Acetate metabolism in Escherichia coli.

Levels of several intermediary metabolites were measured in cells grown in acetate medium in order to test the hypothesis that the glyoxylate cycle is repressed by phosphoenolpyruvate (PEP). Wild-type cells had less PEP than either isocitrate dehydrogenase - deficient cells (which had greater isocitrate lyase activity than the wild type) or isocitrate dehydrogenase - deficient, citrate synthase-deficient cells (which are poorly inducible). Thus induction of the glyoxylate cycle is more complicated than a simple function of PEP concentration. No correlation between enzyme activity and the level of oxaloacetate, pyruvate, or citrate was found either. Citrate was synthesized in citrate synthase-deficient mutants, possibly via citrate lyase.     

Purine base transport in nit-2 mutants of Neurospora crassa.

The nit-2 mutants possess general purine transport activity. Reduced hypoxanthine uptake in germinated conidia of these mutants may be a consequence of their defective purine metabolism.     

Permeability changes in membranes of Neurospora crassa after freezing and thawing.

Conidia of Neurospora crassa which are in different physiological states show different rates of survival after freezing and thawing. [¹⁴C]adenine uptake by frozen and thawed conidia in different physiological states show a correlation with their survival. The uptake method was extended to study the survival of mycelium in log phase and stationary phase. From the uptake data it appears that log phase mycelium is extremely sensitive to all rates of freezing and thawing studied, while the stationary phase mycelium showed slight tolerance to freezing, if freezing was done at a slow rate. A study of the efflux of labeled compounds from the conidia in various physiological states or from the mycelia after freezing and thawing showed that, although efflux followed the same general trend as survival in conidia, it did not relate to the survival in mycelium, suggesting that the death of conidia or mycelium in the freeze-thaw treatments is not due to efflux of compounds.     

Determination of dopamine, homovanillic acid and 3,4-dihydroxyphenylacetic acid in rat brain striatum by high-performance liquid chromatography with electrochemical detection

Two procedures using liquid chromatography with electrochemical detection are described for the determination of dopamine (DA) and its two acidic metabolites, homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), in subregions of rat striatum and nucleus accumbens. A strong cation-exchange column was used for DA analysis and a C₁ reversed-phase column was used for the analysis of the metabolites. Effects of pH, temperature and percentage of methanol on the retention time of HVA and DOPAC were studied. Levels of these compounds in the subregions of rat striatum and nucleus accumbens are reported.     

Glycolytic enzyme activities in murine sarcoma virus-transformed cultures of Balb 3T3. The role of culture density.

The effects of transformation by murine sarcoma virus and of increasing cell density on the activities of several key glycolytic enzymes in Balb 3T3 cells were tested. Hexokinase levels increased with culture density in the uninfected and in the two virus-transformed (HB2 and KA31) cells. Phosphofructokinase did not increase with culture density in the uninfected cells but rose dramatically in dense cultures of virus-transformed cells. 6-Phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase levels were high in sparse cultures of uninfected cells and decreased steadily with increased culture density. Pyruvate kinase levels increased with density only in KA31 cultures. A density-dependent decrease in the level of hexokinase type II with a concomitant increase in type I isozyme was seen in uninfected 3T3 cultures. This change was negligible in HB2 cells.     

Genetic polymorphism of C3 in Papio and Macaca species

C3 phenotypes were examined in species of Papio and Macaca. Baboons (P. cynocephalus) showed extensive polymorphism, with estimated gene frequencies of 0.815, 0.174, and 0.011 for C3*S, C3*F, and C3*F ₁ alleles, respectively. Clear segregation patterns showing codominant inheritance were evident in family studies. Such extensive polymorphism was not observed in M. nemestrina or M. fascicularis. The S gene is the most common allele in all the species studied. The F gene is relatively common in baboons.     

Analysis of neuronal and glial phenotypes in brains of mice deficient in leukemia inhibitory factor

Leukemia inhibitory factor (LIF) can regulate the survival and differentiation of certain neurons and glial cells in culture. To determine the role of this cytokine in the central nervous system in vivo, we examined the brains of young and adult mice in which the LIF gene was disrupted. Immunohistochemical staining of neurons for choline acetyltransferase, tyrosine hydroxylase, serotonin, parvalbumin, calbindin, neuropeptide Y, vasoactive intestinal polypeptide, and calcitonin gene-related peptide revealed no significant differences between null mutant and wild-type (WT) brains. In contrast, analysis of glial phenotypes demonstrated striking deficits in the LIF-knockout brain. Staining with several anti-glial fibrillary acidic protein (GFAP) antibodies showed that the number of GFAP-positive cells in various regions of the hippocampus in the female mutant is much lower than in the WT. The null male hippocampus also displays a significant, though less marked deficit. The number of astrocytes in the mutant hippocampus, as determined by S-100 staining, is not, however, significantly different from WT. In addition, quantification of immunohistochemical staining of female, but not male, mutants reveals a significant deficit in myelin basic protein content in three brain regions, suggesting alterations in oligodendrocytes as well. Thus, while overall brain histology appears normal, the absence of LIF in vivo leads to specific, sexually dimorphic alterations in glial phenotype. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 509–524, 1998     

Affinity ligand selection from a library of small molecules: assay development, screening, and application

A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work.