Reduction of laccase type 1 copper by 3,4-dihydroxyphenylalanine and other catechol derivatives

3,4-Dihydroxyphenylalanine (DOPA) is not a preferred substrate of Rhus vernicifera laccase, as rate constants for the anaerobic reduction of the type 1 cupric atom by L-DOPA (6.3 X 10(1) M-1 s-1), D-DOPA (2.6 X 10(1) M-1 s-1), and L-DOPA methyl ester (2.6 X 10(1) M-1 s-1) are considerably smaller than k1 (catechol) (7 X 10(2) M-1 s-1) and rate constants characteristic of numerous other nonphysiological organic substrates (25 degrees C, pH 7.0, I = 0.5 M). The reactions of DOPA derivatives with laccase are unique, however, in that a two-term rate law pertains: kobsd = k0 + k1[phenol]; k0(L-DOPA) = 7 X 10(-2) s-1. The reactivities of other catechol derivatives (pyrogallol, gallic acid, and methyl gallate) with laccase type 1 copper were also examined.     

Freeze-thaw-refreeze cycle to prepare lymphocytes for HLA antibody detection or tissue typing

Human lymphocytes stored in the frozen state may be thawed, placed on cytotoxicity plates, refrozen, rethawed and used for screening sera or tissue-typing of the cells. The simple procedure described uses only a ?90 °C refrigerator for both freezing and storage of the cells. The technique permits a laboratory to collect a variety of cells over a long period, so that a set of test plates with cells from 10 to 20 donors can be prepared when a convenient number of donor cells are available. Also, the refrozen cells in cytotoxicity test plates may be warmed to the temperature of dry ice for 24 hr, returned to the refrigerator set at a slightly lower temperature, and at a later time, these cells may be thawed and used for serum screening. In view of these results, it appears possible to ship the refrozen cells from one laboratory to another using simple dry ice storage during the transfer. Negative reactions due to soluble antigens in the suspending sera can be obviated by washing out these sera and replacing them with medium 199 or alternatively, fetal calf serum can be used to replace the human serum in the suspending media.     

Severe Metabolic Acidemia in Infants: Clinical and Therapeutic Aspects

A study of 10 infants in severe metabolic acidemia (pH below 7) led to the conclusion that the clinical signs—hyperventilation, coma or lethargy, peripheral vascular collapse, a significantly palpable liver, and abdominal distension—may all be directly related to the metabolic acidemia. In five of 10 infants, an initial erroneous diagnosis of congestive heart failure or pneumonia was made. Dramatic clinical improvement followed correction of the acidemia with rapid intravenous administration of sodium bicarbonate. This rapid administration of sodium bicarbonate was safe, provided hypocalcemia was recognized and treated in its early stages. In severe metabolic acidemia the measurement of blood bicarbonate alone does not reflect adequately the magnitude of the acid-base derangement and repeated measurements of hydrogen ion concentration, Pco₂ and bicarbonates are needed to evaluate and treat such infants correctly.     

Classical plant taxonomic ambiguities extend to the molecular level

Summary The molecular evolution of cytochrome c from angiosperms is compared to that from vertebrates. On the basis of a cladistic analysis from 26 plant species, compared to that from 27 vertebrate species, we find that although the vertebrate sequences yield reasonably well-defined minimal trees that are congruent with the biological tree, the plant sequences yield multiple minimal trees that are not only highly incongruent with each other, but none of which is congruent with any reasonably biological tree. That is, the plant sequence set is much more homoplastic than that of the animal. However, as judged by the relative rate test, the extent of divergence, and degree of functional constraint, cytochrome c evolution in plants does not appear to differ from that of vertebrates.     

