New geochronological, paleoclimatological, and archaeological data from the Narmada Valley hominin locality, central India.

The oldest known fossil hominin in southern Asia was recovered from Hathnora in the Narmada Basin, central India in the early 1980's. Its age and taxonomic affinities, however, have remained uncertain. Current estimates place its maximum age at >236ka, but not likely older than the early middle Pleistocene. The calvaria, however, could be considerably younger. We report recent fieldwork at Hathnora and associated Quaternary type-sections that has provided new geological and archaeological insights. The portion of the exposed 'Boulder Conglomerate' within the Surajkund Formation, which forms a relict terrace and has yielded the hominin fossils, contains reworked and stylistically mixed lithic artifacts and temporally mixed fauna. Three mammalian teeth stratigraphically associated with the hominin calvaria were dated by standard electron spin resonance (ESR). Assuming an early uranium uptake (EU) model for the teeth, two samples collected from the reworked surface deposit averaged 49+/-1ka (83+/-2ka, assuming linear uptake [LU]; 196+/-7ka assuming recent uptake [RU]). Another sample recovered from freshly exposed, crossbedded gravels averaged 93+/-5ka (EU), 162+/-8ka (LU) or 407+/-21ka (RU). While linear uptake models usually provide the most accurate ages for this environment and time range, the EU ages represent the minimum possible age for fossils in the deposit. Regardless, the fossils are clearly reworked and temporally mixed. Therefore, the current data constrains the minimum possible age for the calvaria to 49+/-1ka, although it could have been reworked and deposited into the Hathnora deposit any time after 160ka (given the LU uptake ages) or earlier (given the RU ages). At Hathnora, carbonaceous clay, bivalve shells, and a bovid tooth recovered from layers belonging to the overlying Baneta Formation have yielded (14)C ages of 35.66+/-2.54cal ky BP, 24.28+/-0.39cal ky BP, and 13.15+/-0.34ky BP, respectively. Additional surveys yielded numerous lithics and fossils on the surface and within the stratigraphic sequence. At the foot of the Vindhyan Hills 2km from the river, we recovered a typologically Early Acheulean assemblage comprised of asymmetrical bifaces, large cleavers with minimal working, trihedral picks, and flake tools in fresh condition. These tools may be the oldest Acheulean in the Narmada Valley. Several lithics recovered from the Dhansi Formation may represent the first unequivocal evidence for an early Pleistocene hominin presence in India. In situ invertebrate and vertebrate fossils, pollen, and spores indicate a warm, humid climate during the late middle Pleistocene. High uranium concentrations in the mammalian teeth indicate exposure to saline water, suggesting highly evaporative conditions in the past. Late Pleistocene sediment dated between 24.28+/-0.39cal ky BP and 13.15+/-340ky BP has yielded pollen and spores indicating cool, dry climatic conditions corresponding to Oxygen Isotope Stage 2 (OIS 2). An early Holocene palynological assemblage from the type locality at Baneta shows evidence for relatively dry conditions and a deciduous forest within the region. The Dhansi Formation provisionally replaces the Pilikarar Formation as the oldest Quaternary formation within the central Narmada Basin. The Baneta Formation, previously dated at 70ka to 128ka, correlates with the late Pleistocene and early Holocene. Our results highlight the need for further Quaternary geological and paleoanthropological research within the Narmada Basin, especially because dam construction threatens these deposits.

ESR dating at Mezmaiskaya Cave, Russia.

