bluuebunny/crossref_metadata_2025 huggingface dataset:
Hormone action in newt limb regeneration: insulin and endorphins
Authors: Vethamany-Globus Swani
Published: December 2009

Although several hormones have been linked to newt limb regeneration, a cohesive hypothesis as to how these hormones control the process is yet to emerge. A critical review of the traditional approaches and a reevaluation of currently operative assumptions and interpretations of results precede the data on insulin and β-endorphin. Results from in vivo and in vitro experiments on insulin are summarized, showing that insulin not only promotes various cellular events but also is essential for the expression of the mitogenic effect of nerves on cultured newt limb blastemata. Furthermore, the strong likelihood that insulin may be the common link in promoting limb regeneration in hypophysectomized newts that received pituitary hormone replacement therapy or a nutritional supplement is discussed. The status of β-endorphin in regeneration is also explored. Data are presented to show that vertebrates with regenerating capacity (newts, tadpoles) have higher levels of plasma β-endorphin than that found in species where the capacity to regenerate is either restricted (frogs) or totally lost (mammals). β-Endorphin-like immunoreactivity has been localized in the epidermis of a regenerating newt blastema, as well as in the intermediate lobe of the pituitary gland of axolotl, newt, and Xenopus. A possible opiate connection in vertebrate limb regeneration, in particular, wound healing, is discussed.
The copurification of β-glucosidase, β-xylosidase, and 1,3-β-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58
Authors: Tan Larry U. L. , Mayers Paul , Illing Michelle , Saddler John N.
Published: December 2009

Two enzyme complexes, each with β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21), β-xylosidase (β-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-β-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75 400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the β-xylosidase, β-glucosidase, and 1,3-β-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl β-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the β-glucosidase and 1,3-β-glucanase of the major complex had inhibition constants which differed by more than 80 000 times. In addition, the inhibition on the 1,3-β-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the β-glucosidase, β-xylosidase, and 1,3-β-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.
Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice
Authors: Srivastava U. S. , Sugden E. A. , Majumdar P. K. , Thakur M. L. , Bhatnagar G. M.
Published: December 2009

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate – polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts. In conclusion, it appears that murine muscular dystrophy produces a shorter species of pre-M mRNA via decreased polynucleotide elongation.
A comparative analysis of the amino acid and cDNA sequences of bovine elastin a and chick elastin
Authors: Raju Kaliannan , Anwar Rashid A.
Published: December 2009

A comparative analysis of the amino acid and cDNA sequences of bovine elastin a and chick elastin shows that there are considerable differences between these proteins. There is evidence that duplication of segments of DNA and gene rearrangement have occurred in the gene for chick elastin as compared with the gene for bovine elastin. The length of the polypeptide chain of elastin in both species is similar. Therefore, the duplications are compensated for by deletions in the gene for chick elastin.
Side-chain oxidation of vitamin D3 in mouse kidney mitochondria: effect of the Hyp mutation and 1,25-dihydroxyvitamin D3 treatment
Authors: Jones Glenville , Yip Agatha , Tenenhouse Harriet S.
Published: December 2009

Side-chain oxidation of vitamin D is an important degradative pathway. In the present study we compared the enzymes involved in side-chain oxidation in normal and Hyp mouse kidney. Homogenates of normal mouse kidney catalyze the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3, and 24-oxo-23,25-dihydroxyvitamin D3. After subcellular fractionation, total side-chain oxidative activity, estimated by the sum of the three products synthesized per milligram protein under initial rate conditions, coincided with the mitochondrial enzyme marker succinate–cytochrome-c reductase. Treatment of normal mice with 1,25-dihydroxyvitamin D3 (1.5 ng/g) resulted in an eightfold increase in mitochondrial enzyme activity, with no change in apparent Km but a significant rise in Vmax. With 24,25-dihydroxyvitamin D3 as the substrate, normal renal mitochondria produced 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and the synthesis of these metabolites could be increased sixfold by pretreatment with 1,25-dihydroxyvitamin D3. In the Hyp mouse, the side-chain oxidation pathway showed similar subcellular distribution of enzyme activity. However, product formation from 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 was twofold greater in mutant than in normal mitochondria. Furthermore, 1,25-dihydroxyvitamin D3 pretreatment of Hyp mice resulted in a 3.4-fold increase over basal metabolism of both 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. These results demonstrate that (i) kidneys from normal and Hyp mice possess basal and 1,25-dihydroxyvitamin D3 inducible enzyme system(s) in the mitochondrial fraction, which catalyze the side-chain oxidation of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, and (ii) the Hyp mutation appears to perturb the renal metabolism of both substrates only in the basal state.
Phosphorylation of calcineurin by glycogen synthase (casein) kinase-1
Authors: Singh Toolsee J. , Wang Jerry H.
Published: December 2009

