Effect of various chloride salts on the utilization of phosphorus, calcium, and magnesium

Only part of the effect of dietary protein on urinary calcium excretion can be ascribed to sulfur amino acids. We hypothesized that chloride, another factor often associated with isolated proteins, and another amino acid, lysine, affect utilization of calcium. The effects of supplemental dietary chloride, inorganic or organic, on calcium, phosphorus, and magnesium utilization were studied in two rat studies. Weanling Sprague-Dawley rats were fed semi-purified diets that contained moderate (1.8 mg Cl/g diet) or supplemental (15.5 mg Cl/g diet) chloride as sodium chloride, potassium chloride, or lysine monohydrochloride with or without calcium carbonate for 56 or 119 days. Rats fed supplemental sodium chloride or potassium chloride had higher urinary phosphorus excretion, more efficient phosphorus absorption, but unchanged tissue phosphorus levels after 7 and 16 weeks of dietary treatment as compared to rats fed moderate chloride. Rats fed supplemental sodium chloride or potassium chloride excreted more calcium in urine at 7 weeks and absorbed calcium less efficiently at 16 weeks. Tissue calcium concentrations were unaffected, but total tibia magnesium and plasma magnesium concentrations were lower in rats fed supplemental sodium chloride or potassium chloride than those fed moderate chloride. Lysine chloride with or without additional calcium elevated urinary calcium excretion even more than sodium chloride and potassium chloride ingestion. Rats fed lysine chloride with supplemental calcium had smaller apparent absorption and urinary losses of phosphorus and magnesium after 16 weeks and lower tibia and plasma magnesium concentrations than rats fed lysine chloride.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5’-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate. The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments. Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and “holey” native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane. The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.     

Ancient metalloenzymes as possible markers in molecular archaeology

The successful preparation of an active remnant of Cu,Zn-superoxide dismutase from mummified brain tissue stimulated the isolation of both biochemically and immunologically active alkaline Zn2Mg-phosphatase from antique bone samples of different archaeological sites and age. In particular, specimens from pharaonic Egypt being up to 4000 years of age were used. Gel filtration, ion exchange and affinity chromatographies were employed to optimise the preparation of the ancient enzyme. Compared to the specific activity of alkaline phosphatase from modern autopsy some 50% for a Ptolemaic and 10% for the Old Kingdom enzyme was detectable. The possibility of microbial contamination was checked by employing specific monoclonal antibodies directed against the human bone enzyme. Fortunately, ubiquitously present specified microorganisms on the respective ancient bones did not cross-react with these antibodies while the ancient metalloenzyme reacted with high specificity. Alkaline phosphatase mimicks could be excluded as in the presence of the inhibitors 1,10-phenanthroline and L-homoarginine the enzyme activity was diminished. The presence of ortho-vanadate as a substrate analogon abolished the catalytic function of the enzyme. Likewise, heating to 100 degrees C and replacement of zinc(II) by cadmium(II) resulted in a dramatic loss of activity. In conclusion, alkaline phosphatase appears to be a useful marker enzyme in molecular archaeology.     

Alopecia areata in a rhesus monkey (Macaca mulatta)

BACKGROUND: A 14-year-old female rhesus monkey (Macaca mulatta) of Chinese origin has been suffering from alopecia universalis since childhood. METHODS: Recently, the health status of the animal was recorded comprehensively by detailed clinical examination including hematology and serology supplemented by histological and immunohistochemical investigations of skin biopsies and molecular biological techniques to clarify the causes of the persistent hair loss. RESULTS AND CONCLUSIONS: The hairless gene (hr) nonsense mutation was ruled out by polymerase chain reaction and by sequencing of the corresponding gene. Histological examinations revealed a prominent chronic lymphocytic perifolliculitis and folliculitis affecting anagen stage hair follicles as well as miniaturized hair follicles. Immunohistochemistry using the antibodies CD3, CD20 and CD4 confirmed the diagnosis of a T-cell-mediated autoimmune disease resembling alopecia areata universalis in humans.     

Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography

Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.     

Invasive aspergillosis in a putty-nosed monkey (Cercopithecus nictitans) with adrenocortical Cushing's syndrome

Background  An 18‐year‐old captive female putty‐nosed‐monkey (Cercopithecus nictitans) with a history of long‐term infertility and hyperglucocorticism was euthanized because of perforating thoracic trauma induced by group members and subsequent development of neurological signs. Methods  Complete necropsy and histopathological examination of formalin‐fixed tissue samples was carried out. Results  The monkey showed invasive pulmonary and cerebral infection with Aspergillus fumigatus together with adrenocortical neoplasia and signs of Cushing’s syndrome, such as alopecia with atrophic skin changes, evidence for diabetes mellitus and marked immunosuppression. Conclusions  Spontaneous endocrinopathies are rarely described in non‐human primates. Here we report the first case of spontaneous adrenocortical hyperglucocorticism predisposing to systemic aspergillosis in a putty‐nosed monkey.