Mezmaiskaya Cave has yielded more than 10,000 artifacts, thousands of very well preserved faunal remains, and hominin remains, found in seven Middle Paleolithic (Mousterian) and three Upper Paleolithic levels. A complete Neanderthal infant skeleton was preserved in anatomical juxtaposition lying on a large limestone block, overlain by the earliest Mousterian layer, Layer 3. Twenty-four skull fragments from a 1-2 year-old Neanderthal infant, showing post-mortem deformation, occurred in a pit originating in the Mousterian Layer 2 and penetrating into underlying layers 2A and 2B(1). Bone from Layer 2A was dated by AMS 14C at 35.8-36.3+/-0.5 kyr BP. Direct dating of Neanderthal bone from Layer 3 gave an age of 29 kyr, but that is now considered to be due to contamination by modern carbon. Fourteen large mammal teeth from Layers 2 through 3 have been dated by standard electron spin resonance (ESR). Low U concentrations in both the enamel and dentine ensure that ESR ages do not depend significantly on the U uptake model, but do depend strongly on the sedimentary dose rates. Assuming a sedimentary water concentration equal to 20 wt%, ESR ages for the Mousterian layers range from 36.2 to 73.0+/-5.0 ka.

Analysis of Fusarium graminearum mycotoxins in different biological matrices by LC/MS.

The purpose of this study was to develop an LC/MS assay to accurately detect three mycotoxins produced by Fusarium graminearum in various matrices. Using different LC conditions, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), and zearalenone (ZEN) were detected in four different matrices (fungal liquid cultures, maize grain, insect larvae and pig serum). The sensitivity of MS detection allowed us to detect concentrations as low as 8 ppb of DON and 12 ppb of ZEN. A very small quantity of matrix was therefore necessary for successful analysis of these toxins and a variety of experimental situations were successfully investigated using this technique. Production of 15-ADON and butenolide was monitored in a liquid culture of F. graminearum under controlled conditions. Using simple extraction procedures, the differential accumulation of DON and 15-ADON was followed in inoculated maize genotypes varying in susceptibility to F. graminearum. Toxicokinetic studies were carried out with maize insect pests reared continually on artificial diets containing ZEN and suggested that larvae may possess the ability to degrade ZEN. Finally, persistence of DON was assessed in pigs fed diet supplemented with DON, results indicated that DON accumulates quickly in pig blood and then levels decline progressively for 12 hours thereafter. The LC/MS study reported here is very useful and flexible for the detection of these mycotoxins in different media and at very low concentrations.

A new 1-hydroxy-2,6-pyrazinedione associated with hypovirulent isolates of Sclerotinia minor.

A new 1-hydroxy-2,6-pyrazinedione, sclerominol (1), was isolated from cultures of hypovirulent isolates of Sclerotinia minor, a fungal plant pathogen associated with lettuce drop and other plant diseases. This compound was characterized by NMR, mass spectrometry, and X-ray crystallography. One other 1-hydroxy-2,6-pyrazinedione, flutimide, has been reported. Flutimide has activity as an inhibitor of influenza virus endonuclease, and therefore, sclerominol was evaluated for related biological activity. Sclerominol (1) displayed some activity against cancer cell lines but little activity against three influenza virus strains. The role of 1 in the physiology of hypovirulent isolates of S. minor has not been determined, but 1 has also been recovered from debilitated isolates of S. sclerotiorum.

Improvements in dating tooth enamel by ESR.

ESR dating requires that growth curves be determined by interpreting complex spectra. Spectra, however, can vary significantly in shape and field position between different samples, or occasionally between subsamples, even though the mineralogy remains the same. In some cases, this spectral variability does not affect the resulting accumulated dose calculation. In other cases, signal subtraction may be needed. However, some samples that until recently might have been considered unsuitable for dating are now shown to yield accurate and precise results because a broad interference peak is integral to the hydroxyapatite signal. By studying the spectrum at the Q-band frequency, it can be shown that the interfering signal in most cases is not a problem for dating. A second concern has been that artificially irradiating sample aliquots can introduce a short-lived component that is simply an unstable enhancement of the dating signal. The apparent accumulated dose from growth curves created immediately after irradiation is considerably greater than that after annealing, although the curve's shape remains unchanged. Annealing both the natural and artificially irradiated signal shows the dating signal's lifetime to be greater than 10(10) years.

Oxidative deamination of hydrolyzed fumonisin B(1) (AP(1)) by cultures of Exophiala spinifera.