A previous study demonstrated that calcineurin preparations contain variable amounts of endogenous phosphate. This observation suggests that calcineurin may be regulated by protein phosphorylation. In this study we have used calcineurin as a potential substrate for eight different protein kinases and significant phosphorylation was observed only with glycogen synthase (casein) kinase-1 (CK-1). Analysis by sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed that only subunit A of calcineurin was phosphorylated. The incorporation of 32P into calcineurin catalyzed by CK-1 ranged from 0.4 to 1.5 mol, depending on the preparation of the substrate used. Peptide mapping revealed that two major sites on calcineurin were phosphorylated. No change in calcineurin activity was observed as a result of phosphorylation. The results of this study suggest that CK-1 may be responsible for phosphorylating calcineurin in vivo.
Purification and characterization of catalase-1 from Bacillus subtilis
Authors: Loewen Peter C. , Switala Jacek
Published: December 2009

The catalase activity produced in vegetative Bacillus subtilis, catalase-1, has been purified to homogeneity. The apparent native molecular weight was determined to be 395 000. Only one subunit type with a molecular weight of 65 000 was present, suggesting a hexamer structure for the enzyme. In other respects, catalase-1 was a typical catalase. Protoheme IX was identified as the heme component on the basis of the spectra of the enzyme and of the isolated hemochromogen. The ratio of protoheme/subunit was 1. The enzyme remained active over a broad pH range of 5–11 and was only slowly inactivated at 65 °C. It was inhibited by cyanide, azide, and various sulfhydryl compounds. The apparent Km for hydrogen peroxide was 40.1 mM. The amino acid composition was typical of other catalases in having relatively low amounts of tryptophan and cysteine.
Lipogenesis from ketone bodies in the perfused rat liver: effects of acetate and ethanol
Authors: Endemann Gerda , Goetz Patrick G. , Tomera John F. , Rand William M. , Desrochers Sylvain , Brunengraber Henri
Published: December 2009

The interactions between acetate or ethanol metabolism, lipogenesis, and ketone body utilization have been studied in isolated livers from fed rats perfused with 15 mM glucose and 10 mM acetate or ethanol. The contribution of acetate to ketogenesis is constant; on the other hand, the contribution of ethanol to ketogenesis increases with time, presumably because of the accumulation of acetate in the perfusate. Ketogenesis is decreased in the presence of ethanol (but not acetate), while ketone body utilization is not affected by ethanol or acetate. Acetate contributes one third and ethanol contributes one half of the carbon incorporated into fatty acids and 3-β-hydroxysterols. Only a small fraction (less than 5%) of the incorporation of acetate or ethanol into fatty acids and sterols occurs via transient incorporation into ketone bodies.
The wheat-germ Ec protein is a zinc-containing metallothionein
Authors: Lane Byron , Kajioka Robert , Kennedy Theresa
Published: December 2009

The partial amino-acid sequence of the wheat-germ Ec protein has been published previously. Computer analysis failed to show a significant relation between the amino-acid sequence of Ec and the sequences of other proteins stored in the data base of the National Biomedical Research Foundation, Washington, DC. Visual inspection and comparison of the amino-acid sequence of Ec with the sequences of other proteins rich in half-cystine have revealed that the amino-acid sequence of Ec bears a compelling relation to sequences reported for animal metallothioneins. Experimental evidence is presented which supports the view that Ec is a zinc-containing metallothionein, the first metallothionein from higher plants for which an almost complete amino-acid sequence has been determined.
Cellular compartmentalization of protein kinase activity during the cell cycle
Authors: Fabisz-Kijowska Anna A. , Lumley-Sapanski Katherine , Halleck Margaret S. , Schlegel Robert A.
Published: December 2009

The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.
Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line
Authors: Church Jon G. , Ghosh Shobha , Roufogalis Basil D. , Villalobo Antonio
Published: December 2009

Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [γ-32P]ATP or [γ-32P]GTP as a phosphate donor. In contrast, plasma membranes from normal adult rat liver or fast-growing regenerating liver (24 h after partial hepatectomy) produce significantly less activity for protein phosphorylation and little phosphorylation of the lipids. However, neonatal (24 h old) rat liver plasma membrane preparations show levels of phosphorylation of proteins and lipids intermediate between those in the tumor cell line and normal adult plasma membrane preparations. Phosphatidic acid was identified as one of the 32P-labelled lipids in the tumor plasma membrane chloroform–methanol (2:1, v/v) extract. Phosphorylation of protein was not affected by cAMP or cGMP. However, calcium ion (in the presence or absence of calmodulin) significantly modifies the 32P labelling of a series of proteins in normal tissue but has little effect with the neoplastic preparations. Some plasma membrane proteins were capable of nucleotide binding, instead or in addition to being phosphorylated. Finally, the presence of membrane-bound phosphoprotein phosphatase(s) was also demonstrated in all the preparations examined by means of chase experiments with nonlabelled ATP or GTP, and (or) by the use of the phosphoprotein phosphatase inhibitor, orthovanadate.
Dibutyryl cAMP inhibits expression of transformation-related properties in Kirsten murine sarcoma virus transformed Balb/c-3T3 cells despite continued presence of p21v-Ki-ras
Authors: Ridgway Anthony A.G. , De Vouge Michael W. , Mukherjee Barid B.
Published: December 2009

