modelling and control are reviewed.

Noninvasive detection of active pericardial bleeding using cardiac blood pool scintigraphy.

The diagnosis of active pericardial bleeding has traditionally depended on an invasive documentation by needle aspiration, angiography, or direct inspection. Blood pool scintigraphy performed in 2 patients just before and after the development of hemopericardium revealed unique images in which acute pericardial bleeding manifested itself by an additional blood pool adjacent to the cardiac chambers. With appropriate attention to technical factors, such distinctive images should be highly specific for active bleeding into the pericardial sac.

Regional distribution of pulmonary blood volume: an index of pulmonary capillary wedge pressure determined from blood pool scintigraphy.

Progressive redistribution of pulmonary blood flow to the lung apices occurs with increasing degrees of left ventricular failure, and correlates with increasing pulmonary capillary wedge pressure (PCWP). If similar changes in pulmonary blood volume (PBV) occur, then technetium-99m equilibrium blood pool scintigraphy, by assessing relative distribution of PBV, may allow prediction of PCWP. Therefore 30 patients being monitored with pulmonary artery balloon flotation catheters underwent imaging. PCWP was compared with the average radioactive count density arising from apical (A) and basal (B) regions of interest within the right lung, expressed as an A/B ratio. Correlation was strong for patients imaged erect, either posteriorly (r = 0.864, p = 0.001) or in the 45 degrees left anterior oblique position (r = 0.842, p = 0.001), and only slightly less impressive for patients imaged supine (r = 0.678, p = 0.001). Especially when imaging was performed with patients erect, an A/B ratio greater than unity identified with high sensitivity (100%) and specificity (83 to 88%) an abnormally elevated PCWP (greater than 12 mm Hg). Moreover, directional changes in the A/B ratio reflected concomitant changes in PCWP after intervention. Thus, analysis of lung A/B radioactive count ratios obtained by equilibrium blood pool scintigraphy may be used to evaluate PCWP.

Preparation of serum oxytocin and arginine vasopressin prior to radioimmunoassay: simultaneous extraction and separation on C18 Sep-Pak cartridges.

Oxytocin (OT) and arginine vasopressin (AVP) are often secreted in response to the same stimuli. The hormones are seldom assayed together, however, because of labor intensive sample preparation and the duplicate volumes required. A method has been developed for the simultaneous extraction and separation of OT and AVP from a single serum sample. The method is suited for sample preparation prior to radioimmunoassay (RIA) and reduces sample volume and processing time by 50%. Serum, supplemented with labeled and unlabeled OT and AVP, was adsorbed onto C18 (octadecasilyl-silica, ODS) Sep-Pak cartridges. After washing with phosphosaline and 3% aqueous acetone, OT was eluted with 98% aqueous acetone followed by AVP with 80% acidified (0.02 mol/L HCl) acetone. The recoveries, determined by radioactivity and RIA measurements, were 86 +/- 3% (OT) and 71 +/- 7% (AVP). Cross contamination was less than 10%. Sep-Paks extracted up to 100 pg/mL of the hormones from 10 mL of serum. The method was employed to measure OT and AVP in the pregnant ewe. Both hormones were elevated during salt-loading and dehydration and were decreased by carotid infusions of ethanol.

Absorbed dose calorimetry: bridge nulling and voltage analysis techniques.

A theoretical analysis is made of the conventional calorimeter approach utilizing a two-thermistor Wheatstone bridge--an approach which requires balancing the bridge by adjusting variable resistances. However, with the current availability of computers and digital precision measuring instruments, the conventional calorimeter approach can now be readily replaced by an automated, nonmechanical, electrical systems approach called differential calorimetry. The analytical theory outlining such an approach is also presented.

On the steady-state drift conditions of a water calorimeter in clinical megavoltage photon and electron beams.

We have investigated the characteristic behavior of a water calorimeter in a clinical environment where the temperature is not strictly controlled. Since the ambient temperature plays a crucial role in the successful operation of a calorimeter, its effects were closely monitored. The results were compared with theoretical predictions based on a simple model of heat transfer by conduction. They were found useful in analyzing the radiation dose measurements. In particular, a relationship between the absorbed dose and the pre- and postirradiation thermal drifts was examined. Finally, some suggestions are made concerning the desirable conditions under which a water calorimeter may be operated.

A thermoregulated enclosure for controlling thermal drift in a radiation calorimeter.

