[Study on the association between CYP24A1 genetic polymorphisms and risks related to postmenopausal breast cancer].

Objective: To evaluate the associations between CYP24A1 genetic polymorphisms and related risks on breast cancer among postmenopausal women. Methods: We carried out a population-based case-control study to include 1 134 postmenopausal women (589 cases and 545 controls) from Wuxi, Jiangsu province and to explore the association between CYP24A1 polymorphisms and related risks on breast cancer. Seven CYP24A1 variants (rs2209314, rs2585428, rs2762941, rs3787555, rs4909959, rs912505 and rs927650) were genotyped by Sequenom MassARRAY platform. Logistic regression method was used to estimate the CYP24A1 genetic variants and susceptibility of breast cancer. Loci-loci interactions were evaluated by a generalized multifactor dimensionality reduction (GMDR) method. Results: Result showed that rs2209314, rs2585428, rs2762941, rs3787555, rs4909959, rs912505 and rs927650 of CYP24A1 were not associated with breast cancer under the codominant, dominant, recessive or additive models. Among the population with <80 cm waist circumstance, rs2585428 was associated with the reduced risks on breast cancer (OR=0.64, 95%CI: 0.42-0.96). Similar negative association was observed for rs3787555 (OR=0.58, 95%CI: 0.38-0.87). The genotypes of rs2585428, rs3787555 and rs4909959 showed significant interactions with waist circumstance on the risk of breast cancer. Also, rs2209314, rs3787555 and rs912505 in CYP24A1 could alter the risk of breast cancer by way of loci-loci interaction. Conclusion: CYP24A1 variants rs2585428 and rs3787555 were associated with risks of susceptibility on breast cancer, among postmenopausal women.

Ultrastructure of intestinal mucosa in diarrhea-predominant irritable bowel syndrome.

OBJECTIVES: Impaired intestinal barrier function has been demonstrated in the pathophysiology of diarrhea-predominant irritable bowel syndrome (IBS-D). This study aimed to describe the intestinal ultrastructural findings in the intestinal mucosal layer of IBS-D patients. METHODS: In total, 10 healthy controls and 10 IBS-D patients were analyzed in this study. The mucosa of each patient's rectosigmoid colon was first assessed by confocal laser endomicroscopy (CLE); next, biopsied specimens of these sites were obtained. Intestinal tissues of IBS-D patients and healthy volunteers were examined to observe cellular changes by transmission electron microscopy (TEM). RESULTS: CLE showed no visible epithelial damage or inflammatory changes in the colonic mucosa of IBS-D compared with healthy volunteers. On transmission electron microscopic examination, patients with IBS-D displayed a larger apical intercellular distance with a higher proportion of dilated (>20 nm) intercellular junctional complexes, which was indicative of impaired mucosal integrity. In addition, microvillus exfoliation, extracellular vesicle as well as increased presence of multivesicular bodies were visible in IBS-D patients. Single epithelial cells appeared necrotic, as characterized by cytoplasmic vacuolization, cytoplasmic swelling, and presence of autolysosome. A significant association between bowel habit, frequency of abdominal pain, and enlarged intercellular distance was found. CONCLUSION: This study showed ultrastructural alterations in the architecture of intestinal epithelial cells and intercellular junctional complexes in IBS-D patients, potentially representing a pathophysiological mechanism in IBS-D.

BDNF modulates intestinal barrier integrity through regulating the expression of tight junction proteins.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) may play a vital role in the homeostatic regulation of intestinal barrier integrity. We aimed to investigate the physiological role of BDNF in maintaining the intestinal epithelial barrier using postinflammatory irritable bowel syndrome (PI-IBS) mice and explore the underlying molecular mechanisms using intestinal epithelial cells in vitro. METHODS: Postinflammatory-IBS mice were induced by intrarectal administration of trinitrobenzene sulfonic acid and allowed to recover for 28 days. Frequency of defecation, fecal water content, colonic epithelial integrity and expressions of BDNF and tight junction (TJ) proteins (occludin, ZO-1, claudin-1, claudin-2) of the PI-IBS mice were investigated. Based on the results of animal studies, we further performed RT-PCR and Western blots to assess how BDNF stimulation and BDNF knockdown impacted TJ proteins in the ht-29 intestinal epithelial cells. KEY RESULTS: Water content of stools was significantly increased in the PI-IBS mice compared with controls. Colonic mucosa from the PI-IBS mice displayed epithelial barrier defects and exhibited increased protein expressions of BDNF and claudin-2 and decreased protein expressions of occludin, ZO-1 and claudin-1. Furthermore, a siRNA against BDNF in the ht-29 cells could effectively suppress BDNF gene and protein expressions, and subsequently reduce TJ gene and protein levels. When the ht-29 cells were incubated with different doses of exogenous BDNF, significant increases of occludin, ZO-1 and claudin-1 and decreases of claudin-2 protein were observed. CONCLUSIONS & INFERENCES: BDNF may play a role in regulating intestinal epithelial barrier via affecting the expression of TJ proteins.

