A comparison of the different methods available for determining BCG-macrophage interactions in vitro, including a new method of colony counting in broth

Abstract Different methods of determining BCG viability based on colony forming unit (CFU) counting and radio-isotope labelling were comparatively assessed. These included radio-isotope labelling with [³H]uracil, [³H]uridine, [³H]glycerol, and CFU counting, by both agar plate dilution, and microcolony counting in broth. The sensitivity ranges of the different techniques were determined in both macrophage-free and macrophage-treated systems and used to assess the anti-mycobacterial potential of human monocyte-derived macrophages following BCG infection.     

Survey of normal appearing mouse strain which lacks enzyme and NAD+-linked glycerol phosphate dehydrogenase: Normal pancreatic beta cell function,but abnormal metabolite pattern in skeltal muscle

We studied a mouse doubly homozygous for mutations in the genes encoding malic enzyme (EC1.1.1.40) and cytosolic glycerol phosphate dehydrogenase (EC 1.1.1.8) (cGPD). This mouse, which we call the mmgg mouse and which is the product of intercrosses between the Mod-1 mouse and the BALB/cHeA mouse, lacks activity of both enzymes. Like both parental strains the mmgg mouse is completely normal in appearance. cGPD is one of the two enzymes that catalyze the reactions of the glycerol phosphate shuttle. The activity of the other enzyme of the glycerol phosphate shuttle, mitochondrial glycerol phosphate dehydrogenase (EC 1.1.99.5) (mGPD), is abundant in tissues, such as brain, skeletal muscle and the pancreatic islet, suggesting that the glycerol phosphate shuttle is important in these tissues which rapidly metabolize glucose. Cytosolic malic enzyme activity is important for shuttles which transport NADPH equivalents from mitochondria to the cytosol. The major finding of the study was a highly abnormal metabolite pattern in tissues of the mmgg mouse suggesting a block in the glycerol phosphate shuttle due to cGPD deficiency. The metabolite pattern did not suggest that malic enzyme deficiency caused an abnormality. Tissue levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were only abnormal in skeletal muscle. Glycolytic intermediates, situated at or before the triose phosphates in the pathway, such as fructose bisphosphate and glyceraldehyde phosphate were increased depending on the tissue. Taken together with previous extensive data on the mouse deficient only in cGPD this suggests a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase caused by an abnormally low NAD/NADH ratio resulting from a nonfunctional glycerol phosphate shuttle. Consistent with this idea the lactate/pyruvate ratio was high in skeletal muscle signifying a low cytosolic NAD/NADH ratio. The mmgg mouse was normal in all other factors studied including blood glucose and serum insulin levels, pancreatic islet mass, insulin release from isolated pancreatic islets, as well as the activities of five metabolic enzymes, including mGPD, in liver, kidney, skeletal muscle and pancreatic islets. cGPD enzyme activity was undetectable in pancreatic islets, 0.5% of normal in liver, and 2.1% of normal in kidney and skeletal muscle. Malic enzyme activity was undetectable in these same tissues.     

NaCl stress inhibits maltose fermentation by Saccharomyces cerevisiae

While fermentation of 20 g glucose l^–1 by Saccharomyces cerevisiae was not impaired by high NaCl concentrations, fermentation of 20 g maltose l^–1 was significantly decreased by 0.7 M NaCl, and completely inhibited with 1.4 M NaCl. No glycerol was produced in response to the salt stress when yeast cells were fermenting maltose. Active maltose transport, and not intracellular hydrolysis, was the metabolic step severely impaired by the NaCl stress.     

31P-NMR observation of the temperature and glycerol induced non-lamellar phase formation in wheat thylakoid membranes