Fumonisins are mycotoxins of world-wide distribution in maize infected by the fungus Fusarium verticillioides. They are highly toxic to certain livestock and are potential carcinogens. Exophiala spinifera, a black yeast fungus found on moldy maize kernels, was identified previously as capable of growing on fumonisin B1 as a sole carbon source and thus is a potential source for fumonisin detoxifying enzymes. Pure cultures of E. spinifera transform fumonisin B(1) to the amino polyol AP(1) plus free tricarballylic acid through the activity of a soluble extracellular esterase, and further transformation is evidenced by accumulation in culture supernatant of a less polar compound(s) lacking a fluorescamine-reactive amino group. A free amine is thought to be critical for biological activity of FB(1) or AP(1). As a first step towards characterizing this amine-modifying activity, we investigated the biotransformation of AP(1) by E. spinifera liquid cultures that had been previously grown in liquid medium containing AP(1) as a sole carbon source. Accumulation of AP(1)-derived metabolites was monitored by thin-layer chromatography of culture supernatants, and product metabolites were purified and evaluated by mass spectrometry and nuclear magnetic resonance. Two products of treatment of purified AP(1) with cultures of E. spinifera are shown to be N-acetyl AP(1) and a new compound, 2-oxo-12,16-dimethyl-3,5,10, 14,15-icosanepentol hemiketal (or 2-OP(1) hemiketal).

Identification of a new sphingolipid 3-O-acyl-D-erythro-sphingomyelin in newborn pig and infant plasma.

A new sphingolipid was found in newborn pig plasma at a level of 2.5 +/- 0.4% of total lipids. The compound decreased to less than half that amount by day one of age and virtually disappeared by the fourth week. On thin-layer chromatography (TLC) the new lipid migrated close to phosphatidylethanolamine. The compound was isolated by TLC from the plasma of newborn piglets and identified as a 3-O-acyl-D-erythro-sphingomyelin by chemical and chromatographic techniques, 1H- and 13C-nuclear magnetic resonance and fast-atom bombardment mass spectrometry. Mild alkaline hydrolysis at room temperature gave mainly C16 and C18 fatty acids and sphingomyelin. Subsequent reaction with Ba(OH)2 released long-chain saturated and monounsaturated fatty acids from C14 to C24, and sphingosine which was identified as the erythro configuration by gas chromatography. Less than 1% of the sphingosine was of the C20 isomer. No hydroxy fatty acids were found. The acylated sphingomyelin was only found in plasma lipids of newborn piglets and not in their red blood cell membranes or platelets of newborn piglets, or in sow plasma. This compound was tentatively identified by chromatography in trace amounts in the serum of cord blood of newborn infants, but not in the plasma lipids of adults.

NMR structural studies of fumonisin B1 and related compounds from Fusarium moniliforme.

Fumonisin B1 (FB1) is the primary mycotoxin produced by Fusarium moniliforme and appears to be responsible for the varied toxigenic effects associated with ingestion of this mold, particularly that of the inhibition of sphingolipid biosynthesis. Understanding the structure and biosynthesis of fumonisins is a key factor in determining structure/activity relationships. To this end, Nuclear Magnetic Resonance (NMR) methods have been used to identify various derivatives of FB1, both naturally occurring and synthetic. With accurate chemical shift assignments, NMR may be used to determine the level of impurities in toxicological grade FB1 preparations. Specifically enriched FB1 was prepared from F. moniliforme cultures using 13C-enriched acetate as well as several 13C-enriched amino acids. 13C NMR analysis indicates that the biosynthesis of fumonisins involves the addition of methionine-derived methyl functions, glutamate-derived tricarballylic ester functions and alanine to an 18 carbon hydrocarbon backbone that is likely polyketide in origin. With the goal of obtaining a crystalline compound for the determination of absolute configuration, several derivatives of FB1 have been prepared, and NMR analysis used to determine the relative and absolute configuration of the 10 stereocenters present in this molecule.

Isolation and characterization of new chlamydosporol related metabolites of Fusarium chlamydosporum and Fusarium tricinctum.

