Nω-Hydroxyamino-α-amino acids as a new class of very strong inhibitors of arginases

 The effects of various compounds bearing an N-OH group such as N-hydroxy-guanidines, amidoximes, and hydroxylamines, on bovine and rat liver arginases was studied. Some of these compounds with an l-α-amino acid function at an appropriate distance from the N-OH group acted as strong competitive liver arginase inhibitors, displaying Ki values between 4 and 150 μM. Two compounds, N ^ε-hydroxy-l-lysine and N ^ω-hydroxy-d,l-indospicine, which exhibited Ki values of 4 and 20 μM (at pH 7.4), were the most potent inhibitors of arginase described to date. The distance between the α-amino acid and N-OH functions appeared to be crucial for potent inhibition of arginase, as N ^δ-hydroxy-l-ornithine, which has one -CH₂ group less than N ^ε-hydroxy-l-lysine, exhibited a 37-fold higher Ki value than N ^ε-hydroxy-l-lysine. Based on these results, a model for the interaction of N ^ω-hydroxyamino-l-α-amino acids with the arginase active site is proposed. This model involves the binding of the N-OH group of the inhibitors to the arginase Mn(II) center and suggests that N ^ε-hydroxy-l-lysine is a good transition state analog of arginase.     

Relationship between Poly- β-hydroxybutyrate Production and δ-endotoxin for Bacillus thuringiensis var. kurstaki

A linear relationship between total solid concentration (TSC), δ-endotoxin production [Cry = 0.2795(TSC)−0.2472, R² = 0.8644] and poly-β-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R² = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between δ-endotoxin and PHB accumulation [Cry = 2.1573(PHB)−1.1248, R² = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l⁻¹ was required before the onset of δ-endotoxin production. Revisions requested 28 September 2005 and 4 November 2005; Revisions received 28 October 2005 and 1 February 2006     

Between-tree variations in leaf δ13C of Quercus pubescens and Quercus ilex among Mediterranean habitats with different water availability

In this study, sun leaf carbon isotope composition (δ¹³C) of two co-occurring woody Mediterranean species (Quercus pubescens Willd., a deciduous oak, and Q. ilex L., an evergreen one) was investigated on four sites with different water availability. The total range of δ¹³C values was 4.4 and 3.1‰ for Q. pubescens and Q. ilex respectively. The intra-site variability was about 3‰. Total mean per species was equal. There were significant differences among sites, but at each site means of δ¹³C were not significantly different between species. A simple physiological model predicts no difference in intrinsic water-use efficiency (WUE_i) between evergreen and deciduous oaks. The relationship between site means of δ¹³C and water parameters suggests that there is a leaf functional adjustment with respect to available water resource. No correlation was found between δ¹³C and the contents of any mass-based biochemical constituent. Nevertheless there was a significant correlation between δ¹³C and leaf mass per area of Q. ilex. For both species, there is also a positive correlation between leaf δ¹³C and individual crown area, i.e. a structural characteristic at tree level. Causal relations between δ¹³C and plant-environment interactions are discussed. Received: 25 October 1996 / Accepted: 19 January 1997     

Nocturnal and seasonal patterns of carbon isotope composition of leaf dark-respired carbon dioxide differ among dominant species in a semiarid savanna

