LncRNA SNHG7 is an Oncogenic Biomarker Interacting with MicroRNA-193b in Colon Carcinogenesis.

BACKGROUND: The expression and significance of small nucleolar RNA host gene 7 (SNHG7) in early-stage colon carcinogenesis remains unclear. METHODS: The level of SNHG7 and microRNA-193b (miR-193b) was detected by qRT-PCR in colon tumor tissues and cells. The interaction of SNHG7 and miR-193b and the influence of SNHG7 silencing on colon tumor cells was evaluated. RESULTS: Stepwise upregulated SNHG7 in colon advanced adenomas and early-stage cancer negatively correlates with miR-193b level, the direct interaction was confirmed in vitro. SNHG7 silencing in HT29 cells decreased proliferation and promoted apoptosis by inhibiting K-ras/ERK/cyclinD1. CONCLUSIONS: SNHG7 is an oncogenic biomarker in colon carcinogenesis. The effect may be mediated by interaction with miR-193b.

Is metformin still the most efficacious first-line oral hypoglycaemic drug in treating type 2 diabetes? A network meta-analysis of randomized controlled trials.

OBJECTIVES: The objective of the study is to compare the efficacy of hypoglycaemic drugs for type 2 diabetes mellitus (T2DM) by network meta-analysis of randomized controlled trials (RCTs). METHODS: We compared 11 major oral hypoglycaemic drugs under five categories evaluated by RCTs as drug monotherapy for the patients with T2DM, measuring glycosylated haemoglobin (%) or fasting plasma glucose (mmol L-1 ) as outcomes. RCT quality was assessed with the Cochrane risk of bias tool. Network meta-analysis estimated the mean differences and 95% credible intervals. Subgroup and sensitivity analyses were performed to determine the results robustness. The Grading of Recommendation, Assessment, Development, and Evaluation evidence strength was assessed. RESULTS: Seventy-five RCTs including 33,830 patients were identified. Their study quality was high. Regarding glycosylated haemoglobin, top three anti-diabetics were repaglinide (mean differences -1.39 [95% credible intervals -1.75 to -1.03]), gliclazide (-1.37 [-2.04 to -0.71]) and metformin (-1.13 [-1.37 to -0.90]), against placebo. Regarding fasting plasma glucose, top three anti-diabetics were repaglinide (-2.01 [-2.75 to -0.97]), metformin (-1.72 [-2.16 to -1.27]) and glipizide (-1.57 [-2.44 to -0.64]), against placebo. There was no difference between metformin and repaglinide. Subgroup and sensitivity analyses found the results to be robust. The evidence strength was moderate to high. CONCLUSION: This meta-analysis showed that repaglinide and metformin would be the most efficacious oral drugs for first-line monotherapy of T2DM.

Ventricular shape and relative position abnormalities in preterm neonates.

Recent neuroimaging findings have highlighted the impact of premature birth on subcortical development and morphological changes in the deep grey nuclei and ventricular system. To help characterize subcortical microstructural changes in preterm neonates, we recently implemented a multivariate tensor-based method (mTBM). This method allows to precisely measure local surface deformation of brain structures in infants. Here, we investigated ventricular abnormalities and their spatial relationships with surrounding subcortical structures in preterm neonates. We performed regional group comparisons on the surface morphometry and relative position of the lateral ventricles between 19 full-term and 17 preterm born neonates at term-equivalent age. Furthermore, a relative pose analysis was used to detect individual differences in translation, rotation, and scale of a given brain structure with respect to an average. Our mTBM results revealed broad areas of alterations on the frontal horn and body of the left ventricle, and narrower areas of differences on the temporal horn of the right ventricle. A significant shift in the rotation of the left ventricle was also found in preterm neonates. Furthermore, we located significant correlations between morphology and pose parameters of the lateral ventricles and that of the putamen and thalamus. These results show that regional abnormalities on the surface and pose of the ventricles are also associated with alterations on the putamen and thalamus. The complementarity of the information provided by the surface and pose analysis may help to identify abnormal white and grey matter growth, hinting toward a pattern of neural and cellular dysmaturation.

