Linoleic acid in diets of mice increases total endocannabinoid levels in bowel and liver: modification by dietary glucose.

AIM: Linoleic acid (LA) is an essential fatty acid involved in the biosynthesis of arachidonic acid and prostaglandins. LA is known to induce obesity and insulin resistance. In this study, two concentrations of LA with or without added glucose (G) were fed to mice to investigate their effects on endocannabinoid (EC) biology. MATERIALS AND METHODS: Four groups of C57BL/6 mice were provided with diets containing 1% or 8% LA with or without added G (LAG) for 8 weeks. Body weights, food intake, circulating glucose and insulin levels were measured throughout the study. Following euthanasia, plasma, bowel and hepatic ECs, monoacylglycerol lipase and fatty acid amide hydroxylase protein levels (enzymes responsible for EC degradation) and transcriptional activity of PPARα in liver were quantified. Liver was probed for evidence of insulin receptor activity perturbation. RESULTS: Increasing dietary LA from 1% to 8% significantly increased circulating, small bowel and hepatic ECs. 1%LAG fed mice had lowest feed efficiency, and only liver levels of both ECs were reduced by addition of G. Addition of G to 1% LA diets resulted in elevated monoacylglycerol lipase and fatty acid amide hydroxylase protein levels (p < 0.001 and p < 0.001, respectively) in liver due to increased transcriptional activity of PPARα (p < 0.05). The reduced EC levels with addition of G also correlated with a measure of enhanced insulin action. CONCLUSION: In conclusion, body weight of mice is influenced by the source of calorie intake. Furthermore, tissue EC/g are dependent on tissue-specific synthesis and degradation that are modulated by dietary LA and G which also influence food efficiency, and down-stream insulin signalling pathways. The findings could potentially be useful information for weight management efforts in humans.

Male strategies and Plio-Pleistocene archaeology.

Archaeological data are frequently cited in support of the idea that big game hunting drove the evolution of early Homo, mainly through its role in offspring provisioning. This argument has been disputed on two grounds: (1) ethnographic observations on modern foragers show that although hunting may contribute a large fraction of the overall diet, it is an unreliable day-to-day food source, pursued more for status than subsistence; (2) archaeological evidence from the Plio-Pleistocene, coincident with the emergence of Homo can be read to reflect low-yield scavenging, not hunting. Our review of the archaeology yields results consistent with these critiques: (1) early humans acquired large-bodied ungulates primarily by aggressive scavenging, not hunting; (2) meat was consumed at or near the point of acquisition, not at home bases, as the hunting hypothesis requires; (3) carcasses were taken at highly variable rates and in varying degrees of completeness, making meat from big game an even less reliable food source than it is among modern foragers. Collectively, Plio-Pleistocene site location and assemblage composition are consistent with the hypothesis that large carcasses were taken not for purposes of provisioning, but in the context of competitive male displays. Even if meat were acquired more reliably than the archaeology indicates, its consumption cannot account for the significant changes in life history now seen to distinguish early humans from ancestral australopiths. The coincidence between the earliest dates for Homo ergaster and an increase in the archaeological visibility of meat eating that many find so provocative instead reflects: (1) changes in the structure of the environment that concentrated scavenging opportunities in space, making evidence of their pursuit more obvious to archaeologists; (2) H. ergaster's larger body size (itself a consequence of other factors), which improved its ability at interference competition.

Hadza meat sharing.

In most human foraging societies, the meat of large animals is widely shared. Many assume that people follow this practice because it helps to reduce the risk inherent in big game hunting. In principle, a hunter can offset the chance of many hungry days by exchanging some of the meat earned from a successful strike for shares in future kills made by other hunters. If hunting and its associated risks of failure have great antiquity, then meat sharing might have been the evolutionary foundation for many other distinctively human patterns of social exchange. Here we use previously unpublished data from the Tanzanian Hadza to test hypotheses drawn from a simple version of this argument. Results indicate that Hadza meat sharing does not fit the expectations of risk-reduction reciprocity. We comment on some variations of the "sharing as exchange" argument; then elaborate an alternative based partly on the observation that a successful hunter does not control the distribution of his kill. Instead of family provisioning, his goal may be to enhance his status as a desirable neighbor. If correct, this alternative argument has implications for the evolution of men's work.

