Cerebrotendinous xanthomatosis: case report with evidence of oxidative stress

Cerebrotendinous xanthomatosis is an autosomal recessive disorder of bile acid synthesis, characterized by mutation in the mitochondrial enzyme 27-hydroxylase that leads to an accumulation of cholestanol and cholesterol. Characterized clinically by premature bilateral cataracts, slowly progressive neurological deterioration with dementia, cerebellar and brainstem signs, peripheral neuropathy, and seizures, the disease presents pathologically with lipid granulomata with foamy histiocytes and cholesterol clefts. Replacement therapy with chenodeoxycholic acid slows progression of the disease but does not reverse neurological deficits. Here, we present the case of a 49-year-old woman diagnosed at autopsy with cerebrotendinous xanthomatosis, on the basis of bilateral Achilles tendon granulomas, and typical foamy histiocytic infiltration of the brain, most severe in the dentate nucleus, and a typical clinical presentation. To investigate the pathological manifestations of this disease further, we performed immunohistochemistry for N(epsilon)-(carboxymethyl)-lysine, an indicator of oxidative damage, and found strong labeling of cytoplasmic material within histiocytes. In summary, this case of undiagnosed cerebrotendinous xanthomatosis during life emphasizes the need for a greater awareness of the disease, and early diagnosis and treatment. Further, the involvement of oxidative stress in cerebrotendinous xanthomatosis indicates that combined therapy with chenodeoxycholic acid and antioxidants may improve clinical outcome.     

Risk assessment of transmission of bovine viral diarrhea virus (BVDV) in abattoir-derived in vitro produced embryos

Bovine virus diarrhea virus (BVDV) is a pathogen of the bovine reproductive system causing reduced conception rates, abortions and persistently infected calves. Most if not all strains of BVDV are transmissible by natural mating and AI. For international trade, it is recommended that in vitro fertilized embryos be washed according to the IETS Manual. However, BVDV may not be entirely washed out, resulting in possible transmission risks to recipients. Donor cows, donor bulls and biological agents are all possible sources of contamination. The process for producing in vitro produced (IVP) embryos is complex and non-standard, and some procedures can contribute to spread of BVDV to uninfected embryos. The structure of the zone pellucida (ZP) of IVP embryos permits adherence of BVDV to the ZP. To estimate the risk of producing infected recipients and persistently infected calves from abattoir-derived IVP embryos, a quantitative risk assessment model using Microsoft Excel and Palisade @Risk was developed. Assumptions simplified some of the complexities of the IVP process. Uncertainties due to incomplete or variable data were addressed by incorporating probability distributions in the model. Model variables included: disease prevalence; the number of donor cows slaughtered for ovaries; the number of oocytes collected, selected and cultured; the BVDV status of ovaries, semen, biological compounds and its behavior in the IVP embryo process. The model used the Monte Carlo method to simulate the IVP process. When co-culture cells derived from donor cows of unknown health status were used for in vitro culture (IVC), the probability of a recipient cow at risk of infection to BVDV per oocyte selected for IVP processing averaged 0.0006. However, when co-culture free from BVDV was used, the probability was 1.2 x 10(-5). Thus, for safe international trade in bovine IVP embryos (i.e. negligible risks of transmission of BVDV), co-culture cells, if used during IVC for producing IVP embryos, should be disease-free.     

Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to <Emphasis Type="Italic">Escherichia coli</Emphasis> Genome

Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets (FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P _trc and P _araB ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.     

Critical problems with extraction of ATP for bioluminescence assay of plankton biomass

Recovery of ATP by boiling tris extraction was 90–95 percent greater in 1 liter grab samples than in concentrated net samples. ATP losses were attributed to insulating effects promoted by accumulation of detritus on filters. A series of extractions over a concentration range of whole or size-segregated plankters and cultured algae was made to determine volume of water to be filtered for optimum extraction efficiency. Accuracy of ATP assays was optimized by: (i) using large diameter (i.e. 47 mm) acetate filters; (2) limiting sample volume filtered to 50 ml when particulate organic carbon (POC) exceeded 0.4 mg l^–1; and (3) performing extractions in boiling tris maintained initially on a laboratory hot plate at 400°C as opposed to hot water bath at 100°C.Additional problems were encountered in using published cellular carbon: ATP ratios for conversion of ATP data to biomass as carbon. Ratios of POC: ATP in cultures of sheathed blue-green algae reached 550 : i, while non-sheathed forms yielded ratios near values previously reported for plankton communities. Difficulties in applying a uniform conversion factor may be expected in plankton communities containing significant volumes of sheathed blue-greens.     

Calmodulin and myosin light-chain kinase of rabbit fast skeletal muscle.