MicroRNA-20a-mediated loss of autophagy contributes to breast tumorigenesis by promoting genomic damage and instability.

Gene expression analysis of The Cancer Genome Atlas (TCGA) breast cancer data set show that miR-20a is upregulated in human breast cancer, especially in triple-negative subtype. Gene Set Enrichment Analysis suggests that miR-20a expression negatively correlates with the autophagy/lysosome pathway. We report here that miR-20a inhibits the basal and nutrient starvation-induced autophagic flux and lysosomal proteolytic activity, increases intracellular reactive oxygen species levels and DNA damage response by targeting several key regulators of autophagy, including BECN1, ATG16L1 and SQSTM1. Re-introduction of exogenous BECN1, ATG16L1 or SQSTM1 reverses the inhibitory effect of miR-20a on autophagy and decreases DNA damage. A negative correlation between miR-20a and its target genes is observed in breast cancer tissues. Lower levels of BECN1, ATG16L1 and SQSTM1 are more common in triple-negative cancers than in other subtypes. High levels of miR-20a also associate with higher frequency of copy-number alterations and DNA mutations in breast cancer patients. Further studies in a xenograft mouse model show that miR-20a promotes tumor initiation and tumor growth. Collectively, these findings suggest that miR-20a-mediated autophagy defect might be a new mechanism underlying the oncogenic function of miRNA during breast tumorigenesis.

Vancomycin levels are frequently subtherapeutic in critically ill patients: a prospective observational study.

BACKGROUND: Appropriate utilization of vancomycin is important to attain therapeutic targets while avoiding clinical failure and the development of antimicrobial resistance. Our aim was to observe the use of vancomycin in an intensive care population, with the main focus on achievement of therapeutic serum concentrations (15-20 mg/l) and to evaluate how this was influenced by dose regimens, use of guidelines and therapeutic drug monitoring. METHODS: A prospective observational study was carried out in the intensive care units at two tertiary hospitals in Norway. Data were collected from 83 patients who received vancomycin therapy, half of these received continuous renal replacement therapy. Patients were followed for 72 h after initiation of therapy. Blood samples were drawn for analysis of trough serum concentrations. Urine was collected for calculations of creatinine clearance. Information was gathered from medical records and electronic health records. RESULTS: Less than 40% of the patients attained therapeutic trough serum concentrations during the first 3 days of therapy. Patients with augmented renal clearance had lower serum trough concentrations despite receiving higher maintenance doses and more loading doses. When trough serum concentrations were outside of therapeutic range, dose adjustments in accordance to therapeutic drug monitoring were made to less than half. CONCLUSION: The present study reveals significant challenges in the utilization of vancomycin in critically ill patients. There is a need for clearer guidelines regarding dosing and therapeutic drug monitoring of vancomycin for patient subgroups.

Development of a lytic peptide derived from BH3-only proteins.

Despite great advances in cancer therapy, drug resistance is a difficult hurdle to overcome that requires development of anticancer agents with novel and effective modes of action. In a number of studies, lytic peptides have shown remarkable ability to eliminate cancer cells through a different way from traditional treatments. Lytic peptides are positively charged, amphiphilic, and are efficient at binding and disrupting the negatively charged cell membrane of cancer cells. In this study, we described the anticancer properties of a lytic peptide that was developed on the basis of the alignment of amphiphilic BH3 peptides. Our results demonstrated that the positive charge and conformation constraint were favourable for efficient cancer cell elimination. Artificial BCL-2 homology 3 peptides (ABH3) exhibited effective anticancer effects against a series of cancer cell lines in vitro and in HeLa human cervical tumour xenografts in vivo. ABH3 induced cell death in an apoptosis-independent manner through the lytic properties of the peptide that caused disruption of cell membrane. Our results showed that charge tuning and conformation constraining in a lytic peptide could be applied to optimise the anticancer activity of lytic peptides. These results also suggest that ABH3 may be a promising beginning for the development of additional lytic peptides as anticancer reagents.