We studied the effects of simultaneous treatment with 0.1 mM N6,O2′-dibutyryl cAMP (dbcAMP) and 1 mM theophylline on several transformation-specific properties and on levels of the Kirsten murine sarcoma virus (Ki-MSV) transforming gene product p21v-Ki-ras, in a Ki-MSV-transformed mouse cell line (Balb/c-3T3, clone A31; KA31). The rate of logarithmic growth, cell motility, and final saturation density were reduced in dbcAMP-treated KA31 cultures. Capabilities for anchorage-independent growth were reduced in treated cells, to levels similar to those observed for the untransformed parental A31 cell line. Treatment with dbcAMP had no observable effect on the binding of 125I-labeled epidermal growth factor and did not alter fluorescence staining patterns for actin microfilaments and fibronectin which, although characteristic of normal cells, were also present in KA31 cells. Changes induced by dbcAMP were readily reversible, except for loss of anchorage-independent growth. However, this property was also reversible, provided removal of dbcAMP occurred 48 h prior to inoculation into soft agar medium. Immunoprecipitation with a monoclonal antibody directed against the protein p21v-Ki-ras (Y13-259) revealed the continued presence of this protein in dbcAMP-treated KA31 cells. We, therefore, conclude that cAMP mediates the inhibition of growth-related transformation-specific properties either by acting at steps subsequent to the expression of p21v-Ki-ras or on a pathway independent of p21ras function.
Influence of collagen gel substratum on response to low-density lipoprotein by cultured human skin fibroblasts
Authors: Hoover Gayle A. , McCormick Suzanne , Kalant Norman
Published: December 2009

The effect of low-density lipoprotein (LDL) on accumulation of glycosaminoglycans (GAG) was compared in cultures of human skin fibroblasts on a conventional plastic substratum and in a native type I collagen gel. The 24-h incorporation of [3H]glucosamine and Na235SO4 into GAG secreted into the medium or associated with the substratum and cell surface (SCA) was measured in cells at subconfluent densities. When cells were grown on plastic, 13–25% of the labeled GAG was in the SCA pool. Cells cultured within a collagen gel matrix incorporated three times more [3H]glucosamine and up to five times more [35S]sulfate into this pool. The addition of LDL (300 μg protein/mL) to the medium increased the level of total GAG incorporation of [3H]glucosamine by 40–50% and of [35S]sulfate by 15–20% on both substrata. For cells on plastic the relative increase in the medium and SCA pool was similar, whereas for cells in collagen gel the response to LDL was twice as great in the SCA pool as in the medium. The distribution of GAG types was unaffected by LDL; hyaluronic acid remained the principal GAG in the media pools of both substrata, heparan sulfate remained the main SCA GAG in cultures on plastic, and dermatan sulfate remained the dominant GAG in the SCA pool of collagen gel cultures. LDL degradation was measured at intervals up to 48 h after the addition of 125I-labeled LDL. The rate of accumulation of degraded LDL products was lower in collagen gel cultures, but the final levels achieved were the same in the two substrata. Concentrations of total cell cholesterol were similar, although the increases in free cholesterol induced by LDL were 26% greater in cells within collagen gel than in those on plastic. We conclude that fibroblasts grown within a collagen gel, as compared with those on a plastic substratum, (i) accumulate more GAG that remain attached to the substratum and cell surface; (ii) respond to LDL with a similar degree of increase in GAG accumulation, but more of the increase is found in the substratum and cell surface compartment; and (iii) accumulate more intracellular free cholesterol in response to LDL.
Soluble and membrane-bound polyphosphoinositide phosphohydrolases in mammalian erythrocytes
Authors: Mack Suzanne E. , Palmer Frederick B. St. C.
Published: December 2009