Expression of CD27, CD28 and IL-17A in peripheral blood from patients with colorectal carcinoma.

OBJECTIVE: To compare the different expressions of CD27, CD28, IL-17A, IFN-γ and TNF-α in the peripheral blood sampled from patients with colorectal carcinoma and healthy volunteers. PATIENTS AND METHODS: Vδ2 T cells were isolated from the peripheral blood mononuclear cells (PBMCs) of patients with the colorectal carcinoma (CRC, n = 30) and healthy controls (HC, n = 21). The proportion of CD27, CD28, IL-17A, IFN-γ and TNF-α of Vδ2 T cells was detected by the flow cytometry. RESULTS: We found that the proportion of IL-17A of Vδ2 T cells in PBMCs was higher in the CRC vs. the HC group (p < 0.05). A significant positive correlation was observed between the expression of IFN-γ and TNF-α of Vδ2 T cells. In the CRC patients, the proportions of IL-17A of CD27- Vδ2 T cells and CD28+ Vδ2 T cells were higher than those of CD27+ Vδ2 T cells and CD28- Vδ2 T cells, whereas the expression of IFN-γ and TNF-α of CD27-Vδ2 T cells was lower than that of CD27+ Vδ2 T cells. CONCLUSIONS: Vδ2 T cells from PBMCs had higher expression of IL-17A in CRC patients than that in the HC group. The expression of IFN-γ and TNF-α of Vδ2 T cells from PBMCs was positively correlated. The cytokine profiles of peripheral Vδ2 T cells were likely determined by a CD27 and CD28 involving mechanism.

Genetic diversity and pathogenicity differentiation of Sclerotinia sclerotiorum on rapeseed (Brassica napus L.) in Anhui Province, China.

The pathogenicity of 47 isolates of Sclerotinia sclerotiorum from oilseed rape (Brassica napus L.) in Anhui, China, was tested by detached leaf inoculation using the susceptible rape cultivar Wanyou-14. All isolates were pathogenic to the cultivar and could be grouped into 3 categories based on the lesion length on the leaves tested: weak pathogenicity type, intermediate pathogenicity type, and strong pathogenicity type. This suggested that there was differentiation in the pathogenicity among the strains tested of S. sclerotiorum. Additionally, the intraspecific DNA polymorphisms among 47 strains of S. sclerotiorum were investigated by screening 40 pairs of inter-simple sequence repeat (ISSR) primers. Unweighted pair-group method with arithmetic average cluster analysis of these ISSR data distinguished all strains from each other and revealed considerable genetic variability among them. These strains were classified into 7 clusters according to their branching in the dendrogram, and partial correlation was observed between the genetic polymorphisms and the pathogenicity of S. sclerotiorum strains.

[High glucose promotes gap junctional communication in cultured neonatal cardiac fibroblasts via AMPK activation].

Cardiac fibroblasts are known to be essential for adaptiveresponses in the patho- genesis of cardiovascular diseases, and increased intercellular communication of myocardial cells and cardiac fibroblasts acts as a crucial factor in maintaining the functional integrity of the heart. AMP-activated kinase (AMPK) is a key stress signaling kinase, which plays an important role in promoting cell survival and improving cell function. However, the underlying link between AMPK and gap junctional communication (GJIC) is still poorly understood. In this study, a connection between AMPK and GJIC in high glucose-mediated neonatal cardiac fibroblasts was assessed using fibroblast migration, measurement of dye transfer and connexin43 (Cx43) expression. 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and Compound C (CC) were used to regulate AMPK activity. The levels of cell migration and Cx43 protein expression in neonatal cardiac fibroblasts increased during high glucose treatment, accompanied by developed dye transfer. In addition, high glucose induced abundant phosphorylation of AMPK. Suppression of AMPK phosphorylation using CC reduced dye transfer, cell migration and Cx43 protein expression in neonatal cardiac fibroblasts, whereas the activation of AMPK using AICAR mimicked the high glucose-mediated cell migration, Cx43 protein expression and dye transfer enhancement. AMPK appears to participate in regulating GJIC in high-glucose-treated neonatal cardiac fibroblasts, including cell migration, dye transfer, Cx43 expression and distribution.