Peptide aldehyde inhibitors of bacterial peptide deformylases.

Bacterial peptide deformylases (PDF, EC 3.5.1.27) are metalloenzymes that cleave the N-formyl groups from N-blocked methionine polypeptides. Peptide aldehydes containing a methional or norleucinal inhibited recombinant peptide deformylase from gram-negative Escherichia coli and gram-positive Bacillus subtilis. The most potent inhibitor was calpeptin, N-CBZ-Leu-norleucinal, which was a competitive inhibitor of the zinc-containing metalloenzymes, E. coli and B. subtilis PDF with Ki values of 26.0 and 55.6 microM, respectively. Cobalt-substituted E. coli and B. subtilis deformylases were also inhibited by these aldehydes with Ki values for calpeptin of 9.5 and 12.4 microM, respectively. Distinct spectral changes were observed upon binding of calpeptin to the Co(II)-deformylases, consistent with the noncovalent binding of the inhibitor rather than the formation of a covalent complex. In contrast, the chelator 1,10-phenanthroline caused the time-dependent inhibition of B. subtilis Co(II)-PDF activity with the loss of the active site metal. The fact that calpeptin was nearly equipotent against deformylases from both gram-negative and gram-positive bacterial sources lends further support to the idea that a single deformylase inhibitor might have broad-spectrum antibacterial activity.

A high quality nuclear magnetic resonance solution structure of peptide deformylase from Escherichia coli: application of an automated assignment strategy using GARANT.

The NMR structure of the peptide deformylase (PDF) (1-150) from Escherichia coli, which is an essential enzyme that removes the formyl group from nascent polypeptides and represents a potential target for drug discovery, was determined using 15N/13C doubly labeled protein. Nearly completely automated assignment routines were employed to assign three-dimensional triple resonance, 15N-resolved and 13C-resolved NOESY spectra using the program GARANT. This assignment strategy, demonstrated on a 17 kDa protein, is a significant advance in the automation of NMR data assignment and structure determination that will accelerate future work. A total of 2302 conformational constraints were collected as input for the distance geometry program DYANA. After restrained energy minimization with the program X-PLOR the 20 best conformers characterize a high quality structure with an average of 0.43 A for the root-mean-square deviation calculated from the backbone atoms N, C alpha and C', and 0.81 A for all heavy atoms of the individual conformers relative to the mean coordinates for residues 1 to 150. The globular fold of PDF contains two alpha-helices comprising residues 25-40, 125-138, six beta-strands 57-60, 70-77, 85-88, 98-101, 105-111, 117-123 and one 3(10) helix comprising residues 49-51. The C-terminal helix contains the HEXXH motif positioning a zinc ligand in a similar fashion to other metalloproteases, with the third ligand being cysteine and the fourth presumably a water. The three-dimensional structure of PDF affords insight into the substrate recognition and specificity for N-formylated over N-acetylated substrates and is compared to other PDF structures.

Grandmothering and the evolution of homo erectus.

Despite recent, compelling challenge, the evolution of Homo erectus is still commonly attributed to big game hunting and/or scavenging and family provisioning by men. Here we use a version of the "grandmother" hypothesis to develop an alternative scenario, that climate-driven adjustments in female foraging and food sharing practices, possibly involving tubers, favored significant changes in ancestral life history, morphology, and ecology leading to the appearance, spread and persistence of H. erectus. Available paleoclimatic, environmental, fossil and archaeological data are consistent with this proposition; avenues for further critical research are readily identified. This argument has important implications for widely-held ideas about the recent evolution of long human lifespans, the prevalence of male philopatry among ancestral hominids, and the catalytic role of big game hunting and scavenging in early human evolution.

Primary human hepatocyte culture for the study of HCV.

Research since 1983 has demonstrated that human hepatocytes can be isolated, cultured, and used for biological investigations, including studies of gene transcription and drug metabolism (1,2). In addition, the ability to cyropreserve hepatocytes has facilitated clinical research of hepatitic cell transplantation (3). We have used primary human heptocytes as host tissue for viral infection with hepatitis C. The availability of HCV-infected livers has also allowed for the culturing and analysis of HCV-positive cells. Our laboratory (4) and others (5) have confirmed the ability of these cells to display molecular markers of HCV replication. This chapter will review the basic steps of hepatocyte isolation and culturing and analysis for HCV by RT-PCR. We have also attempted to indicate alternative techniques that may be better suited to an individual investigator's needs.

