Spider silk is protein fibers with extraordinary mechanical properties. Up to now, it is still poorly understood how silk proteins are kept in a soluble form before spinning into fibers and how the protein molecules are aligned orderly to form fibers. Minor ampullate spidroin is one of the seven types of silk proteins, which consists of four types of domains: N-terminal domain, C-terminal domain (CTD), repetitive domain (RP) and linker domain (LK). Here we report the tertiary structure of CTD and secondary structures of RP and LK in aqueous solution, and their roles in protein stability, solubility and fiber formation. The stability and solubility of individual domains are dramatically different and can be explained by their distinct structures. For the tri-domain miniature fibroin, RP-LK-CTD(Mi), the three domains have no or weak interactions with one another at low protein concentrations (<1 mg/ml). The CTD in RP-LK-CTD(Mi) is very stable and soluble, but it cannot stabilize the entire protein against chemical and thermal denaturation while it can keep the entire tri-domain in a highly water-soluble state. In the presence of shear force, protein aggregation is greatly accelerated and the aggregation rate is determined by the stability of folded domains and solubility of the disordered domains. Only the tri-domain RP-LK-CTD(Mi) could form silk-like fibers, indicating that all three domains play distinct roles in fiber formation: LK as a nucleation site for assembly of protein molecules, RP for assistance of the assembly and CTD for regulating alignment of the assembled molecules.

Phaeocystis globosa blooms have frequently occurred along coastal waters and exerted serious impacts on ecological environments by releasing toxic hemolytic substances, forming nuisance foam, and causing oxygen depletion. An actinomycete strain RPS with high algicidal activity against P. globosa was isolated and identified as Streptomyces alboflavus, based on morphology, physiological and biochemical characteristics, and 16S rDNA sequence analysis. RPS lysed 95% of P. globosa within 48 h by releasing an extracellular active substance into the growth medium. The activity of RPS supernatant was sensitive to temperature at and above 50 °C and insensitive to pH from 3 to 11. The molecular weight of the active substance was between 100 Da and 1000 Da, and approximately 90% of it was extracted by ethyl acetate. It was presumed that the active component efficiently inhibited the movement of P. globosa, caused the flagella to fall off the algae, and finally lysed the algal cells. RPS showed a wide target range against harmful algae. S. alboflavus RPS with high algicidal activity and such novel features of temperature and pH sensitivity, low molecular weight, algicidal process, and target range possesses great potential in the biological control of P. globosa blooms.

An assay is described for the enzyme tryptophan decarboxylase from plant cell suspension cultures. It is based on the fluorometric detection of tryptamine by HPLC on a LiChrosorb RP-8 Select B column. Tryptophan decarboxylase from Catharanthus roseus was induced by transferring 14-day-old cells into an induction medium. Optimum activity was found 2 days after transfer, the increase being 5- to 10-fold. When kept at -15 degrees C the crude enzyme lost half its activity in about 7 days. The rate of the decarboxylation reaction was linear for at least 3 h at 35 degrees C.

Recombinant monoclonal antibodies (mAbs) are an emerging therapeutic area. However, there are few reports on disulfide bond assignment of recombinant mAbs. This work describes the complete disulfide bond assignment of a recombinant immunoglobulin G4 (IgG4) mAb. N-ethylmaleimide (NEM) was used to mask free sulfhydryl groups present in the mAb. Digestion of the mAb with endoproteinase Lys-C without disulfide scrambling was achieved by denaturing the mAb in the presence of NEM in guanidine hydrochloride (GuHCl). The Lys-C digest was subsequently reduced with dithiothreitol (DTT). Native and reduced Lys-C digests were mass analyzed by on-line reversed-phase-high-performance liquid chromatography mass spectrometry (RP-HPLC/MS). Disulfide-containing peptides were sequenced by off-line nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoESI-QTOF MS) and N-terminal Edman sequencing for verifying connectivities. The recombinant IgG4 mAb was found to contain the expected disulfide linkages with the proposed method. The NEM alkylating reagent was critical in minimizing disulfide scrambling during the denaturation and digestion of the mAb. This integrated approach, combining MS and N-terminal Edman sequencing, was capable of assigning the disulfide pattern of the IgG4 mAb rapidly and completely, and should be applicable for disulfide bond assignment and structural analysis of other mAbs and large proteins with multiple disulfide bonds.

BACKGROUND

Phytophthora root and stem rot (PRR) of soybean, caused by Phytophthora sojae, is controlled by Rps genes. However, little is known regarding the Rps-induced molecular responses to P. sojae and how they actually overlap. We thus sequenced, analyzed, and compared the transcriptomes of 10 near isogenic lines (NILs), each with a unique Rps gene/allele, and the susceptible parent Williams, pre- and post-inoculation with the pathogen.

RESULTS

A total of 4,330 differentially expressed genes (DEGs) were identified in Williams versus 2,014 to 5,499 DEGs in individual NILs upon inoculation with the pathogen. Comparisons of the DEGs between the NILs and Williams identified incompatible interaction genes (IIGs) and compatible interaction genes (CIGs). Hierarchical cluster and heatmap analyses consistently grouped the NILs into three clusters: Cluster I (RpsRpsRpsRps-induced defense signaling among certain NILs. Gene ontology (GO) analysis revealed associations between members of the WRKY family and incompatible reactions and between a number of phytohormone signaling pathways and incompatible/compatible interactions. These associations appear to be distinguished according to the NIL clusters.

