Comparison of unbiased stereological estimation of total number of cresyl violet stained neurons and parvalbumin positive neurons in the adult human spiral ganglion.

Estimation of total number of neurons in the spiral ganglion (SG) at various ages and their functional status is important as these neurons are constantly exposed to noise and other environmental factors that may lead to neuronal loss with aging due to excitotoxic damage. Parvalbumin (PV) is a calcium-binding protein (CBP), found in highly metabolically active neurons. It helps in buffering cytosolic calcium, which is essential for neurotransmitter release. The neurons in the adult human SG express PV more strongly than other CBPs like calbindin and calretinin. These CBPs can be used as signatures to recognise neurons. In the present study, we quantified the number of neurons expressing PV by unbiased stereology and compared it to the number of neurons stained by cresyl violet (CV), which is a Nissl stain, in the adult human SG. Five adult human cadaveric temporal bones were obtained from the forensic science mortuary, after due clearance from the institute ethics committee. Independent CV stained and PV immunostained sections were used to estimate the total number of neurons (optical fractionator), with StereoInvestigator (SI) software. The estimated total number of SG neurons was 27,485±3251 and 26,705±1823 in the PV and CV stained sections, respectively. There was no significant difference between the estimates (p=0.552). Therefore, CV staining is simpler and more cost effective when estimating neuronal number. Although PV stains spiral ganglion neurons (SGNs) with a greater intensity and provides a functional status, its tedious protocol limits its use for quantification.

Evaluation of Mannose-Binding Lectin is a Useful Approach to Predict the Risk of Infectious Complications Following Autologous Hematopoietic Stem Cell Transplantation.

Hematopoietic stem cell transplantation (HSCT) associated immunocompromised state carries high risk of infectious complications. Mannose-binding lectin (MBL) is an acute phase protein involved in innate immune response. Serum MBL level is genetically determined and quite stable. According to literature, significant association was shown between low MBL concentrations and serious infections. The association between serum MBL level and frequency and severity of infections was studied in 186 patients following autologous HSCT. Double-monoclonal antibody sandwich enzyme-linked immunosorbent assay was used to determine MBL antigen level in sera. MBL levels were measured around 100 days following transplantation, in a period without active infection. Twenty-one patients (11%) were MBL deficient. The median time of first infection and number of infections during the first year post-transplantation were not significantly different between patients with MBL deficiency and those without MBL deficiency. The occurrence and number of infections after HSCT correlated with the MBL/C-reactive protein ratio. The number of severe infections was not higher among those with MBL deficiency. The occurrence of infections after the pre-engraftment period during the first year post-transplantation was significantly different in patient groups separated by MBL cut-off level. The MBL/C-reactive protein ratio might be a useful marker of infectious complications. MBL measurement may be helpful in antibiotic treatment. In case of MBL deficiency, earlier and more intensive treatment may be indicated.

S100A7 has an oncogenic role in oral squamous cell carcinoma by activating p38/MAPK and RAB2A signaling pathway.

Oral cancer consists of squamous cell carcinoma within the oral cavity or on the lip. The clinical prognosis of this cancer is mostly poor owing to delayed diagnosis and a lack of appropriate early detection biomarkers to identify the disease. In the current study, we investigated the role of the S100A7 calcium-binding protein in oral squamous cell carcinoma as an activator of the p38/MAPK and RAB2A signaling pathway. The aim of the present study was to determine whether S100A7 and RAB2A have a role in tumor progression and to assess their potential as early detection biomarkers for oral cancer. This study elucidated the functional and molecular mechanisms of S100A7 and RAB2A activity in oral cancer, leading us to conclude that S100A7 is the major contributing factor in the occurrence of oral cancer and promotes local tumor progression by activating the MAPK signaling pathway via the RAB2A pathway. We hypothesize that S100A7 affects cell motility and invasion by regulating the RAB2A-associated MAPK signaling cascades. Also, the downregulation of S100A7 expression by RNA interference-mediated silencing inhibits oral cancer cell growth, migration and invasion.