Influence of oestradiol on protein expression and methionine utilization during morphogenesis of Paracoccidioides brasiliensis

The temporal sequence of cytosolic protein expression during phase transition of Paracoccidioides brasiliensis was examined. Electrophoretic analysis of cytosol proteins by one-dimensional SDS-PAGE revealed numerous differences between the mycelial and yeast forms as well as alterations induced by 17 beta-oestradiol. Using either protein staining or fluorography of [35S]methionine-labelled proteins 30 phase-specific bands were detected, 12 mycelial-associated bands (range 30 to 140 kDa) and 18 yeast-associated bands (range 22 to 127 kDa). In cells undergoing mycelial to yeast transition after a shift from 25 degrees C to 37 degrees C, the protein patterns showed a temporal progression toward the yeast profile with the accumulation of yeast bands prior to observable morphogenesis. Five novel protein bands (range 23 to 50 kDa) were detected by silver staining during transition. Treatment of temperature-shifted mycelial cultures with 2.6 x 10(-7) M-oestradiol altered observed profiles; 4 of 12 mycelial-associated bands were maintained whereas the appearance of the 5 novel transition bands and 9 of 18 yeast-associated bands was blocked or delayed. Analysis of [35S]methionine-labelled proteins revealed that oestradiol induced label uptake by mycelial cells, blocked the synthesis of a 92 kDa yeast-specific band 72 h into transition, and diminished label incorporation 120 h into transition. In conjunction with these steroid-induced alterations of protein expression, little or no morphological transformation occurred. These results support our hypothesis that, analogous to mammalian steroid receptor action, the functional responses of P. brasiliensis to oestradiol are related to regulation of protein expression, presumably mediated via a specific binding protein-ligand complex.     

Kinetics of the sodium-dependent glutamine transporter in human intestinal cell confluent monolayers.

The intestinal epithelium metabolism of glutamine plays a critical role in inter-organ nitrogen flow. Although it is known that glutamine is the primary oxidative energy source and nucleotide precursor in intestinal cells, the luminal uptake of glutamine by the apical surface of enterocytes is poorly understood. In this study we have uncovered the sodium-dependent transporter system responsible for L-glutamine uptake by the apical membrane of a human intestinal epithelial cell line. The sodium-dependent Michaelis constant (Km) = 247 +/- 45 microM glutamine, and Jmax = 4.44 +/- 0.65 x 10(-9) mole min-1(mg protein)-1 (37 degrees C). Glutamine shares the transporter with alanine, as demonstrated by unlabeled glutamine inhibition of [3H]alanine uptake kinetics with a purely competitive-type inhibition pattern, and glutamine inhibition Ki = 205 +/- 18 microM by Dixon analysis. The inhibition pattern for a series of amino acid analogs indicated that this intestinal apical membrane sodium-dependent transporter for glutamine is distinct from any other transport system found in membranes of non-intestinal cells.     

Effect of harvest intensity and ground flora establishment on inorganic-N leaching from a Sitka spruce plantation in north Wales,UK

Inorganic-N concentrations in soil solution of whole tree harvest (WTH) and conventional fell (CF) plots were monitored for two years before felling and four years after felling. Concentrations in the mineral soil after felling were higher than in standing forest for up to 14 months in both felling treatments. In the WTH plots inorganic-N concentrations then dropped steadily until four years after felling they approached zero. In contrast, inorganic-N concentrations of the CF plots remained comparatively large. Inorganic-N was dominated by nitrate throughout the period of the study, and especially in the mineral horizons.Felling debris was not a source of inorganic-N, unless indirectly through release and mineralisation of soluble organic-N. Vegetation cover, biomass and N content were substantially greater in the WTH plots two to three years after felling, compared with the CF. Vegetation cover and brash cover (slash cover in N. America) were negatively correlated. There was also a negative correlation between inorganic-N concentration in soil water samplers and the vegetation cover within the collection area of, or a 1 m square surrounding, these samplers.Two factors are probably responsible for the reduction in inorganic-N concentrations after felling in the WTH — the rapid re-establishment of vegetation and the lack of a N source in felling debris. In the CF plots, brash prevents re-establishment of vegetation over wide areas for at least four years. However, brash is not directly a source of inorganic-N at this stage.