The use of ash at Late Lower Paleolithic Qesem Cave, Israel-An integrated study of use-wear and residue analysis.

Employing an integrated approach to investigate the use of Late Lower Paleolithic flint tools found at the site of Qesem Cave (Israel), we revealed a particular trace pattern related to the employment of ashes at the site. Using a designated collection of replica items and combining use-wear and residue (morphological analysis, FTIR, SEM-EDX) analyses, we revealed the intentional use of ashes in preserving foods for delayed consumption as well as hide for delayed processing. Our interpretation, we believe is the most plausible one since we were able to delineate the specific use-wear fingerprints of the intentional use of ashes for such purposes, suggesting that our approach might be useful for the recognition of other similar functional-behavioral patterns. Lastly, in support of previous findings at Qesem Cave, our current findings present evidence for the processing of organic matters intentionally mixed with ash, leading us to suggest that the inhabitants of Qesem Cave were proficient not only in the habitual use of fire but also of its main by-product, ash. Hence, we call for a reassessment of the timeline currently assigned to hominins' utilization of ash for storing and processing vegetal foods and hide.

Bone marrow storage and delayed consumption at Middle Pleistocene Qesem Cave, Israel (420 to 200 ka).

Bone marrow and grease constitute an important source of nutrition and have attracted the attention of human groups since prehistoric times. Marrow consumption has been linked to immediate consumption following the procurement and removal of soft tissues. Here, we present the earliest evidence for storage and delayed consumption of bone marrow at Qesem Cave, Israel (~420 to 200 ka). By using experimental series controlling exposure time and environmental parameters, combined with chemical analyses, we evaluated bone marrow preservation. The combination of archaeological and experimental results allowed us to isolate specific marks linked to dry skin removal and determine a low rate of marrow fat degradation of up to 9 weeks of exposure. This is the earliest evidence of such previously unidentified behavior, and it offers insights into the socio-economy of the human groups who lived at Qesem and may mark a threshold to new modes of Palaeolithic human adaptation.

Experimental growing of wild pea in Israel and its bearing on Near Eastern plant domestication.

BACKGROUND AND AIMS: The wild progenitors of the Near Eastern legumes have low germination rates mediated by hardseededness. Hence it was argued that cultivation of these wild legumes would probably result in no yield gain. Based on the meagre natural yield of wild lentil and its poor germination, it was suggested that wild Near Eastern grain legumes were unlikely to have been adopted for cultivation unless freely germinating types were available for the incipient farmers. Unlike wild cereals, data from experimental cultivation of wild legumes are lacking. METHODS: Replicated nurseries of wild pea (Pisum elatius, P. humile and P. fulvum) were sown during 2007-2010 in the Mediterranean district of Israel. To assess the effect of hardseededness on the yield potential, seeds of the wild species were either subjected to scarification (to ensure germination) or left intact, and compared with domesticated controls. KEY RESULTS: Sowing intact wild pea seeds mostly resulted in net yield loss due to poor establishment caused by wild-type low germination rates, while ensuring crop establishment by scarification resulted in net, although modest, yield gain, despite considerable losses due to pod dehiscence. Harvest efficiency of the wild pea plots was significantly higher (2-5 kg seeds h(-1)) compared with foraging efficiency in wild pea populations (ranging from a few grams to 0·6 kg h(-1)). CONCLUSIONS: Germination and yield data from 'cultivation' of wild pea suggest that Near Eastern legumes are unlikely to have been domesticated via a protracted process. Put differently, the agronomic implications of the hardseededness of wild legumes are incompatible with a millennia-long scenario of unconscious selection processes leading to 'full' domestication. This is because net yield loss in cultivation attempts is most likely to have resulted in abandonment of the respective species within a short time frame, rather than perpetual unprofitable cultivation for several centuries or millennia.

Microfaunal remains at Middle Pleistocene Qesem Cave, Israel: Preliminary results on small vertebrates, environment and biostratigraphy.

Preliminary results of the investigation of the microfauna at the Acheulo-Yabrudian Middle Pleistocene site of Qesem Cave, Israel, are presented. Thus far the assemblage includes ca. 10,000 bone and tooth fragments, of which 50% could be identified to the generic and some hundreds to the species level. Based on the current material, the fauna includes the following squamate reptiles: Laudakia sp., Chamaeleo sp., Gekkonidae indet., Lacertidae indet., Scincidae indet., Pseudopus sp., Varanus sp., Colubroidea indet. (at least three species) and micromammals: Suncus etruscus, Crocidura cf. leucodon, Crocidurinae indet. (large form), Chiroptera indet., Sciurus cf. anomalus, Cricetulus cf. migratorius, Microtus guentheri, Nannospalax ehrenbergi, Dipodillus cf. dasyurus, Meriones cf. tristrami, Gerbillidae indet., Mus cf. musculus, Apodemus cf. flavicollis. These results suggest that the fauna includes only taxa that occur recently in the territory of Israel. The ecological preferences of the nearest living relatives of the recorded taxa allow us to infer a paleoenvironment with a mosaic of open and woodland habitats. However, comparing the lower with the upper levels of the microfauna-bearing profile, a slight shift towards more wooded conditions might be detectable. Biostratigraphical inferences from the recorded micromammal taxa cover a rather wide age range, whereas the radiometric (U-series and preliminary TL) dating enable a provisionally estimated date for the microfauna-bearing levels at 360-300 ka. Detailed morphometric comparisons with material from other sites in the region are necessary and may yet provide further insights.

