BBA - General Subjects (v.1474, #1)

Active steroidogenesis in the normal rat skin by Andrzej Slominski; Celso E. Gomez-Sanchez; Mark F. Foecking; Jacobo Wortsman (1-4).
Using the radiolabeled precursors of adrenal steroids 14C-11-deoxycorticosterone (DOC) and 14C-progesterone (14C-PROG) we demonstrate that rat skin can synthesize a number of steroids. TLC separation of labeled metabolites show that among the 14C-steroid products, two co-migrate with corticosterone (B) and 11-dehydrocorticosterone (A) standards. Thus, normal rodent skin possesses steroidogenic activity that can be shown using progesterone or DOC as primary substrates.
Keywords: Steroid; Rat skin; Progesterone; 11-Deoxycorticosterone; 11-Dehydrocorticosterone; Corticosterone;

Differential effects of gemcitabine on ribonucleotide pools of twenty-one solid tumour and leukaemia cell lines by Catharina J.A van Moorsel; Andries M Bergman; Gijsbert Veerman; Daphne A Voorn; Veronique W.T Ruiz van Haperen; Judith R Kroep; Herbert M Pinedo; Godefridus J Peters (5-12).
To gain a more detailed insight into the metabolism of 2′,2′-difluoro-2′-deoxycytidine (dFdC, gemcitabine, Gemzar) and its effect on normal ribonucleotide (NTP) metabolism in relation to sensitivity, we studied the accumulation of dFdCTP and the changes in NTP pools after dFdC exposure in a panel of 21 solid tumour and leukaemia cell lines. Both sensitivity to dFdC and accumulation of dFdCTP were clearly cell line-dependent: in this panel of cell lines, the head and neck cancer (HNSCC) cell line 22B appeared to be the most sensitive, whereas the small cell lung cancer (SCLC) cell lines were the least sensitive to dFdC. The human leukaemia cell line CCRF-CEM accumulated the highest concentration of dFdCTP, whereas the non-SCLC cell lines accumulated the least. Not only the amount of dFdCTP accumulation was clearly related to the sensitivity for dFdC (R=−0.61), but also the intrinsic CTP/UTP ratio (R=0.97). NTP pools were affected considerably by dFdC treatment: in seven cell lines dFdC resulted in a 1.7-fold depletion of CTP pools, in two cell lines CTP pools were unaffected, but in 12 cell lines CTP pools increased about 2-fold. Furthermore, a 1.6–1.9-fold rise in ATP, UTP and GTP pools was shown in 20, 19 and 20 out of 21 cell lines, respectively. Only the UTP levels after treatment with dFdC were clearly related to the amount of dFdCTP accumulating in the cell (R=0.64 (P<0.01)), but not to the sensitivity to dFdC treatment. In conclusion, we demonstrate that besides the accumulation of dFdCTP, the CTP/UTP ratio was clearly related to the sensitivity to dFdC. Furthermore, the UTP levels and the CTP/UTP ratio after treatment were related to dFdCTP accumulation. Therefore, both the CTP and UTP pools appear to play an important role in the sensitivity to dFdC.
Keywords: Gemcitabine sensitivity; dFdCTP accumulation; Ribonucleotide pool;

Glucagon-like peptide-1(7–36)amide (tGLP-1) is inactivated by dipeptidyl peptidase (DPP) IV by removal of the NH2-terminal dipeptide His7-Ala8. We examined the degradation of NH2-terminally modified His7-glucitol tGLP-1 and its insulin-releasing and antihyperglycaemic activity in vivo. tGLP-1 was degraded by purified DPP IV after 4 h (43% intact) and after 12 h, 89% was converted to GLP-1(9–36)amide. In contrast >99% of His7-glucitol tGLP-1 remained intact at 12 h. His7-glucitol tGLP-1 was similarly resistant to plasma degradation in vitro. His7-glucitol tGLP-1 showed greater resistance to degradation in vivo (92% intact) compared to tGLP-1 (27% intact) 10 min after i.p. administration to Wistar rats. Glucose homeostasis was examined following i.p. injection of both peptides (12 nmol/kg) together with glucose (18 mmol/kg). Plasma glucose concentrations were significantly reduced and insulin concentrations elevated following peptides administration compared with glucose alone. The area under the curve (AUC) for glucose for controls (AUC 691±35 mM/min) was significantly lower after administration of tGLP-1 and His7-glucitol tGLP-1 (36 and 49% less; AUC 440±40 and 353±31 mM/min, respectively; P<0.01). This was associated with a significantly higher AUC for insulin (98–99% greater; AUC 834±46 and 838±39 ng/ml/min, respectively; P<0.01) after tGLP-1 and His7-glucitol tGLP-1 administration compared to controls (421±30 ng/ml/min). In conclusion, His7-glucitol tGLP-1 resists plasma DPP IV degradation while retaining potent antihyperglycaemic and insulin-releasing activities in vivo.
Keywords: Glucagon-like peptide-1; His7-glucitol adduct; Peptide degradation; Insulin secretion; Diabetes therapy;

