Isolation and characterization of multiple forms of prunasin hydrolase from black cherry (Prunus serotina Ehrh.) seeds

Three forms of prunasin hydrolase (PH I, PH IIa, and PH IIb), which catalyze the hydrolysis of (R)-prunasin to mandelonitrile and D-glucose, have been purified from homogenates of mature black cherry (Prunus serotina Ehrh.) seeds. Hydroxyapatite chromatography completely resolved PH I from PH IIa and PH IIb. PH IIa and IIb, which coeluted on hydroxyapatite, were resolved by gel filtration. PH IIa was a dimer with a native molecular weight of 140,000. Both PH I and PH IIb were monomeric with molecular weights of 68,000. The isozymes appeared to be glycoproteins based on their binding to concanavalin A-Sepharose 4B with subsequent elution by alpha-methyl-D-glucoside. When presented several potential glycosidic substrates, these enzymes exhibited a narrow specificity towards (R)-prunasin. Km values for (R)-prunasin for PH I, PH IIa, and PH IIb were 1.73, 2.3, and 1.35 mM, respectively. PH I and PH IIb possessed fivefold greater Vmax/Km values than PH IIa. Ortho- and para-nitrophenyl-beta-D-glucosides were hydrolyzed at the same active site. All forms had a pH optimum of 5.0 in citrate-phosphate buffer. PH I and PH IIb were competitively inhibited by castanospermine with Ki values of 0.19 and 0.09 mM, respectively. PH activity was not stimulated by any metal ion tested and was unaffected by diethyldithiocarbamate, o-phenanthroline, 2,2'-dipyridyl, and EDTA.     

Immunological characterization of maize starch branching enzymes

Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties.     

Effect of myosin heavy chain composition of muscles on meat quality in Laiwu pigs and Duroc

In order to explain the mechanism of high meat quality in Laiwu pigs and investigate the relation between myosin heavy chains (MyHC) composition and meat quality, meat quality analysis was conducted and mRNA expression of MyHC I, IIa, IIx, IIb was quantified by real-time fluorescence PCR in longissimus muscle (LM) and semimembranous muscle of Laiwu pigs and Duroc. The result indicated that, compared with Duroc, mRNA expression of MyHC IIa, IIx in LM and semimembranous muscle of Laiwu pigs was significantly increased, mRNA expression of MyHC IIb was dramatically decreased. However, the expression of MyHC I was not significantly affected by breeds. The correlation between mRNA expression of MyHC I, IIa, IIx in LM and meat color, pH value, marbling, intramuscular fat content was positive, but shear value of LM was negative. The relation between MyHC IIb mRNA expression and marbling, intramuscular fat content was dramatically negative, whereas shear value was strikingly positive, as well as fiber diameter, but without reaching statistical significance. Therefore, the composition of MyHC I, IIa, IIx, IIb affected meat quality, furthermore, expression of MyHC I, IIa, IIx, IIb mRNA prominently influenced meat characteristics, especially edible quality of muscle, suggesting that mRNA expression level of MyHC I, IIa, IIx, IIb can exactly and impersonally estimate meat quality.     

Immunochemical quantification of sarcoplasmic reticulum Ca-ATPase, of calsequestrin and of parvalbumin in rabbit skeletal muscles of defined fiber composition