Phosphatases and phosphodiesterases that hydrolyse polyphosphoinositides are described in both membrane and cytosol fractions of human, pig, rat, rabbit, and sheep erythrocytes using exogenous substrates. With suitably optimized assay conditions, Ca2+-dependent phosphatidylinositol bisphosphate (PIP2) phosphodiesterase activity was found in the hemoglobin-free cytosol fraction, as well as the membrane. Membrane activity is completely dependent upon Triton X-100 and salt and inhibited by cetyltrimethylammonium bromide (CTAB), while the soluble activity requires CTAB and is inhibited by Triton. A low Ca2+-dependent PIP2 phosphatase activity, not present in other tissues, was also detected. The cation-independent phosphatidylinositol phosphate (PIP) phosphatase is localized in the membrane in most species, while the diesterase and the PIP2 phosphatases (both Mg2+ and Ca2+ dependent) are localized in the cytosol. Rat and rabbit erythrocytes are atypical in having a substantial proportion of their Mg2+ -dependent PIP2 phosphatase activities in the membrane. All activities are lowest in sheep erythrocytes, except the PIP phosphatase, most of which is soluble in this species. Ca2+-dependent PIP2 phosphatase activity is not correlated with the activity or subcellular distribution of any of the other hydrolases and seems to be a separate enzyme. All the phosphoinositide hydrolase activities, particularly the diesterase, are orders of magnitude lower in erythrocytes than in other tissues. Both soluble and membrane diesterase activities are lost as erythrocytes age. Soluble polyphosphoinositide diesterase does not seem to be active with membrane-bound substrate, since pig and sheep erythrocytes that have negligible membrane activity do not respond to Ca2+ loading, yet have substantial diesterase activity in the cytosol. This supports the view that the diesterase is not physiologically functional in normal erythrocytes.
The intracellular pH of white blood cells: measurement and regulation
Authors: Grinstein Sergio
Published: December 2009

This review summarizes recent progress in our understanding of the mechanisms of intracellular pH regulation, with particular emphasis on Na+–H+ countertransport. Methods developed for the measurement of cytoplasmic pH in small cells and their application to mammalian lymphocytes and neutrophils are described. The basic properties of the Na+–H+ antiport in these cells and its regulation by growth factors and tumor promoters will be discussed briefly. Finally, the significance of cytoplasmic pH regulation to cell function and survival will be considered.
Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates
Authors: Boneh Avihu , Tenenhouse Harriet S.
Published: December 2009

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 ± 10 μM) and the highest apparent maximal velocity (1146 ± 116 pmol P/(mg protein∙min)) of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 μM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4α-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa)), particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.
Intracellular Ca2+ shift and signal transduction from the tubulovesicular portion of gastric parietal cells during gastrin stimulation or Ca2+ ionophore treatment: comparison between luminescent and fluorescent probes, and electron probe X-ray microanalyzer
Authors: Tsunoda Yasuhiro , Takeda Hiroshi , Otaki Toshihiro , Asaka Masahiro , Nakagaki Ikuko , Sasaki Sadao
Published: December 2009

In gastrin-stimulated, aequorin-loaded parietal cells from guinea pig gastric mucosa, a rapid but transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i), owing to Ca2+ released from the store(s), and a more prolonged Ca2+ entry from outside the cells were observed. However, there was a little increase in [Ca2+]i when similar measurements were assessed by quin 2 or fura-2 in physiological saline. However, depletion or elimination of Na+ from the incubation medium caused a significant increase in the [Ca2+]; response to gastrin as measured by quin 2. These findings suggest that aequorin and quin 2 (or fura-2) provide information about different aspects of Ca2+ homeostasis and that there is an inhomogeneity of [Ca2+]i in the cytoplasm during gastrin stimulation. By the gastrin stimulation, the intracellular Ca2+ gradients were shifted from the unidentified portion(s) to the restricted apical cytoplasm, as determined by electron probe X-ray microanalysis. Therefore, localization and identification of the source of intracellular Ca2+ as a pool were determined by an X-ray microanalyzer. In the resting state, the tubulovesicle had high Ca2+ concentration compared with the level in the apical cytoplasm. Cells treated with the Ca2+ ionophore ionomycin had a decreased tubulovesicular Ca2+ level, followed by a reciprocal increase in area of the canalicular membrane. The secretory canaliculus in stimulated cells had lower Ca2+ or higher K+ and Cl− concentrations than that of tubulovesicles or cytoplasm in the resting state, respectively. These findings suggest that the Ca2+ pool of the parietal cell is in the tubulovesicles and (or) luminal cell membrane and that the Ca2+ released from the store(s) may mediate a flow of K+ or Cl− into the secretory canaliculus.
Distinctive multiple thymidine kinase species of the aquatic filamentous fungus Achlya ambisexualis
Authors: Lam F. K. , Jing G. J. , Leung W.-C.
Published: December 2009