Guttiferone K impedes cell cycle re-entry of quiescent prostate cancer cells via stabilization of FBXW7 and subsequent c-MYC degradation.

Cell cycle re-entry by quiescent cancer cells is an important mechanism for cancer progression. While high levels of c-MYC expression are sufficient for cell cycle re-entry, the modality to block c-MYC expression, and subsequent cell cycle re-entry, is limited. Using reversible quiescence rendered by serum withdrawal or contact inhibition in PTEN(null)/p53(WT) (LNCaP) or PTEN(null)/p53(mut) (PC-3) prostate cancer cells, we have identified a compound that is able to impede cell cycle re-entry through c-MYC. Guttiferone K (GUTK) blocked resumption of DNA synthesis and preserved the cell cycle phase characteristics of quiescent cells after release from the quiescence. In vehicle-treated cells, there was a rapid increase in c-MYC protein levels upon release from the quiescence. However, this increase was inhibited in the presence of GUTK with an associated acceleration in c-MYC protein degradation. The inhibitory effect of GUTK on cell cycle re-entry was significantly reduced in cells overexpressing c-MYC. The protein level of FBXW7, a subunit of E3 ubiquitin ligase responsible for degradation of c-MYC, was reduced upon the release from the quiescence. In contrast, GUTK stabilized FBXW7 protein levels during release from the quiescence. The critical role of FBXW7 was confirmed using siRNA knockdown, which impaired the inhibitory effect of GUTK on c-MYC protein levels and cell cycle re-entry. Administration of GUTK, either in vitro prior to transplantation or in vivo, suppressed the growth of quiescent prostate cancer cell xenografts. Furthermore, elevation of FBXW7 protein levels and reduction of c-MYC protein levels were found in the xenografts of GUTK-treated compared with vehicle-treated mice. Hence, we have identified a compound that is capable of impeding cell cycle re-entry by quiescent PTEN(null)/p53(WT) and PTEN(null)/p53(mut) prostate cancer cells likely by promoting c-MYC protein degradation through stabilization of FBXW7. Its usage as a clinical modality to prevent prostate cancer progression should be further evaluated.

[Factors associated with adherence of highly active antiretroviral therapy among 386 HIV/AIDS patients in 3 provinces of China].

OBJECTIVE: To investigate adherence and the influence factors among patients who are on antiretrovirus therapy (ART) of 3 provinces in China. METHODS: This study selected 18-year-old and older AIDS patients as the survey objects who initiated anti-retrovirus therapy between April and September of 2014 and kept on the treatment for one year in Yunnan,Sichuan,and Hu'nan province. Information of demography, treatment and social support was collected by questionnaires. Adopt SSRS questionnaire to calculate the information of objective support, subjective support and utilization of social support. χ(2) test and logistic regression were performed to examine relationships between factors and adherence. RESULTS: A total of 386 patients with medication were investigated. Among them, there were 365 (94.6%) cases who were compliant to the ART, and 357 (92.5%) cases can take their pills on time, and 29 (7.5%) cases take their medication in excess of the prescribed time more than two hours. Social support score was 27.2 ± 7.3, including objective support score (5.6 ± 2.7), subjective support score (16.1 ± 4.8), and utilization of social support (5.5 ± 1.9). Multivariate logistic regression analysis revealed that adherence was significantly associated with the correct cognition of medication (OR (95%CI): 3.24 (1.17-9.00)), the self-awareness of the drug regimen was not complexity (OR (95%CI): 9.34 (3.27-26.68)), taking medication 1 time a day (OR (95%CI): 4.00 (1.35-11.84)), and the batter utilization of social support (OR (95%CI): 1.49 (1.06-2.01)). Married/cohabiting or farmers tend to be nonadherenced, while the OR (95%CI) was 0.24 (0.08-0.67) and 0.23 (0.08-0.69), respectively. CONCLUSION: The patients among these provinces were compliant to the ART, and most of them can take their pills on time. The social support score of these patients was lower than normal person. Participants who have correct cognition of medication, thinking their drug regimen was not complexity; Taking medication 1 time a day or have high level of utilization of social support showed a significantly higher level of self-reported adherence.