Antibodies directed against purified Ca-ATPase from sarcoplasmic reticulum, calsequestrin and parvalbumin from rabbit fast-twitch muscle were raised in sheep. The specificity of the antibodies was shown by immunoblot analysis and by enzyme-linked immunoadsorbent assays (ELISAs). IgG against the sarcoplasmic reticulum Ca-ATPase inhibited the catalytic activities of Ca-ATPase from fast-twitch (psoas, tibialis anterior) and slow-twitch (soleus) muscles to the same degree. In non-equilibrium competitive ELISAs the anti(Ca-ATPase) IgG displayed a slightly higher affinity for the Ca-ATPase from fast-twitch muscle than for that from slow-twitch muscle. This suggests a fiber-type-specific polymorphism of the sarcoplasmic reticulum Ca-ATPase. Quantification of Ca-ATPase, calsequestrin and parvalbumin in various rabbit skeletal muscles of histochemically determined fiber composition was achieved by sandwich ELISA. Ca-ATPase was found to be 6-7 times higher in fast than in slow-twitch muscles. A slightly higher concentration was found in fast-twitch muscles with a higher percentage of IIb fibers when compared with fast-twitch muscles with a higher percentage of IIa fibers. Thus Ca-ATPase is distributed as follows, IIb greater than or equal to IIa much greater than I. Calsequestrin was uniformly distributed in fast-twitch muscles independently of their IIa/IIb fiber ratio and displayed 50% lower concentrations in slow than in fast-twitch muscles (IIb = IIa greater than I). Parvalbumin contents were 200-300-fold higher in fast than in slow-twitch muscles. Significantly lower parvalbumin concentrations were found in fast-twitch muscles with a higher percentage of IIa fibers than in fast-twitch muscles with a higher percentage of IIb fibers (IIb greater than IIa much greater than I).     

Morphologic differences in skeletal muscle with age in normally active human males and their well-trained counterparts

In this study we elucidate the interaction of physical activity with aging as regards skeletal muscle fiber distribution and size. Thirty-three male athletes and 42 normally active counterparts served as subjects. They were assigned to younger (less than 25.5 years) and older (greater than 25.5 years) subgroups. Serial cross-sections from muscle biopsy samples (musculus vastus lateralis) were stained to distinguish fiber type: fast glycolytic (type IIb), fast oxidative-glycolytic (type IIa), or slow oxidative (type I). We also measured fiber diameters. A greater mean diameter of type I fibers was seen in older as opposed to younger athletes. Older controls had a smaller mean diameter of type IIb fibers than did younger controls. Athletes had a smaller mean percentage of type IIa fibers and a greater mean percentage of type I fibers than did controls. There was a greater mean percentage of type I fibers in older as opposed to younger controls, but this was not the case in athletes. Athletes may have larger fibers and a greater percentage of type I fibers at the expense of type IIa fibers. Atrophy of fibers with aging might be retarded by training, which might also reduce the age-associated rate of type IIb percentage loss and type I percentage gain.     

Glucocorticoid-induced muscle atrophy prevention by exercise in fast-twitch fibers

Exercise has been shown to be effective in preventing glucocorticoid-induced atrophy in muscles containing high proportions of type II or fast-twitch fibers. This investigation was undertaken to further evaluate this response in type IIa and IIb fibers, determined by histochemical staining for myofibrillar adenosinetriphosphatase with alkaline and acid preincubation. Steroid [cortisol acetate (CA), 100 mg/kg body wt] and exercise (running 90 min/day, 29 m/min) treatments were initiated simultaneously for 11 consecutive days in female rats. Fiber distribution and area measurements were performed in a deep and superficial region of plantaris muscle. The exercise regimen spared approximately 40% of the CA-induced plantaris muscle atrophy. In the deep region, the fiber population, which contained approximately 13% type I (slow-twitch), 24% type IIa, and 63% IIb fibers, was not affected by either treatment. In the superficial section, which consisted solely of type II fibers, the proportion of type IIa fibers was higher (27 vs. 9%, P less than 0.01) in the steroid- than in the vehicle-treated groups. Within each region, type IIa fibers were less susceptible to atrophy than type IIb fibers, and within each fiber type, the deep region had less atrophy than the superficial region. Type I fibers were unchanged by steroid treatment. For type IIa fibers, exercise prevented 100% of the atrophy in the deep region and 50% in the superficial region. For type IIb fibers, the activity spared 67 and 40% of the atrophy in these same regions, respectively. These results show that glucocorticoids are capable of changing the myosin phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

B cell response to surface IgM cross-linking identifies different prognostic groups of B-chronic lymphocytic leukemia patients