Down-regulation of 67LR reduces the migratory activity of human glioma cells in vitro.

OBJECTIVES: Glioma is the most common brain tumor in central nervous system. Traditional therapies are not effective to cure this disease. Experimental evidence indicates that the 67 kDa elastin-laminin receptor (67LR) subunit is a high-affinity non-integrin laminin-binding protein that is over-expressed on the tumor cell surface in a variety of human carcinomas, and directly correlates with a higher proliferation rate of malignant cells and tendency to metastasize. However, little is known of the expression and function of 67LR in glioma cells. METHODS: In this study, we estimated whether 67LR was constitutively over-expressed in high-grade astrocytomas by immunohistochemical staining and Western blotting, and investigated the role of a low level of 67LR expression in glioma cell line-U251 by constructing an interfering RNA expression plasmid. RESULTS: The results showed that the 67LR had an enhanced over-expression in high-grade astrocytomas against normal brain tissues samples, and that the migratory activity of glioma cells was reduced after the down-regulation of the 67LR gene by RNAi. DISCUSSION: It was hypothesized that a low level of 67LR expression could reduce migratory activity of glioma cells, which further proved that 67LR played an important role in glioma invasion by mediating tumor cell functions leading to sarcomata. This study provided a new alternative to gene therapy for glioma treatment.

Dynamic CT perfusion imaging in subarachnoid hemorrhage-related vasospasm.

BACKGROUND AND PURPOSE: Nimodipine is a therapy that reduces morbidity and mortality in patients with subarachnoid hemorrhage (SAH), though the mechanisms by which it does so are not well understood. In a rabbit model of SAH, we studied the effects of nimodipine by using functional CT imaging. We hypothesized that the nimodipine treatment group would have (1) increased mean basilar artery diameter, (2) less diminished cerebral blood flow (CBF) following vasospasm, and (3) better neurologic outcomes. METHODS: SAH was induced in 26 New Zealand White rabbits randomized to 2 groups: treated (nimodipine) or control (no treatment). CT perfusion and CT angiography were used to measure CBF and basilar artery diameter at baseline, 10, 30, and 60 minutes after SAH, and on days 3, 5, 7, 9, and 16. Neurologic assessments were performed on each day of scanning. RESULTS: Basilar artery diameter in the treated group was greater than in the control group post-SAH (P < .05). When vasospasm was >15%, CBF in the nimodipine group was significantly greater than in the control group in the brain stem, cerebellum, parieto-occipital cerebrum, and deep gray matter (P < .05). Neurologic scores in the nimodipine group were significantly better than in the control group on days 5 and 9 (P < .05). CONCLUSION: Animals treated with nimodipine showed (1) increased mean basilar artery diameter, (2) improved neurologic outcome, and (3) increased mean CBF despite no significant difference in the incidence and severity of delayed vasospasm. These data provide a basis for future studies comparing the efficacy of new treatments for SAH to that of nimodipine.

Effects of dietary restriction on insulin resistance in obese mice.