Grandmothering, menopause, and the evolution of human life histories.

Long postmenopausal lifespans distinguish humans from all other primates. This pattern may have evolved with mother-child food sharing, a practice that allowed aging females to enhance their daughters' fertility, thereby increasing selection against senescence. Combined with Charnov's dimensionless assembly rules for mammalian life histories, this hypothesis also accounts for our late maturity, small size at weaning, and high fertility. It has implications for past human habitat choice and social organization and for ideas about the importance of extended learning and paternal provisioning in human evolution.

Predictable deuteration of recombinant proteins expressed in Escherichia coli.

Random deuteration of recombinant proteins in Escherichia coli is widely used for protein structure determination by nuclear magnetic resonance (NMR). It is desirable to predict accurately the degree of deuteration because each NMR experiment benefits from a different level of deuteration. The method described here which uses [2H]2O and glucose as the sole carbon and deuterium sources is an alternative for a previously published procedure using acetate and [2H]2O (Venter et al., J. Biomol. NMR 5, 339-344, 1995) and it is of advantage for proteins that do not express well using acetate. While the deuteration degree with acetate is approximately linear with the [2H]2O content in the medium, the use of glucose leads to deviations up to 19%, which is analyzed systematically here. With [2H]2O as the sole deuterium source 0-86% of the chemically nonexchangeable hydrogen atoms can be deuterated. Higher levels of deuteration require perdeuterated glucose in combination with [2H]2O. As an example, recombinant peptide deformylase from Bacillus subtilis was overexpressed, deuterated to various degrees, purified, and analyzed by mass spectrometry and NMR.

SCH 43478 and analogs: in vitro activity and in vivo efficacy of novel agents for herpesvirus type 2.

SCH 43478 and analogs are a class of non-nucleoside antiviral agents that have potent and selective activity against herpes simplex virus type 2 (HSV-2). The IC50 for these compounds in plaque reduction analysis using Vero cells ranges from 0.8 to 2.0 microg/ml. All compounds have a LC50 > 100 microg/ml in cytotoxicity analysis. Mechanism of action studies suggest that these molecules have an effect on the transactivation of viral immediate early (alpha) gene expression. Time of addition studies indicate that antiviral activity of these analogs is limited to the initial 2-3 h after infection and is not due to inhibition of viral adsorption or penetration. Analysis of HSV protein expression demonstrates that SCH 49286 inhibits the accumulation of viral immediate early (alpha) gene products. SCH 43478 demonstrates statistically significant efficacy (P < 0.05) in the guinea pig genital model of HSV infection. Following subcutaneous administration in a therapeutic treatment regimen, SCH 43478 (90 mg/kg/day) is efficacious in reducing the number and severity of lesions and the neurological complications of acute HSV infection. Thus, SCH 43478 and analogs are anti-herpesvirus agents with a unique mechanism of action.

SCH 48973: a potent, broad-spectrum, antienterovirus compound.

SCH 48973 is a novel molecule with potent, selective, antienterovirus activity. In assays of the cytopathic effect against five picornaviruses, SCH 48973 had antiviral activity (50% inhibitory concentrations [IC50s]) of 0.02 to 0.11 microg/ml, with no detectable cytotoxicity at 50 microg/ml. SCH 48973 inhibited 80% of 154 recent human enterovirus isolates at an IC50 of 0.9 microg/ml. The antiviral activity of SCH 48973 is derived from its specific interaction with viral capsid, as confirmed by competition binding studies. The affinity constant (Ki) for SCH 48973 binding to poliovirus was 8.85 x 10(-8) M. In kinetic studies, a maximum of approximately 44 molecules of SCH 48973 were bound to poliovirus capsid. SCH 48973 demonstrated efficacy in a murine poliovirus model of enterovirus disease. SCH 48973 increased the survival of infected mice when it was administered orally at dosages of 3 to 20 mg/kg of body weight/day. Oral administration of SCH 48973 also reduced viral titers in the brains of infected mice. On the basis of its in vitro and in vivo profiles, SCH 48973 represents a potential candidate for therapeutic intervention against enterovirus infections.