On the basis of responses to surface IgM (sIgM) cross-linking, B cells from 41 patients with B-chronic lymphocytic leukemia were categorized as 15 nonresponders (group I) and 26 responders (group II). The latter cases were subclassified as those seven where proliferation was induced (subgroup IIa) and the remaining 19 in whom apoptosis occurred (subgroup IIa). Signal disruption in group I was confirmed by the absence of Ca2+ mobilization. Activation of PI3K was constitutive in subgroup IIa, but not in subgroup IIb, and that of Akt induced by anti-mu in subgroup IIa, but not in subgroup IIb. Among the MAPK, ERK was more highly activated relative to p38 in subgroup IIa, whereas activation of p38 predominated over that of ERK in subgroup IIb. For subgroup IIb cells, based on tyrosine phosphorylation and translocation into lipid rafts, sIgM signaling was shown to be enhanced by Zap70. The different consequences of signaling through sIgM were associated with biological prognosis indicators. These included high levels of CD38, lack of mutations in the IgVH chain genes, preferential usage of full-length CD79b, and severe clinical stage. Thus, modification of sIgM-induced signaling could be a therapeutic approach.     

Nitrofurazone-reducing enzymes in E. coli and their role in drug activation in vivo.

Earlier work showed that Escherichia coli contains at least two enzymes which reduce nitrofurazone and other nitrofuran derivatives. One of these enzymes is lacking in some nitrofurazone-resistant mutant strains. We now report that there are three separable nitrofuran reductases in this organism: reductase I (mol. wt. approximately 50 000, insensitive to O2), reductase IIa (mol. wt. approximately 120 000, inhibited by oxygen), reductase IIb (mol. wt. approximately 700 000, inhibited by O2). Unstable metabolites formed during the reduction of nitrofurazone by preparations containing reductases IIa and IIb produce breaks in DNA in vitro. In vivo experiments with nitrofurazone-resistant strains, which lack reductase II but contain reductases IIa and IIb, demonstrated that lethality, mutation, and DNA breakage are all greatly increased when cultures are incubated under anaerobic conditions, i.e., conditions such that reductase II is active. These results provide further evidence for the importance of reductive activation of nitrofurazone.     

Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms

Three forms (I, IIa and IIb) of phospholipase C, hydrolyzing specifically inositol phospholipids, were resolved from human platelet cytosol and partially purified by DEAE-cellulose, phenyl-Sepharose, Ultrogel ACA-44 and hydroxylapatite column chromatographies. All three forms exhibited pH optimum at 6.5 - 7.0 in the presence of deoxycholate and their molecular weights were 67,000 (form I), 120,000 (IIa) and 70,000 (IIb). These enzymes hydrolyzed both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in Ca2+-dependent manner; their maximal activities for phosphatidylinositol hydrolysis were obtained at 10(-4) to 10(-3) M Ca2+, whereas phosphatidylinositol 4,5-bisphosphate was preferentially hydrolyzed at lower concentration of Ca2+.     

Multiple forms of alkaline phosphatase in untreated and 5-bromo-2'-deoxyuridine-treated choriocarcinoma cells

The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2′-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)]were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K_m value for p-nitrophenyl phosphate, were more sensitive to inhibition by l-leucine, levamisole, l-p-bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000, 240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.     

Escherichia coli membrane proteins with an affinity for deoxyribonucleic acid.

From the membrane fraction of Escherichia coli K-12 strain, four protein fractions (peaks I, IIa, IIb, and III) which have affinity for deoxyribonucleic acid (DNA) have been isolated. The molecular weights of these proteins are between 12,000 and 8,000. Only the peak III fraction contains a protein that binds preferentially to single-stranded DNA, whereas the others contain proteins that bind also to double-stranded DNA. The binding activity of the peak IIb protein is inhibited in the presence of polyuridylic acid. Peak I and peak IIa protein fractions behave like hydrophobic proteins.     

