Analysis of Carnosine, Homocarnosine, and Other Histidyl Derivatives in Rat Brain

Isocratic reverse-phase analytical HPLC has been used to examine naturally occurring imidazoles of rat brain. Elution of brain extracts with a phosphate buffer mobile phase from columns packed with Hypersil ODS (5 microns) resulted in good separation of the well-documented brain imidazole-containing dipeptides carnosine and homocarnosine. Measured concentrations corresponded to published values. Several further peaks observed had properties consistent with those of N-acetyl derivatives of compounds related to carnosine and homocarnosine. N-Acetyl forms not commercially available were prepared and their identities verified by nuclear magnetic resonance spectroscopy. A number of these had chromatographic properties identical to those of compounds in brain extracts. Fractions corresponding to some of the peaks were examined using staining systems specific for certain chemical features and compared with results obtained for commercial or synthetic standards. The results of these tests supported the chromatographic data. Thus, chromatographic and microchemical evidence is presented for the existence of N-acetyl forms of histidine, 1-methylhistidine, carnosine, anserine, and homocarnosine in rat brain.     

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP. When guanosine and adenine were added together with tiazofurin, the differentiation of K562 was prevented, the concentration of diacylglycerol was maintained at control values, and the reduction in the concentration of cGMP was partially prevented. Other inducers of differentiation which acted by different mechanisms, caused similar changes in the concentrations of diacylglycerol and cGMP.     

Use of in vivo and in vitro assays for the characterization of mammalian excision repair and isolation of repair proteins

Elucidation of the molecular mechanism of mammalian nucleotide excision repair requires the availability of purified proteins, DNA substrates with defined lesions and suitable repair assays. Repair assays introduced in recent years vary from testing individual steps and successions of steps in vitro to systems that closely reflect the entire process in vivo. In the first part of this review, an in vivo microinjection system is discussed. The second part of the article reviews an in vitro system for study of repair synthesis promoted by cell extracts. Both systems can be utilized as assays during the purification of protein factors that complement repair-defective xeroderma pigmentosum cells. The effect of purified repair proteins from other organisms on mammalian repair is also considered.     

Factors regulating tropane-alkaloid production in a transformed root culture of a Datura candida × D. aurea hybrid

Using in combination an analysis of (i) the levels of enzyme activities present, (ii) the pool sizes of metabolic intermediates and end products and (iii) the effects of feeding metabolic intermediates, the limitations ? flux into tropane alkaloids in a Datura root culture have been examined. This culture, produced by transforming a Datura candida × D. aurea hybrid with Agrobacterium rhizogenes, is found to be highly competent in the biosynthesis of both hyoscyamine and scopolamine as well as a wide range of other hygrine-derived alkaloids. It has been found that, of six enzymes which are involved in this pathway, the two initial activities, ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), are present at potentially flux-limiting levels, in contrast to those other enzymes assayed which act further down the pathway. An additional limitation to flux, involving the supply of activated acids for condensation with tropine to form the identified tropoyl and tigloyl derivatives, is also indicated from the observed effect of feeding free acids. The relative contribution to flux limitation caused by these two interacting phenomena is inferred from an analysis of the changing relative levels of metabolic intermediates and end products as cultures mature.     

Inhibition of duck hepatitis B virus replication by purine 2',3'-dideoxynucleosides

Primary hepatocyte cultures from duck hepatitis B virus (DHBV) infected ducklings were used to evaluate the antiviral activity of purine and pyrimidine 2',3'-dideoxynucleosides. The purine 2',3'-dideoxynucleosides were very effective inhibitors of hepadnavirus replication, whereas the pyrimidine dideoxynucleosides were not. 2',3'-Dideoxyguanosine and 2,6-diaminopurine 2',3'-dideoxyriboside (ddDAPR) were the most effective antiviral agents studied. ddDAPR given intramuscularly twice daily at 10 mg/kg rapidly cleared DHBV-DNA from the sera of persistently infected ducklings but this effect was not permanent.     