Flint mining in prehistory recorded by in situ-produced cosmogenic 10Be.

The development of mining to acquire the best raw materials for producing stone tools represents a breakthrough in human technological and intellectual development. We present a new approach to studying the history of flint mining, using in situ-produced cosmogenic 10Be concentrations. We show that the raw material used to manufacture flint artifacts approximately 300,000 years old from Qesem Cave (Israel) was most likely surface-collected or obtained from shallow quarries, whereas artifacts of the same period from Tabun Cave (Israel) were made of flint originating from layers 2 or more meters deep, possibly mined or quarried by humans.

Uranium series dates from Qesem Cave, Israel, and the end of the Lower Palaeolithic.

Israel is part of a geographical 'out of Africa' corridor for human dispersals. An important event in these dispersals was the possible arrival of anatomically modern humans in the Levant during the late Middle Pleistocene. In the Levant the Lower Palaeolithic ends with the Acheulo-Yabrudian complex, characterized by technological developments, including the introduction of technological innovations such as the systematic production of blades and the disappearance of hand-axes. These reflect new human perceptions and capabilities in lithic technology and tool function. Qesem Cave, discovered in 2000, has a rich, well-preserved Acheulo-Yabrudian deposit holding great promise for providing new insights into the period. Here we report the dates of this deposit obtained by uranium isotopic series on associated speleothems and their implications. The results shed light on the temporal range of the Acheulo-Yabrudian and the end of the Lower Palaeolithic, suggesting a long cultural phase between the Lower Palaeolithic Acheulian and the Middle Palaeolithic Mousterian phases, starting before 382 kyr ago and ending at about 200 kyr ago.

Charge displacements in interfacial layers containing reaction centers.

Reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides were oriented in phospholipid interfacial layers adsorbed to a Teflon film separating two electrolyte-filled compartments of a Teflon cell. Light-induced voltage changes were measured as a function of time across electrodes immersed in the cell compartments. The experimental system is characterized both experimentally and theoretically to relate the measured signals to the light-induced displacement currents in the reaction centers. Mathematical relations between the measured signals and the distances and geometries of the charge-transfer reactions are derived. At pH 8.0 the reaction centers were found to be oriented with approximately 60% of the population oriented with the donor facing the aqueous phase. The density of the reaction centers in the layer was approximately 10(11) cm-2, which is close to that found in the native system. Reconstitution of the secondary quinone, QB, in 90% of the RCs was achieved with an approximately 100-fold excess of ubiquinone in the vesicle preparation.

Quantitation of metabolic compartmentation in hyperammonemic brain by natural abundance 13C-NMR detection of 13C-15N coupling patterns and isotopic shifts.

In the present study, the removal of cerebral ammonia by glutamine synthetase (GS) and by reductive amination of 2-oxoglutarate by glutamate dehydrogenase in the presence of an amino donor group, was determined in hyperammonemic rabbit brains. The 15N enrichments of brain metabolite alpha-amino and amide positions of glutamine, glutamate, and alanine were determined by the indirect detection of 15N-labeled compounds of the 13C-15N spin coupling patterns of natural abundance 13C-NMR spectra. The 13C-NMR spectra of brain extracts were obtained from rabbits infused with 15NH4Cl with or without intraperitoneal infusion of the GS inhibitor, L-methionine DL-sulfoximine, in a reasonable acquisition time period. When 15NH4Cl was infused, [5-15N]glutamine and [2-15N]glutamine concentrations reached 5.2 mumol/100 mg protein and 3.6 mumol/100 mg protein, respectively, which indicates the relatively high activity of reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. The low concentration of [2-15N]glutamate, which is about 30% of that of [2-15N]glutamine obtained in this study, suggests that very little glutamine serves as a precursor of neuronal glutamate. When GS was inhibited by L-methionine DL-sulfoximine, a flux of 15NH4+ via the residual activity of GS was accompanied by an apparent increase of [2-15N]glutamate and [15N]alanine concentrations (2.9 mumol/100 mg protein and 1.8 mumol/100 mg protein, respectively). These findings and those obtained from 13C-13C isotopomer analysis (Lapidot and Gopher, 1994b) suggest that astrocytic 2-oxoglutarate is partially utilized (together with an amino group donor) as a precursor for neuronal glutamate in the hyperammonemic brain when GS is inhibited. This process can partly replace GS activity in metabolizing ammonia in the hyperammonemic rabbit brain.

Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors.

In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB1 or CB2--the two cannabinoid receptors identified thus far--with Ki values of 472 +/- 55 and 1400 +/- 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of delta 9-tetrahydrocannabinol (delta 9-THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of delta 9-THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than delta 9-THC.

Omeprazole has no effect on acetaminophen kinetics in the rat.

We investigated in the rat the effect of the H+/H(+)-ATPase inhibitor, omeprazole, on the kinetics of acetaminophen. Two groups of rats were treated with either omeprazole 50 mg/kg/day or the vehicle for 7 days. On day seven, the pharmacokinetic parameters of acetaminophen clearance were determined in the conscious rat. Elimination rate constant, elimination half life time, clearance and volume of distribution of acetaminophen were not disturbed by omeprazole. It is concluded that in the rat, omeprazole does not affect acetaminophen kinetics and, therefore, will not increase susceptibility to acetaminophen toxicity.

Cerebral metabolic compartmentation. Estimation of glucose flux via pyruvate carboxylase/pyruvate dehydrogenase by 13C NMR isotopomer analysis of D-[U-13C]glucose metabolites.

A method is presented for determining the compartmentation of amino acid metabolism in the brain. 13C NMR spectroscopy, and more specifically, homonuclear 13C-13C spin coupling patterns of 13C-labeled amino acids were used to measure the relative flux of label from D-[U-13C]glucose through the anaplerotic pathway versus the oxidative pathway. Glucose flux through the pyruvate carboxylase pathway was quantitated following primed dose constant infusion of D-[U-13C]glucose to young rabbits at a rate of 1 mg/kg body weight per min. We demonstrate, for the first time, that multiplet spectra of three adjacent 13C isotopomer in 1,2,3-13C3 in glutamine and glutamate, which are derived from [1,2,3-13C3]pyruvate, present different isotopomer populations in glutamine in comparison to that in glutamate. This is due to two different metabolic compartments characterized by the presence or absence of glutamine synthetase activity and two different tricarboxylic acid cycles, one preferentially mediated by pyruvate carboxylase and the other by pyruvate dehydrogenase. Our results indicate that the anaplerotic pathway accounts for 34% of glutamine synthesis and only 16% of glutamate and gamma-aminobutyric acid syntheses in metabolic and isotopic steady state conditions. These results support the concept, and provide a quantitative measure, that glutamine and/or tricarboxylic acid cycle intermediates are supplied by astrocytes to neurons to replenish the neurotransmitter pool of gamma-aminobutyric acid and glutamate.

Determination of fructose metabolic pathways in normal and fructose-intolerant children: a 13C NMR study using [U-13C]fructose.

An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-[U-13C]fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of 13C NMR spectra of plasma glucose. The conversion parameters in the control and HFI children were estimated on the basis of doublet/singlet values of the plasma beta-glucose C-1 splitting pattern as a function of the rate of fructose infusion (0.26-0.5 mg/kg per min). Significantly lower values (approximately 3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitative determination of the metabolic pathways of fructose conversion to glucose was derived from 13C NMR measurement of plasma [13C]glucose isotopomer populations. The finding of isotopomer populations of three adjacent 13C atoms at glucose C-4 (13C3-13C4-13C5) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only approximately 50% of the total amount of hepatic fructose conversion to glucose. It is suggested that phosphorylation of fructose 1-phosphate to fructose 1,6-bisphosphate by 1-phosphofructokinase occurs in human liver (and intestine) when fructose is administered nasogastrically; 47% and 27% of the total fructose conversion to glucose in controls and in HFI children, respectively, takes place by way of this pathway. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of [13C]glucose formation from a "trace" amount (approximately 20 mg/kg) of [U-13C]fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism.

Microbial synthesis of L-[15N]leucine L-[15N]isoleucine, and L-[3-13C]-and L-[3'-13C]isoleucines studied by nuclear magnetic resonance and gas chromatography-mass spectrometry.

The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.

Effects of streptozotocin-induced diabetes on drug metabolism in rats.

The metabolism of salicylic acid was investigated in male normal and diabetic rats. The results showed a decrease in urinary excretion of salicylic-glucuronic acid in diabetic animals and this decrease is statistically significant (P less than or equal to 0.01), while the urinary excretion of salicyluric acid in the diabetic group was higher than in controls and statistically significant (P less than or equal to 0.001). There was no marked difference between normal and diabetic rats in total urinary excretion of salicylic acid and metabolites.