GW627368X inhibits proliferation and induces apoptosis in cervical cancer by interfering with EP4/EGFR interactive signaling.

PGE2, the major product of cyclooxygenases implicated in carcinogenesis, is significantly upregulated in cervical cancer. PGE2 via prostanoid receptor EP4 stimulates proliferation and motility while inhibiting apoptosis and immune surveillance. It promotes angiogenesis by stimulating the production of pro-angiogenic factors. The present study demonstrates GW627368X, a highly selective competitive EP4 antagonist, which hinders cervical cancer progression by inhibiting EP4/epithelial growth factor receptor (EGFR) interactive signaling. GW627368X reduced protein kinase A (PKA) phosphorylation which in turn leads to decreased cAMP response element-binding protein (CREB) activation. Decreased PKA phosphorylation also directly enhanced Bax activity and in part reduced glycogen synthase kinase 3 (GSK3)ß phosphorylation. Owing to the interactive signaling between EP4 and EGFR, GW627368X lowered EGFR phosphorylation in turn reducing Akt, mitogen-activated protein kinase (MAPK) and GSK3ß activity significantly. Sublethal dose of GW627368X was found to reduce the nuclear translocation of ß-catenin in a time dependent manner along with time-dependent decrease in cytoplasmic as well as whole-cell ß-catenin. Decreased CREB and ß-catenin transcriptional activity restricts the aberrant transcription of key genes like EP4, cyclooxygenase (COX)-2, vascular endothelial growth factor and c-myc, which ultimately control cell survival, proliferation and angiogenesis. Reduced activity of EGFR resulted in enhanced expression of 15-hydroxyprostaglandin dehydrogenase increasing PGE2 degradation thereby blocking a positive feedback loop. In xenograft model, dose-dependent decrease in cancer proliferation was observed characterized by reduction in tumor mass and volume and a marked decrease in Ki67 expression. A diminished CD31 specific staining signified decreased tumor angiogenesis. Reduced expression of pAkt, pMAPK, pEGFR and COX-2 validated in vitro results. GW627368X therefore effectively inhibits tumor survival, motility, proliferation and angiogenesis by blocking EP4/EGFR interactive signaling. EP4 is a potent therapeutic target in cervical cancer and can be explored in combination with conventional therapies to attain superior outcomes and to overcome complications associated with organ toxicities, therapeutic resistance and disease relapse.

BI-69A11 enhances susceptibility of colon cancer cells to mda-7/IL-24-induced growth inhibition by targeting Akt.

BACKGROUND: Akt and its downstream signalling pathways contribute to the aetiology and progression of colorectal carcinoma (CRC). Targeting the Akt pathway is an attractive strategy but few chemotherapeutic drugs have been used to treat CRC with only limited success. BI-69A11, a small molecule inhibitor of Akt, efficiently inhibits growth in melanoma cells. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 promotes cancer-selective apoptosis when delivered by a tropism-modified replication incompetent adenovirus (Ad.5/3-mda-7). However, Ad.5/3-mda-7 displays diminished antitumour efficacy in several CRC cell lines, which correlates with the expression of K-RAS. METHODS: The individual and combinatorial effect of BI-69A11 and Ad.5/3-mda-7 in vitro was studied by cell viability, cell cycle, apoptosis and invasion assays in HT29 and HCT116 cells containing wild type or mutant K-ras, respectively. In vivo HT29 tumour xenografts were used to test the efficacy of the combination treatment. RESULTS: BI-69A11 inhibited growth and induced apoptosis in CRC. However, combinatorial treatment was more effective compared with single treatment. This combination showed profound antitumour and anti angiogenic effects in vitro and in vivo by downregulating Akt activity. CONCLUSIONS: BI-69A11 enhances the antitumour efficacy of Ad.5/3-mda-7 on CRC overexpressing K-RAS by inducing apoptosis and regulating Akt activity thereby warranting further evaluation in treating CRC.

