13C isotopomer analysis of glutamate by J-resolved heteronuclear single quantum coherence spectroscopy

13C NMR isotopomer analysis is a powerful method for measuring metabolic fluxes through pathways intersecting in the tricarboxylic acid cycle. However, the inherent insensitivity of 13C NMR spectroscopy makes application of isotopomer analysis to small tissue samples (mouse tissue, human biopsies, or cells grown in tissue culture) problematic. (1)H NMR is intrinsically more sensitive than 13C NMR and can potentially supply the same information via indirect detection of 13C providing that isotopomer information can be preserved. We report here the use of J-resolved HSQC (J-HSQC) for 13C isotopomer analysis of tissue samples. We show that J-HSQC reports isotopomer multiplet patterns identical to those reported by direct 13C detection but with improved sensitivity.     

Contribution to the Biology of the Vermilion Snapper, Rhomboplites aurorubens, in Trinidad and Tobago, West Indies

Reproduction and growth of the vermilion snapper, Rhomboplites aurorubens, were studied in Trinidad and Tobago. The smallest individual caught measured 145mm total length (TL) and all fish appeared to be mature. It was not possible to precisely determine size at first maturity due to the use of macroscopic techniques. The smallest spent male and female measured 181 and 211mm TL respectively, suggesting a size at first maturity below these sizes. Spawning occurred throughout the year, with a period of peak spawning from about June to November in the rainy season when river runoff increased. Sagittal otolith sections were used for age determination and the opaque ring, which was counted as the annulus, was deposited from January to May in the dry season. A total of 11 age groups between the ages of 2–12 years (155–505mm total length) were found. The von Bertalanffy growth parameters were: L=532mm, K=0.13y^–1, and t₀=–0.17, where L is the asymptotic length, K is the growth coefficient and t₀ is the theoretical age at zero length. The relationship between weight (WT) and length (TL) was WT=3.43×10^–5 TL^2.82. Vermilion snapper in this study area appears to grow slower and attain a smaller asymptotic length, but has a longer lifespan than found in populations in higher latitudes. This may be attributed to different levels of exploitation, which may be higher in the latter areas.     

A (13)C isotopomer kinetic analysis of cardiac metabolism: influence of altered cytosolic redox and [Ca(2+)](o)

Rat hearts were perfused with mixtures of [3-(13)C]pyruvate and [3-(13)C]lactate (to alter cytosolic redox) at low (0.5 mM) or high (2.5 mM) Ca(2+) concentrations to alter contractility. Hearts were frozen at various times after exposure to these substrates, were extracted, and were then analyzed by (13)C NMR spectroscopy. The time-dependent multiplets observed in the (13)C NMR resonances of glutamate in all hearts and in malate and aspartate in hearts perfused with high-pyruvate/low-lactate concentrations were analyzed using a kinetic model of the tricarboxylic acid (TCA) cycle. The analysis showed that TCA cycle flux (V(TCA)) and exchange flux (V(X)) that involved cycle intermediates were both sensitive to cell redox and altered Ca(2+) concentration, and the ratio of these fluxes (V(X)/V(TCA)) varied >10-fold.     

Preliminary in vitro studies on two potent, water-soluble trimethoprim analogues with exceptional species selectivity against dihydrofolate reductase from Pneumocystis carinii and Mycobacterium avium

2,4-Diamino-5-[3',4'-dimethoxy-5'-(5-carboxy-1-pentynyl)]benzylpyrimidine (6) and 2,4-diamino-5-[3',4'-dimethoxy-5'-(4-carboxyphenylethynyl)benzylpyrimidine (7) were synthesized from 2,4-diamino-5-(5'-iodo-3',4'-dimethoxybenzyl)pyrimidine (9) via a Sonogashira reaction with appropriate acetylenic esters followed by saponification, and were tested as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat in comparison with the widely used antibacterial agent 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine (trimethoprim, TMP). The selectivity index (SI) for each compound was calculated by dividing its 50% inhibitory concentration (IC(50)) against rat DHFR by its IC(50) against Pc, Tg, or Ma DHFR. The IC(50) of 6 against Pc DHFR was 1.0 nM, with an SI of 5000. Compound 7 had an IC(50) of 8.2 nM against Ma DHFR, with an SI of 11000. By comparison, the IC(50) of TMP was 12000 nM against Pc, 300 nM against Ma, and 180000 against rat DHFR. The potency and selectivity values of 6 and 7 were not as high against Tg as they were against Pc or Ma DHFR, but nonetheless exceeded those of TMP. Because of the outstanding selectivity of 6 against Pc and of 7 against Ma DHFR, these novel analogues may be viewed as promising leads for further structure-activity optimization.     