Two hybridization events define the population structure of Trypanosoma cruzi

Genetic variation in Trypanosoma cruzi is likely a key determinant in transmission and pathogenesis of Chagas disease. We have examined nine loci as markers for the extant T. cruzi strains. Four distinct alleles were found for each locus, corresponding to the sequence classes present in the homozygous discrete typing units (DTUs) I, IIa, IIb, and IIc. The alleles in DTUs IIa and IIc showed a spectrum of polymorphism ranging from DTU I-like to DTU IIb-like, in addition to DTU-specific sequence variation. DTUs IId and IIe were indistinguishable, showing DTU homozygosity at one locus and heterozygosity with DTU IIb and IIc allelic sequences at eight loci. Recombination between the DTU IIb and IIc alleles is evidenced from mosaic polymorphisms. These data imply that two discrete hybridization events resulted in the formation of the current DTUs. We propose a model in which a fusion between ancestral DTU I and IIb strains gave rise to a heterozygous hybrid that homogenized its genome to become the homozygous progenitor of DTUs IIa and IIc. The second hybridization between DTU IIb and IIc strains that generated DTUs IId and IIe resulted in extensive heterozygosity with subsequent recombination of parental genotypes.     

Diaphragm capillarity and oxidative capacity during postnatal development.

In the cat diaphragm, fiber capillarity, cross-sectional area, and succinate dehydrogenase (SDH) activity were measured across the first 6 wk of postnatal development. Fibers were classified as type I, IIa, IIb, or IIc on the basis of staining for myofibrillar adenosinetriphosphatase (ATPase). Capillaries were identified in sections stained for ATPase at pH 4.2. Fiber cross-sectional areas and SDH activities were quantified using an image-processing system. During postnatal development, the proportions of type I fibers increased while type II fibers decreased. At birth, all type II fibers were IIc. From the 1st to the 2nd postnatal wk, the proportion of type IIc fibers decreased while the numbers of IIa and IIb increased. Thereafter the proportion of type IIb fibers continued to increase while the number of IIa steadily declined. At birth, capillarity, cross-sectional areas, and SDH activities of type I and II fibers were low compared with other postnatal age groups. Fiber cross-sectional areas increased progressively with age. The number of capillaries surrounding type I and II fibers increased markedly by the 2nd wk and then continued to increase at a slower rate. The number of capillaries per fiber area reached a peak by the 2nd wk and then declined as fiber cross-sectional area increased. Postnatal changes in capillarity depended on fiber type, being greatest in IIb. SDH activities of type I and II fibers were initially low during the first 2 postnatal wk and then peaked by the 3rd wk. After the 6th wk, fiber SDH activities decreased to adult values. Among the type II fibers, IIb showed the greatest change in SDH activity during early postnatal development.     

Diversity of neurodegenerative processes in the model of brain cortex tissue ischemia

Stroke is known to induce massive cell death in the ischemic brain. Either necrotic or apoptotic types of cell death program were observed in neurons in zone of ischemia. We suggest that spatial heterogeneity of glucose and oxygen distribution plays a crucial role in this phenomenon. In order to elucidate the role of glucose and oxygen in ischemic neurons choice of cell death pathway, conditions corresponding to different areas of insult were reproduced in vitro in the model of surviving brain cortex tissue slices. Three zones were modeled in vitro by varying glucose and oxygen concentration in surviving slices incubation media. Modeled ischemic area I (MIA I) was corresponded to the center of suggested ischemic zone where the levels of glucose and oxygen were considered to be extremely low. MIA II was assigned as intermediate area where oxygen concentration was still very low but glucose was present (this area was also divided into two sub-areas MIA IIa and MIA IIb with physiologically low (5 mM) and normal (10 mM) level of glucose respectively). MIA III was considered as a periphery area where glucose concentration was close to physiological level and high level of ROS production had been induced by reoxygenation after anoxia. Analysis of molecular mechanisms of cell death in MIA I, IIa, IIb and III was carried out. Cell death in MIA I was found to proceed by necrotic manner. Apoptosis characterized by cyt c release, caspase 3 activation and internucleosomal DNA fragmentation was observed in MIA III. Cell death in MIA II was accompanied by several (not all) hallmarks of apoptosis. Mechanisms of cell death in MIA IIa and MIA IIb were found to be different. Internucleosomal DNA fragmentation in MIA IIa but not in MIA IIb was sensitive to glycine (5 mM), inhibitor of NMDA receptor MK-801 (10 μM) and PTP inhibitor cyclosporine A (10 μM). Activation of caspase 3 was detected in MIA IIb but not in MIA IIa. However cytochrome c release was observed neither in MIA IIa nor in MIA IIb. In MIAs II–III apoptosis was accompanied by uncoupling of oxidative phosphorylation, which was induced by rise of intracellular Ca²⁺ and intensive ROS production. Results obtained in present study allow us to propose existence of at least four molecular pathways of cell death development in brain ischemic zone. The choice of cell death pathway is determined by oxygen and glucose concentration in the particular area of the ischemic zone.     