Investigation of spectroscopic properties, structure and luminescence spectra of Sm3+ doped zinc bismuth silicate glasses.

The glasses with compositions 20ZnO·(79.5-x)Bi2O3·xSiO2·0.5Sm2O3 (10≤x≤50, mol%) have been synthesized using normal melt-quench technique. Optical absorption and fluorescence spectra of the glasses were recorded at ambient temperature. Judd-Ofelt (J-O) theory has been successfully applied to characterize the absorption and luminescence spectra of these glasses. From the measured intensities of absorption bands of these glasses, the Judd-Ofelt parameters, Ωλ (λ=2, 4, 6) have been evaluated. The variation of Ω2 with Bi2O3 content has been attributed to changes in the asymmetry of the ligand field at the rare earth (RE) ion site (due to structural change) and to changes in RE-O covalency, whereas the variation of Ω6 is found to be strongly dependent on nephlauxetic effect. The shift of the hypersensitive band shows that the covalency of the RE-O decreases with decrease in Bi2O3 content in the host glass. Also, using J-O theory various radiative properties like spontaneous emission probability (Arad), radiative life time (τr), fluorescence branching ratio (ßr) and stimulated emission cross-section (σ) for various emission bands of these glasses in the visible spectral region have been determined. A close correlation is observed between the Bi2O3 content and the spectroscopic, radiative and structural properties of the prepared glasses. The values of radiative properties indicated that 4G5/2→6H7/2 and 4G5/2→6H9/2 transitions responsible for orange luminescence might be used in the development of materials for LED's and other optical devices in the visible region.

Staurosporine induces necroptotic cell death under caspase-compromised conditions in U937 cells.

For a long time necrosis was thought to be an uncontrolled process but evidences recently have revealed that necrosis can also occur in a regulated manner. Necroptosis, a type of programmed necrosis is defined as a death receptor-initiated process under caspase-compromised conditions. The process requires the kinase activity of receptor-interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and mixed lineage kinase domain-like protein (MLKL), as a substrate of RIPK3. The further downstream events remain elusive. We applied known inhibitors to characterize the contributing enzymes in necroptosis and their effect on cell viability and different cellular functions were detected mainly by flow cytometry. Here we report that staurosporine, the classical inducer of intrinsic apoptotic pathway can induce necroptosis under caspase-compromised conditions in U937 cell line. This process could be hampered at least partially by the RIPK1 inhibitor necrotstin-1 and by the heat shock protein 90 kDa inhibitor geldanamycin. Moreover both the staurosporine-triggered and the classical death ligand-induced necroptotic pathway can be effectively arrested by a lysosomal enzyme inhibitor CA-074-OMe and the recently discovered MLKL inhibitor necrosulfonamide. We also confirmed that the enzymatic role of poly(ADP-ribose)polymerase (PARP) is dispensable in necroptosis but it contributes to membrane disruption in secondary necrosis. In conclusion, we identified a novel way of necroptosis induction that can facilitate our understanding of the molecular mechanisms of necroptosis. Our results shed light on alternative application of staurosporine, as a possible anticancer therapeutic agent. Furthermore, we showed that the CA-074-OMe has a target in the signaling pathway leading to necroptosis. Finally, we could differentiate necroptotic and secondary necrotic processes based on participation of PARP enzyme.

The guanine-quadruplex structure in the human c-myc gene's promoter is converted into B-DNA form by the human poly(ADP-ribose)polymerase-1.

The important regulatory role of the guanine-quadruplex (GQ) structure, present in the nuclease hypersensitive element (NHE) III(1) region of the human c-myc (h c-myc) gene's promoter, in the regulation of the transcription of that gene has been documented. Here we present evidences, that the human nuclear poly(ADP-ribose)polymerase-1 (h PARP-1) protein participates in the regulation of the h c-myc gene expression through its interaction with this GQ structure, characterized by binding assays, fluorescence energy transfer (FRET) experiments and by affinity pull-down experiments in vitro, and by chromatin immunoprecipitation (ChIP)-qPCR analysis and h c-myc-promoter-luciferase reporter determinations in vivo. We surmise that h PARP-1 binds to the GQ structure and participates in the conversion of that structure into the transcriptionally more active B-DNA form. The first Zn-finger structure present in h PARP-1 participates in this interaction. PARP-1 might be a new member of the group of proteins participating in the regulation of transcription through their interactions with GQ structures present in the promoters of different genes.