Fusarium chlamydosporum strain T-826 isolated from corn in the USA produced chlamydosporol and two analogs which have been identified by various spectroscopic techniques as: 7,8-dihydro-5-hydroxy-4-methoxy-trans-7,8-dimethyl-2H,5H-pyrano(4, 3-b)pyran-2-one (or isochlamydosporol) and 4-methoxy-5-hydroxymethyl-6-(3-butan-2-ol)-2H-pyran-2-one (or chlamydospordiol). Chlamydosporol (compound a + b) chlamydospordiol (compound c) and isochlamydosporol (compound d) were produced together (up to 6000 micrograms/g) by 3 out of 11 isolates of F. chlamydosporum and by 3 out of 24 isolates of F. tricinctum from various substrates and geographic origin. Three isolates of F. chlamydosporum and one isolate of F. tricinctum produced only chlamydospordiol and 2 isolates of F. tricinctum produced chlamydosporol (a + b), and chlamydospordiol (c).

Ion transport and methane production in Methanobacterium thermoautotrophicum.

In Methanobacterium thermoautotrophicum, the protonmotive force for the H+-translocating ATPase consists mainly of a transmembrane electrical gradient (Deltapsi). These cells do not establish a significant transmembrane pH gradient (inside alkaline) and, in fact, if the suspending medium is of pH >/= 7.0, the pH gradient may be reversed-i.e., inside acid with respect to the extracellular pH. These studies show by both 23Na NMR and 22Na+ distribution that Na+ extrusion with the generation of Deltapsi precedes methanogenesis in Mb. thermoautotrophicum. It is calculated that the newly established Na+ gradients increase Deltapsi by approximately 50 mV (inside negative). There is no detectable H+ extrusion during methane synthesis; instead there is a high rate of H+ consumption for methane synthesis and an increase in internal pH. This was supported by 31P NMR experiments, which showed an internal pH shift from 6.8 to 7.6. With the cells maintained at an external pH of 7.2, the initial transmembrane pH gradient of -0.4 (inside acid) at 60 degrees C is equivalent to Deltapsi of + 27 mV (inside positive); after 20 min of incubation, the transmembrane pH gradient is + 0.4 (inside alkaline), which at 60 degrees C is equivalent to Deltapsi of -27 mV (inside negative). Actively respiring cells generated a protonmotive force of -198 mV. It is proposed that energy for CO2 reduction to the level of formaldehyde (the first step in methane synthesis) in Mb. thermoautotrophicum is derived from the Deltapsi generated by electrogenic Na+ extrusion. The protonmotive force required for ATP synthesis consists primarily of Deltapsi and appears to be the result of both an electrogenic Na+ extrusion and a pH gradient (inside alkaline) which develops during methanogenesis.

ESR dating tooth enamel from the Paleolithic site at Longola, Zambia.

A single radiation-sensitive ESR signal at g = 2.0018 occurs in fossil tooth enamel, but not in modern teeth. In dating fossil teeth, the equivalent radiation dose (AD) needed to produce the observed ESR signal is the integral with respect to time of the natural, environmental dose rate (ED) experienced by the tooth during burial. Since the age depends on the U uptake history assumed, three dates are normally calculated assuming early U uptake (EU), continuous (linear) U uptake (LU), or recent U uptake (RU). Generally the LU ages agree best with known ages determined by other methods, but the EU and RU ages are respectively the minimum and maximum ages. Longola Spring Mound, in Central Zambia, contains a Late Stone Age collection occurs on the mound surface. Embedded in layer near the base is a much older layer containing Middle Stone Age artifacts and bone material. Four ungulate teeth collected from the lower layer were ESR dated. EU, LU, and RU ages for each tooth agree very closely, but ages range from 14 to 96 ka. Although the layer may be a two component deposit with teeth averaging 18 +/- 2 ka and 91 +/- 3 ka, high sedimentary Th concentrations and ESR isochrons suggest that gamma ext dose estimates are in error. LU dates estimated from isochron plots average 204 +/- 86 ka, while LU ages calculated with the average isochron-derived gamma ext = 10.79 +/- 1.89 mrad/a average 220 +/- 62 ka. More excavation and dating are necessary to determine if the isochron data is reasonable.(ABSTRACT TRUNCATED AT 250 WORDS)

ESR isochron dating for teeth: a brief demonstration in solving the external dose calculation problem.