The C isotope composition of leaf dark-respired CO₂ (δ¹³C_l) integrates short-term metabolic responses to environmental change and is potentially recorded in the isotopic signature of ecosystem-level respiration. Species differences in photosynthetic pathway, resource acquisition and allocation patterns, and associated isotopic fractionations at metabolic branch points can influence δ¹³C_l, and differences are likely to be modified by seasonal variation in drought intensity. We measured δ¹³C_l in two deep-rooted C₃ trees (Prosopis velutina and Celtis reticulata), and two relatively shallow-rooted perennial herbs (a C₃ dicot Viguiera dentata and a C₄ grass Sporobolus wrightii) in a floodplain savanna ecosystem in southeastern Arizona, USA during the dry pre-monsoon and wet monsoon seasons. δ¹³C_l decreased during the nighttime and reached minimum values at pre-dawn in all species. The magnitude of nocturnal shift in δ¹³C_l differed among species and between pre-monsoon and monsoon seasons. During the pre-monsoon season, the magnitude of the nocturnal shift in δ¹³C_l in the deep-rooted C₃ trees P. velutina (2.8 ± 0.4‰) and C. reticulata (2.9 ± 0.2‰) was greater than in the C₃ herb V. dentata (1.8 ± 0.4‰) and C₄ grass S. wrightii (2.2 ± 0.4‰). The nocturnal shift in δ¹³C_l in V. dentata and S. wrightii increased to 3.2 ± 0.1‰ and 4.6 ± 0.6‰, respectively, during the monsoon season, but in C₃ trees did not change significantly from pre-monsoon values. Cumulative daytime net CO₂ uptake was positively correlated with the magnitude of the nocturnal decline in δ¹³C_l across all species, suggesting that nocturnal δ¹³C_l may be controlled by ¹³C/¹²C fractionations associated with C substrate availability and C metabolite partitioning. Nocturnal patterns of δ¹³C_l in dominant plant species in the semiarid savanna apparently have predictable responses to seasonal changes in water availability, which is important for interpreting and modeling the C isotope signature of ecosystem-respired CO₂.     

Vertical canopy gradients in δ13C correspond with leaf nitrogen content in a mixed-species conifer forest

Stable carbon isotope composition varies markedly between sun and shade leaves, with sun leaves being invariably more enriched (i.e., they contain more¹³C). Several hypotheses have emerged to explain this pattern, but controversy remains as to which mechanism is most general. We measured vertical gradients in stable carbon isotope composition (δ¹³C) in more than 200 trees of nine conifer species growing in mixed-species forests in the Northern Rocky Mountains, USA. For all species except western larch, δ¹³C decreased from top to bottom of the canopy. We found that δ¹³C was strongly correlated with nitrogen per unit leaf area (N _area), which is a measure of photosynthetic capacity. Usually weaker correlations were found between δ¹³C and leaf mass per area, nitrogen per unit leaf mass, height from the ground, or depth in the canopy, and these correlations were more variable between trees than for N _area. Gradients of δ¹³C (per meter canopy depth) were steeper in small trees than in tall trees, indicating that a recent explanation of δ¹³C gradients in terms of drought stress of upper canopy leaves is unlikely to apply in our study area. The strong relationship between N _area and δ¹³C here reported is consistent with the general finding that leaves or species with higher photosynthetic capacity tend to maintain lower CO₂ concentrations inside leaves. We conclude that photosynthetic capacity is a strong determinant of δ¹³C in vertical canopy profiles, and must be accounted for when interpreting δ¹³C values in conifer forests.     

Biophysical parameters as informative tools for elucidating the causes of root growth retardation under stressful conditions

The root growth rate in barley (Hordeum vulgare L.) seedlings was measured in parallel with temporal changes in longitudinal (δl) and transverse (δD/D) cell-wall extensibilities and membrane hydraulic conductivity (L _p) in the root extension zone. The root growth rate and biophysical parameters examined were sensitive to UV-B irradiation of shoots or roots and to excessive content of ammonium, glutamate, or nickel in the nutrient medium. The root responses to the above treatments were compared with the effects of abscisic acid, salicylate, hydrogen peroxide, diethylstilbestrol, α-naphthyl acetate, oryzalin, and ionomycin. The progressive reduction of root growth under the action of various stressors was accompanied by typical temporal patterns of the growth zone parameters: the δl extensibility declined monotonically, while δD/D and L _p changed nonmonotonically, exhibiting the reversion from the initial decrease to the eventual increase above the control values. The decline of δl indicated that the root growth suppression was mainly due to changes in cell-wall mechanical properties caused probably by disorganization of cortical microtubules. It was found that the decline in δD/D and L _p was caused primarily by the appearance of oxidative stress, disorders in cytoplasmic H⁺ homeostasis in root cells, and the consequent transient activation of the plasmalemmal H⁺-pump. Conversely, the increase in δD/D and L _p upon the abrupt retardation of root growth was presumably caused by the increase in cytoplasmic Ca²⁺ content, disassembling of cortical microtubules, and by partial inhibition of the plasmalemmal H⁺-pump. The reversion of δD/D and L _p changes upon progressive reduction of root growth can be used as an indicator to distinguish moderate and severe stress conditions in the root growth zone. Furthermore, this reversion indicates the increasing disbalance in the homeostasis of reactive oxygen species, cytosolic Ca²⁺, and cytosolic H⁺ upon severe stress.     