Effect of Bi2O3 on spectroscopic and structural properties of Er3+ doped cadmium bismuth borate glasses.

Glasses with composition 20CdO·xBi(2)O(3)·(79.5-x)B(2)O(3) (15≤x≤35, x in mol%) containing 0.5 mol% of Er(3+) ions were prepared by melt-quench technique (1150°C in air). The amorphous nature of the glasses was confirmed by X-ray diffraction. The spectroscopic properties of the glasses were investigated using optical absorption spectra and fluorescence spectra. The phenomenological Judd-Ofelt intensity parameters Ω(λ) (λ=2, 4, 6) were determined from the spectral intensities of absorption bands in order to calculate the radiative transition probability (A(R)), radiative life time (τ(R)), branching ratios (ß(R)) for various excited luminescent states. Using the near infrared emission spectra, full width at half maxima (FWHM), stimulated emission cross-section (σ(e)) and figure of merit (FOM) were evaluated and compared with other hosts. Especially, the numerical values of these parameters indicate that the emission transition (4)I(13/2)→(4)I(15/2) at 1.506 µm in Er(3+)-doped cadmium bismuth borate glasses may be useful in optical communication.

Necroptosis: biochemical, physiological and pathological aspects.

Programmed cell death is a key component of tissue homeostasis, normal development and wide variety of diseases. Conventional view refers to programmed cell death form as caspase-mediated apoptosis while necrosis is considered as an accidental and unwanted cell demise, carried out in a non-regulated manner and caused by extreme conditions. However, accumulating evidences indicate that necrotic cell death can also be a regulated process. The term necroptosis has been introduced to describe a cell death receptor-induced, caspase-independent, highly regulated type of programmed cell death process with morphological resemblance of necrosis. Necroptosis recently has been found to contribute to a wide range of pathologic cell death forms including ischemic brain injury, neurodegenerative diseases and viral infection, therefore a better understanding of the necroptotic signaling machinery has clinical relevance.

Regulation of malignant phenotype and bioenergetics by a π-electron donor-inducible mitochondrial MgATPase.

The recognition of poly ADP-ribose transferase-1 (PARP-1) as an ATP sensor receiving this energy source by way of a specific adenylate kinase ATP wire (AK) from mitochondrial ATP synthase (F0F1), and directly regulating cellular mRNA and DNA synthesis, was the first step towards the identification of an effect by PARP-1 that is of fundamental significance. The molecular target of AK-ATP is Arg 34 of the Zn finger I of PARP-1, which is also a site for cation-π interactions as a target of π-electron donors. We now identify this π-electron receptor site as the second active center of PARP-1 which by interaction with a π-electron donor-inducible MgATPase reversibly controls a malignant vs. non-malignant phenotype through energizing the NADH➝NADP+ transhydrogenase, a reaction which is the metabolic connection of PARP-1 to cell function. The specific enzyme-inducing action of the π-electrons is executed by the PARP-1 -topoisomerase I - DNA complex of the nuclei regulating both the nature and the quantity of cellular enzymes that constitute cell-specific physiology.

Dependence of trans-ADP-ribosylation and nuclear glycolysis on the Arg 34-ATP complex of Zn2+ finger I of poly-ADP-ribose polymerase-1.