1H-NMR and Raman studies on perforating trauma-induced cataract formation in a mouse lens by Koji Nakamura; Young Mee Jung; Seiichi Era; Masaru Sogami; Yukihiro Ozaki; Akihiko Takasaki (23-30).
In order to provide new insight into the molecular mechanism of perforating trauma-induced cataract formation in an 8-week-old ddY mouse lens, we performed an in situ investigation into changes in the water-protein and/or protein-protein interactions by using 500 MHz 1H-NMR spectroscopy, and into structural alterations in lens proteins by using Raman spectroscopy. Cross-relaxation times of water protons in the perforated opaque lens were considerably shorter than those in the intact transparent lens, whereas there was no significant difference in water content, suggesting a drastic change in water-protein and protein-protein interactions in the perforated lens. In addition, there was no significant difference in the intensity ratios of several key Raman bands between intact and perforated lenses, indicating that no significant local and overall conformational changes in lens protein itself occur in the perforated lens. The present 1H-NMR and Raman results lead us to the conclusion that changes leading to lens opacification in the perforating trauma-induced cataract appear to involve the rapid formation of immobile large lens protein aggregates without formation of intra- and intermolecular disulfide linkages, and rapid increase in a fraction of bound water associated with large protein aggregates.
Keywords: Cataract; Cross-relaxation time; Lens (mouse); NMR; Raman spectroscopy; Water-protein interaction;

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell to cell signalization and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate (HSPG) in immature rat Sertoli cells. The present study focused on the qualitative and quantitative expression of three membrane HSPG, syndecan-1, syndecan-4 and glypican-1 in Sertoli cells of 20-day-old rat. A semi-quantitative multiplex RT-PCR strategy was developed to appreciate the effect of PKC activation on the mRNA expression of the three HSPG. Our data show that the syndecan-1 and glypican-1 mRNA expression is increased by the phorbol myristate acetate (PMA) suggesting a regulation of their expression by the phosphatidyl inositol pathway, as previously hypothesized (Fagen et al., Biochim. Biophys. Acta, 1472 (1999) 250–261). In addition, a physiological effector of the PKC as ATP gave similar effects. Thus, this over-expression could be related with paracrine factors secreted by germ cells.
Keywords: Syndecan; Glypican; Rat Sertoli cell; RT-PCR analysis; Protein kinase C;