Regulation of translation of the head protein of T4 bacteriophage by specific binding of EF-Tu to a leader sequence

Recent evidence indicates that translation elongation factor Tu (EF-Tu) has a role in the cell in addition to its well established role in translation. The translation factor binds to a specific region called the Gol region close to the N terminus of the T4 bacteriophage major head protein as the head protein emerges from the ribosome. This binding was discovered because EF-Tu bound to Gol peptide is the specific substrate of the Lit protease that cleaves the EF-Tu between amino acid residues Gly59 and lle60, blocking phage development. These experiments raised the question of why the Gol region of the incipient head protein binds to EF-Tu, as binding to incipient proteins is not expected from the canonical role of EF-Tu. Here, we use gol-lacZ translational fusions to show that cleavage of EF-Tu in the complex with Gol peptide can block translation of a lacZ reporter gene fused translationally downstream of the Gol peptide that activated the cleavage. We propose a model to explain how binding of EF-Tu to the emerging Gol peptide could cause translation to pause temporarily and allow time for the leader polypeptide to bind to the GroEL chaperonin before translation continues, allowing cotranslation of the head protein with its insertion into the GroEL chaperonin chamber, and preventing premature synthesis and precipitation of the head protein. Cleavage of EF-Tu in the complex would block translation of the head protein and therefore development of the infecting phage. Experiments are presented that confirm two predictions of this model. Considering the evolutionary conservation of the components of this system, this novel regulatory mechanism could be used in other situations, both in bacteria and eukaryotes, where proteins are cotranslated with their insertion into cellular structures.     

Increased hepatic fructose 2,6-bisphosphate after an oral glucose load does not affect gluconeogenesis

The generally accepted metabolic concept that fructose 2,6-bisphosphate (Fru-2,6-P2) inhibits gluconeogenesis by directly inhibiting fructose 1,6-bisphosphatase is based entirely on in vitro observations. To establish whether gluconeogenesis is indeed inhibited by Fru-2,6-P2 in intact animals, a novel NMR method was developed using [U-13C]glucose and 2H2O as tracers. The method was used to estimate the sources of plasma glucose from gastric absorption of oral [U-13C]glucose, from gluconeogenesis, and from glycogen in 24-h fasted rats. Liver Fru-2,6-P2 increased approximately 10-fold shortly after the glucose load, reached a maximum at 60 min, and then dropped to base-line levels by 150 min. The gastric contribution to plasma glucose reached approximately 50% at 30 min after the glucose load and gradually decreased thereafter. Although the contribution of glycogen to plasma glucose was small, glucose formed from gluconeogenesis was substantial throughout the study period even when liver Fru-2,6-P2 was high. Liver glycogen repletion was also brisk throughout the study period, reaching approximately 30 micromol/g at 3 h. These data demonstrate that Fru-2,6-P2 does not inhibit gluconeogenesis significantly in vivo.     

Cell wall reactive proteins in the coat and wall of maize pollen: potential role in pollen tube growth on the stigma and through the style

The surface of a pollen grain consists of an outermost coat and an underlying wall. In maize (Zea mays L.), the pollen coat contains two major proteins derived from the adjacent tapetum cells in the anthers. One of the proteins is a 35-kDa endoxylanase (Wu, S. S. H., Suen, D. F., Chang, H. C., and Huang, A. H. C. (2002) J. Biol. Chem. 277, 49055-49064). The other protein of 70 kDa was purified to homogeneity and shown to be a beta-glucanase. Its gene sequence and the developmental pattern of its mRNA differ from those of the known beta-glucanases that hydrolyze the callose wall of the microspore tetrad. Mature pollen placed in a liquid medium released about nine major proteins. These proteins were partially sequenced and identified via GenBank trade mark data bases, and some had not been previously reported to be in pollen. They appear to have wall-loosening, structural, and enzymatic functions. A novel pollen wall-bound protein of 17 kDa has a unique pattern of cysteine distribution in its sequence (six tandem repeats of CX3CX10-15) that could chelate cations and form signal-receiving finger motifs. These pollen-released proteins were synthesized in the pollen interior, and their mRNA increased during pollen maturation and germination. They were localized mainly in the pollen tube wall. The pollen shell was isolated and found to contain no detectable proteins. We suggest that the pollen-coat beta-glucanase and xylanase hydrolyze the stigma wall for pollen tube entry and that the pollen secrete proteins to loosen or become new wall constituents of the tube and to break the wall of the transmitting track for tube advance.