The environmental signals of stable carbon isotope in various tree-ring components of Pinus tabulaeformis

Chinese pine (Pinus tabulaeformis) trees were sampled in the Helan Mountain, northwest China. The stable carbon isotope (δ¹³C) values of whole wood, holocellulose and alpha-cellulose in tree rings over 30 years (1968–1997) were measured to study the δ¹³C response of different tree-ring components to past environmental change. There were obvious differences in the δ¹³C values of the three components. The Pearson correlation coefficient between the δ¹³C of alpha-cellulose and that of holocellulose was 0.547 (ρ < 0.01); between alpha-cellulose and whole wood, the coefficient was −0.126 (ρ > 0.10); between holocellulose and whole wood, the coefficient was −0.056. Correlation function analyses indicated that the δ¹³C content of tree-ring alpha-cellulose correlated strongly with the average temperature from June to August (r = 0.427, ρ < 0.05), more than that of holocellulose (0.324, ρ < 0.10) or total wood (−0.245, ρ > 0.10). Significant correlations were observed between δ¹³C of tree-ring alpha-cellulose and the precipitation from the current year’s February to July (r = −0.514, ρ < 0.01) that were much higher than that of holocellulose (−0.481, ρ < 0.05) or total wood (−0.249, ρ > 0.10). A significant correlation (−0.545, ρ < 0.01) was also found between the ring width and the δ¹³C residual chronologies. These results suggest that more past environmental information is retained in the δ¹³C of tree-ring alpha-cellulose. Thus, the δ¹³C of alpha-cellulose of tree rings is the most suitable among the studied parameters for reconstructing the past climatic conditions during the growing season. The δ¹³C values of other organic compounds in Pinus tabulaeformis xylem were affected by the external environment after carbon was fixed from the atmosphere.     

Inhibitors of N α-acetyl-l-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency

A series of N ^α-acyl (alkyl)- and N ^α-alkoxycarbonyl-derivatives of l- and d-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N ^α-acetyl-l-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N ^α-acetyl-l-ornithine to l-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC₅₀ values in the μM range toward ArgE, indicating that they are moderately strong inhibitors. N ^α-chloroacetyl-l-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC₅₀ value of 85 μM while N ^α-trifluoroacetyl-l-ornithine (1f), N ^α-ethoxycarbonyl-l-ornithine (2b), and N ^α-acetyl-d-ornithine (1a) weakly inhibited ArgE activity providing IC₅₀ values between 200 and 410 μM. Weak inhibitory potency toward Bacillus subtilis-168 for N ^α-acetyl-d-ornithine (1a) and N ^α-fluoro- (1f), N ^α-chloro- (1g), N ^α-dichloro- (1h), and N ^α-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC₅₀ values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that ArgE is likely the bacterial enzymatic target.     