Serine and glycine transport in fetal ovine hepatocytes by Michael R Narkewicz; Gayle Jones; Denise Morales (41-46).
The role of hepatic serine and glycine transport in the regulation of the biosynthesis of serine by the fetal liver has not been studied. The goal of this study was to characterize serine and glycine transport and utilization at physiologic concentrations in primary cultures of fetal ovine hepatocytes. Primary culture of hepatocytes from mid gestation (≈90 days) and term (≈135 days) fetal sheep were studied after overnight serum free culture. At both gestational ages, the initial rate for sodium dependent 300 μM serine transport (1697±131 pmoles/min/mg protein at mid, 1765±544 at term) was fourfold greater than sodium dependent 300 μM glycine transport (309±54 at mid, 579±252 at term). At physiologic concentrations (300 μM), 69±7% of serine and 49±8% of glycine transport was sodium dependent. At term, sodium dependent serine transport has a V max of 1751±348 pmoles/min/mg protein and a K m of 159±111 μM. Sodium independent serine transport has a V max of 904±185 and a K m of 416±188 μM. Sodium dependent glycine transport has a V max of 410±69 and a K m of 2290±895 μM while sodium independent glycine transport exhibits non-saturable kinetics. Glycine (300 μM) sodium dependent transport was not inhibited by methyl-AIB while sodium dependent 300 μM serine transport was inhibited (31%). n-Ethylmaleimide inhibited sodium dependent serine and glycine transport by 36±9% and 37±2% respectively in term hepatocytes. Cysteine inhibited sodium dependent serine transport by 37%. Sodium independent glycine transport at 300 μM was higher in low glucose (1.1 mM) medium (881±76 pmoles/min/mg protein) compared to high glucose (5.5 mM) medium (510±60 P=0.004). There were no significant differences in serine or glycine incorporation into RNA, DNA, glycogen or lipid and protein. The predominance of serine transport over glycine at physiologic concentrations suggests that inward cellular amino acid transport of serine and glycine is not likely to be a regulatory mechanism that would favor serine biosynthesis in fetal ovine hepatocytes.
Keywords: Serine; Glycine; Amino acid transport; Hepatocyte; Fetal;

As encountered with a plethora of other natural products, the antioxidant activity of β-carotene has been proposed as one of the mechanisms by which diets rich in this pro-vitamin A active carotenoid apparently afford chemoprevention. Here, we report the ability of β-carotene to alter endogenous reactive oxygen levels and antioxidant defences within non-stressed ‘differentiated’ monolayers of an intestinal epithelial cell line (Caco-2) and to subsequently effect resistance to general oxidative insult. The differentiated monolayers efficiently absorbed β-carotene. Between 3 and 8 days post confluence, cultures exhibited a progressive increase in antioxidant enzyme activity and a corresponding reduction to intracellular ROS levels. The profile for antioxidant enzyme activity was unaffected by sustained daily supplementation with β-carotene. However, after two daily treatments with 50 μM β-carotene intracellular ROS levels were significantly reduced and there was a trend towards reduced intracellular ROS within monolayers subject to five daily treatments with 0.5 and 5 μM β-carotene. Prolonged supplementation with 0.1 and 0.5 μM β-carotene or short supplementation periods with 5 and 50 μM β-carotene did not alter susceptibility to H2O2. However, cultures treated daily between 3 and 8 days post confluence with 5 or 50 μM β-carotene exhibited enhanced LDH release, increased non-adherence and enhanced Trypan blue staining when challenged with 10 mM H2O2. In the absence of H2O2, the β-carotene treatments were not overtly toxic to the monolayers. These results indicate that β-carotene does not enhance antioxidant defences within Caco-2 monolayers. The enhancement of H2O2 toxicity by persistent, high doses of β-carotene may contribute to the failure of this carotenoid to protect high risk individuals from certain degenerative conditions.
Keywords: β-Carotene; Antioxidant; Reactive oxygen species; Membrane damage; Cell culture; Caco-2;

Crotamine, a neurotoxin present in the venom of the South American rattlesnake Crotalus durrisus terrificus exists as several polymorphic variants, as demonstrated by recombinant DNA technology (Smith and Schmidt, Toxicon 28 (1990) 575–585). We have isolated native crotamine by chromatography on Sephadex G75, and have purified two crotamine isoforms (F2 and F3) by a single step of RP-HPLC. Native crotamine and RP-HPLC fractions F2 and F3 produced skeletal muscle spasms and spastic paralysis in mice. At low glucose concentrations (2.8–5.6 mmol/l), none of the crotamines altered the insulin secretion by rat isolated islets. In the presence of 16.7 mmol glucose/l, F2 (5 μg/ml), but not F3, increased insulin secretion two-fold, whereas native crotamine (1.5, 5 and 16.5 μg/ml) potentiated the secretion dose-dependently. The increase in insulin secretion induced by F2 fraction (5 μg/ml) was similar to that obtained with 16.5 μg of native crotamine/ml. These results indicate that the mode of action of the F2 and F3 isoforms in β-cells is different from that in muscle cells. This difference may be related to the binding affinity of each isoform for the Na+ channels located in the β-cell membrane. Crotamine isoforms may be valuable tools for studying the involvement of Na+ channels in the mechanism of insulin secretion.
Keywords: Crotalus durrisus terrificus; Crotamine; Venom; Pancreatic β-cell; Insulin secretion; Na+ channel;