Four thymidine kinase (TK) species were found in the cell extract of the filamentous fungus Achyla ambisexualis. They differed by their relative electrophoretic mobilities (Rm) on polyacrylamide gel in nondenaturing buffer and are designated as the Rm 0.30, 0.45, 0.65, and 0.85 TK species. The Rm 0.30 activity was readily solubilized from the mycelia by freeze–thaw treatment, whereas the other TK species were solubilized by additional treatment with nonionic detergent nonidet P-40. The Rm 0.30, 0.65, and 0.85 TK species utilized only ATP as a phosphate donor and dT as a phosphate acceptor. However, the Rm 0.45 species utilized all four ribonucleoside triphosphates as phosphate donors and dC, in addition to dT, as phosphate acceptors. Therefore, this TK species is a deoxypyrimidine kinase. All four TK activities were inhibited by the feedback inhibitor dTTP. However, only the Rm 0.45 TK activity was inhibited by dCTP. The characterization of distinctive multiple TK species in A. ambisexualis will facilitate the studies using this fungus as a transformation host cell for vectors carrying TK as a reported gene.
A superoxide anion induced DNA strand-break metabolic pathway in human leukocytes: effects of vanadate
Authors: Birnboim H. C.
Published: December 2009

Superoxide anion (O2−) is an active oxygen species found in virtually all cells grown in the presence of oxygen. In vivo, the highest concentration of this oxygen radical is found after granulocytes have been exposed to particles or the tumor promoter, phorbol myristate acetate. O2− is released from the cell as a "respiratory burst," which is followed shortly by the appearance of strand breaks in the DNA of the producing cell. In the present report, we have continued our investigation into the mechanism by which extracellular O2− causes breakage of intracellular DNA. Although hydrogen peroxide is present and could also cause strand breaks, its effects are eliminated by the addition of catalase. When the amount of O2− is increased threefold by adding glucose to the medium, the number of breaks increases only slightly, suggesting that the number of breaks that could be induced is limited. The strand-break process is abruptly interrupted by the addition of metabolic poisons such as ionophore A23187, fluoride, or 2-deoxyglucose, but ATP does not appear to be involved. The number of O2−-induced strand breaks is increased in the presence of sodium orthovanadate and decreased by A23187. Orthovanadate prevents the inhibition caused by A23187. Reaction of O2− with orthovanadate itself appears not to be responsible for the enhancement of breaks by orthovanadate. We propose that orthovanadate exerts its effect by acting as an inhibitor of a phosphoprotein phosphatase and that A23187 acts to deplete intracellular Ca2+. These data support our hypothesis that the O2− radical causes strand breaks not by attacking the DNA but rather by activating a specific metabolic DNA strand-break pathway.
Changes in the cooperativity of diaphragm-associated acetylcholinesterase induced by monovalent (Na+, Li+) cations
Authors: Tsakiris Stylianos , Kouniniotou-Krontiri Panagiota
Published: December 2009

Acetylcholinesterase (AChE) activity has been determined using homogenized rat diaphragm and soluble AChE from the eel Electrophorus electricus, using as a substrate different amounts of acetylthiocholine in the presence or absence of 115 mM NaCl or LiCl. With LiCl the KM values derived from Lineweaver–Burk plots are found to be 470–650 and 1045–1425 μM without cations or with NaCl. The cooperativity of the enzyme is increased when cations are added to the homogenate, as demonstrated by changes of the Hill coefficient. With soluble AChE, only Li+ is able to produce this effect. Preincubation of the soluble enzyme at low pH (5.5) and a change to a higher value (8.7–9.4) causes a decrease of the Hill coefficient with Li+ only; this effect is not detected using the homogenate. Our results suggest the following. (i) Li+ may neutralize negative charges of AChE more successfully than does Na+, resulting in higher activity, stabilization, and cooperativity of the enzyme, (ii) The KM values calculated at high substrate concentrations (>200 μM) indicate that the substrate affinity of AChE can be increased only by Li+ binding on the enzyme, (iii) Changes in pH can modulate the cooperativity and may denature allosteric sites on the enzyme that bind Li+. (iv) Membrane, cations and (or) cellular factor(s) may regulate the cooperativity and substrate affinity of AChE, when they have been affected by pH changes.
Electrochemical differentiation between photosystem I and II in thylakoid membranes
Authors: Mimeault Murielle , Carpentier Robert
Published: December 2009

The current–voltage relationship of a thylakoid-containing electrochemical cell of small volume (80 μL) was determined in the dark and under illumination. The photocurrent produced was dependent on pH, on the type of artificial electron acceptor or inhibitor added, and on the electrode potential. Specific monitoring of photosystem-I and -II electron-transfer activity was possible. Photosystem II produced maximal photoeffect at pH 6.0, and the photocurrent–voltage behavior was monophasic. In contrast, photocurrent induction by photosystem I was maximal at pH 8.0 and a biphasic photocurrent–voltage dependency was obtained, presumably as a result of a redox potential barrier located on the donor side of photosystem I. It was concluded that the three-electrode system used is appropriate for measurement of the partial reactions of photosynthesis as well as the coupled photoreactions I and II using the same type of experiment.
Host binding proteins and bacterial adhesion: ecology and binding model
Authors: Ceri Howard , Westra Yolande
Published: December 2009