In many cases, development of insulin resistance has been linked to obesity and may contribute to mechanism of aging. The role of diet, irrespective of degree of obesity, in modulating insulin resistance and development of age degeneration disease remains uncertain. Lowered blood glucose levels are commonly associated with diet restriction (DR), which is an intervention shown to successfully retard aging and age associated disease. The effects of DR on blood glucose and insulin resistance were measured in yellow obese (A(vy)/A), lean black (a/a) mice and in another common inbred strain (B6C3F1) (at three different ages). The yellow obese mice become diabetic as a result of an insulin receptor defect which is not clearly understood. Insulin responses and radioinsulin binding were assayed in yellow obese and lean black mice fed either ad libitum (AL) or DR diets (YAL, BAL, YDR and YAL, respectively) at four different circadian intervals. The B6C3F1 controls were fed either AL (CAL) or DR (CDR) and measures were made at six circadian stages and three different ages. Within 23 days, DR produced a significant loss in body weight and a time-dependent 22-55% reduction in basal blood glucose levels in the yellow obese mice. Additionally, exogenous insulin produced circadian stage dependent (at the time of food intake) reductions in blood glucose in the YDR animals that were not present in YAL animals. (125)I-Insulin binding in liver was increased nearly 2-fold in YDR and BDR mice during the time of day that animals were active and eating. (125)I-Insulin binding was two-fold-higher in CDR mice at 4, 12 and >24 months of age. Binding decreased as a function of age in both the CAL and CDR animals. However, even in the >24 month group the CDR animals were found to have levels of binding that were as high as those found in younger CAL liver. The mechanism of action appears to be through resolution of insulin resistance by modulating an insulin receptor defect.

Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent.

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT --> TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10(-6), Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.

N-(2-chloroethyl)-N-nitrosourea tethered to lexitropsin induces minor groove lesions at the p53 cDNA that are more cytotoxic than mutagenic.

Many different N-chloroethyl-N-nitrosourea (CENU) derivatives have been synthesized in an attempt to minimize carcinogenic activity while favoring antineoplastic activity. CENU derivatives linked to the dipeptide lexitropsin (lex) showed significant changes in groove- and sequence-selective DNA alkylation inducing thermolabile N3-alkyladenines (N3-Alkyl-As) at lex equilibrium binding sites. CENU-lex sequence specificity for DNA alkylation was determined using 32P-end-labeled restriction fragments of the p53 cDNA. The adducted sites were converted into single-strand breaks by sequential heating at neutral pH and exposure to piperidine. To establish the mutagenic and lethal properties of CENU-lex-specific lesions, a yeast expression vector harboring a human wild-type p53 cDNA was treated in vitro with CENU-lex and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutants were isolated from independent ade- transformants. The results revealed that: (a) CENU-lex preferentially induces N3-Alkyl-A at specific lex equilibrium binding sites, the formations of which are strongly inhibited by distamycin; (b) reactivity toward Gs is still present, albeit to a lesser extent when compared to N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea and to CENU; (c) 91% of the 49 CENU-lex p53 mutations (45 of 49) were bp substitutions, 29 of which were GC-->AT transitions, mainly at 5' purine G sites; (d) all AT-targeted mutations but one were AT-->TA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GC-->AT transitions were observed, being a real (n = 3, P < 0.0002) CENU-lex mutation hot spot; and (f) a shift in DNA alkylation sites between lesion spectra induced by CENU-lex and N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea was associated with an increased lethality and a decreased mutagenicity, whereas no dramatic change in mutational specificity was observed. Hence, it is tempting to conclude that, in this experimental system, N3-Alkyl-A is more lethal than mutagenic, whereas O6-alkylguanine is a common premutational lesion formed at non-lex binding sites. These results suggest that CENU derivatives with virtually absolute specificity for A residues would make targeting of lethal, nonmutagenic lesions at A+T-rich regions possible, and this may represent a new strategy for the development of new chemotherapeutic agents with a higher therapeutic index.

Quantitative and qualitative analysis of DNA methylation at N3-adenine by N-methyl-N-nitrosourea.

The sequence-specific alkylation of DNA by N-methyl-N-nitrosourea (MNU) has been demonstrated for the minor groove N3-methyladenine (N3-MeAde) adduct using neutral thermal hydrolysis and polyacrylamide sequencing gels. The ratio of relative yields of N7- and N3-MeAde and N7-methylguanine (N7-MeGua) is approximately 0.03:0. 15:1.00, respectively, on the basis of the gel data, and these values are comparable to relative yields determined by bulk digestion of MNU-methylated DNA when HPLC was used to analyze the individual adducts. In contrast to the methylation at N7-guanine (N7-Gua) by MNU, alkylation at Ade shows minimal sequence selectivity. Similar to the methylation at N7-Gua, formation of N3-MeAde by MNU is inhibited by 50-200 mM concentrations of NaCl and DNA binding cations, including distamycin and spermine. However, N3-MeAde formation at Ade residues within methidiumpropyl-EDTA-Fe(II) footprinted distamycin DNA affinity binding regions is selectively inhibited at low concentrations of distamycin relative to Ade sites outside of ligand binding regions, and N7-Gua within or outside the distamycin binding regions. HPLC analysis shows that distamycin also quantitatively inhibits the production of N3-methylguanine when calf thymus DNA is treated with MNU or methyl methanesulfonate. The specific inhibitory effect of distamycin, which binds in the minor groove at Ade/Thy-rich sequences, provides additional evidence that the predominant DNA lesion detected at Ade by sequencing gel analysis involves minor groove N3-MeAde modifications.