The behavioral ecology of modern hunter-gatherers, and human evolution.

Modern day hunter-gatherers are an obvious source of information about human life in the past. But can modern people really tell us anything about other hominids, those represented only in the fossil record? In a world of state governments and a global economy, can present-day foragers even tell us much about life before agriculture? Some behavioral ecologists think so. Their findings show (1) that foraging practices are closely related to the character and distribution of local resources, (2) that men, women and children react to foraging opportunities quite differently, and (3) that sex and age difference in these reactions have important social causes and consequences. Some results directly challenge long-held views about hunter-gatherer economics and social organization, and the scenarios of human evolution based on them.

Antipicornavirus activity of SCH 47802 and analogs: in vitro and in vivo studies.

SCH 47802 and its derivatives are potent inhibitors of enteroviruses in vitro. The IC50 for SCH 47802 ranges from 0.03 to 10 micrograms/ml when tested against a spectrum of enteroviruses in plaque reduction assays. The compounds have in vitro therapeutic indices of at least 81 based on viral cytopathic effect (CPE) assays. The in vitro activity of SCH 47802 translates into in vivo activity in the murine model of poliovirus encephalitis. In an oral dosing regimen, SCH 47802 protects mice from mortality at 60 mg/kg per day. Consistent with the in vivo efficacy, pharmacokinetic analyses after oral dosing with SCH 47802 demonstrate serum levels of the compound above the in vitro IC50 for poliovirus for at least 4 h. SCH 47802 and its active analogs stabilize poliovirus to thermal inactivation indicating that the compounds bind to the virus capsid. Mechanistic studies with poliovirus indicate that SCH 47802 acts early in viral infection. This series of molecules represents potential candidates for the treatment of human enterovirus infections.

Comparison of the structure of human recombinant short form stromelysin by multidimensional heteronuclear NMR and X-ray crystallography.

Stromelysin-1 is a matrix metalloprotease that has been implicated in a number of degenerative diseases. Here we present the refined NMR solution structure of the catalytic domain of stromelysin-1 complexed with a small inhibitor and compare it to the X-ray crystal structure of the same complex. The structures are similar in global fold and show an unusual bottomless S1' subsite. There are differences, however, in the least well defined regions, Phe83-Ile89, His224-Phe232 and Pro249- Pro250, reflecting the lack of NOE data and large B-factors. The region His224-Phe232 contains residues of the S1' subsite and, consequently, small differences are observed in this subsite. Hydrogen-bond data show that, in contrast to the crystal structure, the solution structure lacks a hydrogen bond between the amide of Tyr223 and the carbonyl of the P3' residue. Analysis of bound water shows two tightly bound water molecules both in the solution and the crystal structure; neither of these waters are in the inhibitor binding site.

The nuclear-magnetic-resonance solution structure of the mutant alpha-amylase inhibitor [R19L] Tendamistat and comparison with wild-type Tendamistat.

The recombinant mutant alpha-amylase inhibitor [R19L]Tendamistat, with Arg19 replaced by Leu, was prepared and its NMR solution structure determined. Based on complete sequence-specific 1H-NMR assignments, 845 nuclear Overhauser effect upper-distance constraints and 156 dihedral angle constraints were collected using two-dimensional homonuclear 1H-NMR experiments. The structure was calculated with the program DIANA, using the redundant dihedral angle constraints strategy for improved convergence. For restrained energy minimization, the programs FANTOM and AMBER were used. The wild-type NMR solution structure was similarly recalculated from the previously published input of conformational constraints [Kline, A., Braun, W. & Wüthrich, K. (1988) J. Mol. Biol. 204, 675-724]. For each protein a group of 20 conformers represents a well-defined solution structure, with average root-mean-square-distance values for the backbone atoms of the individual conformers relative to the mean coordinates of 50 pm. The two structures are nearly identical to each other and to the previously published Tendamistat structures in solution and in crystals. The only significant difference is strictly localized near the single amino acid substitution in the presumed active site -Trp18-Arg(Leu)-Tyr-, i.e. Leu19 and Tyr20 are more precisely defined in the solution structure of [R19L]Tendamistat than the corresponding residues Arg19 and Tyr20 in wild-type Tendamistat.