The ability to control thermal drifts is essential in operating a calorimeter. We investigated a thermal enclosure, which envelops the calorimeter with temperature-regulated air, thus thermally isolating the calorimeter from the room. The desired temperature in the enclosure is controlled by a control circuit and a thermoelectric device, which works as a Peltier effect heat pump. In this report, the details of the enclosure design and construction are presented with actual performance evaluations.

Purification and active-site characterization of equine plasma amine oxidase.

An improved purification scheme for an amine oxidase from equine plasma (EPAO), a nonruminant source, is described and the protein's active-site is characterized. EPAO is dimeric and contains one Type-2 Cu(II) ion per monomer. The EPAO Cu(II) site is spectroscopically very similar to the Cu(II) sites in other amine oxidases. Unlike the extensively investigated nonruminant amine oxidase from porcine plasma, EPAO does not display half-of-the-sites reactivity; titrations with p-nitrophenylhydrazine and phenylhydrazine indicate two active cofactors per dimer. This cofactor is determined to be the same as that of other copper-containing amine oxidases, 6-hydroxydopa quinone (topa quinone). Upon anaerobic reduction with substrate at ambient temperature, the EPR spectrum of EPAO exhibits a sharp signal at g congruent to 2, attributable to the topa semiquinone. Equine plasma amine oxidase possesses novel in vitro substrate specificity; while other mammalian amine oxidases oxidize norepinephrine only slowly or not at all, EPAO displays significant activity toward this biogenic amine.

Clenbuterol: a substitute for anabolic steroids?

Clenbuterol is a recently popular drug used by athletes in many sports for its purported anabolic effects and reduction of subcutaneous fat. It is a beta-2 (beta 2) agonist prescribed overseas as a bronchodilator, but not approved for use in this country. It is on the banned substance list of the United States Olympic Committee. To avoid any erosion of confidence, physicians caring for athletes need accurate information regarding clenbuterol. Such information is unavailable within the routine medical environs. A review of the literature of animal husbandry reveals that this drug, when administered in doses far greater than those required for bronchodilation, does indeed increase the deposition rate of lean mass and retard adipose gain. There are no human studies available. Animal studies were conducted on laboratory and slaughter stock. No investigation into long-term cardiovascular side effects has been undertaken. The rate of extrapolation from animal studies to unsupervised human usage is alarming. If this category of drugs does preserve lean mass in humans, there are legitimate medical applications. Trials of efficacy and safety are needed.

Physicochemical characteristics of patent blue violet dye.

Physicochemical data for patent blue violet dye (I) are reported. The pKa for protonation of the first diethylanilino group was 2.78 +/- 0.03. The absorptivity values calculated for a 1% (w/v) solution of previously dried I at pH 7.4 were 1650, 170, and 250 at 638, 412, and 309 nm, respectively. A table of wavelength maxima and observed solution color as a function of pH and Ho and five spectra of I at certain pH and Ho values are included. The solution chemistry of I is explained, and a scheme showing its two protonated carbonium ions and its triphenylcarbinol derivative is presented. The distribution coefficients of I in n-octanol or chloroform and pH 7.4 phosphate buffer systems were 0.013 and 0.12, respectively. The approximate solubilities at 25 degrees of I in six organic solvents and the solubility analysis of I in distilled water are reported. Results of the latter analysis suggest that I forms a lyotropic mesophase in high aqueous concentrations. Compound I is poorly lipid soluble. Samples of 1.000% I in 0.9% NaCl, formulated with and without 1% (v/v) benzyl alcohol and autoclaving, varied not more than 5% from the initial I content during storage in the dark and under constant fluorescent light at 25 +/- 5 degrees for 20 months. Data from the TLC of I in several eluents indicated a high degree of purity of the dye. The half-lives for the loss of color in 5 X 10(-4)% I solutions in potassium hydroxide solutions of pH 13.7, 12.7, 11.3, and 10.0 were 1.2 hr, 17.0 hr, 9.5 days, and 180 days, respectively. The fraction of I bound to 4% (w/v) human serum albumin at 37 degrees and pH 7.4 ranged from 0.05 to 0.83, corresponding to unbound I in the postdialysis concentration range of 1.7 X 10(-4) to 2.0% (w/v). A Scatchard plot of the albumin binding data of I revealed one high-affinity binding site, K = 6235 M-1, and five low-affinity sites, with average affinity constants of 33 M-1. The data support the fact that the spectrophotometric determination of I at 639 +/- 2 nm appears to comprise a stability-indicating assay.