Defining the involvement of specific recognition and (or) adhesion molecules in the precise association formed between cells of an organism during development or between bacteria and specific host tissues has become a focus of extensive research. The possibility that the same molecules responsible for cellular adhesion in the host may also play a major role in determining host–bacterial interactions is now becoming more evident. The following review looks at the interaction of a group of host binding proteins, including lectins, fibronectin, and laminin, with respect to their specific association with bacteria. This information is dealt with both from the perspective of the ecology of the host and its autochthonous and pathogenic bacterial populations, as well as in terms of the difficulties in defining the nature of ligand associations even in the more simplified bacterial–host interaction.
Transactivation by the adenovirus E1A gene
Authors: Nevins Joseph R. , Raychaudhuri Pradip , Yee Amy S. , Rooney Robert J. , Kovesdi Imre , Reichel Ronald
Published: December 2009

The 289aa product of the adenovirus E1A gene mediates the transcriptional activation of the set of early viral genes as well as several cellular genes. The E1A protein is not a DNA binding protein but, rather, acts indirectly to achieve the activation. The process of viral gene activation involves the use of cellular transcription factors, and in at least one case, in vivo assays have demonstrated a stimulation of stable promoter complex formation as a function of the E1A gene product. Analysis of transcription factors in nuclear extracts has identified a cellular factor, termed E2F, with specificity for the viral E2 promoter. The concentration of this factor increases as a result of the action of E1A. This increase in DNA binding activity does not require protein synthesis, thus indicating an E1A-mediated modification of a pre-existing factor. The E2F factor has been purified to homogeneity and is a polypeptide of 54 000 molecular weight. Analysis of an additional viral promoter, the E4 promoter, has identified a protein that interacts with sequences critical for transcription. This factor, termed E4F, is also increased as a function of the E1A product. The E4F factor has also been purified to homogeneity and has a molecular weight of 50 000. Therefore, the coordinate control of transcription by the E1A gene product involves the activation of multiple promoter specific factors.
Transcriptional activation of heat-shock genes in eukaryotes
Authors: Tanguay Robert M.
Published: December 2009

Prokaryotes and eukaryotes respond to thermal or various chemical stresses by the rapid induction of a group of genes collectively referred to as the heat shock genes. In eucaryotes, the expression of these genes is primarily regulated at the transcriptional level. The early observations that transfected heat shock genes were inducible in heterologous systems suggested the existence of common regulatory elements in these ubiquitous genes. Sequence analysis of cloned Drosophila heat shock genes revealed a conserved 14 base pair (bp) inverted repeat, which is essential for heat induction. This regulatory sequence, referred to as the heat shock element (HSE), is found in multiple imperfect copies upstream of the TATA box of all heat shock genes. While studies in heterologous systems indicated that a single copy of HSE was sufficient for inducibility, further analysis in homologous assays suggests that multiple HSE can act in a cooperative way and that the efficiency of transcriptional activation is related, within limits, to the number of HSE. Comparative analysis of heat shock genes reveals that HSE can be positioned at different distances from the TATA box in either orientation, a behavior reminiscent of enhancer elements. However, the presence of HSE does not necessarily confer heat inducibility, as shown by their presence in the constitutively expressed but non-heat-inducible homologous cognate genes.Footprinting and nuclease mapping have been used to show that a protein factor (HSTF: heat shock transcription factor) binds to the HSE element, activating heat shock gene transcription in a dose-dependent manner. The recent progress in the isolation and characterization of HSTF in Drosophila, yeast, and human cells is reviewed. Finally, different models suggested to account for the positive regulation of heat shock genes by the HSTF are presented.
Principles underlying the use of plant cell fermentation for secondary metabolite production
Authors: Constabel F.
Published: December 2009

This review on plant cell fermentation for secondary metabolite production covers cell differentiation, product synthesis and accumulation, compartmentation, material cultured, and culture conditions employed. While product formation, including industrial scale operations, is well advanced, the induction of product formation is still the subject of much present-day research. Analysis of the competence of cells for product formation when associated with structural differentiation is lacking and will require genetic engineering technology.
Use of protoplast fusion systems to study organelle genetics in a commercially important crop
Authors: Kemble Roger J. , Barsby Tina L.
Published: December 2009