The radiation sensitive ESR signal at g = 2.0018 in fossil enamel can be used to date teeth absolutely, providing a suitable U uptake model can be selected, and the external dose rate, Dext, can be accurately calculated. Apart from the uptake model, the most obvious uncertainty lies in Dext. With the isochron method, Dext can be derived easily during the ESR analysis. By plotting the AD vs. the total internal dose rate for teeth where multiple subsamples have been analyzed, a regression line, the isochron, can be determined for each model age calculation. Each isochron slope represents the sample age, while its y-intercept gives the total external dose, Aext.

Structure of a novel cofactor containing N-(7-mercaptoheptanoyl)-O-3-phosphothreonine.

The cofactor required in the methylcoenzyme M methylreductase reaction was shown to be a large molecule with an Mr of 1149.21 in the free acid form. The cofactor, named MRF for methyl reducing factor, was identified from analyses by fast atom bombardment mass spectrometry and 1H, 13C, and 31P NMR spectroscopy as uridine 5'-[N-(7-mercaptoheptanoyl)-O-3-phosphothreonine-P-yl(2-acetamido- 2-deoxy- beta-mannopyranuronosyl)(acid anhydride)]-(1----4)-O-2-acetamido-2-deoxy- alpha-glucopyranosyl diphosphate. MRF contains N-(7-mercaptoheptanoyl)threonine O-3-phosphate (HS-HTP) [No11, K. M., Rinehart, K. L., Tanner, R. S., & Wolfe, R. S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4238-4242] and is linked to C-6 of 2-acetamido-2-deoxymannopyranuronic acid of the UDP-disaccharide through a carboxylic-phosphoric anhydride linkage. It is postulated that this bond is responsible for the instability of the molecule and its hydrolysis during isolation. Analyses of Eadie and Hofstee plots of the methylcoenzyme M methylreductase reaction indicate that MRF has a 6-fold lower Km(app) than HS-HTP and a 50% greater Vmax. This suggests that the UDP-disaccharide moiety may be of importance in the binding of MRF to the enzyme active site.

Structure determination of the UDP-disaccharide fragment of cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum.

The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5'-(O-2-acetamido-2-deoxy-beta-manno-pyranuronosyl acid (1----4)-2-acetamido-2-deoxy-alpha-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex.

Structure of purified cytoplasmic cofactor from Methanobacterium thermoautotrophicum.

The reduction of methylcoenzyme M to methane is known to require a heat stable and oxygen sensitive cofactor. Recently it has been shown that the active site of this cofactor is 7-mercaptoheptanoylthreonine phosphate. The present study shows that in the complete structure of this cofactor 7-mercaptoheptanoylthreonine phosphate is linked by pyrophosphate to two N-acetyl-glucosamine residues and an unidentified terminal group R with m/z 214. By fast-atom-bombardment mass spectrometry the intact cofactor, isolated as the mixed disulfide with 2-mercaptoethanol, was shown to have a molecular weight of 1084.5. The pyrophosphate bond is quite labile and undergoes hydrolysis or prolonged storage. This lability of the pyrophosphate bond may explain why the intact cofactor has not been isolated until now.

Structure of two new aminophospholipids from Methanobacterium thermoautotrophicum.