High-level Expression of an α-l-arabinofuranosidase from Thermotoga maritima in Escherichia coli for the Production of Xylobiose from Xylan

To efficiently produce xylobiose from xylan, high-level expression of an α-l-arabinofuranosidase gene from Thermotoga maritima was carried out in Escherichia coli. A 1.5-kb DNA fragment, coding for an α-l-arabinofuranosidase of T. maritima, was inserted into plasmid pET-20b without the pelB signal sequence leader, and produced pET-20b-araA1 with 8 nt spacing between ATG and Shine–Dalgarno sequence. A maximum activity of 12 U mg⁻¹ was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-20b-araA1. The over-expressed α-l-arabinofuranosidase was purified 13-fold with a 94% yield from the cellular extract of E. coli by a simple heat treatment. Production of xylooligosaccharides from corncob xylan by endoxylanase and α-l-arabinofuranosidase was examined by TLC and HPLC: xylobiose was the major product from xylan at 90 °C and its proportion in the xylan hydrolyzates increased with the reaction time. Hydrolysis with in the xylanase absence of α-l-arabinofuranosidase gave only half this yield. Revisions requested 27 October 2005; Revisions received 5 September 2005     

Staphylococcus aureus in chronic and recurrent infections

Ninety-fourStaphylococcus aureus strains isolated from chronic and recurrent skin and respiratory tract infections were investigated for several virulence factor expressions. Production of protein A was noticed in all of the tested strains in amounts from less than 0.1 to more than 2.5 ng per 10⁶ bacterial cells. The percentage of the extracellularly produced protein A was found to lie between 4.5 and 27.8%. Two strains (both from the respiratory tract) produced more than 50 % of protein A in the extracellular form and one strain did not produce any detectable amount of the extracellular protein A; 99 % of the tested strains produced the clumping factor, 96% staphylocoagulase, 79 % staphylokinase and 90 % gelatinolytic activity; 79 % produced α-toxin exclusively or in combination with δ- or β-toxin; 8 % of strains produced β-toxin. There were differences in β-toxin production between strains from the respiratory tract (5 %) and skin infections (25 %). δ-Toxin was produced by 53 % of the strains. In each of the tested strains a complex of virulence factors was detected. The importance of inactivated extracellular products (especially α- and δ-toxin and in the case of skin infections also β-toxin) as components of staphylococcal whole-cell vaccine was suggested. Dedicated to Professor C. John on the occasion of his 75th birthday     

Complexity,polymorphism, and recombination of mouse T-cell receptor α gene families

Genomic DNA from a large panel of inbred strains of mice were hybridized sequentially with 15 V_α, 2 V_δ, 1 C_α, and 1 C_δ probes. Most of the V_α probes detected a high degree of plymorphism and have allowed the definition of five mouse T-cell receptor α (Tcr _α) haplotypes. One of these haplotypes (Tcr _α ^e ) appears to arise from a recombination between theTcr _α ^b andTcr _α ^a haplotypes, the latter being the most frequently found in the conventional inbred strains. This recombination event clearly indicates that the members of at least 11 V_α subfamilies are not closely linked but highly interspersed with one another on chromosome 14.     

Investigation of mercury concentrations in fur of phocid seals using stable isotopes as tracers of trophic levels and geographical regions

Recent studies have shown that the complementary analysis of mercury (Hg) concentrations and stable isotopic ratios of nitrogen (δ¹⁵N) and carbon (δ¹³C) can be useful for investigating the trophic influence on the Hg exposure and accumulation in marine top predators. In this study, we propose to evaluate the interspecies variability of Hg concentrations in phocids from polar areas and to compare Hg bioaccumulation between both hemispheres. Mercury concentrations, δ¹⁵N and δ¹³C were measured in fur from 85 individuals representing 7 phocidae species, a Ross seal (Ommatophoca rossii), Weddell seals (Leptonychotes weddellii), crabeater seals (Lobodon carcinophagus), harbour seals (Phoca vitulina), grey seals (Halichoerus grypus), ringed seals (Pusa hispida) and a bearded seal (Erignathus barbatus), from Greenland, Denmark and Antarctica. Our results showed a positive correlation between Hg concentrations and δ¹⁵N values among all individuals. Seals from the Northern ecosystems displayed greater Hg concentrations, δ¹⁵N and δ¹³C values than those from the Southern waters. Those geographical differences in Hg and stable isotopes values were likely due to higher environmental Hg concentrations and somewhat greater number of steps in Arctic food webs. Moreover, dissimilarities in feeding habits among species were shown through δ¹⁵N and δ¹³C analysis, resulting in an important interspecific variation in fur Hg concentrations. A trophic segregation was observed between crabeater seals and the other species, resulting from the very specific diet of krill of this species and leading to the lowest observed Hg concentrations.     