The enzyme glucokinase (GK) (EC plays an important role in the control of glucose homeostasis. Qualitative and/or quantitative variations in GK enzyme have been postulated by previous studies to explain why dietary carbohydrate utilisation is lower in gilthead seabream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss) than in common carp (Cyprinus carpio). In this study, we report the isolation and characterisation of a full-length cDNA coding for GK in these teleosts. Amino acid sequences derived from these cDNA clones are highly similar to other vertebrate GKs. These findings, including a detailed phylogenetic analysis, reveal that GK gene highly homologous to mammalian GK exists in these fish species with similar tissue specific expression (mainly liver).
Keywords: Fish nutrition; Dietary carbohydrate; Glucose phosphorylation;

In vitro recycling of α-D-ribose 1-phosphate for the salvage of purine bases by Laura Mascia; Mario Cappiello; Silvia Cherri; Piero Luigi Ipata (70-74).
In this paper, we extend our previous observation on the mobilization of the ribose moiety from a purine nucleoside to a pyrimidine base, with subsequent pyrimidine nucleotides formation (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273–281). The data show that, at least in vitro, also the reverse process is possible. In rat brain extracts, the activated ribose, stemming from uridine as ribose 1-phosphate, can be used to salvage adenine and hypoxanthine to their respective nucleotides. Since the salvage of purine bases is a 5-phosphoribosyl 1-pyrophosphate-dependent process, catalyzed by adenine phosphoribosyltransferase and hypoxanthine guanine phosphoribosyltransferase, our results imply that Rib-1P must be transformed into 5-phosphoribosyl 1-pyrophosphate, via the successive action of phosphopentomutase and 5-phosphoribosyl 1-pyrophosphate synthetase; and ,in fact, no adenosine could be found as an intermediate when rat brain extracts were incubated with adenine, Rib-1P and ATP, showing that adenine salvage does not imply adenine ribosylation, followed by adenosine phosphorylation. Taken together with our previous results on the Rib-1P-dependent salvage of pyrimidine nucleotides, our results give a clear picture of the in vitro Rib-1P recycling, for both purine and pyrimidine salvage.
Keywords: Ribose 1-phosphate; Ribose 5-phosphate; Purine salvage; Rat brain;

Deficiencies of glycolytic enzymes as a possible cause of hemolytic anemia by Michael V. Martinov; Andrew G. Plotnikov; Victor M. Vitvitsky; Fazoil I. Ataullakhanov (75-87).
The critical minimum values of Na,K-ATPase and glycolytic enzyme activities at which the erythrocyte viability is lost were calculated using the mathematical model of the erythrocyte, which included all reactions of glycolysis, adenylate metabolism, ionic balance, and osmotic regulation of erythrocyte volume. The criterion for cell death was an increase in its volume to the level at which it is sequestrated from the circulation or is lysed. In hemolytic anemia associated with hexokinase or pyruvate kinase deficiency, activities of these enzymes measured in patient erythrocytes appeared to be close to the calculated critical values. By contrast, in hemolytic anemia associated with phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, activities of these enzymes measured in patient erythrocytes were significantly greater than the calculated critical values. In this case, if the deficient enzyme were stable, i.e. its activity in the cell were low, but constant in time, the deficiency observed would not account for the erythrocyte destruction observed and the development of hemolytic anemia. It was shown, however, that in phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, hemolytic anemia can arise because of the instability of these enzymes in time.
Keywords: Erythrocyte; Glycolysis; Enzyme deficiency; Hemolytic anemia; Mathematical model;

