Study of developmental changes on hexoses metabolism in rat cerebral cortex

We have studied the developmental changes of glucose, mannose, fructose and galactose metabolism in rat cerebral cortex. As the animals aged, glucose, mannose and fructose oxidation to CO₂ increased, whereas galactose oxidation decreased. Lipid synthesis from glucose and fructose also increased with age, that from mannose decreased and galactose did not change. Cytochalasin B, a potent non-competitive inhibitor of sodium-independent glucose transport, significantly impaired glucose, mannose and galactose metabolism, but had no effect on fructose metabolism. Both galactose or fructose did not change, whereas mannose declined the glucose metabolism. Glucose decreased fructose, galactose and mannose metabolism. Our results show that besides glucose, the metabolism of mannose, galactose and fructose present developmental changes from fetal to adult age, and reinforce the literature data indicating that mannose and galactose are transported by glucose carriers, while fructose is not.     

Developmental changes in cyclin B1 and cyclin-dependent kinase 1 (CDK1) levels in the different populations of spermatogenic cells of the post-natal rat testis

Spermatogenesis is a highly ordered process which requires mitotic and meiotic divisions. In this work, we studied the relative changes in the levels of the two components of the M-phase promoting factor (MPF): the regulatory subunit cyclin B1 (CycB1) and its catalytic subunit cdk1, in spermatogenic cells of rats between 16 and 90 days of life. A multivariate flow cytometry analysis of forward scatter (FSC), side scatter (SSC) and DNA content was used to identify six populations of rat germ cells: spermatogonia with preleptotene spermatocytes, young pachytene spermatocytes, middle to late pachytene spermatocytes, secondary spermatocytes with doublets of round spermatids, round spermatids, and elongated spermatids. For any population studied no significant difference in the relative cellular content of CycB1 or cdk1 proteins between animals of different ages was observed. By contrast, CycB1 and cdk1 levels were different between the different populations of germ cells. CycB1 and cdk1 were rather high in young pachytene spermatocytes and culminated in late spermatocytes, i.e. just before the first meiotic division. The relative levels of the two proteins remained high in secondary spermatocytes then decreased in round spermatids at the exit of meiosis. Similar results were obtained by Western-blot analysis of total proteins obtained from lysates of elutriated fractions of spermatocytes and spermatids. MPF activity was assessed in lysates of germ cells from 32-day-old rats or adult animals using p13suc1 agarose and histone H1 as an exogenous substrate. H1 kinase activity was higher in pachytene spermatocytes than in round spermatid fractions from both adult and young rats. These results indicate that the meiotic G2/M transition is associated to high levels of CycB1 and cdk1 leading to high MPF activity irrespective of the age of the animals.     

Characterization of an echinocandin B-producing strain blocked for sterigmatocystin biosynthesis reveals a translocation in the stcW gene of the aflatoxin biosynthetic pathway

Echinocandin B (ECB), a lipopolypeptide used as a starting material for chemical manufacture of the anti-Candida agent LY303366, is produced by fermentation using a strain of Aspergillus nidulans. In addition to ECB, the wild-type strain also produces a significant level of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Characterization of a mutant designated A42355-OC-1 (OC-1), which is blocked in ST biosynthesis, was the result of a chromosomal translocation. The chromosomal regions containing the breakpoints of the translocation were isolated and DNA sequencing and PCR analysis of the chromosomal breakpoints demonstrated the translocation occurred within the stcW gene of the ST biosynthetic pathway, resulting in disruption of the open reading frame for this gene. Biochemical feeding studies indicate the involvement of this gene product in the conversion of averufin to 1-hydroxy versicolorone. This work demonstrates an effective synergy between classical strain improvement methods and molecular genetics. Journal of Industrial Microbiology & Biotechnology (2000) 25, 333–341. Received 27 April 2000/ Accepted in revised form 25 November 2000     

IκB/NF-κB mediated cisplatin resistance in HeLa cells after low-dose γ-irradiation is associated with altered SODD expression