The causes for barley root growth retardation in the presence of ammonium and glutamate

In barley (Hordeum vulgare L.) seedlings, the rate of root growth, osmotic pressure (Π), hydraulic conductance (L _p), and longitudinal (δl) and transverse (δD/D) extensibility of root cells were measured. The seedlings were grown on Knop solution with nitrate or without nitrate with addition of 5–10 mM NH₄⁺ or 0.5–1.0 mM glutamate. Root growth retardation on the 1st–4th days of exposure to NH₄⁺ was determined by a decrease in δl in the zone of elongation, whereas root thickening was evidently related to an increase in Π. Biphasic dynamics of δl in the presence of NH₄⁺ was imitated by medium acidification near the root surface to pH 3.7, which confirms a conclusion, we have done earlier, about a non-monotonous pH-dependence of longitudinal extensibility. Root growth retardation during the first day of exposure to Glu was also determined by a decrease in the δl, which was, however, accompanied by an increase in the δD/D and L _p. Fast Glu-induced changes of measured root parameters were imitated by root exposure to oryzalin, ionomycin, and inhibitors of the H⁺-pump. It was supposed that a decrease in δl in the presence of NH₄⁺ and Glu was related to cortical microtubule disorganization with the involvement of cytosolic calcium Ca_cyt²⁺. A decrease in the δD/D and L _p in the presence of NH₄⁺ was related to apoplast acidification and a high activity of the plasmalemmal H⁺-pump. An increase in the δD/D and L _p in the presence of Glu indicates the inhibition of the plasmalemmal H⁺-pump. On the 2nd–4th days of exposure to Glu, root growth ceased, as distinct from treatment with NH₄⁺. This complete root growth inhibition by Glu was possibly related to a rapid uptake of Ca²⁺ through Glu-sensitive Ca²⁺-channels, Ca²⁺-dependent inhibition of the plasmalemmal H⁺-pump, and a decrease in mitotic activity.     

SPARTA+: a modest improvement in empirical NMR chemical shift prediction by means of an artificial neural network

NMR chemical shifts provide important local structural information for proteins and are key in recently described protein structure generation protocols. We describe a new chemical shift prediction program, SPARTA+, which is based on artificial neural networking. The neural network is trained on a large carefully pruned database, containing 580 proteins for which high-resolution X-ray structures and nearly complete backbone and ¹³C^β chemical shifts are available. The neural network is trained to establish quantitative relations between chemical shifts and protein structures, including backbone and side-chain conformation, H-bonding, electric fields and ring-current effects. The trained neural network yields rapid chemical shift prediction for backbone and ¹³C^β atoms, with standard deviations of 2.45, 1.09, 0.94, 1.14, 0.25 and 0.49 ppm for δ¹⁵N, δ¹³C’, δ¹³C^α, δ¹³C^β, δ¹H^α and δ¹H^N, respectively, between the SPARTA+ predicted and experimental shifts for a set of eleven validation proteins. These results represent a modest but consistent improvement (2–10%) over the best programs available to date, and appear to be approaching the limit at which empirical approaches can predict chemical shifts.     

