Effect of insulin and glucagon on accumulation of uroporphyrin and coproporphyrin from 5-aminolevulinate in hepatocyte cultures

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.     

Dissecting the binding energy epitope of a high-affinity variant of human growth hormone: cooperative and additive effects from combining mutations from independently selected phage display mutagenesis libraries

Phage display mutagenesis is a widely used approach to engineering novel protein properties and is especially powerful in probing structure-function relationships in molecular recognition processes. The relative contributions of additive and cooperative binding forces and the influence of conformational diversity in producing a novel protein-protein interface is investigated using as a model an ultra-high-affinity receptor binding variant of human growth hormone (hGHv) that has been previously affinity matured. The modular aspect of how the mutations were grouped in the phage display libraries and combined allowed for a systematic probing of the inherent functional cross-talk between the different secondary structure elements that make up the remodeled hGHv binding surface. We performed an alanine scanning analyses of 35 hGHv residues and determined the kinetics of each variant by surface plasmon resonance (SPR). This analysis showed that there is a significant difference between the additive and cooperative binding forces existing among the selected residues in each library module, and the binding advantage of these residues is maximized over the original wild-type residue when in the context of the other mutations in the library. The degree to which residues in a particular mutagenesis library display binding cooperativity characteristics is generally correlated with the conformational plasticity of the polypeptide chain. Additionally, these cooperativity effects change when the mutations from one library are combined with the mutations from one or several of the other separate libraries. This supports the idea that significant functional cross-talk exists between the combined library modules that can affect the binding energetics of individual residues over a large distance.     

Prepartum plasma estradiol and postpartum cortisol, but not oxytocin, are associated with interindividual and breed differences in the expression of maternal behaviour in sheep

Consistent, individual differences in the expression of maternal behaviour have been described in several species including the sheep. The neuroendocrine mechanisms underlying the onset of maternal behaviour in the sheep have been described, although the relationship between hormonal events and individual differences in behaviour has yet to be determined. In this study, we examined whether the individual differences in plasma estradiol, progesterone, oxytocin and cortisol concentrations were related to observed individual and breed differences in maternal behaviours in two breeds of sheep (Scottish Blackface and Suffolk) known to differ in maternal behavioural expression. Maternal estradiol concentration increased rapidly before parturition and was higher in Blackface ewes than Suffolk ewes. Plasma progesterone declined before parturition and was higher in Suffolk than Blackface ewes. Prepartum estradiol, but not progesterone, was related to individual differences in maternal grooming of the lamb. Plasma oxytocin did not differ between breeds in late gestation. There was a tendency for oxytocin to be higher in Blackface than Suffolk ewes immediately after birth. However, there were no significant relationships between prepartum or postpartum oxytocin and any maternal behaviours. Plasma cortisol was higher in Blackface than Suffolk ewes in the last days of pregnancy but rose in both breeds over the last 24 h before parturition and did not differ at delivery. Cortisol peripartum was negatively related to individual differences in maternal affiliative behaviours. These data suggest that estradiol, and potentially cortisol, may mediate individual differences in maternal behaviour in sheep.     

Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism

The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice.     

The effects of a nap opportunity in quiet and noisy environments on driving performance

While napping has previously been shown to alleviate the effects of sleep loss, before advocating the use of naps in transport accident campaigns it is necessary to consider whether a nap opportunity in a noisy uncomfortable environment can produce the same benefits as a nap opportunity in conditions that are conducive to sleep. To examine this, eight participants drove a driving simulator for 50 min at 11:00 h on three different test days. The simulator used has previously been found to be sensitive to the effects of sleep loss, alcohol consumption, and time of day. All three sessions were conducted after one night of sleep loss. Prior to driving during each session the participants either had a 60 min nap opportunity in a quiet or noisy environment, or no nap opportunity. Driving performance and reaction time while driving were measured, as were subjective sleepiness and ratings of sleep quality. No significant benefits of nap opportunities on driving performance were found. Levels of subjective sleepiness were not affected by the nap opportunity condition; however, sleep was rated as more refreshing and restful after a nap in a quiet environment compared to noisy environment. The measures of effect size reported suggest further research is required to unequivocally test the effects of nap opportunities on driving ability.     

Dam inactivation in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>: prevalence among diverse hyperinvasive lineages

Background  

DNA adenine methyltransferase (Dam) activity is absent in many, but not all, disease isolates of Neisseria meningitidis, as a consequence of the insertion of a restriction endonuclease-encoding gene, the 'dam replacing gene' (drg) at the dam locus. Here, we report the results of a survey to assess the prevalence of drg in a globally representative panel of disease-associated meningococci.     

The human pathogen Leishmania donovani secretes a histidine acid phosphatase activity that is resistant to proteolytic degradation

Promastigotes of all pathogenic Leishmania species secrete acid phosphatase (SAcP) activity during their growth in vitro. It has been suggested that this enzyme may play a role in the survival of the parasite within its sandfly-vector host. To carry out such functions, SAcP would have to be relatively resistant to endogenous sandfly gut-proteases. Therefore, the current study was undertaken to ascertain whether L. donovani SAcP activity was affected by treatment with various proteases. Native L. donovani SAcP was treated with a variety of serine-, thiol-, metallo- and mixed-proteases and subsequently assayed for enzymatic activity. Of the eleven proteases tested, only bromelain and subtilisin treatments caused a pronounced reduction in SAcP activity. Treatment of SAcP with seven out of the remaining nine proteases, resulted in an overall enhancement in SAcP enzymatic activity ranging from approximately 10% (e.g. with trypsin) to > or = 90% (e.g. with ficin). The resistance of the Leishmania SAcP to various proteases may prolong its functional life within the sandfly gut and help to facilitate parasite infection in this host.     

Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.