Electrophoretic analysis of plasma membrane proteins of wild-type and differentiation-deficient mutant strains ofPodospora anserina

In the fungusPodospora anserina, themodC mutations inhibit the sexual female organ differentiation. Previous results have suggested that the plasma membrane of this mutant may be altered. Proteins solubilized from highly purified plasmalemma from a wild-type and threemodC mutant strains were studied by one- and two-dimensional electrophoresis. Isoelectric focusing revealed in the wild-type strain about 80 polypeptide spots whose molecular weight ranged from 15,000 to 80,000 daltons; 65% of the spots were between 20,000 and 60,000 daltons, and 60% were in the 6.5–7.5 pH zone. The only difference inprotein noted between wild-type and the threemodC mutants was that the threemodC mutants lacked a plasmalemma protein of M_r=42,000 and of pI=6.7. These results suggest a close relationship between plasma membrane proteins and differentiation in this eukaryotic organism.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Quantitation of cell proliferation,colony formation,and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers

Summary A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), γ-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-epoxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.     

High resolution nuclear magnetic resonance studies of nigeran

Polymer motion in solution can be studied by ¹³CNMR relaxation methods, which provide information about the correlation time for C-H vectors. ¹³C-Relaxation and Nuclear Overhauser Enhancement (NOE) data may frequently be combined to determine the dipole-dipole relaxation contribution. An alternative method is proposed based on a comparison of the proton spin-lattice relaxation rates of the centre proton resonances of an unlabelled molecule with the relaxation rates of the ¹³C satellites (from ¹³C labelled molecules).Selectively labelled nigeran which is an alternating $\text{1 → 3 and 1 → 4 α-}\text{d}\text{-glucan}$ has been investigated. The discussion in terms of the occurrence of different motions for each of the two units of the polymer requires an unambiguous assignment of the two anomeric carbons. For this reason a detailed assignment of the ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectra of nigeran in dimethylsulphoxide-d₆ is described, based on T₁ and NOE measurements in addition to selective homonuclear and heteronuclear spin decoupling experiments. These values are correlated with a conformation estimated by HSEA hard-spheres calculation. The measurements of the relaxation parameters for labelled and unlabelled compounds which provide an alternative determination of the ¹³C-¹H dipole-dipole relaxation contribution in a macromolecule agree well with ¹³C-{¹H} NOE experiments.     

A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor

A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C.     

Glutamate Dehydrogenase Reaction as a Source of Glutamic Acid in Synaptosomes

The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM 15NH4Cl and then utilizing gas chromatography-mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of [15N]glutamate and [15N]aspartate from 5 mM 15NH4Cl was approximately 0.2 nmol/min/mg of protein, a value much less than flux through glutaminase (4.8 nmol/min/mg of protein) but greater than flux through glutamine synthetase (0.045 nmol/min/mg of protein). Addition of 1 mM 2-oxoglutarate to the medium did not affect the rate of [15N]glutamate formation. O2 consumption and lactate formation were increased in the presence of 5 mM NH3, whereas the intrasynaptosomal concentrations of glutamate and aspartate were unaffected. Treatment of synaptosomes with veratridine stimulated reductive amination of 2-oxoglutarate during the early time points. The production of ([15N]glutamate + [15N]aspartate) was enhanced about twofold in the presence of 5 mM beta-(+/-)-2-aminobicyclo [2.2.1]heptane-2-carboxylic acid, a known effector of glutamate dehydrogenase. Supplementation of the incubation medium with a mixture of unlabelled amino acids at concentrations similar to those present in the extracellular fluid of the brain had little effect on the intrasynaptosomal [glutamate] and [aspartate]. However, the enrichment in these amino acids was consistently greater in the presence of supplementary amino acids, which appeared to stimulate modestly the reductive amination of 2-oxoglutarate. It is concluded: (a) compared with the phosphate-dependent glutaminase reaction, reductive amination is a relatively minor pathway of synaptosomal glutamate synthesis in both the basal state and during depolarization; (b) NH3 toxicity, at least in synaptosomes, is not referable to energy failure caused by a depletion of 2-oxoglutarate in the glutamate dehydrogenase reaction; and (c) transamination is not a major mechanism of glutamate nitrogen production in nerve endings.     

Tissue distribution,metabolism, and elimination of perfluorooctanoic acid in male and female rats

The elimination, tissue distribution, and metabolism of [1-¹⁴C]perfluorooctanoic acid (PFOA) was examined in male and female rats for 28 days after a single ip dose (9.4 μmol/kg, 4 mg/kg). A sex difference in urinary elimination of PFOA-derived ¹⁴C was observed. Female rats eliminated PFOA-derived radioactivity rapidly in the urine with 91% of the dose being excreted in the first 24 hr. In the same period, male rats eliminated only 6% of the administered ¹⁴C in the urine. The sex-related difference in urinary elimination resulted in the observed difference in the whole-body elimination half-life (t_1/2) of PFOA in males (t_1/2 = 15 days) and females (t_1/2 < 1 day). Analysis of PFOA-derived ¹⁴C in tissues showed that the liver and plasma of male rats and the liver, plasma, and kidney of female rats were the primary tissues of distribution. The relatively high concentration of PFOA in the male liver was further examined using an in situ nonrecirculating liver perfusion technique. It was shown that 11% of the PFOA infused was extracted by the liver in a single pass. The ability of the liver to eliminate PFOA into bile was examined in rats whose renal pedicles were ligated to alleviate sex differences in the urinary excretion of PFOA. In a 6-hr period following IP administration of PFOA, there was no apparent difference in biliary excretion, where both males and females eliminated less than 1% of the PFOA dose via this route. We hypothesized that the sex difference in the persistence of PFOA was due to a more rapid formation of a PFOA-containing lipid (i.e., a PFOA-containing mono-, di-, or triacylglycerol, cholesteryl ester, methyl ester, or phospholipid) in the male rat. Also, the increased urinary elimination of PFOA in females may have been due to increased metabolism to a PFOA-glucuronide or sulfate ester. However, no evidence that PFOA is conjugated to form a persistent hybrid lipid was obtained, nor were polar metabolites of PFOA in urine or bile detected. In addition, daily urinary excretion of fluoride in male and female rats before or after PFOA treatment were similar, suggesting that the parent compound is not defluorinated. Thus, the more rapid elimination of PFOA from female rats is not due to formation of a PFOA metabolite.     

Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Human τ Detection

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.     

Increased Neopterin Levels in Brains of Patients with Human Immunodeficiency Virus Type 1 Infection

Postmortem levels of native neopterin (D-erythro-neopterin) were measured in cerebral cortical samples from 44 human immunodeficiency virus type 1-infected and eight uninfected, nonneurological control patients. Cerebral cortical gray and white matter neopterin levels for the controls ranged from 0.5 to 7.2 pmol/mg of protein in contrast to neopterin levels in brains of the virus-infected patients, which frequently were more than threefold and occasionally more than 30-fold higher than mean control levels. Cortical neopterin levels did not correlate with severity of the acquired immunodeficiency syndrome dementia complex, but subcortical levels correlated with the presence of active human immunodeficiency virus type 1 infection, as reflected by pathological evidence of multinucleated giant cell encephalitis. Evidence of opportunistic cytomegalovirus infections in approximately 25% of the human immunodeficiency virus type 1-infected patients was associated with enhanced levels of neopterin in frontal cortex.