Climate signals in the ring widths and stable carbon, hydrogen and oxygen isotopic composition of Larix decidua growing at the forest limit in the southeastern European Alps

The southeastern border of the European Alps is not well resourced with high-resolution climate proxies and experiences a distinct climatic regime from the northern and western Alpine zones. Here, we present new high-resolution climatic proxies (AD 1907–2006) from ring widths and stable carbon (δ¹³C), non-exchangeable hydrogen (δ²H) and oxygen (δ¹⁸O) isotope ratios of cellulose extracted from Larix decidua tree rings, growing at the forest limit in the southeastern European Alps (Slovenia). δ¹³C, δ²H and δ¹⁸O are strongly (p < 0.001) and positively correlated with each other. June temperature has the strongest control on tree ring width (TRW), while later summer conditions (July–August) influence the stable isotope composition. All four proxies are strongly correlated (r > 0.4; p < 0.001) with summer temperature and also sunshine hours, while precipitation has less impact. A combination of TRW and δ¹³C provides the greatest potential for reconstructing past temperatures (June–August) with significant (p < 0.001) correlations with gridded temperatures extending across a very large part of southern and western Europe west of the Carpathian Mountains. The water isotopes (oxygen and hydrogen) record conditions in the Adriatic and Mediterranean, which are the source area for the air masses that bring precipitation to this region giving strong correlations with temperatures in southern Italy and the western part of the Balkan Peninsula. Combining proxies with different spatial and temporal signals allows the strength and spatial footprint of climate signals to be enhanced. These findings open new perspectives for climate reconstruction in the southeastern European Alps and Western Balkans.     

Spatial and temporal trends in stable carbon and oxygen isotope ratios of juvenile winter flounder otoliths

Isotopic ratios of fish otoliths have been used in numerous studies as natural tags or markers to aid in the study of connectivity among fish populations. We investigated the use of spatial and temporal changes in the stable carbon and oxygen isotope ratios of otoliths to differentiate juvenile habitats of winter flounder (Pseudopleuronectes americanus). Young-of-the-year (YOY) juvenile winter flounder were collected annually over a three-year period from 18 stations along the coast of Rhode Island, USA. Sagittal otoliths were removed from fish and analyzed for stable carbon (¹³C/¹²C or δ¹³C) and oxygen (¹⁸O/¹⁶O or δ¹⁸O) isotope ratios using continuous flow isotope ratio mass spectrometry. Differences in isotope ratios were observed among stations and along salinity gradients in the Narragansett Bay estuary and an estuarine river system (Narrow River). Overall, the isotope ratio patterns observed among stations were consistent over the three sampling years; however, differences were noted in isotope ratios and the magnitude of the isotope ratio gradients among years. Significant positive correlations were noted between salinity and δ¹³C for two of the three years. For each of the three years sampled there was a highly significant positive correlation (2002, r = 0.93, P < 0.01; 2003, r = 0.85, P < 0.01; 2004, r = 0.97, P < 0.01) between δ¹⁸O and the salinity of the collection site. Also, there was a significant negative correlation between the number of months of above average river flow and δ¹⁸O for the three sampling years (r = 0.99, P < 0.05). These findings suggest that yearly changes in the volume of freshwater inputs to these estuarine habitats may be related to the differences observed in otolith δ¹⁸O isotope ratios. Because of these year-to-year differences, sampling of each cohort may be necessary in order to use this isotopic technique for winter flounder connectivity studies.     

The Polarity of Lipid-Exposed Residues Contributes to the Functional Differences between Torpedo and Muscle-Type Nicotinic Receptors

A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues αS287, αC412, βY441, γM299, γS460, δM293, δS297 and δN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [¹²⁵I]-α-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC₅₀), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC₅₀. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.     