Protoplast fusion can overcome the sexual incompatibility barriers that exist between different plant species and can give rise to novel plants that cannot be produced in nature. It therefore provides a new source of genetic variation, particularly within the cytoplasmic organelles. We review the mixtures, rearrangements, and recombinations of mitochondrial and chloroplast genomes in plants regenerated from protoplast fusion products. Particular emphasis is placed on canola fusion systems that are being utilized in the commercial production of hybrid seed for a worldwide market.
Taurine uptake by normal and cystic fibrosis fibroblasts
Authors: Thompson Geoffrey N.
Published: December 2009

Taurine deficiency recently has been proposed to be clinically significant in cystic fibrosis (CF). Uptake of [14C]taurine by four cystic fibrosis (CF) and three control fibroblast lines was examined to determine whether a generalized defect in taurine transport could contribute to the deficiency. The time course of uptake was linear up to 20 h and was similar in both CF and control fibroblasts. Taurine was avidly retained after uptake, and the effect of metabolic (chlorpromazine) and competitive (hypotaurine, L-leucine) inhibitors was similar in both CF and control cells. In contrast, while taurine uptake in a calcium-free medium was impaired in both CF and control fibroblasts, the impairment was significantly less in CF cells. The findings suggest that a generalized abnormality in taurine transport is unlikely to be responsible for the taurine deficiency in CF.
Inhibition of lymphocyte 5′-nucleotidase by lectins: effects of lectin specificityand cross-linking ability
Authors: Sharom Frances J. , Lamb Mary P. , Kupsh Christine C. , Head Susan
Published: December 2009

5′-Nucleotidase, an integral glycoprotein enzyme of the lymphocyte plasma membrane, is inhibited cooperatively by the lectin concanavalin A. Because divalent succinyl-concanavalin A is a poor enzyme inhibitor, both binding and lectin-induced cross-linking of 5′-nucleotidase may be necessary for inhibition. Succinyl-concanavalin A does not compete with concanavalin A for binding to the enzyme; however, maleyl-concanavalin A, another poor inhibitor, competes effectively with the parent lectin. Thus, maleyl-concanavalin A binds to the same site as concanavalin A but causes little inhibition, whereas succinyl-concanavalin A does not bind to this site. The monovalent lectin from Ricinus communis (RCA-60) is a more effective enzyme inhibitor than the related divalent lectin (RCA-120), and inactivation of the second low-affinity sugar binding site on RCA-60 does not abolish inhibition, suggesting that multivalent cross-linking is not required for 5′-nucleotidase inhibition. Peanut and wheat germ agglutinins do not inhibit the enzyme, whereas lectins from lentil, pea, soybean, Griffonia simplicifolia, and Phaseolus vulgaris inhibit 5′-nucleotidase with various degrees of effectiveness. The only lectin showing strong positive cooperativity in its interaction with 5′-nucleotidase is concanavalin A.
The independent regulation of and tRNAAsn synthesis during Friend cell erythroid differentiation
Authors: Kleiman Lawrence , Schmedt Erich , Miller Harvey
Published: December 2009

In this report, we have compared the changes in the production of [Formula: see text] (initiator tRNAMet) and tRNAAsn, which occur during erythroid differentiation in the Friend erythroleukemia cell. The relative steady-state concentration of these two tRNAs (relative to the total tRNA population) was measured by aminoacylation. The results show that while the relative steady-state concentration of [Formula: see text] changes very little in the cytoplasmic tRNA population, the relative concentration of tRNAAsn decreases during the first two days of differentiation and then undergoes an increase. This difference in the behavior of these two tRNAs is also seen when their relative concentrations in newly synthesized tRNA is examined. When tRNA is labeled with tritiated uridine for 24 h in vivo prior to isolation, the hybridization of this labeled tRNA to filter-bound tRNA genes shows that the relative concentration of [Formula: see text] in newly synthesized tRNA changes very little, while the relative concentration of newly synthesized tRNAAsn again decreases through the first 2 days of differentiation, and then undergoes a smaller increase. Thus, the production of these two tRNAs appears to be independently regulated. Independent regulation of synthesis is also observed when examining the production of these two tRNAs in isolated nuclei. During erythroid differentiation, the relative synthesis of [Formula: see text] (relative to total nuclear RNA synthesis) remains constant, while the relative synthesis of tRNAAsn undergoes periodic increases and decreases in value.
Identification of glucose and nucleoside transport proteins in neonatal pig erythrocytes using monoclonal antibodies against band 4.5 polypeptides of adult human and pig erythrocytes
Authors: Craik James D. , Good A. Heather , Gottschalk Russell , Jarvis Simon M. , Paterson Alan R. P. , Cass Carol E.
Published: December 2009

