Noninvasive paternity assignment in Gombe chimpanzees

The relative success of chimpanzee male mating strategies, the role of male dominance rank and the success of inbreeding avoidance behaviour can only be assessed when paternities are known. We report the probable paternities of 14 chimpanzees included in a long‐term behavioural study of chimpanzees (Pan troglodytes schweinfurthii) at Gombe National Park, Tanzania. DNA samples were collected noninvasively from shed hair and faeces and genotyped using 13–16 microsatellite loci characterized in humans. All 14 offspring could be assigned to fathers within the community. While there is a positive relationship between male rank and reproductive success, we demonstrate that a range of male mating strategies (possessiveness, opportunistic mating and consortships) can lead to paternity across all male ranks. Several adult females were at risk of breeding with close male relatives. Most successfully avoided close inbreeding but in one case a high‐ranking male in the community mated with his mother and produced an offspring. In contrast to recent data on chimpanzees (P. t. verus) from the Taï forest, Côte d’Ivoire, no evidence of extra‐group paternity was observed in our study. Reanalysis of Taï data using a likelihood approach casts doubt on the occurrence of extra‐group paternity in that community as well.     

Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing

Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.     

The oxidative and nitrosative chemistry of the nitric oxide/superoxide reaction in the presence of bicarbonate

The primary product of the interaction between nitric oxide (NO) and superoxide () is peroxynitrite (ONOO-), which is capable of either oxidizing or nitrating various biological substrates. However, it has been shown that excess NO or can further react with ONOO- to form species which mediate nitrosation. Subsequently, the controlled equilibrium between nitrosative and oxidative chemistry is critically dependent on the flux of NO and. Since ONOO- reacts not only with NO and but also with CO2, the effects of bicarbonate () on the biphasic oxidation profile of dihydrorhodamine-123 (DHR) and on the nitrosation of both 2,3-diaminonaphthalene and reduced glutathione were examined. Nitric oxide and were formed with DEA/NO [NaEt2NN(O)NO] and xanthine oxidase, respectively. The presence of did not alter either the oxidation profile of DHR with varying radical concentrations or the affinity of DHR for the oxidative species. This suggests that the presence of CO2 does not affect the scavenging of ONOO- by either NO or. However, an increase in the rate of DHR oxidation by ONOO- in the presence of suggests that a CO2-ONOO- adduct does play a role in the interaction of NO or with a product derived from ONOO-. Further examination of the chemistry revealed that the intermediate that reacts with NO is neither ONOO- nor cis-HOONO. It was concluded that NO reacts with both trans-HOONO and a CO2 adduct of ONOO- to form nitrosating species which have similar oxidation chemistry and reactivity with and NO.     

Prediction of inhibitor binding free energies by quantum neural networks. Nucleoside analogues binding to trypanosomal nucleoside hydrolase

A computational method has been developed to predict inhibitor binding energy for untested inhibitor molecules. A neural network is trained from the electrostatic potential surfaces of known inhibitors and their binding energies. The algorithm is then able to predict, with high accuracy, the binding energy of unknown inhibitors. IU-nucleoside hydrolase from Crithidia fasciculata and the inhibitor molecules described previously [Miles, R. W. Tyler, P. C. Evans, G. Furneaux R. H., Parkin, D. W., and Schramm, V. L. (1999) Biochemistry 38, xxxx-xxxx] are used as the test system. Discrete points on the molecular electrostatic potential surface of inhibitor molecules are input to neural networks to identify the quantum mechanical features that contribute to binding. Feed-forward neural networks with back-propagation of error are trained to recognize the quantum mechanical electrostatic potential and geometry at the entire van der Waals surface of a group of training molecules and to predict the strength of interactions between the enzyme and novel inhibitors. The binding energies of unknown inhibitors were predicted, followed by experimental determination of K(i)() values. Predictions of K(i)() values using this theory are compared to other methods and are more robust in estimating inhibitory strength. The average deviation in estimating K(i)() values for 18 unknown inhibitor molecules, with 21 training molecules, is a factor of 5 x K(i)() over a range of 660 000 in K(i)() values for all molecules. The a posteriori accuracy of the predictions suggests the method will be effective as a guide for experimental inhibitor design.     

Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal β-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.     

Localisation and characterisation of cell wall mannan polysaccharides in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Polysaccharides containing -1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.Abbreviations BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - PACE polysaccharide analysis by carbohydrate gel electrophoresis     

Alterations in corticospinal excitability with imposed vs. voluntary fatigue in human hand muscles.

We aimed to determine whether postexercise depression of motor-evoked potentials (MEPs) could be demonstrated without voluntary muscle activation in humans. Voluntary fatigue was induced with a 2-min maximal voluntary contraction (MVC) of the first dorsal interosseous (FDI) muscle. On another occasion, "electrical fatigue" was induced with trains of shocks delivered for 2 min over the FDI motor point. Five of the twelve subjects also underwent "sequential fatigue" consisting of a 2-min MVC of FDI followed by 20 min of rest and then 2 min of motor point stimulation. Voluntary fatigue induced MEP depression that persisted for at least 20 min. Electrical fatigue induced a transient MEP facilitation that subsided 20 min after the stimulation and became depressed within 30 min. Thus MEP depression can be induced by both voluntary and electrical fatigue. With electrical fatigue, the initial depression is "masked" by transient MEP facilitation, reflecting cortical plasticity induced by the prolonged electrical stimulation. MEP depression probably reflects tonic afferent input from the exercising muscle that alters cortical excitability without altering spinal excitability.     

Evidence for a transition state analog, MgADP-aluminum fluoride-acetate, in acetate kinase from Methanosarcina thermophila

Aluminum fluoride has become an important tool for investigating the mechanism of phosphoryl transfer, an essential reaction that controls a host of vital cell functions. Planar AlF(3) or AlF(4)(-) molecules are proposed to mimic the phosphoryl group in the catalytic transition state. Acetate kinase catalyzes phosphoryl transfer of the ATP gamma-phosphate to acetate. Here we describe the inhibition of acetate kinase from Methanosarcina thermophila by preincubation with MgCl(2), ADP, AlCl(3), NaF, and acetate. Preincubation with butyrate in place of acetate did not significantly inhibit the enzyme. Several NTPs can substitute for ATP in the reaction, and the corresponding NDPs, in conjunction with MgCl(2), AlCl(3), NaF, and acetate, inhibit acetate kinase activity. Fluorescence quenching experiments indicated an increase in binding affinity of acetate kinase for MgADP in the presence of AlCl(3), NaF, and acetate. These and other characteristics of the inhibition indicate that the transition state analog, MgADP-aluminum fluoride-acetate, forms an abortive complex in the active site. The protection from inhibition by a non-hydrolyzable ATP analog or acetylphosphate, in conjunction with the strict dependence of inhibition on the presence of both ADP and acetate, supports a direct in-line mechanism for acetate kinase.