How well can we quantify dust deposition to the ocean?

Deposition of continental mineral aerosols (dust) in the Eastern Tropical North Atlantic Ocean, between the coast of Africa and the Mid-Atlantic Ridge, was estimated using several strategies based on the measurement of aerosols, trace metals dissolved in seawater, particulate material filtered from the water column, particles collected by sediment traps and sediments. Most of the data used in this synthesis involve samples collected during US GEOTRACES expeditions in 2010 and 2011, although some results from the literature are also used. Dust deposition generated by a global model serves as a reference against which the results from each observational strategy are compared. Observation-based dust fluxes disagree with one another by as much as two orders of magnitude, although most of the methods produce results that are consistent with the reference model to within a factor of 5. The large range of estimates indicates that further work is needed to reduce uncertainties associated with each method before it can be applied routinely to map dust deposition to the ocean. Calculated dust deposition using observational strategies thought to have the smallest uncertainties is lower than the reference model by a factor of 2-5, suggesting that the model may overestimate dust deposition in our study area.This article is part of the themed issue 'Biological and climatic impacts of ocean trace element chemistry'.

Effects of feeding lutein on production performance, antioxidative status, and milk quality of high-yielding dairy cows.

This experiment was conducted to determine the influences of supplementing different levels of an additive containing lutein in the diet of Chinese Holstein lactating cows on production performance, antioxidative plasma metabolites, and milk quality. This study was performed on 60 multiparous Holstein dairy cows in peak lactation. The cows were randomly allocated to 1 of 4 homogeneous treatments, with lutein preparation (extracted from marigolds; effective lutein content was 2%) added at levels of 0, 100, 150, and 200 g/d per head, with the actual available amounts being 0, 2, 3, and 4 g of lutein/d per head, respectively. The experiment lasted for 13 wk, with the first week for adaptation. Milk yield and milk compositions were recorded weekly, and milk concentrations of lutein, dry matter intake, and antioxidative blood index were analyzed in the first, fourth, seventh, and thirteenth week of the study. The results showed that adding lutein in the diet had no effect on dry matter intake compared with the control group; however, it slowed down the trend of decline in milk yield, and had a linear incremental effect on milk yield with increasing concentration of lutein. Dietary lutein tended to quadratically increase the percentage of milk fat, and linearly increased milk lactose concentration, with the highest value when treated at 200 g of lutein preparation/d per head, and decreased somatic cell count, with the lowest values when treated with 150 and 200 g of lutein preparation/d per head. The concentration of lutein in milk linearly increased with the incorporation of the additive, with a value of 0.59, 0.70, 1.20, and 1.50 µg/100mL when treated with 0, 100, 150, and 200 g/d, respectively. Total plasma antioxidant capacity tended to linearly increase in cows fed lutein preparation, whereas plasma superoxide dismutase and glutathione peroxidase activities did not differ significantly. In conclusion, addition of lutein in the diet could improve the production performance and health status of dairy cows.

Peptides-staple method development and its application in cancer therapy.

α-Helixes are important structural motifs of protein three dimension structures and are largely involved in protein- protein interactions. This review covers the recent advances of the peptide stabilizing methodologies and introduces their applications in cancer research.

HBx-mediated miR-21 upregulation represses tumor-suppressor function of PDCD4 in hepatocellular carcinoma.