³¹P-NMR spectra at 162 MHz were used to monitor phase changes of wheat thylakoid membranes as a function of temperature. At room temperature the³¹P-NMR line was a superposition of anisotropic component characteristic of phospholipid lamellar phase and isotropic line due to inorganic phosphorus or small membrane vesicles arising as an effect of preparation. For temperatures higher than +35 °C an increase of the isotropic component occurs, which is irreversible as the sample is cooled. For the temperatures between +55 °C and +60 °C the presence of the hexagonal phase cylinders is suggested, as monitored by phosphorus lineshape. However, the addition of glycerol stimulates a formation of the isotropic phase. The effect of reconstitution of freeze-dried thylakoid membranes by addition of water or water-glycerol medium to the sample was examined. As lyophilizate was gradually diluted, the increase of isotropic line component was observed. For thylakoid membranes suspended in D₂O at the highest dilution examined, the line contribution due to small membrane fragments is not greater than 50%, but in presence of glycerol, this contribution could reach 70%. This suggests that the presence of glycerol increases the formation of the small membrane particles as the thylakoid membrane is reconstituted from lyophilizate. The wheat thylakoid membranes reconstituted from lyophilizate show, in comparison to native membranes, the increased contribution of small membrane vesicles. Moreover, the³¹P -NMR spectra suggest the appearance of the hexagonal phase cylinders even at +50 °C.Abbreviations DGDG digalactosyldiacylglycerol - DLPC dilinoleoyl phosphatidylcholine - DLPE dilinoleoyl phosphatidylethanolamine - EDTA ethylenediamine-tetraacetic acid - MGDG monogalactosyldiacylglycerol - NMR nuclear magnetic resonance - PC phosphatidylcholine - PG phosphatidylglycerol - PSII photosystem II - TGDG trigalactosyldiacylglycerol - Tris Tris-(hydroxymethyl)-aminomethan - S/N signal to noise ratio     

Growth of an Insect Trypanosomatid at 37 C in a Defined Medium*

SYNOPSIS. A defined medium for an insect trypanosomatid, “Leptomonas pessoai” (probably a member of the genus Herpetomonas) isolated from the reduviid Zelus leucogrammus, allows growth up to 37 C. No marked differences were evident for growth at 37 C. The requirements for amino acids, vitamins, purine, and hemin, and pH range were like those established for Crithidia fasciculata. Again like C. fasciculata, it remains alive at 4 C for at least 3 months in a çlycerolated defined medium.     

Therapeutic targeting of the p53 tumor suppressor gene

The p53 tumor suppressor gene is a logical target for cancer therapy. Several therapeutic strategies can be envisioned based upon recent advances concerning structure and function of the p53 protein, its interaction with cellular and viral proteins and its roles in repairing DNA, regulating cell division and promoting apoptosis.     

Zinc-induced self-assembly of goat brain tubulin: some novel aspects

Unlike normal microtubule assembly, the $\text{in vitro}$ assembly of DEAE-purified goat brain tubulin in presence of Zn(II) is not inhibited by suprastoichiometric concentrations of antimicrotubular drugs like colchicine and podophyllotoxin. However, assembly in the presence of Zn(II) is inhibited by vinblastine. Vinblastine sensitivity of the assembly process depends on the Mg(II) concentration in the assembly medium. Like normal microtubules, Zn(II)-induced polymers are sensitive to cold. The polymers assembled in presence of Zn(II) are readily disassembled on treatment with Zn(II)-chelators like EDTA or o-phenanthroline, indicating that the binding of Zn(II) to tubulin is essential for maintaining the polymeric structure.     

ABHD4 regulates adipocyte differentiation in vitro but does not affect adipose tissue lipid metabolism in mice

Alpha/beta hydrolase domain-containing protein 4 (ABHD4) catalyzes the deacylation of N-acyl phosphatidyl-ethanolamine (NAPE) and lyso-NAPE to produce glycerophospho-N-acyl ethanolamine (GP-NAE). Through a variety of metabolic enzymes, NAPE, lyso-NAPE, and GP-NAE are ultimately converted into NAE, a group of bioactive lipids that control many physiological processes including inflammation, cognition, food intake, and lipolysis (i.e., oleoylethanolamide or OEA). In a diet-induced obese mouse model, adipose tissue Abhd4 gene expression positively correlated with adiposity. However, it is unknown whether Abhd4 is a causal or a reactive gene to obesity. To fill this knowledge gap, we generated an Abhd4 knockout (KO) 3T3-L1 pre-adipocyte. During adipogenic stimulation, Abhd4 KO pre-adipocytes had increased adipogenesis and lipid accumulation, suggesting Abhd4 is responding to (a reactive gene), not contributing to (not a causal gene), adiposity, and may serve as a mechanism for protecting against obesity. However, we did not observe any differences in adiposity and metabolic outcomes between whole-body Abhd4 KO or adipocyte-specific Abhd4 KO mice and their littermate control mice (both male and female) on chow or a high-fat diet. This might be because we found that deletion of Abhd4 did not affect NAE such as OEA production, even though Abhd4 was highly expressed in adipose tissue and correlated with fasting adipose OEA levels and lipolysis. These data suggest that ABHD4 regulates adipocyte differentiation in vitro but does not affect adipose tissue lipid metabolism in mice despite nutrient overload, possibly due to compensation from other NAPE and NAE metabolic enzymes.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.