The H-bonded complex of ATP with Arg 34 of Zn2+ finger I of poly-ADP-ribose polymerase-1 (PARP-1) determines trans-oligo-ADP-ribosylation from NAD+ to proteins other than PARP-1. This mechanism was tested in lysolecithin fractions of non-malignant and cancer cells separately and after their recombination. Cellular PARP-1 activity was recovered when the centrifugal sediment was recombined with the supernatant fraction containing cellular ADP-ribose oligomer acceptor proteins. Combination of the matrix fraction (Mx) of cancer cells (lacking OXPHOS) with its supernatant had the same PARP-1 activity as the Mx alone. The supernatant of non-malignant cells was replaced by glycolytic enzymes as ADP-ribose acceptor. The hexokinase activity of the supernatant increased when OXPHOS of intact cells was uncoupled by carbonyl cyanide 4-(trifluoro methoxy) phenylhydrazone. trans-ADP-ribosylation was demonstrated by polyacrylamide gel electrophoresis.

Neurological features and enzyme therapy in patients with endocrine and exocrine pancreas dysfunction due to CEL mutations.

OBJECTIVE: To further define clinical features associated with the syndrome of diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl-ester lipase (CEL) gene and to assess the effects of pancreatic enzyme substitution therapy. RESEARCH DESIGN AND METHODS: Nine patients with CEL gene mutation, exocrine deficiency, and diabetes were treated and followed for 30 months. RESULTS: Treatment improved symptoms in seven of nine patients. Exocrine and endocrine function assessed by fecal elastase and A1C were not affected, although fecal lipid excretion was reduced. Vitamin E was low in all patients but increased with treatment (P < 0.001 at 30 months) and improved in five subjects. A predominantly demyelinating neuropathy was seen in a majority of patients, and carpal tunnel syndrome was common. CONCLUSIONS: Pancreatic enzyme substitution alleviated symptoms and malabsorption and normalized vitamin E levels. Glycemic control was not significantly affected. The CEL syndrome seems associated with a demyelinating neuropathology.

Identification of poly(ADP-ribose) polymerase-1 as the OXPHOS-generated ATP sensor of nuclei of animal cells.

Our results show that in the intact normal animal cell mitochondrial ATP is directly connected to nuclear PARP-1 by way of a specific adenylate kinase enzymatic path. This mechanism is demonstrated in two models: (a) by its inhibition with a specific inhibitor of adenylate kinase, and (b) by disruption of ATP synthesis through uncoupling of OXPHOS. In each instance the de-inhibited PARP-1 is quantitatively determined by enzyme kinetics. The nuclear binding site of PARP-1 is Topo I, and is identified as a critical "switchpoint" indicating the nuclear element that connects OXPHOS with mRNA synthesis in real time. The mitochondrial-nuclear PARP-1 pathway is not operative in cancer cells.

Modulation of the homocysteine-betaine relationship by methylenetetrahydrofolate reductase 677 C->t genotypes and B-vitamin status in a large-scale epidemiological study.

CONTEXT: Betaine is formed from the essential nutrient choline or is supplied from the diet. It serves as a substrate in the betaine-homocysteine methyltransferase reaction and thereby provides methyl groups for the homocysteine-methionine cycle, which is regulated by enzymes dependent on folate, vitamin B12, riboflavin (vitamin B2), or vitamin B6. OBJECTIVE: We investigated how betaine affected total homocysteine (tHcy) concentration within the frame of variable B-vitamin status and according to the methylenetetrahydrofolate reductase (MTHFR) 677C->T genotype. DESIGN/SETTING/PATIENTS: This is a population-based study with a cross-sectional design. It includes 10,601 healthy men and women aged 50-64 yr. OUTCOME MEASURES: Plasma samples were analyzed for tHcy, betaine, choline, dimethylglycine, riboflavin, and vitamin B6, whereas folate and vitamin B12 were analyzed in serum. RESULTS: Betaine was a strong determinant of plasma tHcy in subjects with low serum folate and the MTHFR TT genotype. The association was further strengthened at low levels in the circulation of the other B-vitamins (B2, B6, and B12). Thus, in subjects with the combination of serum folate in the lowest quartile, low vitamin B2, B6, and B12 status, and the MTHFR TT genotype, the difference in tHcy (mean, 95% confidence interval) across extreme plasma betaine quartiles was 8.8 (1.3-16.2) micromol/liter. CONCLUSION: Betaine may thus be an important one-carbon source, particularly in MTHFR 677 TT subjects with inadequate B-vitamin status.

