Australian prehistory: new aspects of antiquity.

Human occupation of greater Australia occurred by 50,000 years ago, probably through deliberate voyaging by a small group of people. Later migrations, if any, are unlikely to have significantly changed the original genetic and cultural make-up While early Aborigines may have hunted extinct megafauna, the data do not support a rapid "Pleistocene overkill" hypothesis. Finally, aspects of Australian Aboriginal economy, especially plant utilization, and technology-the small tool tradition, ground stone hatchets and boomerangs-are of considerable antiquity and probably originated locally.

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.

Enhanced malignant progression of mouse skin tumors by the free-radical generator benzoyl peroxide.

Chemical carcinogenesis in mouse skin can be divided into the processes of initiation, promotion, and progression. The free-radical generator benzoyl peroxide is moderately active during the promotion stage. Repetitive treatment of mouse benign skin tumors (papillomas) with benzoyl peroxide (20 mg, twice weekly) increased the number of cumulative carcinomas per group by 325% and the number of keratoacanthomas by 44% compared to tumor-bearing Sencar mice treated with the promoter 12-O-tetradecanoylphorbol-13-acetate. The lack of increase in the number of cumulative papillomas per group due to benzoyl peroxide treatment suggests that benzoyl peroxide enhanced the progression of preexisting papillomas. The ability of benzoyl peroxide to enhance the progression of benign tumors to cancer should be considered when determining the human risk from exposure to this widely used chemical agent; in addition, biological assays specifically testing malignant progression may be essential and beneficial for determining an agent's carcinogenic risk.

Initiation, promotion and complete carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine or ethylnitrosourea in the Sencar mouse skin tumorigenesis model.

Five doses of either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ethylnitrosourea (ENU) were tested as complete carcinogens, tumor initiators and tumor promoters in the SENCAR skin tumorigenesis model. As tumor initiators, MNNG-induced papillomas at all doses tested, while ENU was active from 10-40 mumol. As complete carcinogens, MNNG from doses of 0.5-5.0 mumol and ENU from doses of 10 mumol-40 mumol were potent inducers of both papillomas and carcinomas indicating that these agents are active as both tumor initiators and tumor promoters.

Malignant progression of mouse skin papillomas treated with ethylnitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, or 12-O-tetradecanoylphorbol-13-acetate.

Mouse skin tumors were induced by a single topical application of 7,12-dimethylbenzanthracene (DMBA), followed by biweekly promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). After 20 weeks of promotion, mice were treated twice weekly for 2 weeks with either ethylnitrosourea (ENU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or TPA. Thereafter all groups were treated biweekly with TPA. The ENU-treated group had a higher percentage of animals with carcinomas and developed 217% more cumulative carcinomas per group than TPA-treated controls. The percentage of mice with carcinomas and the cumulative number of carcinomas per group in MNNG-treated mice was higher than TPA-treated controls but was less than ENU-treated mice. The ratio of cumulative carcinomas to cumulative papillomas in ENU treated, MNNG-treated and TPA-treated mice was 16%, 9% and 6%, respectively. Histological examination of tumors remaining at the termination of the experiment revealed the presence of keratoacanthomas, some of which stained positive for gamma-glutamyltransferase (GGT), in the ENU-treated and MNNG-treated, but not the TPA-treated groups. The fact that no new papillomas developed during the progression stage indicated that enhanced carcinogenesis resulted from the progression of pre-existing tumors. Enhanced progression of benign skin tumors in mice by only a few treatments of an agent may serve as a potential model for studies into the mechanisms and the inhibition of malignant progression. The model also allows for a comparison of the potency of agents in enhancing malignant progression.

Synthesis and radiolabeling of heterocyclic food mutagens.

The imidazoquinoline and imidazoquinoxaline food mutagens found in cooked meat are being synthesized by unambiguous methods that allow for the preparation of sufficient quantities of material for biological studies. These methods avoid difficult separations of regioisomeric mixtures of products and are designed to allow incorporation of specific high level tritium labeling.

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.

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.

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.

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.

Determination of the nuclear-magnetic-resonance solution structure of cardiotoxin CTX IIb from Naja mossambica mossambica.

The NMR structure of cardiotoxin CTX IIb from Naja mossambica mossambica in aqueous solution was determined from a total of 593 nuclear Overhauser enhancement distance constraints and 135 dihedral angle constraints, which were collected using two-dimensional homonuclear 1H-NMR experiments. Structure calculations were performed with the program DIANA, using the redundant dihedral angle constraints strategy for improved convergence, followed by restrained energy minimization with the program FANTOM and a modified version of the program AMBER. The CTX IIb structure is represented by a group of 20 conformers with an average root-mean-square deviation relative to the mean solution structure of 0.072 nm for the backbone atoms, and 0.116 nm for all heavy atoms. The molecular structure of CTX IIb is characterized by a three-stranded beta-sheet made up of residues 20-26, 32-39 and 48-54, and a two-stranded beta-sheet composed of residues 1-5 and 10-14. A cluster of four disulfide bonds, 3-21, 14-38, 42-53 and 54-59, form the core of the molecule and crosslink the individual polypeptide strands. The NMR structure is similar to the previously reported X-ray crystal structure of the cardiotoxin CTX VII4 from the same species. Differences between the two structures were noted in the tips of the two loops formed by residues 6-9 and 27-31, which connect the beta-strand 1-5 with 10-14, and 20-26 with 32-39, respectively. For these loops the NMR data also indicate significantly increased dynamic disorder in the solution structure. These observations are discussed with respect to earlier suggestions by others that these two loops are essential structural elements for function and specificity of a wide variety of homologous toxins.

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.

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.

Hunting income patterns among the Hadza: big game, common goods, foraging goals and the evolution of the human diet.

The assumption that large mammal hunting and scavenging are economically advantageous to hominid foragers is examined in the light of data collected among the Hadza of northern Tanzania. Hadza hunters disregard small prey in favour of larger forms (mean adult mass greater than or equal to 40 kg). Here we report experimental data showing that hunters would reduce their mean rates if they included small animals in the array they target. Still, daily variance in large animal hunting returns is high, and the risk of failure correspondingly great, significantly greater than that associated with small game hunting and trapping. Sharing large kills reduces the risk of meatless days for big game hunters, and obviates the problem of storing large amounts of meat. It may be unavoidable if large carcasses cannot be defended economically against the demands of other consumers. If so, then large prey are common goods. A hunter may gain no consumption advantage from his own big game acquisition efforts. We use Hadza data to model this 'collective action' problem, and find that an exclusive focus on large game with extensive sharing is not the optimal strategy for hunters concerned with maximizing their own chances of eating meat. Other explanations for the emergence and persistence of this practice must be considered.

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.

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.

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.