Mesozooplankton of the Kosi Bay lakes,South Africa

The Kosi coastal lake system, a chain of four interconnected basins, is located in the subtropical north-eastern corner of South Africa. Little information is available on zooplankton of the system and the main aim of this study is to report on zooplankton samples collected during 2002 and 2003. The set of samples consists of seasonal, subsurface mesozooplankton samples that were collected during nighttime in each of the lakes. A well-developed salinity gradient was evident along the interconnected lakes in the subsurface water during all seasons, ranging from freshwater in the upper lake Amanzamnyama to a maximum of 22 recorded in Lake Makhawulani. The zooplankton community structures of the lakes reflected the salinity gradient of the system, with some coastal marine taxa recorded in the lakes closer to the mouth and only freshwater taxa recorded in Lake Amanzamnyama. Mesozooplankton diversity and abundance were relatively low compared to other estuarine systems along the eastern coast of South Africa. The dominant taxa were calanoid copepods Acartiella natalensis and Pseudodiaptomus stuhlmanni and the mysid Mesopodopsis africana in the lower lakes, whereas cyclopoids Mesocyclops sp. and Thermocyclops sp. dominated the freshwater lake Amanzamnyama.     

Stability of emulsions stabilised by two physiological surfactants: L-alpha-phosphatidylcholine and sodium taurocholate

The emulsion phase formed within the stomach and duodenum during digestion of a fatty meal has been modelled using two physiological surfactants, the phospholipid L-alpha-phosphatidylcholine (PC) and the bile salt sodium taurocholate (NaT). Upon dilution of the phospholipid stabilised emulsions with a solution of NaT the bile salt became incorporated into the oil/water interface imparting a negative charge to the droplet surface. The magnitude of the droplet microelectrophoretic mobility for the mixed PC and NaT system was 47% of that found for emulsion droplets stabilised by NaT alone. But the electrostatic repulsion between droplets was not sufficient to account for the observed improvement in emulsion stability to coalescence. It is suggested that a residual liquid crystalline phospholipid interface is present imparting a significant steric component to the stabilisation of the emulsions droplets.     

An in vivo 15 N NMR study of agropine synthesis in transformed root cultures of Nicotiana tabacum

The biosynthesis of the opine agropine in transformed Nicotiana tabacum L. root cultures was studied using in vivo 15 N nuclear magnetic resonance (NMR) spectroscopy. Roots were incubated with [ 15 N]ammonium and/or [ 15 N]nitrate, and the incorporation of the label into agropine, conjugated polyamines, Γ -aminobutyric acid (GABA), glutamate (Glu), glutamine (Gln) and nicotine was monitored by NMR. The largest labelled pool was agropine in cells grown on a 15 N-enriched medium and its synthesis was maintained, to the detriment of the Gln pool, under conditions of nitrogen (N) starvation. These observations indicate that the synthesis of agropine is not tightly regulated and that it represents a significant sink for carbon (C) and N in the plant. The addition of α -naphthaleneacetic acid (NAA) and kinetin to the growth medium caused de-differentiation of the root cultures and perturbation of secondary N metabolism. The amount of agropine relative to Gln increased but the total accumulation of agropine decreased, in part because of the increase in the synthesis of conjugated polyamines. These results show that agropine biosynthesis perturbs both primary and secondary N metabolism, and that the perturbation differs according to the culture conditions and the imposed stress.     

Lacustrine growth of juvenile pink salmon and a comparison with sympatric sockeye salmon

The growth rates of naturally sympatric juvenile pink Oncorhynchus gorbuscha and sockeye Oncorhynchus nerka salmon were compared in a common lacustrine environment in south‐west Alsaka, an unusual opportunity given the normal disparity in freshwater residence time of these two species. Fork length ( L _F) frequency distributions of juvenile pink salmon caught in the lake during the summer in 1991 and 1999–2003 indicated a growth rate of 0·54 mm day⁻¹, 54% greater than the estimated growth rate of juvenile sockeye salmon sampled from 1958 to 2003 (0·35 mm day⁻¹). Examination of daily growth rings on otoliths indicated that pink salmon in Lake Aleknagik grew an average of 1·34 mm day⁻¹ in 2003 but sockeye salmon grew only 0·63 mm day⁻¹(average specific growth rates were 3·0 and 1·8% day⁻¹, respectively). Pink salmon increased from c . 32 mm L _F and 0·2 g at emergence to 78 mm L _F and 3·0 g within 3–4 weeks. After experiencing these rapid growth rates, the pink salmon appeared to leave the lake by late July in most years. The diets of pink and sockeye salmon in the littoral zone of the lake were very similar; >80% of the stomach contents consisted of adult and pupal insects and the remainder was zooplankton. This high degree of diet overlap suggested that the observed differences in growth rate were not attributable to variation in prey composition.     

Inactivation of Prions and Amyloid Seeds with Hypochlorous Acid

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥10³- to 10⁶-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~10^5.75-fold and >10⁴-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids.