Dysmenorrhea is painful menstrual cramps, which negatively impacts the quality of life of those diagnosed. The paper reviews traditional Chinese medicine's treatment of dysmenorrhea through the use of combination-herbal-formula therapeutics. These herbal treatments are effective for dysmenorrhea with minimal side effects. Pharmacological studies suggest Chinese herbal dysmenorrhea therapies likely decrease prostaglandin levels, modulate nitric oxide, increase plasma beta-endorphin (beta-EP) levels, block calcium-channels and improve microcirculation. Conventional therapy for dysmenorrhea, which usually includes non-steroidal antiinflammatory drugs (NSAIDs), provides symptomatic relief but has increasing adverse effects with long-term use. Therefore, Chinese herbal medicines, including simple herbal and combination formulas, are perhaps the ideal therapeutics of choice.

The neuropeptide alpha-melanocyte stimulating hormone [alpha-MSH(1-13)] occurs in the pituitary, brain, skin and other tissues and receptors for this molecule are likewise widespread. In previous research, this tridecapeptide, which shares its amino acid sequence with ACTH(1-13), was shown to have both potent antipyretic activity and a role in the endogenous control of the febrile response. alpha-MSH(1-13) and its COOH-terminal tripeptide were subsequently found to inhibit inflammation induced by general stimuli such as topical application of an irritant. The aim in the present experiments was to determine if these peptides can inhibit acute inflammatory responses induced in mice by injection of individual cytokines, endogenous pyrogen (EP), a natural cytokine mixture, and other mediators of inflammation. Inflammation induced in the mouse ear by rIL-1 beta, rIL-6 or rTNF-alpha was inhibited by alpha-MSH and a D-valine-substituted analog of alpha-MSH(11-13) whereas substantial doses of alpha-MSH(1-13) did not alter inflammation induced by LTB4, PAF and IL-8. Both peptides inhibited edema caused in the mouse paw by local injection of EP. The results indicate that alpha-MSH molecules antagonize the actions of certain cytokine mediators of inflammation, consistent with previous observations of anti-cytokine activity of these peptides. Failure to inhibit edema caused by LTB4, PAF and IL-8 suggests that, in inflammation induced by general stimuli, such as EP, the peptides act prior to the release of these mediators of the inflammatory response. Because of the anticytokine/anti-inflammatory actions of the alpha-MSH molecules they may be useful in understanding the cytokine network and for treatment of inflammatory diseases.

Ovariectomized female rats subcutaneously (SC) injected or intracerebrally implanted with estradiol benzoate (EB), and given progesterone SC were used as experimental animals to assess the effects of the beta-endorphin (beta-EP) neuronal system on lordosis behavior. In intraventricular (IV) injection of beta-EP at the onset of sc EB priming, the lordosis behavior was significantly (p < 0.001) facilitated. In contrast, the lordosis behavior was significantly (p < 0.001) inhibited by IV injection of naloxone, an opioid receptor antagonist. beta-EP facilitation of lordosis was observed exclusively within the initial stage of estrogen action. The behavior was significantly (p < 0.001) facilitated by IV injection of beta-EP given with an intracerebral implantation of crystalline EB into the septal-preoptic regions. However, the lordosis behavior was significantly (p < 0.001) inhibited by beta-EP when EB was implanted into the ventromedial hypothalamus. Animals receiving EB implants into the mesencephalic reticular formation were not affected by beta-EP. The present study suggests that the beta-EP neuronal system stimulates sexual receptivity through an action on the central nervous system in relation to the site of estrogen-initial activation to induce the lordosis reflex. The sites of beta-EP action may be the estrogen receptive septal-preoptic and hypothalamic regions; the former for facilitatory effect and the latter for inhibitory effect.

Yttrium-90 (90Y), a pure beta emitter, is an attractive radionuclide for radioimmunotherapy of cancer. Therapeutic management requires quantitative imaging to measure the pharmacokinetics of the radionuclide in the patient for radiation dosimetric calculations. The bremsstrahlung emissions can be utilized to acquire an image of beta sources using a gamma camera. Quantitation of 90Y by bremsstrahlung imaging is difficult because of poor image quality that results from septal penetration and scatter secondary to the broad bremsstrahlung energies. In this work, quantitative methods for bremsstrahlung imaging of 90Y sources that involved the use of (a) a Wiener filter to deconvolve the septal penetration and scatter while suppressing image noise, and (b) the geometric mean of the conjugate view (GM) and effective point source (EPS) methods to quantify activities were investigated. An abdominal phantom was prepared with 90Y activities in the liver, spleen, tumors, and background volumes that were similar to those observed in patient studies. A twofold improvement in resolution recovery for full width at tenth maximum of the line spread function at 11 cm depth in water was achieved using Wiener restoration. Definition of the organ and tumor edges was greatly enhanced and cross talk between adjacent sources was suppressed after Wiener restoration. These improvements in image quality led to more accurate estimation of organ and tumor activities. Using the optimum attenuation correction method for GM and EPS quantitation of filtered bremsstrahlung images, estimates of individual activities (< or = 17% error) and cumulated activities (< or = 8% error) in all of the sources were accurate except for a tumor of 2 cm diameter. The results of this study provide the basis for a method to quantify beta source distribution and demonstrate the potential use of bremsstrahlung imaging in clinical settings.

The extracellular polysaccharides (EPSs) produced by 37 isolates presently classified as Butyrivibrio species (or more specifically as Butyrivibrio fibrisolvens) were purified from glucose-grown cultures. The neutral sugar compositions of these EPSs were determined by both thin-layer and gas-liquid chromatographic techniques. Results showed that while the neutral sugar composition of the EPS was constant for a given strain, it varied considerably between strains. In addition, several acidic components in the EPS, of both known and unknown structure, were detected artifactually as acetylated lactones, the acetylated alditols derived from these lactone(s), or both. Two novel components, L-altrose and the acidic sugar 4-O-[1-carboxyethyl]-D-galactose, were common constituents of the EPS from some strains of B. fibrisolvens. These and other EPS compositional features were used to sort isolates of B. fibrisolvens into groups which may have taxonomic significance. A scheme for sorting isolates into these groups, and the relative relationships between groups, is proposed.

Many food-grade bacteria produce exopolysaccharides (EPS) that affect the texture of fermented food products and that may be involved in probiotic properties. Propionibacterium freudenreichii is a Gram-positive food-grade bacterium with reported probiotic capabilities that is widely used as starter in Swiss-type cheese. In this study, 68 strains of P. freudenreichii were screened for the beta-glucan capsular phenotype by immunoagglutination with a specific antibody and for the presence of the gtf gene coding for polysaccharide synthase. All strains were positive for PCR amplification with gtf gene-specific primers, but the presence of beta-glucan capsular EPS was detected for only 35% of the strains studied. Disruption of gtf in P. freudenreichii revealed that gtf is a unique gene involved in beta-glucan capsular EPS production in P. freudenreichii. The gtf gene was transferred into and expressed in Lactococcus lactis, in which it conferred an agglutination-positive phenotype. Expression of the gtf gene was measured by performing quantitative reverse transcription-PCR assays with RNA from four capsular and three noncapsular strains. A positive correlation was found between the beta-glucan capsular phenotype and gtf gene expression. Sequencing of the region upstream of the gtf open reading frame revealed the presence of an insertion element (IS element) in this upstream region in the four strains with the beta-glucan capsular phenotype. The role of the IS element in the expression of neighboring genes and its impact on interstrain variability of the P. freudenreichii capsule phenotype remain to be elucidated.

A few phytophagous hemipteran species such as the glassy-winged sharpshooter, Homalodisca vitripennis, (Germar), subsist entirely on xylem fluid. Although poorly understood, aspects of the insect's salivary physiology may facilitate both xylem-feeding and transmission of plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease of grape and other scorch diseases in many important crops. X. fastidiosa colonizes the anterior foregut (precibarium and cibarium) of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored in part by an exopolysaccharide (EPS) matrix that is reported to have a β-1,4-glucan backbone. Recently published evidence supports the following, salivation-egestion hypothesis for the inoculation of X. fastidiosa during vector feeding. The insect secretes saliva into the plant and then rapidly takes up a mixture of saliva and plant constituents. During turbulent fluid movements in the precibarium, the bacteria may become mechanically and enzymatically dislodged; the mixture is then egested back out through the stylets into plant cells, possibly including xylem vessels. The present study found that proteins extracted from dissected H. vitripennis salivary glands contain several enzyme activities capable of hydrolyzing glycosidic linkages in polysaccharides such as those found in EPS and plant cell walls, based on current information about the structures of those polysaccharides. One of these enzymes, a β-1,4-endoglucanase (EGase) was enriched in the salivary gland protein extract by subjecting the extract to a few, simple purification steps. The EGase-enriched extract was then used to generate a polyclonal antiserum that was used for immunohistochemical imaging of enzymes in sharpshooter salivary sheaths in grape. Results showed that enzyme-containing gelling saliva is injected into xylem vessels during sharpshooter feeding, in one case being carried by the transpiration stream away from the injection site. Thus, the present study provides support for the salivation-egestion hypothesis.

The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n = 6) with non-decidualized samples (n = 5), and b) the decidualized EP (n = 6) with decidualization-matched IUP (n = 6) samples using an Affymetrix gene array platform, with Ingenuity Pathway Analysis, combined with quantitative RT-PCR. Expression of PRL and IGFBP1 was used to confirm the degree of decidualization in each group. There were no differences in PRL or IGFBP1 expression in the decidualization-matched samples but a marked reduction (P<0.001) in the non-decidualized samples. Decidualization was associated with increased expression of 428 genes including SCARA5 (181-fold), DKK1 (71-fold) and PROK1 (32-fold), and decreased expression of 230 genes including MMP-7 (35-fold) and SFRP4 (21-fold). The top canonical pathways associated with these differentially expressed genes were Natural Killer Cell and Wnt/b-Catenin signaling. Local trophoblast was associated with much less alteration of endometrial gene expression with an increase in 56 genes, including CSH1 (8-fold), and a reduction in 29 genes including CRISP3 (8-fold). The top associated canonical pathway was Antigen Presentation. The study of endometrium from tubal EP may promote novel insights into genes involved in decidualization and those influenced by factors from neighboring trophoblast. This has afforded unique information not highlighted by previous studies and adds to our understanding of the endometrium in early pregnancy.

The exopolysaccharide (EPS) produced by Lactobacillus plantarum YW32 was purified and characterized, and the in vitro bioactivities of the purified EPS were also evaluated. The EPS had a molecular weight of 1.03×10(5) Da, and it consisted of mannose, fructose, galactose and glucose in an approximate molar ratio of 8.2:1:4.1:4.2. Microstructural studies of the EPS demonstrated a web-like structure composed of compact ropes, and presence of many homogeneous rod-shaped lumps. The EPS also showed high thermal stability with a degradation temperature of 283.5°C. Furthermore, the EPS at a dose of 5mg/ml had strong scavenging abilities toward hydroxyl (77.5%) and superoxide radicals (66.5%). The EPS exhibited a concentration-dependent inhibitory effect on the formation of biofilms by several pathogenic bacteria, including Escherichia coli O157, Shigella flexneri CMCC (B), Staphylococcus aureus AC1 and Salmonella typhimurium S50333. In vitro antitumor assay of the EPS showed that it had good inhibitory activity against colon cancer HT-29 cells. These characteristics and bioactivities of the EPS would make it a promising candidate for use as a potential food adjunct in foods with healthy properties.

The high mobility group box-BBB (Akt) pathways play a crucial role in tumorigenesis and development of many malignant tumors. Ethyl pyruvate (EP), a potent inhibitor of HMGBEP on gastric cancer growth in vitro and in vivo. Human gastric adenocarcinoma tissues of different grades (N=45) were collected. The expression of HMGBEP, the expression of HMGBEP on cell proliferation, invasion, cell cycle distribution and apoptosis were assessed. A subcutaneous xenograft tumor model was established, validating the effects of EP on tumor growth in vivo. The expression of HMGBEP decreased the expression of HMGBEP could inhibit tumor cell proliferation and invasion, induce cell cycle arrest and apoptosis and slow the growth of xenograft tumors. In conclusion, HMGBEP administration inhibited gastric cancer growth via regulation of the HMGBEP may play a critical role in the treatment of cancer in conjunction with other therapeutic agents.

This study was designed to synthesize, characterize and investigate the drug inclusion property of a series of novel cationic beta-cyclodextrin polymers (CPbetaCDs). Proposed water-soluble polymers were synthesized from beta-cyclodextrin (beta-CD), epichlorohydrin (EP) and choline chloride (CC) through a one-step polymerization procedure by varying molar ratio of EP and CC to beta-CD. Physicochemical properties of the polymers were characterized with colloidal titration, nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC) and aqueous solubility determination. The formation of naproxen/CPbetaCDs inclusion complexes was confirmed by NMR and fourier transform infrared spectroscopy (FT-IR). Cationic beta-CD polymers showed better hemolytic activities than parent beta-CD and neutral beta-CD polymer in hemolysis test. The morphological study of erythrocytes revealed a cell membrane invagination induced by the cationic groups. The effects of molecular weight and charge density of the polymers on their inclusion and release performance of naproxen were also investigated through phase-solubility and dissolution studies. It was found that the cationic beta-CD polymers with high molecular weight or low charge density exhibited better drug inclusion and dissolution abilities.

Alkaline phosphatase (AP) was covalently linked to the two antitumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), forming conjugates that could bind to antigen-positive tumor cells. The conjugates were able to convert the prodrugs, mitomycin phosphate (MOP) and etoposide phosphate (EP), into an active mitomycin C derivative, mitomycin alcohol, and etoposide, respectively. MOP and EP were less toxic to cultured cells from the H2981 lung adenocarcinoma than their respective hydrolysis products, mitomycin alcohol and etoposide, by a factor greater than 100, and they were also less toxic in mice. Pretreatment of H2981 cells with L6-AP greatly enhanced the cytotoxic effects of MOP and EP, while 1F5-AP caused no such enhancement. A strong antitumor response was observed in H2981-bearing mice that were treated with L6-AP followed 24 h later by either MOP or a combination of MOP and EP. This response was superior to that of MOP or combinations of MOP and EP given alone.

Wheat is prone to strawbreaker foot rot (eyespot), a fungal disease caused by Oculimacula yallundae and O. acuformis. The most effective source of genetic resistance is Pch1, a gene derived from Aegilops ventricosa. The endopeptidase isozyme marker allele Ep-D1b, linked to Pch1, has been shown to be more effective for tracking resistance than DNA-based markers developed to date. Therefore, we sought to identify a candidate gene for Ep-D1 as a basis for a DNA-based marker. Comparative mapping suggested that the endopeptidase loci Ep-D1 (wheat), enp1 (maize), and Enp (rice) were orthologous. Since the product of the maize endopeptidase locus enp1 has been shown to exhibit biochemical properties similar to oligopeptidase B purified from E. coli, we reasoned that Ep-D1 may also encode an oligopeptidase B. Consistent with this hypothesis, a sequence-tagged-site (STS) marker, Xorw1, derived from an oligopeptidase B-encoding wheat expressed-sequence-tag (EST) showed complete linkage with Ep-D1 and Pch1 in a population of 254 recombinant inbred lines (RILs) derived from a cross between wheat cultivars Coda and Brundage. Two other STS markers, Xorw5 and Xorw6, and three microsatellite markers (Xwmc14, Xbarc97, and Xcfd175) were also completely linked to Pch1. On the other hand, Xwmc14, Xbarc97, and Xcfd175 showed recombination in the W7984 x Opata85 RIL population suggesting that recombination near Pch1 is reduced in the Coda/Brundage population. In a panel of 44 wheat varieties with known eyespot reactions, Xorw1, Xorw5, and Xorw6 were 100% accurate in predicting the presence or absence of Pch1 whereas Xwmc14, Xbarc97, and Xcfd175 were less effective. Thus, linkage mapping and a germplasm survey suggest that the STS markers identified here should be useful for indirect selection of Pch1.

The mechanical properties of the aorta play a major role in the regulation of blood pressure and cardiac performance. The effect of sympathetic stimulation on the mechanical properties of the human abdominal aorta was studied in 19 healthy volunteers, divided into young (25 +/- 2 years) and elderly individuals (69 +/- 2 years) of both sexes. A non-invasive ultrasonic echo-tracking system for measurement of systolic/diastolic variation of aortic diameter in combination with intra-aortic pressure measurements was used to determine wall mechanics. The pressure-diameter (P-D) relationship and the distensibility indices, stiffness (beta) and pressure strain elastic modulus (Ep) of the abdominal aorta were obtained. Measurements were made at rest and during sympathetic stimulation induced by lower body negative pressure (LBNP). As a sign of sympathetic activation, the peripheral resistance increased by 74-96% (P < 0.001) during LBNP. However, the mechanical properties of the abdominal aorta remained unaltered, as estimated either from the P-D relationship or from the indices Ep and beta, both in the young (rest: Ep = 0.53 +/- 0.18, beta = 4.5 +/- 1.5; LBNP: Ep = 0.51 +/- 0.15, beta = 4.5 +/- 1.2, NS) and in the elderly (rest: Ep = 2.17 +/- 0.70, beta = 17.6 +/- 5.8; LBNP: Ep = 2.11 +/- 0.60, beta = 16.9 +/- 3.9, NS). In conclusion, this investigation shows that LBNP-induced sympathetic activation does not change aortic wall mechanics. Thus, sympathetic modulation of the aortic smooth muscle contractile activity seems to be unimportant in the blood pressure regulation.

BACKGROUND

Few studies have examined the emotional approach to coping on diabetes outcomes. This study examined the relationship between emotional coping and diabetes knowledge, medication adherence and self-care behaviors in adults with type 2 diabetes.

METHODS

Data on 378 subjects with type 2 diabetes recruited from two primary care clinics in the southeastern United States were examined. Previously validated scales were used to measure coping, medication adherence, diabetes knowledge and diabetes self-care behaviors (including diet, physical activity, blood sugar testing and foot care). Multiple linear regression was used to assess the independent effect of coping through emotional approach on medication adherence and self-care behaviors while controlling for relevant covariates.

RESULTS

Significant correlations were observed between emotional coping [as measured by emotional expression (EE) and emotional processing (EP)] and self-care behaviors. In the linear regression model, EP was significantly associated with medication adherence [β -0.17, 95% confidence interval (CI) -0.32 to -0.015], diabetes knowledge (β 0.76, 95% CI 0.29 to 1.24), diet (β 0.52, 95% CI 0.24 to 0.81), exercise (β 0.51, 95% CI 0.19 to 0.82), blood sugar testing (β 0.54, 95% CI 0.16 to 0.91) and foot care (β 0.32, 95% CI -0.02 to 0.67). On the other hand, EE was associated with diet (β 0.38, 95% CI 0.13 to 0.64), exercise (β 0.54, 95% CI 0.27 to 0.82), blood sugar testing (β 0.42, 95% CI 0.09 to 0.76) and foot care (β 0.36, 95% CI 0.06 to 0.66), but it was not associated with diabetes knowledge.

CONCLUSIONS

These findings indicate that coping through an emotional approach is significantly associated with behaviors that lead to positive diabetes outcomes.

In this study, we analyzed the EEG oscillatory activity induced during a simple visual task, in search of spectral correlate(s) of attention. This task has been previously analyzed by conventional event-related potential (ERP) computation, and Slow Potentials (SPs) were seen to be highly variable across subjects in topography and generators [Basile LF, Brunetti EP, Pereira JF Jr, Ballester G, Amaro E Jr, Anghinah R, Ribeiro P, Piedade R, Gattaz WF. (2006) Complex slow potential generators in a simplified attention paradigm. Int J Psychophysiol. 61(2):149-57]. We obtained 124-channel EEG recordings from 12 individuals and computed latency-corrected peak averaging in oscillatory bursts. We used current-density reconstruction to model the generators of attention-related activity that would not be seen in ERPs, which are restricted to stimulus-locked activity. We intended to compare a possibly found spectral correlate of attention, in topographic variability, with stimulus-related activity. The main results were (1) the detection of two bands of attention-induced beta range oscillations (around 25 and 21 Hz), whose scalp topography and current density cortical distribution were complex multi-focal, and highly variable across subjects (topographic dispersion significantly higher than sensory-related visual theta induced band-power), including prefrontal and posterior cortical areas. Most interesting, however, was the observation that (2) the generators of task-induced oscillations are largely the same individual-specific sets of cortical areas active during the pre-stimulus baseline. We concluded that attention-related electrical cortical activity is highly individual-specific, and possibly, to a great extent already established during mere resting wakefulness. We discuss the critical implications of those results, in combination with results from other methods that present individual data, to functional mapping of cortical association areas.

BACKGROUND

The source and regulatory mechanisms that elevate beta-endorphin (beta-EP) approximately twofold higher than circulating plasma levels in the colostrum of lactating mothers are still unknown, and no studies have examined beta-EP availability previously during maturation phases of human milk. Therefore, the aim of this study was to determine whether concentrations of beta-EP vary over time between colostrum, transitional, and mature breast-milk and to evaluate whether this depends on the method of delivery.

METHODS

Mothers of healthy full-term and pre-term newborn infants who planned to breast-feed their newborn infants were considered for this study. They were consecutively recruited in one of 3 groups of 14, according to delivery method: group 1, vaginal delivery at term (gestational age 40.2 +/- 0.3 weeks; birth weight, 3.48 +/- 0.09 kg); group 2, preterm vaginal delivery (gestational age, 35.6 +/- 0.3 weeks; birth weight, 2.49 +/- 0.08 kg); and group 3, at-term elective cesarean section (gestational age, 39.0 +/- 0.3 weeks; birth weight, 3.32 +/- 0.14 kg). Three consecutive breast milk samples were obtained on the fourth day after birth, before each mother's discharge, and thereafter on the 10th and 30th postpartum days, close to expression of the colostrum, transitional, and mature milk production phases, respectively, to test beta-EP concentrations (beta-Endorphin 125I RIA; INCSTAR Corporation, Stillwater, MN). Data are presented as mean +/- standard deviation. Statistical comparison of beta-EP concentration among the three lactating mother groups was performed using the Kruskal-Wallis nonparametric test. In addition, to test the hypothesis of a trend toward smaller values with time of beta-EP, the authors computed within each mother group a P value per trend (Kruskal-Wallis test) of beta-EP concentration averages on the 4th, 10th, and 30th days, respectively. Student's t test for independent samples was used for the analysis of the other data. The 0.05 significance level was used in the statistical analysis. All computations were made by computer.

RESULTS

Colostrum beta-EP concentrations on the fourth postpartum day of group 1 and group 2 mothers who were delivered of a neonate vaginally, at term, or prematurely were significantly higher (P < 0.01) than colostrum levels of group 3 mothers who underwent cesarean section. Group 2 mothers who were delivered of a neonate vaginally and prematurely presented the highest beta-EP concentrations (P < 0.05), lasting until the transitional milk phase (10th day). No significant differences were found across all 3 groups of lactating mothers in mature milk (30th day) beta-EP concentrations. In addition, the beta-EP trend toward smaller values with time within each of the three groups on days 4, 10, and 30 was statistically significant (P < 0.01 per trend).

CONCLUSIONS

It is hypothesized that elevated beta-EP concentrations in colostrum and transitional milk of mothers who were vaginally delivered of infants may contribute to postnatal fetal adaptation, to overcoming birth stress of natural labor and delivery, and at the same time to the postnatal development of several related biologic functions of breast-fed infants.

Hepatitis B virus (HBV) infection is a major risk factor worldwide for the development of hepatocellular carcinoma (HCC). Integrated HBV DNA fragments, often highly rearranged, are frequently detected in HCC. In woodchuck, the viral enhancer plays a central role in hepatocarcinogenesis, but in humans the mechanism of HBV oncogenesis has not been established. In this study we investigated the status of the viral enhancer in two human HCC cell lines, Hep3B and PLC/PRF/5 each containing one or more integrated HBV DNA fragments. Active enhancer was defined by virtue of its protein occupancy as determined by genomic in vivo DMS footprinting. In PLC/PRF/5 cells, the HBV DNA was integrated in a cellular gene at chromosome 11q13, at a locus reported to be amplified in many tumors. We show here that in both cell lines, the integrated HBV DNA fragments contain an active enhancer-I. In particular, the occupation of the two previously defined basic enhancer elements, E and EP, was prominent. While in both cell lines the same protein binds to the EP elements, the E element, however, is occupied in a cell-line specific manner. In PLC/PRF/5 but not Hep3B, the prominent binding of an undefined protein was detected. Our data suggest that this protein is likely to be the fetoprotein transcription factor (FTF). The finding that enhancer sequences are conserved and functional in different cell lines suggests a selection pressure for their long-term maintenance. We therefore propose that the HBV enhancer-I might play a role in hepatocellular carcinogenesis.

Podocytes possess receptors for a variety of hormones. The following receptors whose stimulation results in increased cAMP levels have been detected in podocytes: adrenergic beta(2) receptor, dopamine D(1) receptor, prostaglandin IP and EP(4) receptors, and parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor. Besides activating protein kinase A, increased levels of cAMP depolarize podocytes via opening of chloride channels. Relatively little is known about the impact of the cAMP pathway on podocyte function. Results obtained in a limited number of studies indicate that cAMP in podocytes may regulate cell morphology, actin assembly, and matrix production. In addition, cAMP seems to attenuate the action of hormones, which activate the Ca(2+)/protein kinase C pathway. Effects of the cAMP pathway on further aspects of podocyte biology, such as contractility, phosphorylation state of slit membrane-associated proteins, glomerular permeability, cell cycle control, and synthesis of reactive oxygen species can be anticipated from studies on other cell types and from studies on isolated glomeruli. In summary, the data available indicate that the cAMP pathway affects several aspects of podocyte biology in an overall glomerulo-protective manner.

Sinorhizobium fredii HH103 produces cyclic beta glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgsColon, two colonslacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs::Omega) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.

OBJECTIVE

Hyperglycemia has been associated with an increased risk of cardiovascular morbidity and mortality. Although numerous studies have demonstrated that hyperglycemia is associated with the atherosis component of atherosclerosis, limited studies have addressed the independent role of hyperglycemia in the pathophysiology of sclerotic vascular disease. We hypothesized that hyperglycemia, as assessed by hemoglobin A1c (HbA1c), would be independently associated two common indices of arterial stiffness (pressure-strain elastic modulus (Ep) and Young's elastic modulus (YEM)).

METHODS

We examined the cross-sectional association between HbA1c and arterial stiffness using B-mode ultrasound examination of the carotid artery in 9050 participants from the community-based Atherosclerosis Risk in Communities (ARIC) study. We used multivariable linear and logistic regression models to characterize the association between HbA1c and increased Ep and YEM.

RESULTS

Higher values of HbA1c were associated in a graded fashion with increased arterial stiffness (P-trend < 0.001 for both EP and YEM). After adjusting for traditional risk factors, increasing HbA1c deciles were significantly associated with elevated EP (OR for the highest decile of HbA1c compared to the lowest, 2.01, 95% CI: 1.30, 3.11) and YEM (OR = 1.71, 95% CI: 1.15, 2.55).

CONCLUSIONS

Elevated HbA1c is associated with measures of increased arterial stiffness, even after accounting for arterial wall thickness. This is consistent with the hypothesis that hyperglycemia contributes to arterial stiffness beyond its effects on atherosis and suggests that hyperglycemia is associated with altered material within the arterial wall.

A genome-wide transcriptional profile of Bradyrhizobium japonicum, the nitrogen-fixing endosymbiont of the soybean plant, revealed differential expression of approximately 15% of the genome after a 1 mM treatment with the phytohormone indole-3-acetic acid (IAA). A total of 1,323 genes were differentially expressed (619 up-regulated and 704 down-regulated) at a two-fold cut off with q value ≤ 0.05. General stress response genes were induced, such as those involved in response to heat, cold, oxidative, osmotic, and desiccation stresses and in exopolysaccharide (EPS) biosynthesis. This suggests that IAA is effective in activating a generalized stress response in B. japonicum. The transcriptional data were corroborated by the finding that stress tolerance of B. japonicum in cell viability assays was enhanced when pre-treated with 1 mM IAA compared to controls. The IAA treatment also stimulated biofilm formation and EPS production by B. japonicum, especially acidic sugar components in the total EPS. The IAA pre-treatment did not influence the nodulation ability of B. japonicum. The data provide a comprehensive overview of the potential transcriptional responses of the symbiotic bacterium when exposed to the ubiquitous hormone of its plant host.

While antibodies are a major extracellular tool of the highest specificity to answer important biomedical questions, the improvements in electroporation discussed below may make it feasible to also use antibodies as an intracellular deletion tool to study (a) viruses inside the cell, (b) cancer cells, (c) signal transduction, (d) genetics, (e) metabolism, and (f) other structures and mechanisms. Already, others have succeeded in depositing macromolecules, including antibodies (Abs), and nucleic acids inside cells, using many techniques, including electroporation (EP). However, EP has limitations that have precluded its widespread use, particularly its high kill rate for cells and the low percentage of cells that are able to incorporate macromolecules. If these limitations could be overcome for Abs and nucleic acids, then it would be practical to use them as highly specific probes for intracellular molecules. In our experiments using EP, we were able to largely prevent lethality for cells during EP by employing a commercially available cold-storage solution for organ transplants containing high K(+) and Mg(++) (ViaSpan, Belzer UW cold-storage solution, DuPont Pharmaceuticals). This solution decreased cell death after standard EP by an average of 50% for a number of cell lines. Viability of WISH cells after EP approached 100%. In transfection studies, ViaSpan medium strongly increased both P3HR1 cell survival as well as the total number of cells transfected with DNA for green fluorescent protein (GFP). In additional experiments with Abs, we were able to strongly increase the percent of cells that incorporated Ab by using two serial EPs. This enhanced the intracellular protection by Abs against viruses in Vero cells from 64% to a maximum of 98%. We were able to further simplify the EP technique by using unpurified antiserum in place of purified IgG. Thus, this EP technique offers multiple advantages: simplicity, high cell viability, high effectiveness, high specificity, rapid action, usefulness with adherent or non-adherent cells, and no requirement for purification of antibodies from antiserum.

New Delhi metallo-β-lactamases (NDMs), the recent additions to metallo-β-lactamases (MBLs), pose a serious public health threat due to its highly efficient hydrolysis of β-lactam antibiotics and rapid worldwide dissemination. The MBL-hydrolyzing mechanism for carbapenems is less studied than that of penicillins and cephalosporins. Here, we report crystal structures of NDM-1 in complex with hydrolyzed imipenem and meropenem, at resolutions of 1.80-2.32 Å, together with NMR spectra monitoring meropenem hydrolysis. Three enzyme-intermediate/product derivatives, EI1, EI2, and EP, are trapped in these crystals. Our structural data reveal double-bond tautomerization from Δ2 to Δ1, absence of a bridging water molecule and an exclusive β-diastereomeric product, all suggesting that the hydrolytic intermediates are protonated by a bulky water molecule incoming from the β-face. These results strongly suggest a distinct mechanism of NDM-1-catalyzed carbapenem hydrolysis from that of penicillin or cephalosporin hydrolysis, which may provide a novel rationale for design of mechanism-based inhibitors.