Regioselective effect of zwitterionic DNA substitutions on DNA alkylation: evidence for a strong side chain orientational preference.

The incorporation of zwitterionic residues (5-substituted omega-aminoalkyl-2'-deoxypyrimidines) into DNA has been reported to bend DNA as measured by aberrant gel mobility [Strauss et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9515-9520]. Herein we report that DNA methylation by N-methyl-N-nitrosourea at N7-guanine is regioselectively inhibited by point substitutions of the zwitterionic residues 5-(6-aminohexyl)-2'-deoxycytidine, 5-(6-aminohexyl)-2'-deoxyuridine, or 5-(3-aminopropyl)-2'-deoxyuridine. No inhibition is observed for DNA methylation by dimethyl sulfate. On the basis of inhibition patterns for methylation with the different zwitterionic substitutions and the different length tethers, the omega-aminoalkyl side chains prefer to adopt a conformation that points them toward the 3'-base. Molecular modeling grid searches, coupled with energy minimizations, and simulated annealing molecular dynamics studies indicate that unfavorable steric interactions with the 5'-base and backbone, as well as stabilizing electrostatic interactions with electronegative atoms on the 3'-side, are responsible for the observed conformational preference. No evidence for association of the cationic side chain with the phosphate backbone is observed. The observed bending of DNA induced by the tethered ammonium ions may simply arise from their localization in the major groove.

The design of agents to control DNA methylation adducts. Enhanced major groove methylation of DNA by an N-methyl-N-nitrosourea functionalized phenyl neutral red intercalator.

An N-methyl-N-nitrosourea (MNU) moiety [CH3N(N=O)C(=O)NH-] linked to the C4'-position of the 5-substituted phenyl ring of phenyl neutral red (PNR), 2-methyl-3-amino-5-[p-[[2-[(N-nitroso-N-methylcarbamoyl)amino]ethy l] carbamoyl]phenyl]-7-(dimethylamino)phenazenium chloride (MNU-PNR), has been synthesized as an approach to design a molecule that will deliver alkylating agents with some preference to guanine (Gua) in the major groove of DNA. The PNR nucleus was chosen because previous studies suggested the following: (1) PNR binds with a slight preference for G/C rich sequences; and (2) PNR intercalates into DNA from the major groove with the 5-phenyl ring pointing out into the major groove (Müller, W., Bünemann, H., and Dattagupta, N. (1975) Eur. J. Biochem. 54, 279-291). It is demonstrated that MNU-PNR yields 2.6 and 6.0 times more N7-methylguanine (7-MeGua) than MNU at low salt (10 mM Tris buffer) and high salt (10 mM Tris buffer + 200 mM NaCl), respectively. It is also shown that the ratio of 7-MeGua (a major groove adduct) to N3-methyladenine (a minor groove adduct) is approximately 5 times higher for MNU-PNR than for MNU. The yield of the 7-MeGua adduct is decreased by the coaddition of a nonmethylating analogue of MNU-PNR or NaCl, but increased in the presence of the minor groove intercalator, ethidium bromide. Using a 32P-end-labeled restriction fragment, the enhanced methylation by MNU-PNR at 7-Gua is confirmed, and it is demonstrated that the sequence-dependent formation of 7-MeGua from MNU-PNR is the same as that seen with MNU. UV, circular dichrosism, and viscosity studies are consistent with MNU-PNR binding to DNA via an intercalation-based process.

Effects of ischemia-hypoxia induced by interruption of uterine blood flow on fetal rat liver and brain enzyme activities and offspring behavior.