Fractionated -irradiation (15 × 2 Gy in 3 weeks) induces a cellular resistance in HeLa cells against cisplatin exposure but not against irradiation. The mechanisms underlying this cellular resistance are associated with major changes in the TNFR1-dependent transduction pathway. The resistant HeLa/B cells exhibit increased levels of NF-B with temporally independent regulation of the subunits NF-B50 and NF-B65. Blocking IB degradation by the proteasome inhibitor PSI, which abolishes the release of the active NF-B protein, induces cell death much more effectively in the parental than in the resistant HeLa/B cells. The translocation of NF-B does not seem to be affected in a similar manner since masking of the translocation sequence by NF-B SN50 enhances cisplatin toxicity to the same degree in both cell lines and overcomes drug resistance. Changes in upstream signaling are suggested by increased sensitivity of the parental HeLa cells to cisplatin in the presence of neutralizing anti-TNFR1. In HeLa/B cells, reduced expression of the 50 kDa silencer of death domain, SODD, is accompanied by constitutive overexpression of a 40–42 kDa SODD-like protein. A possible involvement of SODD in cisplatin resistance is discussed, which may shift the balance between life and death in the TNF receptor pathway to increased NF-B activation.     

N-acetyl- -arginine ethyl ester synthesis catalysed by bovine trypsin in organic media

The bovine trypsin-catalysed synthesis of N-acetyl- -arginine ethyl ester from N-acetyl- -arginine and ethanol was studied in various organic solvents (dimethyl sulfoxide, dioxane, dimethylformamide, acetonitrile, acetone, tetrahydrofuran, chloroform, toluene, carbon tetrachloride, cyclohexane and n-hexane). The highest yield was achieved in acetonitrile after incubation for 6 or 24 h. The optimal conditions for ester synthesis in acetonitrile for 6 h were as follows: 5.0 mM N-acetyl- -arginine, 10.0 M ethanol, 7.2 mg trypsin, 2.87% water, total volume 10.3 ml, pH 7.0 and 30°C. The hydrolytic activity of trypsin was determined after incubation for 6 days, when 87.7% of the original activity remained, suggesting that acetonitrile caused little inactivation of the enzyme. The synthetic reaction resulted in a maximal 79.3% conversion under optimized conditions after incubation for 48 h.     

Alternating Planarity/Nonplanarity in n-Doped Odd-Membered, All-Trans Polyenes: Molecular Structures of NaCnHn+2 (n = 3, 5, 7, and 9)

The electronic structures of small, odd-membered, all-trans polyenes doped with one Na atom at various positions have been investigated using Hartree-Fock and density functional (B3LYP) theory with a 6-31G(d) basis set. Two distinctly different structural motifs have been identified. In one motif, the dopant atom interacts with an allylic polyene unit in a 4-electron interaction that results in a planar polyene backbone. The other motif has the dopant atom interacting with a pentadienyl polyene unit in a 6-electron interaction, which produces a significantly warped polyene backbone. Independent of structural motif at the doping site, the portion of the polyene structure outside the interaction region remains largely undisturbed in terms of planarity and bond length alternation. For a particular formula unit and potential energy surface, the stationary points corresponding to minima and transition states are remarkably close in energy despite the pronounced changes that occur in the dihedral angles of the polyene backbone at the dopant sites. Whereas internal and external instabilities are found in the Hartree-Fock wavefunctions for NaC₇H₉ and NaC₉H₁₁ structures, the restricted B3LYP wavefunctions are stable for all structures investigated.Electronic Supplementary Material available.     

Differential effect of schisandrin B and dimethyl diphenyl bicarboxylate (DDB) on hepatic mitochondrial glutathione redox status in carbon tetrachloride intoxicated mice