Fiber number and type composition in extensor digitorum longus, soleus, and diaphragm muscles with aging in Fisher 344 rats

Histochemical (M-ATPase) fiber typing was done on extensor digitorum longus, (EDL), soleus (SOL), and diaphragm (DIA) muscles of barrier-reared Fisher 344 rats obtained at four different ages (3, 9, 28, and 30 months) from the colonies of the National Institute of Aging. In the EDL there are no differences in the percent of type I fibers among the four age groups. The percent of type IIa and IIb fibers also showed no difference between the 3 and 30 month age groups. There was no apparent trend for an increase or decrease in the percent of type IIa or IIb fibers between the four age groups. In both the SOL and DIA muscles the percent of type I fibers was greater in the aged than in the young groups. The percent of type IIa fibers was lower in the 30 month group than in the younger groups for both muscles. The percent of type IIb (DIA) and IIc (SOL) fibers did not change between groups. Total fiber number per cross section of muscle showed no change in the EDL over this age range or in the SOL after 9 months of age. These findings bring into question published results that imply that decreasing fiber number and preferential loss of type II (a and b) fibers are typical aging phenomena.     

Melatonin increases the survival time of animals with untreated mammary tumours: Neuroendocrine stabilization

Mitochondrial respiratory rates and regulation by phosphate acceptors were studied on permeabilized fiber bundles differing in their myosin heavy chain profiles. The acceptor control ratio, an indicator of oxidation to phosphorylation coupling, and mitochondrial K_m for ADP were the highest in type I, intermediate in mixed IIa/IIx and the lowest in IIx and predominantly IIb fiber bundles. A functional coupling between mitochondrial creatine kinase and oxidative phosphorylation occurred in type I and IIa/IIx fiber bundles, exclusively. Our study suggests that mitochondrial functioning in fast IIa fibers is closer to that of the slow/I than fast IIx or IIb fibers. (Mol Cell Biochem 276: 15–20, 2005)     

Molecular determinants of pH sensitivity of the type IIa Na/P(i) cotransporter

Type II Na/P(i) cotransporters play key roles in epithelial P(i) transport and thereby contribute to overall P(i) homeostasis. Renal proximal tubular brush border membrane expresses the IIa isoform, whereas the IIb isoform is preferentially expressed in small intestinal brush border membrane of mammals. IIa and IIb proteins are predicted to contain eight transmembrane domains with the N- and C-terminal tails facing the cytoplasm. They differ in their pH dependences: the activity of IIa increases at higher pH, whereas the IIb shows no or a slightly opposite pH dependence. To determine the structural domains responsible for the difference in pH sensitivity, mouse IIa and IIb chimeras were constructed, and their pH dependence was characterized. A region between the fourth and fifth transmembrane domains was required for conferring pH sensitivity to the IIa-mediated Na/P(i) cotransport. Sequence comparison (IIa versus IIb) of the third extracellular loops revealed a stretch of three charged amino acids in IIa (REK) replaced by uncharged residues in IIb (GNT). Introduction of the uncharged GNT sequence (by REK) in IIa abolished its pH dependence, whereas introduction of the charged REK stretch in IIb (by GNT) led to a pH dependence similar to IIa. These findings suggest that charged residues within the third extracellular loop are involved in the pH sensitivity of IIa Na/P(i) cotransporter.