Cytochalasin B and nitrobenzylthioinosine (NBMPR), which inhibit membrane transport of glucose and nucleosides, respectively, have served as photoaffinity ligands that become covalently linked at inhibitor binding sites on transporter-associated proteins. Thus, when membranes from erythrocytes of neonatal pigs with site-bound [3H]cytochalasin B or [3H]NBMPR were irradiated with uv light, two labeled membrane polypeptides (peak Mr values: 55 000 and 64 000, respectively) were identified. Treatment of the photolabeled membranes with endoglycosidase F increased the mobility of [3H]cytochalasin B- and [3H]NBMPR-labeled material (peak Mr values: 44 000 and 57 000, respectively) and limited digestion with trypsin yielded different polypeptide fragments (Mr values: 18 000 – 23 000 and 43 000, respectively). Identification of the photolabeled polypeptides as transporter components was established using monoclonal antibodies (MAbs) raised against partially purified preparations of band 4.5 from erythrocytes of adult pigs and humans. MAbs 65D4 and 64C7 (anti-human band 4.5), raised in this study, reacted with [3H]cytochalasin B-labeled material from membranes of human erythrocytes and bound to permeabilized erythrocytes but not to intact cells. MAb 65D4 also bound to erythrocytes of mice and neonatal pigs and to a variety of cultured cells (mouse, human, rat), including AE1 mouse lymphoma cells, which lack an NBMPR-sensitive nucleoside transporter. Also employed was MAb 11C4 (anti-pig band 4.5), which recognizes the NBMPR-binding protein of erythrocyte membranes from adult pigs. When membrane proteins from neonatal and adult pigs were subjected to electrophoretic analysis and blots were probed with different MAbs, MAb 65D4 (anti-human band 4.5) bound to material that comigrated with [3H]cytochalasin B-labeled polypeptides (band 4.5) from neonatal, but not adult, pig erythrocytes, whereas MAb 11C4 (anti-pig band 4.5) bound to material that comigrated with [3H]NBMPR-labeled band 4.5 polypeptides of erythrocytes from both neonatal and adult pigs. These results, which indicate structural differences in the cytochalasin B- and NBMPR-binding proteins of pig erythrocytes, establish the presence of both proteins in erythrocytes of neonatal pigs and suggest that only the NBMPR-binding protein is present in erythrocytes of adult pigs.
Lipid analysis of skeletal muscle from pigs susceptible to malignant hyperthermia
Authors: Fletcher Jeffrey E. , Rosenberg Henry , Michaux Kirsten , Cheah Khay S. , Cheah Anne M.
Published: December 2009

Previous studies demonstrated that lipid profiles of humans and pigs susceptible to malignant hyperthermia (MH) differ from those of normal humans and pigs. Lipid extraction techniques retaining in vivo lipid profiles most closely were used in the present study to determine if stimulation of lipolysis by the processes of homogenization or extraction might account for the reported differences in lipid profiles. No differences were observed among three genotypes of British Landrace pigs with respect to cholesterol levels, triglyceride levels, or total lipid phosphorus values of whole muscle (longissimus dorsi). Phospholipid distributions were the same for all three groups. Individual free fatty acids and fatty acids acylated to triglycerides were similar among the genotypes. These results do not support altered lipid profiles in vivo in MH-susceptible swine. Previously used homogenization and extraction procedures most likely affect the lipolytic activity to a different extent in muscle from MH-susceptible pigs and normal pigs.
Carrier-dependent and carrier-independent uptake of myo-inositol in cultured retinal pigment epithelial cells: activation by heat and concentration
Authors: Khatami Mahin
Published: December 2009

Transport of myo-inositol (MI) was studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. At low external concentrations (0.01–1 mM), uptake appeared to follow saturation kinetics, although the reciprocal forms of the rate equations did not fit either Lineweaver–Burk or Eadie–Hofstee plots. Increasing external concentrations dramatically changed the pattern of MI entry. At two to three orders of magnitude higher than physiological concentrations, a second saturation occurred (pseudo saturation). Cells incubated with 20 μM [3H]MI for 60 min had a ratio of intracellular to extracellular radioactivity ≥ 8, indicating active transport.MI transport reduction by Na+ replacement or inhibitors (phlorizin, ouabain, amiloride, KSCN, iodoacetamide, MI analogues) was greater when RPE cells were incubated with low (20–400 μM) than with high (10–20 mM) MI concentrations. Cells incubated with 20 μM MI at 53 or 65 °C showed increased transport (up to 560%) compared with cells at 22 °C. The effect on MI uptake (20 μM) of Na+ replacement also was reduced at 53 °C. The uptake of MI involved at least two transport systems. The major mechanism at low external MI concentrations (physiological levels) was a carrier-mediated active process. At high external MI levels, uptake occurred by a diffusion process. A lipotropic effect of MI may be responsible for this increased rate of diffusion.