Quantitative correlation between cellular proliferation and nuclear poly (ADP-ribose) polymerase (PARP-1).

Treatment of cells with lysophosphatidyl choline and centrifugal extraction can separate poly (ADP-ribose) synthetase (PARP-1) and DNA synthetase activities, permitting the experimental analysis and comparison of both multienzyme systems. Only PARP-1 is being assayed by our system. Ca(2+) and Mg(2+) have minor activating effects, and added histones are without activating action. Short end-blocked dsDNAs at nM concentrations and spermine at mM concentrations are maximally activating coenzymes of poly (ADP-ribose) synthesis. Comparison of non-proliferating non-malignant cells with rapidly growing cancer cells demonstrates that rates of poly (ADP-ribose) synthesis and DNA synthesis are highest in pre-confluent non-malignant cells and in proliferating cancer cells, and lowest in contact-inhibited non-malignant cells. Rates of poly (ADP-ribose) synthesis correlate with the number of enzymatically activable PARP-1 molecules per cell, determined under Vmax conditions where activity is linearly proportional to enzyme protein. Contact-inhibited non-malignant cells exhibit only trans-ADP-ribosylation that is not affected by ATP, while rapid growth, especially in cancer cells, demonstrates extensive auto-poly (ADP)-ribosylation that is strongly inhibited by ATP at concentrations present in cells exhibiting normal bioenergetics. Rates of mRNA synthesis in non-proliferating non-malignant cells and in cancer cells were indistinguishable, indicating that the differences observed between cellular phenotypes are most likely due to reassembly of PARP-1 molecules in nuclei to homo-dimers (in cancer cells) and hetero-dimers (in non-cancer cells). A specific inhibitor and an inactivator of PARP-1 each inhibit DNA synthesis when intact cancer cells are pretreated with these drugs. Direct addition of these drugs to permeabilized cells performing DNA synthesis has no effect on DNA synthesis. The most striking diagnostic signal for cancer cells is activation of PARP-1 and of DNA synthesis.

Mutations in the CEL VNTR cause a syndrome of diabetes and pancreatic exocrine dysfunction.

Dysfunction of the exocrine pancreas is observed in diabetes, but links between concurrent exocrine and endocrine pancreatic disease and contributing genetic factors are poorly characterized. We studied two families with diabetes and exocrine pancreatic dysfunction by genetic, physiological and in vitro functional studies. A genome-wide screen in Family 1 linked diabetes to chromosome 9q34 (maximal lod score 5.07). Using fecal elastase deficiency as a marker of exocrine pancreatic dysfunction refined the critical chromosomal region to 1.16 Mb (maximal lod score 11.6). Here, we identified a single-base deletion in the variable number of tandem repeats (VNTR)-containing exon 11 of the carboxyl ester lipase (CEL) gene, a major component of pancreatic juice and responsible for the duodenal hydrolysis of cholesterol esters. Screening subjects with maturity-onset diabetes of the young identified Family 2, with another single-base deletion in CEL and a similar phenotype with beta-cell failure and pancreatic exocrine disease. The in vitro catalytic activities of wild-type and mutant CEL protein were comparable. The mutant enzyme was, however, less stable and secreted at a lower rate. Furthermore, we found some evidence for an association between common insertions in the CEL VNTR and exocrine dysfunction in a group of 182 unrelated subjects with diabetes (odds ratio 4.2 (1.6, 11.5)). Our findings link diabetes to the disrupted function of a lipase in the pancreatic acinar cells.

Betaine: a key modulator of one-carbon metabolism and homocysteine status.