The effects of acute perinatal ischemia-hypoxia on fetal liver and brain energy metabolism, fetal brain total free fatty acid concentration and subsequent offspring behavior were investigated in rats. Ischemia-hypoxia was induced at term either by ligation of the uterine blood vessels or submersion of the entire uterine horn in warmed saline. Fetuses of the adjacent horn served as within-dam controls for all assessments and fetuses of dams which had not undergone the surgical stress served as independent controls for enzyme assays. Ischemia-hypoxia was associated with reduced activity of fatty acid synthase in the liver and brain. Total free fatty acid concentration significantly increased in the fetal hypoxic brain. Pups not used for enzyme analyses were cross-fostered for behavioral assessments. Relative to the enzymatic alterations, there were few behavioral alterations associated with ischemia-hypoxia. At postnatal day 30, rats made hypoxic by ligation of the uterine blood vessels had decreased caudate nucleus and brain stem weights relative to within-dam controls. At postnatal day 85, rats made hypoxic by submersion of the uterine horn had decreased olfactory bulb weight. The results of this study indicate an initial acute response to a brief period of ischemia-hypoxia at term pregnancy in the fetal rat brain and liver.

Characterisation of circulating chromogranin A in human cancer patients.

The structure of circulating chromogranin A (CgA) of phaeochromocytoma patients was characterised and compared with that of CgA extracted from tumours. Size exclusion chromatography experiments provided evidence that CgA is present in the blood of different patients, as well as in tumour extracts, as multiple forms having different hydrodynamic sizes of 600 kDa (CgA-I), 100 kDa (CgA-II) and 55 kDA (CgA-III). The amount of each CgA form as a proportion of the total antigenic material was different in different patients. Western blot analysis of chromatographic fractions indicated that these forms are made up by polypeptides of similar molecular weight (about 60-70 kDa). All CgA forms express the epitopes recognised by two monoclonal antibodies (A11 and B4E11), directed against residues 68-70 and 81-90 of human CgA. However, their relative immunoreactivity was markedly different. No evidence for the presence of multimeric complexes in the CgA-I fraction was obtained by various immunological and biochemical methods. These results suggest that circulating CgA in phaeochromocytoma patients consists of at least three forms that appear to be made up by polypeptides with similar molecular weight and different hydrodynamic properties and immunoreactivity. We hypothesise that different conformations and shapes contribute to the heterogeneity of circulating CgA.

Reaction of N-(2-chloroethyl)-N-nitrosoureas with DNA: effect of buffers on DNA adduction, cross-linking, and cytotoxicity.

N-(2-Chloroethyl)nitrosoureas (CNU) are clinically used anticancer drugs whose cytotoxicity is associated with the generation of DNA interstrand cross-links. While studying the sequence selectivity for a series of CNU, a dramatic increase in the formation of N7-alkyldeoxyguanosine was observed when Tris buffer was used rather than phosphate or cacodylate buffers. Moreover, the formation of N7-alkyldeoxyguanosine lesions continues in Tris long after all of the CNU has hydrolyzed. These effects are not seen with the monofunctional alkylating analogues, e.g., N-methyl- and N-(2-hydroxyethyl)-N-nitrosourea. In order to determine if the nature of the CNU-mediated DNA damage was altered by Tris, studies were initiated on the following: (1) alkylation of N7-G in end-labeled DNA restriction fragments; (2) covalent modification of DNA with [ethyl-3H]-N-(2-chloroethyl)-N-nitrosourea; and (3) cytotoxicity in L1210 cells. The data presented demonstrate that Tris increases the yield of the "normal" CNU monofunctional cross-linked adducts, i.e., N7-(2-hydroxyethyl)deoxyguanosine, N7-(2-chloroethyl)deoxyguanosine, O6-(2-chloroethyl)deoxyguanosine, and bifunctional adducts, i.e., 1-(deoxycytid-3-yl)-2-(deoxyguanosin-1-yl)ethane and 1,2-bis(deoxyguanosin-7-yl)ethane. In addition, CNU appears to react with Tris to give a long-lived alkylating intermediate that affords large amounts of DNA adducts not seen with CNU in the absence of Tris. However, in vivo toxicity of CNU in L1210 cells is not affected by the presence of Tris, indicating that the reaction pathway(s) responsible for cross-linking is not significantly sensitive to the nature of the buffer.

[Mechanism and prevention of hemolysis in jaundiced infants in phototherapy].