The methanogenic bacterium Methanobacterium thermoautotrophicum (A.T.C.C. 29183) was shown to contain two new aminophospholipids. These are 2-aminoethyl phosphate ester of diphytanylglycerol diether and a sugar containing bisdiphytanyldiglycerol tetraether. The two aminophospholipids were stable to acid methanolysis except for the sugar on the bisdiphytanyldiglycerol tetraether. Strong acid (6 M-HCl) hydrolysed the alkyl ether and aminophosphate ester bonds. The structure of the phosphate linkage was demonstrated by 31P n.m.r., and the 2-ethanolamine structure was elucidated by 1H- and 13C-n.m.r. spectroscopy and by fast-atom-bombardment m.s.

The role of tetrahydromethanopterin and cytoplasmic cofactor in methane synthesis.

A fraction previously isolated from acid-treated supernatant fraction of Methanobacterium thermoautotrophicum by DEAE-Sephadex chromatography [Sauer, Mahadevan & Erfle (1984) Biochem. J. 221, 61-97] which was absolutely required for methane synthesis, has been separated into two compounds, tetrahydromethanopterin (H4MPT) and an as-yet-unidentified cofactor we call 'cytoplasmic cofactor'. H4MPT was identified by its u.v. spectrum and by 13C- and 1H-n.m.r. spectroscopy. The reduction of 2-(methylthio)ethanesulphonic acid (CH3-S-CoM) to methane by the membrane fraction from M. thermoautotrophicum was completely dependent on the addition of cytoplasmic cofactor. Methane synthesis from CO2, however, was only partially dependent on cofactor addition, and 57% of the original activity was retained in its absence. The kinetics of 14C labelling were consistent with the scheme methyl-H4MPT----CH3-S-CoM----methane, as has been proposed. This is the first time that direct experimental evidence has been presented to show that the proposed methyl transfer from H4MPT to coenzyme M (HS-CoM) actually occurs.

13C NMR study of the biosynthesis of toxins by Fusarium graminearum.

13C NMR spectroscopic investigations on the biosynthesis of mycotoxins produced by Fusarium graminearum (M69) were carried out through the incorporation of [1-13C]- and [2-13C]acetate precursors. The major secondary metabolites produced by this species in still culture were deoxynivalenol (3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-one), 15-acetyldeoxynivalenol, zearalenone, and butenolide. [1-13C]- and [2-13C]acetate were incorporated in alternate carbon atoms in zearalenone, consistent with the head to tail condensation of nine acetate units. The trichothecenes were enriched in a manner consistent with the condensation of three mevalonate units. 13C/13C couplings, observed between C-5 and C-12, as well as between C-6 and C-15 of 15-acetyldeoxynivalenol, confirms the current hypothesis of formation of the trichothecene ring system by cyclization of farnesyl pyrophosphate. The incorporation pattern in ergosterol is also consistent with a mevalonate origin, while the adjacent incorporation of acetate methyl groups in butenolide suggests a glutamate precursor. The degree of enrichment in the secondary metabolites, which ranged from 3 to 10% at each carbon site, was observed in the 13C NMR spectra of the crude fungal extracts to be dependent on the timing of acetate addition to the culture. The specific toxins produced together with the quantity of each, were also found to be dependent on the timing of acetate addition. Competition between the three biosynthetic pathways of secondary metabolism, i.e. polyketide, mevalonate, and amino acid for the labeled acetate in this organism is a complex function of culture conditions.

Membrane Organization of the Desiccation-Tolerant Moss Tortula ruralis in Dehydrated States.

Membrane organization of the desiccation tolerant moss Tortula ruralis was studied in several intensely dehydrated states (75% relative humidity [RH], 90% RH, plasmolysis in molar salt, freezing to -20 degrees C) by (31)P nuclear magnetic resonance and ultrastructural analyses. Both methods revealed that even at 75% RH (-400 bars), the moss cellular membranes retained extended phospholipid bilayers. Ultrastructural analyses of the fully hydrated moss showed an extensive proliferation of membrane vesicles in the endoplasmic reticulum. During dehydration, these vesicles form layers of membrane under the plasmalemma and in some cases appear to fuse with the surface membrane. This suggests that these vesicles may serve as a reservoir of membranes to accommodate for membrane surface area changes during desiccation and subsequent rehydration.