The hepatitis B virus (HBV) X protein (HBx) has a key role in the molecular pathogenesis of HBV-related hepatocellular carcinoma (HCC). However, the mechanism of HBx-mediated hepatocarcinogenesis remains to be elucidated. In this study, we aimed to better understand the effects of HBx on gene-expression profiles that participate in hepatocarcinogenesis and the mechanism by which HBx regulates these genes. Differentially expressed genes between L02-HBx and L02-Vector control cells were identified by microarray and validated using quantitative real-time PCR. HBx upregulates 456 genes and downregulates 843 genes, including programmed cell death 4 (PDCD4). PDCD4 was downregulated in clinical HCC specimens and the downregulation of PDCD4 in HCC is correlated with HBx. Furthermore, overexpression experiments in HCC cells proved that PDCD4 has strong tumor-suppressive effects both in vitro and in vivo, and may induce cell apoptosis to suppress the development of HCC. HBx induces expression of DNA methyltransferases (DNMTs), but failed to change the methylation status of the PDCD4 promoter. HBx downregulates PDCD4 expression at least partially through miR-21. Taken together, this study reported for the first time that HBx downregulates PDCD4 and upregulates miR-21 expression. The overexpression of PDCD4 could suppress tumorigenicity. The deregulation of PDCD4 by HBx through miR-21 represents a potential novel mechanism of the downregulation of PDCD4 in HBV-related HCC and provides new insights into HCC development.

Plasma frequency and dielectric function dependence on doping and temperature for p-type indium phosphide epitaxial films.

The optical properties of p-type InP epitaxial films with different doping concentrations are investigated by infrared absorption measurements accompanied by reflection and transmission spectra taken from 25 to 300 K. A complete dielectric function (DF) model, including intervalence band (IVB) transitions, free-carrier and lattice absorption, is used to determine the optical constants with improved accuracy in the spectral range from 2 to 35 µm. The IVB transitions by free holes among the split-off, light-hole, and heavy-hole bands are studied using the DF model under the parabolic-band approximation. A good understanding of IVB transitions and the absorption coefficient is useful for designing high operating temperature and high detectivity infrared detectors and other optoelectronic devices. In addition, refractive index values reported here are useful for optoelectronic device designing, such as implementing p-InP waveguides in semiconductor quantum cascade lasers. The temperature dependence of hole effective mass and plasma frequency is also reported.

Cdk-mediated phosphorylation of Chk1 is required for efficient activation and full checkpoint proficiency in response to DNA damage.

Here, we show that activation of the checkpoint effector kinase Chk1 in response to irradiation-induced DNA damage is minimal in G1, maximal during S-phase and diminishes as cells enter G2. In addition, formation of irradiation-induced replication protein A (RPA)-coated single-stranded DNA (RPA-ssDNA), a structure required for ATM and Rad3-related (ATR)-Chk1 activation, occurs in a broadly similar pattern. Cyclin-dependent kinase (Cdk) activity is thought to promote RPA-ssDNA formation by stimulating DNA strand resection at double-strand breaks (DSBs), providing one possible mechanism of imposing cell cycle dependence on DNA damage signaling. However, it has recently been shown that Chk1 itself is also subject to Cdk-mediated phosphorylation at serines 286 and 301 (S286 and 301). We show that Chk1 S301 phosphorylation increases as cells progress through S and G2 and that both Cdk1 and Cdk2 are likely to contribute to this modification in vivo. We also find that substitution of S286 and S301 with non-phosphorylatable alanine residues strongly attenuates DNA damage-induced Chk1 activation and G2 checkpoint proficiency, but does not eliminate the underlying cell cycle dependence of Chk1 regulation. Taken together, these data indicate that Cdk activity regulates multiple steps in the DNA damage response pathway including full activation of Chk1 and checkpoint proficiency.