It has been reported that the prolonged phototherapy results in decreased glutathione reductase (GR) activity in red blood cells. We found short-term phototherapy had the same side-effect. Besides it aggravates hemolysis of newborns during the therapy. This side-effect can be prevented by oral administration of Vit B2. Eighteen jaundiced infants who were given Vit B2 5mg three times a day during the phototherapy, and the control group of 16 patients were not given Vit B2. The results showed that the decrease of hemoglobin and the time of jaundice disappearance during the phototherapy were more favourable in the oral Vit B2 group than those of the control. These results indicated that the short-term phototherapy not only results in decreased GR activity in red blood cell, but also results in hemolysis of the newborn. This side-effect can be prevented by oral administration of Vit B2.

Acute toxicity of ganciclovir: effect of dietary restriction and chronobiology.

The effect of diet, age and time of dose delivery on the mortality of female B6C3F1 mice from ganciclovir sodium (DHPG) was determined for both single (SD; 400 mg DHPG/kg, ip) and multiple doses (MD; same dose ip for 10 additional days) of the drug. Young (7-10 months) and middle-aged (MA; 19-22 months) mice (B6C3F1), both fed ad lib. (AL) and calorie restricted (CR), were dosed at 0, 6, 12 and 18 hr after lights on (HALO; SD study) and at 12.00 hr (MD study). The SD study mortality rate was 38% (AL) and 1.7% (CR) (P < 0.00001). Mortality was 53% (AL, young; P < 0.00001), over 20% (AL, MA), over 1.8% (CR, MA; P = 0.00004) or more than 1.7% (CR, young; P = 0.00002). Effects were independent of lean body mass differences between AL and CR mice. In the SD study, comparing AL mice only, the greatest mortality was seen in young mice at 6 HALO, (73%; P = 0.0034) and lowest mortality in MA mice at 12 HALO (8%; P = 0.026), whereas in the MD study mortality was 63% AL and 33% CR (P = 0.015). By age, MD mortality was 80% (AL, young; P = 0.0035), 50% (CR, MA), 47% (AL, MA), and 15% (CR, young; P = 0.0013). CR protected both young and MA mice in SD and young mice in MD. Lowest mortality for AL was at 12 HALO. It is suggested that dosing at 12 HALO may protect by decreasing DHPG uptake during a period of minimal DNA synthesis in the affected organ(s). CR and timing of DHPG dose may obviate the necessity to discontinue DHPG because of toxicity in humans. The most significant finding of this study is the impact of diet on mortality.

Groove- and sequence-selective alkylation of DNA by sulfonate esters tethered to lexitropsins.

A series of sulfonate esters that are attached to a noncationic minor-groove-binding N-methylpyrrole dipeptide (Lex) related to netrospin have been synthesized. The compounds prepared differ in two respects: (1) the length [(CH2)2 vs (CH2)8] of the tether between the DNA affinity binding portion of the molecule and the sulfonate ester and (2) whether a methyl group [MeOSO2(CH2)n-Lex] or the dipeptide including the aliphatic tether [MeSO2O(CH2)n-Lex] is covalently transferred to the DNA. The DNA-cleavage patterns of these bimolecular alkylating compounds have been mapped in 32P-end-labeled restriction fragments using neutral thermal hydrolysis and alkali treatment to expose single-strand breaks at bases with thermally labile modifications. In contrast to the alkylation of DNA by simple alkyl alkanesulfonate esters, that predominantly yield major-groove alkylation at N7-guanine, the modification of DNA by MeOSO2(CH2)n-Lex and MeSO2O(CH2)n-Lex occurs primarily at N3-adenine residues associated with previously footprinted Lex DNA affinity binding regions. The ratio for the formation of N3-methyladenine (minor groove) to N7-methylguanine (major groove) in calf thymus DNA is 1:7 for dimethyl sulfate, while only the former adenine product is observed with MeSO2O(CH2)n-Lex indicating the change in groove specificity. DNA cleavage by MeOSO2(CH2)n-Lex and MeSO2O(CH2)n-Lex is efficiently inhibited by the coaddition of distamycin; however, only the DNA damage generated by the latter is blocked by NaCl. As expected, increasing the length of the (CH2)n tether from n = 2 to n = 8 moves the alkylation site by 1-2 base pairs further from the affinity binding domain. Finally, a comparison of the methylation patterns of MeOSO2(CH2)n-Lex as a function of tether length provides an insight into Lex sequence and orientational preferences.