Production,partial purification and characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase from <Emphasis Type="Italic">Penicillium ulaiense</Emphasis>

Penicillium ulaiense is a post-harvest pathogenic fungus that attacks citrus fruits. The objective of this work was to study this microorganism as an α-l-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. α-l-Rhamnosidase was produced in a stirred-batch reactor using rhamnose as the main carbon source. The kinetic parameters for the growth of the fungi and for the enzyme production were calculated from the experimental values. A method for partial purification, including (NH₄)₂SO₄ precipitation, incubation at pH 12 and DEAE-sepharose chromatography yielded an enzyme with very low β-glucosidase activity. The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis–Menten constants for the hydrolysis of p-nitrophenyl-α-l-rhamnoside were V _max = 26 ± 4 IU ml⁻¹ and K _m  = 11 ± 2 mM. The enzyme showed good thermostability up to 60°C and good operational stability in white wine. Co²⁺ affected positively the activity; EDTA, Mn²⁺, Mg²⁺, dithiotreitol and Cu²⁺ reduced the activity by different amounts, and Hg²⁺ completely inhibited the enzyme. The enzyme showed more activity on p-nitrophenyl-α-l-rhamnoside than on naringin. According to these results, this enzyme has potential for use in the food and pharmacy industries since P. ulaiense does not produce mycotoxins.     

Biosynthesis,processing, and extracellular release of α-l-fucosidase in lymphoid cell lines of different genetic origins

In humans, the quantity of α-l-fucosidase in serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. Lymphoid cell lines derived from individuals with either low, intermediate, or high α-l-fucosidase in serum were established. Steady-state levels of intracellular and extracellular α-l-fucosidase as well as rates of synthesis and secretion of enzyme overlapped among the cell lines. Thus,_vivo} serum phenotypes were not expressed in this system. No appreciable differences in the qualitative processing of newly made α-l-fucosidase were observed among these lymphoid cell lines. Cells pulse-labeled with³⁵S-methionine from 0.25 to 2 hr had an intracellular form of enzyme with aM _r=58,000. Cells pulsed for 1.5 hr and chased for 21 hr with unlabeled methionine had an intracellular form ofM _r=60,000 and an extracellular form ofM _r=62,000. All three enzyme forms were glycoproteins with a common polypeptide chain ofM _r=52,000 but with different carbohydrate moieties. No evidence for a high molecular mass precursor form of α-l-fucosidase was found. Fucosidosis is a rare, inherited disease in which α-l-fucosidase activity in tissues and body fluids is low or absent. The mutations for fucosidosis and the serum polymorphism map separately. Lymphoid cells from two siblings with fucosidosis had 8-fold to 341-fold less intracellular α-l-fucosidase protein with 11-fold to 56-fold lower specific activities than control cells. Residual mutant enzyme was a glycoprotein with a polypeptide chain virtually the same size (M _r=52,000) as control enzyme. However, residual mutant enzyme was hypoglycosylated and hypersecreted as compared to control enzyme. This research was supported by National Institutes of Health Grant DK 32161.     

Subunit Regulation of the Human Brain α1E Calcium Channel

The α₁ subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α₂δ and β subunits. α_1E channels directed with the expression of Ba²⁺ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α_1E with α_2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α_1E with β_2a shifted the peak current relationship by −10 mV, and strongly reduced Ba²⁺ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba²⁺ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β_2a and the neuronal α_2bδ subunits increased by ≈10-fold whole-cell Ba²⁺ currents although coinjection with either β_2a or α_2bδ alone failed to significantly increase α_1E peak currents. Coexpression with β_2a and α_2bδ yielded Ba²⁺ currents with inactivation kinetics similar to the β_2a induced currents, indicating that the neuronal α_2bδ subunit has little effect on α_1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit genes. The slower inactivation was unique to α_1E/β_2a currents. Coexpression with β_1a, β_1b, β₃, and β₄, yielded faster-inactivating Ba²⁺ currents than currents recorded from the α_1E subunit alone. Furthermore, α_1E/α_2bδ/β_1a; α_1E/α_2bδ/β_1b; α_1E/α_2bδ/β₃; α_1E/α_2bδ/β₄ channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The β subunit-induced changes in the properties of α_1E channel were comparable to modulation effects reported for α_1C and α_1A channels with β₃≈β_1b > β_1a≈β₄≫β_2a inducing fastest to slowest rate of whole-cell inactivation. Received: 27 March 1997/Revised: 10 July 1997