Delayed onset of hyperglycaemia in a mouse model with impaired glucagon secretion demonstrates that dysregulated glucagon secretion promotes hyperglycaemia and type 2 diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is caused by relative deficiency of insulin secretion and is associated with dysregulation of glucagon secretion during the late stage of diabetes development. Like insulin secretion from beta cells, glucagon secretion is dependent on calcium signals and a calcium sensing protein, synaptotagmin-7. In this study, we tested the relative contribution of dysregulated glucagon secretion and reduced insulin release in the development of hyperglycaemia and type 2 diabetes by using synaptotagmin-7 knockout (KO) mice, which exhibit glucose intolerance, reduced insulin secretion and nearly abolished Ca(2+)-stimulated glucagon secretion. METHODS: We fed the synaptotagmin-7 KO and control mice with a high-fat diet (HFD) for 14 weeks, and compared their body weight, glucose levels, glucose and insulin tolerance, and insulin and glucagon secretion. RESULTS: On the HFD, synaptotagmin-7 KO mice showed progressive impairment of glucose tolerance and insulin secretion, along with continued maintenance of a low glucagon level. The control mice were less affected in terms of glucose intolerance, and showed enhanced insulin secretion with a concurrent increase in glucagon levels. Unexpectedly, after 14 weeks of HFD feeding, only the control mice displayed resting hyperglycaemia, whereas in synaptotagmin-7 KO mice defective insulin secretion and reduced insulin sensitivity were not sufficient to cause hyperglycaemia in the absence of enhanced glucagon secretion. CONCLUSIONS/INTERPRETATION: Our data uncover a previously overlooked role of dysregulated glucagon secretion in promoting hyperglycaemia and the ensuing diabetes, and strongly suggest maintenance of adequate regulation of glucagon secretion as an important therapeutic target in addition to the preservation of beta cell function and mass in the prevention and treatment of diabetes.

Influence of formulation properties on chemical stability of captopril in aqueous preparations.

The influence of various formulation properties on the chemical stability of captopril in aqueous media at pH 3 was investigated, in order to reformulate and increase the shelf-life of an oral mixture of the drug. At this pH, chemical stability is improved by an increase in drug concentration (1-5 mg/ml) and a decrease in temperature (5-36 degrees C), the latter demonstrated by a linear Arrhenius-plot. The activation energy is low (Ea = 10.2 kcal/mol), thus the Q10 value is only 1.8 in pure aqueous solutions. The degradation at the lowest concentration investigated in pure aqueous solution apparently follows zero order kinetics. The reaction order is changed at higher concentrations. We are presenting a hypothesis of intramolecular proton transfer from the thiol to the ionized carboxylic group as the initial step in the oxidative degradation pathways of captopril. Long-term stability of 1 mg/ml captopril in aqueous solutions at pH 3, stored at 36 degrees C for one year, shows that the sugar alcohol sorbitol accelerates degradation of the drug while Na-EDTA at a concentration as low as 0.01% is sufficient to stabilize these samples. Purging with N2-gas prior to storage is not essential for drug stability, as long as Na-EDTA is present. Only at a low level of Na-EDTA (0.01%) combined with a high level of sorbitol (35%), purging with N2-gas appears to have a small effect. The destabilizing effect of sugar alcohols is confirmed by accelerated degradation also in the presence of glycerol. The efficient stabilization in the presence of Na-EDTA at a low concentration indicates that the metal-ion-catalyzed oxidation pathway dominates the chemical degradation process at low pH, although several mechanisms seem to be involved depending on excipients present.

Study of the functional role of Bcl-2 family proteins in regulating Ca(2+) signals in apoptotic cells.

Recently, it was found that Bcl-2 family proteins can affect the apoptotic process by modifying Ca(2+) released from the ER (endoplasmic reticulum). In this review, we summarize the evidence that Bcl-2 and Bax can modulate Ca(2+) mobilization from the ER to the cytosol and mitochondria. We also found evidence that both Bcl-2 and Bax can interact with IP(3)Rs (InsP(3) receptors) to modify the Ca(2+) efflux from the ER. On the basis of our findings, we suggest that Bax may interact with IP(3)Rs to facilitate the release of Ca(2+) from the ER during apoptosis.

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis.

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).

The C2B domain of synaptotagmin I is a Ca2+-binding module.

Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.

Replication protein A interactions with DNA. III. Molecular basis of recognition of damaged DNA.

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.