The effects of schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, and dimethyl diphenyl bicarboxylate (DDB), a synthetic intermediate of schisandrin C (also a dibenzocyclooctadiene derivative), on hepatic mitochondrial glutathione redox status in control and carbon tetrachloride (CCl₄)-intoxicated mice were examined. Treating mice with Sch B or DDB at a daily oral dose of 1 mmol/kg for 3 d did not produce any significant alterations in plasma alanine aminotransferase (ALT) and sorbital dehydrogenase (SDH) activities. CCl₄ treatment caused drastic increases in both plasma ALT and SDH activities in mice. Pretreating mice with Sch B or DDB at the same dosage regimen significantly suppressed the CCl₄-induced increase in plasma ALT activity, with the inhibitory effect of Sch B being much more potent. Sch B, but not DDB, pretreatment could also decrease the plasma SDH activity in CCl₄-intoxicated mice. The lowering of plasma SDH activity, indicative of hepatoprotection against CCl₄ toxicity, by Sch B pretreatment was associated with an enhancement in hepatic mitochondrial glutathione redox status as well as an increase in mitochondrial glutathione reductase (mtGRD) activity in both non-CCl₄ and CCl₄-treated mice. DDB pretreatment, though enhancing both hepatic mitochondrial glutathione redox status and mtGRD activity in control animals, did not produce any beneficial effect in CCl₄-treated mice. The difference in hepatoprotective action against CCl₄ toxicity between Sch B and DDB may therefore be related to their ability to maintain hepatic mitochondrial glutathione redox status under oxidative stress condition.     

Salmonella typhimurium forms adenylcobamide and 2-methyladenylcobamide, but no detectable cobalamin during strictly anaerobic growth

Under microaerophilic conditions Salmonella typhimurium LT2 synthesizes cobalamin, during which 5,6-dimethylbenzimidazole is formed from riboflavin. We report here that in an anoxic environment S. typhimurium did not form cobalamin, but rather adenylcobamide, 2-methyladenylcobamide, and cobyric acid. This indicated that S. typhimurium, like other microorganisms that synthesize 5,6-dimethylbenzimidazole from riboflavin, requires oxygen for the formation of the cobalamin base. Received: 29 June 1999 / Accepted: 29 September 1999     

Antigenicity of human melanoma cells transfected to express the B7-1 co-stimulatory molecule (CD80) varies with the level of B7-1 expression

 The aim of this study was to compare the antigenicity of human melanoma cells molecularly modified by particle-mediated gene transfer to have transient or stable expression of the B7-1 co-stimulatory molecule (CD80). The unmodified melanoma cells (mel5, m21) had no constitutive expression of B7-1, but 22%–28% of cells had transient B7-1 expression 24 h following transfection with cDNA for B7-1 (mel5-B7, m21-B7). In addition, 85%–90% of cells had stable B7-1 expression following transfection with cDNA for B7-1 and in vitro culture under selection conditions (mel5-B7neo, m21-B7neo). Allogeneic HLA-unmatched normal donor peripheral blood mononuclear cells (PBMC) secreted greater amounts of granulocyte/macrophage-colony-stimulating factor (GM-CSF) when incubated for 3 days with m21-B7neo than did PBMC incubated with m21-B7, which, in turn, secreted greater amount of GM-CSF than PBMC incubated with m21. Similarly, cell-mediated cytotoxicity against unmodified melanoma cells by PBMC co-cultured for 5 days with the modified or unmodified melanoma cells was proportional to the level of B7-1 expression on the stimulating cells. This cytolytic activity had both an HLA-class-I-restricted and an HLA-class-I-unrestricted component. Following 5 days of co-culture, PBMC expression of CD28, the ligand for B7-1, was down-regulated in proportion to the level of B7-1 expression on the stimulating melanoma cells. Thus, particle-mediated gene delivery of cDNA for B7-1 into human melanoma cells increased expression of functional B7-1 and enhanced the antigenicity of the gene-modified cells in proportion to their level of B7-1 expression. Received: 14 October 1999 / Accepted: 3 December 1999     

Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-kappaB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (Ras(DN)), constitutively active MEK1 (MEK(CA)), dominant negative IkappaB kinase 2 (IKK(DN)), and constitutively active IKK2 (IKK(CA)). Inhibiting ERK activity by Ras(DN) overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEK(CA) remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-kappaB pathway with IKK(DN) virus suppressed the pit-forming activity of OCLs and NF-kappaB activation by IKK(CA) expression upregulated it without affecting their survival. Interleukin 1alpha (IL-1alpha) strongly induced ERK activation as well as NF-kappaB activation. Ras(DN) virus partially inhibited ERK activation, and OCL survival promoted by IL-1alpha. Inhibiting NF-kappaB activation by IKK(DN) virus significantly suppressed the pit-forming activity enhanced by IL-1alpha. These results indicate that ERK and NF-kappaB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-kappaB regulates osteoclast activation for bone resorption.