A quantitative analysis of the metabolic pathways of hepatic glucose synthesis in vivo with 13C-labeled substrates.

A quantitative analysis of the major metabolic pathways of hepatic glucose synthesis in fasted rats was conducted. [2-13C]Acetate was administered intraintestinally into awake fasted rats. 13C NMR and GC-MS analysis were used to quantitate the isotopic enrichments of glutamate, glutamine, lactate, alanine and the newly synthesized liver glucose. By measuring the ratio of carbon atoms in glutamate molecules derived from acetyl-CoA to carbon atoms in the glucose molecule derived from oxaloacetate and gluconeogenic substrates, such as lactate and alanine, the relative activities of the Krebs cycle and gluconeogenesis were quantified. Our results indicate that the percentage of glucose carbons originating by 'metabolic exchange' with the oxaloacetate pool, via the Krebs cycle, is less than 7%.

Metabolic pathways leading to liver glycogen repletion in vivo, studied by GC-MS and NMR.

A quantitative analysis of the pathways leading to glycogen repletion in rats was conducted. [U-13C]Glucose was administered intra-intestinally into awake fasted animals. The distribution of glucose isotopomers derived from liver glycogen, liver extracts and plasma was performed by GC-MS and 13C NMR. The potential gluconeogenic precursors for liver glycogen, lactate, alanine, glutamate and glutamine, were also analyzed. The amount of glycogen that is synthesized by the direct pathway was found to be 35%. The 13C enrichment of liver lactate, alanine and glucose is similar, indicating that they are the major precursors for liver glycogen synthesis via the indirect pathway. Our results demonstrate that after 24 h fasting, when glucose is supplied, gluconeogenesis from endogenous sources is not shut off.

The effect of an applied electric field on the charge recombination kinetics in reaction centers reconstituted in planar lipid bilayers.

Reaction Centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides were incorporated in planar bilayers made from monolayers derived from liposomes reconstituted with purified RCs. The photocurrents associated with the charge recombination process between the reduced primary quinone (QA-) and the oxidized bacteriochlorophyll donor (D+) were measured as a function of voltage (-150 mV less than V less than 150 mV) applied across the bilayer. When QA was the native ubiquinone (UQ) the charge recombination was voltage independent. However, when UQ was replaced by anthraquinone (AQ), the recombination time depended on the applied voltage V according to the relation tau = 8.5 X 10(-3) eV/0.175S. These results were explained by a simple model in which the charge recombination from UQ- proceeds directly to D+ while that from AQ occurs via a thermally activated intermediate state, D+I-QA, where I is the intermediate acceptor. The voltage dependence arises from an electric field induced change in the energy gap, delta G0, between the states D+I-QA and D+IQA-. This model is supported by the measured temperature dependence of the charge recombination time, which for RCs with AQ gave a value of delta G0 = 340 +/- 20 meV. In contrast, delta G0 for RCs with UQ as the primary acceptor, is sufficiently large (approximately 550 meV) so that even in the presence of the field, the direct pathway dominates. The voltage dependence shows that the electron transfer from I- to QA is electrogenic. From a quantitative analysis of the voltage dependence on the recombination rate it was concluded that the component of the distance between I and QA along the normal to the membrane is about one-seventh of the thickness of the membrane. This implies that the electron transfer from I to Q contributes at least one-seventh to the potential generated by the charge separation between D+ and QA-.

The effect of membrane potential on the redox state of cytochrome b561 in antimycin-inhibited submitochondrial particles.

The oxidation of cytochrome b561 by ATP was measured in submitochondrial particles inhibited by antimycin. The redox potential of the bulk (M phase) was controlled by the ratio of fumarate:succinate, and the oxidation of cytochrome b was calculated and expressed as a change in redox potential (Eh) measured in millivolts. The oxidation of cytochrome b561 is an energy-driven reaction affected only by the delta psi component of the proton motive force. The oxidation (measured in millivolts) is a function of the phosphate potential, reaching a maximal value of 40 mV at delta G'ATP less than - 12 kcal/mole. The maximal measured value of ATP-dependent delta psi was 100 mV. Thus only a fraction of the membrane potential effects the redox state of cytochrome b561. In contrast to the ATP-induced oxidation of cytochrome b561, cytochrome b566 is in redox equilibrium with fumarate succinate either in the presence or in the absence of ATP. The selective oxidation of b561 is explained within the term of the Q cycle as a reflection of delta psi on the electron electrochemical potential. The positive electric potential of the C phase causes cytochrome b566 to act as oxidant with respect to cytochrome b561. In the presence of antimycin cytochrome b561 cannot equilibrate with the quinone and undergoes oxidation, while cytochrome b566 reequilibrates with the quinone and thus regains redox equilibrium with the fumarate succinate redox buffer.