Betaine serves as a methyl donor in a reaction converting homocysteine to methionine, catalysed by the enzyme betaine-homocysteine methyltransferase. It has been used for years to lower the concentration of plasma total homocysteine (tHcy) in patients with homocystinuria, and has recently been shown to reduce fasting and in particular post-methionine load (PML) tHcy in healthy subjects. Betaine exists in plasma at concentrations of about 30 micromol/L; it varies 10-fold (from 9 to 90 micromol/L) between individuals, but the intra-individual variability is small. Major determinants are choline, dimethylglycine and folate in plasma, folic acid intake and gender. Recent studies have demonstrated that plasma betaine is a stronger determinant of PML tHcy than are vitamin B6 and folate. The betaine-PML tHcy relationship is attenuated after supplementation with B-vitamins, and is most pronounced in subjects with low folate. Betaine shows a weaker association with fasting tHcy (than with PML tHcy), and also this association is most pronounced in subjects with low folate. In pregnancy, plasma betaine declines until gestational week 20, and thereafter remains constant. From gestational week 20 onwards, fasting tHcy shows a strong inverse association with plasma betaine, and betaine becomes a stronger predictor than folate of fasting tHcy. To conclude, betaine status is a component of an individual's biochemical make-up with ramifications to one-carbon metabolism. Betaine status should be investigated in pathologies related to altered metabolism of homocysteine and folate, including cardiovascular disease, cancer and neural tube defects.

The influence of ATP on poly(ADP-ribose) metabolism.

ATP affects poly(ADP-ribose) metabolism at two distinct sites: it inhibits poly(ADP-ribose) polymerase-1 and activates the glycohydrolase directly. The inhibitory site of ATP on poly(ADP-ribose) polymerase-1 was identified by amino acid exchange mutation to be at the arginine 34 residue in the first Zn2+ finger. Mutation of 138 arginine residue of Zn2+ finger 2 had negligible influence on the inhibitory action of ATP, pinpointing arginine 34 of the first Zn2+ finger as the specific ATP site. The glycohydrolase protein was activated by ATP when the substrate was a long-chain ADP-ribose polymer, but not with a short-chain substrate. Isolated cell nuclei also responded to both inhibition of poly(ADP-ribose) polymerase by ATP and to poly(ADP-ribose) glycohydrolase activation by ATP, demonstrating that enzymological results can be extrapolated to cellular systems. The activation of poly(ADP-ribose) polymerase in nuclei by an alkylating drug was completely suppressed by ATP, demonstrating that the bioenergetic competence of cells can regulate the cytocidal action of DNA alkylating drugs. The potential significance of bioenergetic regulation of poly(ADP-ribose) metabolism is proposed.

Betaine concentration as a determinant of fasting total homocysteine concentrations and the effect of folic acid supplementation on betaine concentrations.

BACKGROUND: Remethylation of homocysteine to methionine can occur through either the folate-dependent methionine synthase pathway or the betaine-dependent betaine-homocysteine methyltransferase pathway. The relevance of betaine as a determinant of fasting total homocysteine (tHcy) is not known, nor is it known how the 2 remethylation pathways are interrelated. OBJECTIVE: The objectives of the study were to examine the relation between plasma betaine concentration and fasting plasma tHcy concentrations and to assess the effect of folic acid supplementation on betaine concentrations in healthy subjects. DESIGN: A double-blind randomized trial of 6 incremental daily doses of folic acid (50-800 microg/d) or placebo was carried out in 308 Dutch men and postmenopausal women (aged 50-75 y). Fasted blood concentrations of tHcy, betaine, choline, dimethylglycine, and folate were measured at baseline and after 12 wk of vitamin supplementation. RESULTS: Concentrations of tHcy were inversely related to the betaine concentration (r = -0.17, P < 0.01), and the association was independent of age, sex, and serum concentrations of folate, creatinine, and cobalamin. Folic acid supplementation increased betaine concentration in a dose-dependent manner (P for trend = 0.018); the maximum increase (15%) was obtained at daily doses of 400-800 microg/d. CONCLUSIONS: The plasma betaine concentration is a significant determinant of fasting tHcy concentrations in healthy humans. Folic acid supplementation increases the betaine concentration, which indicates that the 2 remethylation pathways are interrelated.