1. It is confirmed that myosin light-chain kinase is a protein of mol.wt. about 80,000 that is inactive in the absence of calmodulin. 2. In the presence of 1 mol of calmodulin/mol of kinase 80-90% of the maximal activity is obtained. 3. Crude preparations of the whole light-chain fraction of rabbit fast-skeletal-muscle myosin contain enough calmodulin to activate the enzyme. A method for the preparation of calmodulin-free P light chain is described. 4. A procedure is described for the isolation of calmodulin from rabbit fast skeletal muscle. 5. Rabbit fast-skeletal-muscle calmodulin is indistinguishable from bovine brain calmodulin in its ability to activate myosin light-chain kinase. The other properties of these two proteins are also very similar. 6. Rabbit fast-skeletal-muscle troponin C was about 10% as effective as calmodulin as activator for myosin light-chain kinase. 7. By chromatography on a Sepharose-calmodulin affinity column evidence was obtained for the formation of a Ca2+-dependent complex between calmodulin and myosin light-chain kinase. 8. Troponin I from rabbit fast skeletal muscle and histone IIAS were phosphorylated by fully activated myosin light-chain kinase at about 1% of the rate of the P light chain.     

Properties and attempted culture of Pasteuria penetrans, a bacterial parasite of root-knot nematode (Meloidogyne javanica)

When they were subjected to a range of physical and chemical treatments, spores of Pasteuria penetrans showed properties similar to those of other endospore-forming bacteria. The spores did not take up some stains, were resistant to desiccation and sonication and showed extrusion of spore contents ('spore popping') on prolonged exposure to 0.1% KMnO₄ in 0.3 n HNO₃. Calcium and dipicolinic acid (DPA) were present at concentrations of 0.28% and 0.96% of the spore dry weight respectively, giving a Ca: DPA molar ratio of 1.2. The infectivity of P. penetrans spores was reduced to a low level after heating at 100°C for 5 min, but spore attachment was not markedly affected by heating at 100°C for 15 min. Evidence for the presence of catalase in P. penetrans spores was equivocal because the low levels of catalase activity observed in spore suspensions may have been due to contamination from catalase-positive nematode tissue. When P. penetrans spores were exposed to a range of substances known to act as germinants for spores of Bacillus spp., germination or loss of refractility was not observed by phase microscopy. In vitro culture of P. penetrans was attempted by inoculating either spores or vegetative mycelial bodies onto a diverse range of simple and complex media and incubating them in aerobic, reduced oxygen, anaerobic and increased CO₂ environments. Signs of spore germination or growth of vegetative stages were never observed.     

γ-VINYL GABA: EFFECTS OF CHRONIC ADMINISTRATION ON THE METABOLISM OF GABA AND OTHER AMINO COMPOUNDS IN RAT BRAIN

Rats were given γ-vinyl GABA (4-amino-hex-5-enoic acid), a new irreversible inhibitor of GABA aminotransferase (GABA-T), by daily subcutaneous injection (100mgkg) for 11 days. Amino acids were quantitated in the brains of the γ-vinyl GABA-treated and control animals 24 h after the last injection, and enzyme activities of GABA-T and glutamic acid decarboxylase (GAD) were measured. Chronic administration of γ-vinyl GABA produced a 150% increase in brain GABA content, along with marked increases in the contents of B-alanine and homocarnosine. Brain GABA-T activity was reduced by 26%, and GAD activity was reduced by 22%. In addition, γ-vinyl GABA caused a marked increase in hypotaurine content in rat brain, suggesting that it acts as an inhibitor of hypotaurine dehydrogenase, and it produced significant decreases in brain contents of glutamine and threonine. Although it is an effective GABA-T inhibitor, γ-vinyl GABA apparently affects several other brain enzymes as well, and it may not be an ideal drug for elevating brain GABA levels in man.     

The Bacillus subtilis DivIVA protein has a sporulation-specific proximity to Spo0J

The Bacillus subtilis DivIVA protein controls the positioning of the division site and the relocation of the chromosome during sporulation. By performing coimmunoprecipitation experiments, we demonstrated that a myc-DivIVA protein is in proximity to FtsZ and MinD during vegetative growth and Spo0J during the first 120 min of sporulation.     

Soluble derivatives of the beta amyloid protein precursor of Alzheimer's disease are labeled by antisera to the beta amyloid protein

The amyloid deposited in Alzheimer's disease (AD) is composed primarily of a 39-42 residue polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). In previous studies, we and others identified full-length, membrane-associated forms of the beta APP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble approximately 125 and approximately 105 kDa forms of the beta APP found in human cerebrospinal fluid are specifically labeled by several different antisera to the beta AP. This finding indicates that both soluble derivatives contain all or part of the beta AP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.