c-Series gangliosides in extraneural tissues from young and adult rats were examined using thin-layer chromatographic (TLC) immunostaining with a specific monoclonal antibody A2B5. The composition of c-series gangliosides significantly differed among tissues. In adult rats, while liver tissue contained GT1c, GQ1c, and GP1c, renal tissue had GT3 as the major c-series ganglioside with GT2 in a lesser amount. Pancreatic tissue expressed c-series gangliosides that consisted of GT3, GT2, GQ1c, and GP1c. In other tissues including adrenal, thyroid, and eye lens, GT3 constituted the main c-series ganglioside species. While total ganglioside contents of extraneural tissues were much lower than that of brain tissue, the proportions of c-series gangliosides to total gangliosides were higher in many extraneural tissues. Interestingly, eye lens had the highest GT3 content among rat tissues examined. The compositions and concentrations of c-series gangliosides in liver and kidney significantly differed between 5-day-old and 7-week-old rats, suggesting the development-dependent expression of c-series gangliosides in these tissues. These results suggest that the expression of c-series gangliosides in extraneural tissues is regulated in a tissue-specific manner.
Keywords: Ganglioside; C-series ganglioside; Glycolipid; A2B5; Extraneural tissue;

Effects of 6-formylpterin, a xanthine oxidase inhibitor and a superoxide scavenger, on production of nitric oxide in RAW 264.7 macrophages by Hiroko Mori; Toshiyuki Arai; Kiichi Hirota; Hisanari Ishii; Nobuyuki Endo; Keisuke Makino; Kazuhiko Fukuda (93-99).
As well as superoxide generated from neutrophils, nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in macrophages plays an important role in inflammation. We previously showed that 6-formylpterin, a xanthine oxidase inhibitor, has a superoxide scavenging activity. In the present study, to elucidate other pharmacological activities of 6-formylpterin, we investigated the effects of 6-formylpterin on production of nitric oxide (NO) in the murine macrophage cell line RAW 264.7 stimulated by lipopolysaccharide (LPS) and interferon-γ (INF-γ). 6-Formylpterin suppressed the expression of iNOS, and it also inhibited the catalytic activity of iNOS, which collectively resulted in the inhibition of NO production in the stimulated macrophages. However, 6-formylpterin did not scavenge the released NO from an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). These results indicate that 6-formylpterin inhibits pathological NO generation from macrophages during inflammation, but that it does not disturb the physiological action of NO released from other sources.
Keywords: 6-Formylpterin; Inducible nitric oxide synthase; Nitric oxide; Spin trap; Macrophage;

Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum by Ehtishamul Haq; Sadhna Sharma; Gopal K Khuller (100-106).
A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalytic (C) subunits were purified to homogeneity from soluble extract of Microsporum gypseum. Purified enzyme showed a final specific activity of 277.9 nmol phosphate transferred min−1 mg protein−1 with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa and 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa subunit was found to be the regulatory (R) subunit of PKA holoenzyme as determined by its immunoreactivity and the isoelectric point of this subunit was 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sepharose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, compatible with its tetrameric structure, consisting of two regulatory subunits (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein acceptors in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had apparent K m values of 71 μM and 25 μM for ATP and kemptide, respectively. Phosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmaleimide (NEM) which shows the involvement of sulfhydryl groups for the activity of PKA. PKA phosphorylated a number of endogenous proteins suggesting the multifunctional role of cAMP dependent protein kinase in M. gypseum. Further work is under progress to identify the natural substrates of this enzyme through which it may regulate the enzymes involved in phospholipid metabolism.
Keywords: cAMP; cAMP-dependent protein kinase (PKA); Microsporum gypseum;

Helicobacter pylori-antigen-binding fragments expressed on the filamentous M13 phage prevent bacterial growth by Jun Cao; Yi-qian Sun; Thomas Berglindh; Björn Mellgård; Zhao-qi Li; Bibbi Mårdh; Sven Mårdh (107-113).
Colonization of the human stomach by Helicobacter pylori is associated with the development of gastritis, duodenal ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori-antigen-binding single-chain variable fragments (ScFv) were derived from murine hybridomas producing monoclonal antibodies and expressed as a g3p-fusion protein on a filamentous M13 phage. The recombinant ScFv-phage reacted specifically with a 30-kDa monomeric protein of a H. pylori surface antigen preparation and by means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant phage exhibited a bacteriocidal effect and inhibited specifically the growth of all the six strains of H. pylori tested. When H. pylori was pretreated with the phage 10 min before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced (P<0.01). The results suggest that genetic engineering may be used to generate therapy-effective phages.
Keywords: Bactericidal; Helicobacter pylori infection; Monoclonal antibody; Mouse model; Phage specificity; Recombinant bacteriophage; Single-chain fragment variable (ScFv);