Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.     

Characteristics of brain Kv1 channels tailored to mimic native counterparts by tandem linkage of alpha subunits: implications for K+ channelopathies

Most neuronal Kv1 channels contain Kv1.1, Kv1.2 alpha, and Kvbeta2.1 subunits, yet the influences of their stoichiometries on properties of the (alpha)(4)(beta)(4) variants remain undefined. cDNAs were engineered to contain 0, 1, 2, or 4 copies of Kv1.1 with the requisite number of Kv1.2 and co-expressed in mammalian cells with Kvbeta2.1 to achieve "native-like" hetero-oligomers. The monomeric (Kv1.1 or 1.2), dimeric (Kv1.1-1.2 or 1.2-1.2), and tetrameric (Kv1.1-(1.2)(3)) constructs produced proteins of M(r) approximately 62,000, 120,000, and 240,000, which assembled into (alpha)(4)(beta)(4) complexes. Each alpha cRNA yielded a distinct K(+) current in oocytes, with voltage dependence of activation being shifted negatively as the Kv1.1 content in tetramers was increased. Channels containing 1, 2, or 4 copies of Kv1.1 were blocked by dendrotoxin k (DTX)(k) with similarly high potencies, whereas Kv(1.2)(4) proved nonsusceptible. Accordingly, Kv1.2/beta2.1 expressed in baby hamster kidney cells failed to bind DTX(k); in contrast, oligomers containing only one Kv1.1 subunit in a tetramer exhibited high affinity, with additional copies causing modest increases. Thus, one Kv1.1 subunit largely confers high affinity for DTX(k), whereas channel electrophysiological properties are tailored by the content of Kv1.1 relative to Kv1.2. This notable advance could explain the diversity of symptoms of human episodic ataxia I, which is often accompanied by myokymia, due to mutated Kv1.1 being assembled in different combinations with wild-type and Kv1.2.     

Differential steric effects in the axial ligation of pyridine and imidazole to α- and β-alkylcobinamides

One subclass of B₁₂-requiring enzymes is now known to bind their B₁₂ coenzymes “base-off,” with a histidine residue from the protein supplying an imidazole ligand to the cobalt center. Recent results from Sirovatka and Finke (J.M. Sirovatka and R.G. Finke, J.Am. Chem. Soc. 119, (1997) 3057) show that imidazole has an extraordinary trans effect on the mode of carbon–cobalt bond cleavage in coenzyme B₁₂ analogs, compared to pyridine or the natural 5,6-dimethylbenzimidazole ligand, and it was suggested that a differential steric effect could, in part, account for the uniqueness of the imidazole ligand. Such a differential steric effect for imidazole and pyridine is now demonstrated by studies of the thermodynamics of ligation of these ligands to the α and β diastereomers of two alkylcobinamides (RCbi⁺s, derivatives of cobalamins which lack the normal axial nucleotide) based on the known differences in steric crowding of the α (“lower”) and β (“upper”) axial ligand positions of cobalt corrinoids. Imidazole binds more tightly than pyridine to both diastereomers of NCCH₂Cbi⁺ and CF₃Cbi⁺, in all cases due to a more favorable entropy change, which is the result of lowered steric interference with corrin side chain thermal motions.     

Glycerol Phosphate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4-fold during development, and at 120 days cells of this type had 1.4-fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10-day-old rats were 1.4-fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20-, 60-, and 120-day-old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin-related membrane vesicles.     

Species distribution and <Emphasis Type="Italic">in vitro</Emphasis> antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasis

Background  

In Tanzania, little is known on the species distribution and antifungal susceptibility profiles of yeast isolates from HIV-infected patients with primary and recurrent oropharyngeal candidiasis.     

Age- and Gender-Based Studies of Trace Metal Levels and Various Enzymes Associated with Myocardial Infarction

The present investigation deals with the determination of various serum enzymes known to be elevated during myocardial infarction (MI) and estimation of selected metals like Cu, Cr, Co, Fe, Pb, and Mg by flame atomic absorption spectrophotometry. The data obtained thereby were processed for the determination of correlation coefficient matrix among the cardiac enzymes and the serum metals. The study evidenced the accumulation of Pb during MI and reduction in the level of Fe. A significant negative correlation was observed between Cu and creatine kinase-MB. The data were also segregated into various groups to study the influence of age and gender on the levels of selected parameters. In both the genders, the age of the patients was found to be correlated significantly with various cardiac enzymes. In case of male patients, the most significant correlation was observed between age and blood sugar at random. The other significant correlations among the male patients included Cr–CPK, Cr–creatine kinase-MB, Fe–age, and others. In female patients, the pairs of studied parameters that exhibited significant correlations included age–lactic dehydrogenase, creatine phosphokinase isoenzyme–aspartate aminotransferase, lactic dehydrogenase–creatine phosphokinase isoenzymes, Pb-Fe, and Cu-Co in addition to others.     

Differential protein expression profile in the liver of pikeperch (Sander lucioperca) larvae fed with increasing levels of phospholipids

A comparative proteomic approach was used to assess the protein expression profile in the liver of 34 days old pikeperch larvae fed from day 10 post hatching, with three isoproteic and isolipidic formulated diets varying by their phospholipid (PL) contents (% dry diet weight): 1.4% (PL1), 4.7% (PL5) and 9.5% (PL9). Using 2D-DIGE minimal labelling of liver extracts, we were able to show 56 protein spots with a differential intensity (p < 0.05) depending on the dietary PL content. Among these spots, 11 proteins were unambiguously identified using nanoLC-MS/MS tandem mass spectrometry. In the PL9 larvae, our results indicate that the glycolytic pathway could be down-regulated due to the under-expression of the fructose biphosphate aldolase B and the phosphoglucomutase 1. Meanwhile, propionyl coenzyme A carboxylase (a gluconeogenic enzyme) was under-expressed. In addition, another gluconeogenic and lipogenic enzyme, pyruvate carboxylase, was identified in 3 different spots as being under-expressed in fish fed with the intermediate PL level (PL5). A high PL content increased the expression of sarcosine dehydrogenase, an enzyme involved in methionine metabolism, along with vinculin, a structural protein. Moreover, several stress proteins (glutathione S-transferase M, glucose regulated protein 75 and peroxiredoxin-1) were modulated in response to the dietary PL level and fatty acid composition. In the larvae fed with the lowest dietary PL content (PL1), over-expression of both GSTM and GRP75 might indicate a cellular stress in this experimental treatment, while the under-expression of Prx1 might indicate a lower defence against oxidative stress. In conclusion, this nutriproteomic approach showed significant modifications of protein expression in the liver of pikeperch larvae fed different PL contents, highlighting the importance of these nutrients and their influence on metabolism processes and on stress response.