CONCLUSIONS

This study characterized genes and multiple branches of putative regulatory networks associated with resistance to P. sojae in ten soybean NILs, and depicted functional "fingerprints" of individual Rps-mediated resistance responses through comparative transcriptomic analysis. Of particular interest are dramatic variations of detected DEGs, putatively involved in ethylene (ET)-, jasmonic acid (JA)-, (reactive oxygen species) ROS-, and (MAP-kinase) MAPK- signaling, among these soybean NILs, implicating their important roles of these signaling in differentiating molecular defense responses. We hypothesize that different timing and robustness in defense signaling to the same pathogen may be largely responsible for such variations.

The Mann-Whitney U-test is ubiquitous in statistical practice for the comparison of measures of location for two samples where the assumption of normality is questionable. Frequently, one has replicate data for each individual in a group and would like to compare measures of central tendency between groups without assuming normality. For this purpose, we present a generalization of the Mann-Whitney U-test for clustered data. The test is performed by computing zc = (Wc - mu c)/sigma c, approximately N(0, 1) under H0, where Wc, mu c are the observed and expected Mann-Whitney U-statistic based on a comparison of all pairs of replicates in the two groups and sigma c is the standard deviation of Wc that is modified to account for clustering effects within a cluster. We obtain an explicit variance formula that is a function of four clustering parameters. We validate the properties of the test procedure in a simulation study. We illustrate the methods with an example comparing the baseline Humphrey visual field between two treatment groups in a randomized clinical trial of patients with retinitis pigmentosa (RP).

Due to the large variability in the reported contribution of bladder dysfunction to postprostatectomy incontinence and the impact this dysfunction may have on the outcome of selected treatment, we retrospectively reviewed the videourodynamic findings of bladder and sphincteric function in patients with postprostatectomy incontinence. The contributions of bladder and sphincteric causes of incontinence are determined. Ninety-two patients had multichannel videourdynamic testing performed as part of a comprehensive evaluation for incontinence at least 1 year after prostatectomy. Using a 6-French double-lumen catheter in the bladder and a 10-French catheter in the rectum, all pressures were recorded continuously while in the upright position. Valsalva leak point pressures (VLPP) were measured in the absence of a bladder contraction at a 150-ml volume and at 50-ml increments thereafter until maximum functional capacity was reached. Bladder compliance and bladder capacity were determined and the presence of detrusor instability (DI) was documented. Sixty-five patients (71%) presented after radical prostatectomy (RP) and 27 patients (29%) after transurethral resection of the prostate (TURP). The predominant urodynamic finding was sphincteric incompetence as VLPP were obtained in 85 patients (92%) and ranged from 12 to 120 cm water. DI was a common finding, occurring in 34 patients (37%), and classified as follows: a) phasic instability in 22/34, b) tonic instability in 3/34, and c) mixed phasic and tonic instability in 9/34. However, we found DI to be the sole cause of incontinence in only 3/92 patients (3.3%). There was no statistically significant difference in the incidence of sphincteric incompetence after RP or TURP; however, TURP patients had a higher incidence of DI, which was statistically significant (P=0.019). There was no correlation of incontinence severity and VLPP when comparing preoperative pad usage to VLPP < or =70 or>> or =71 cm water. Although bladder dysfunction may be contributing problem in patients with postprostatectomy incontinence, it is rarely the only mechanism for this disorder. VLPP does not correlate with incontinence severity. Although sphincteric incompetence is the most common mechanism contributing to incontinence after prostatectomy, bladder dysfunction may coexist or be an isolated cause of postprostatectomy incontinence. Therefore, urodynamic studies are important to illustrate the exact cause(s) of incontinence in each individual patient after prostatectomy.

We aimed to evaluate the efficacy and safety of oxycodone/acetaminophen (O/A) and codeine/acetaminophen (C/A) vs. conventional therapy (CT) without opioids in older women suffering from osteoarthritis (OA)-related pain, sub-optimally responsive to prior conventional treatments. We performed a 6 week, randomized, single blind, controlled study in three nursing homes. We enrolled 154 women with painful OA. They were assigned to treatment with O/A (n=52) and C/A (n=52) vs. CT (n=50). We evaluated at baseline and at week 6: average pain in the last week (mean pain, MeP), pain at rest (RP), pain in movement (MP) (numeric rating scale, NRS); depressive symptoms (Beck Depression Inventory-II, BDI-II); functional status (activities of daily living, ADL) and cognitive status (mini mental state evaluation, MMSE). We considered the adverse events (AEs) in the study period. At week 6, MeP, RP and MP were significantly reduced in all three groups (p<0.001); compared to CT, O/A and C/A were associated with greater reductions in MeP (p<0.001 and p=0.004, respectively), in RP (p=0.028 and p=0.032, respectively) in MP (p<0.001 and p=0.002, respectively) and with significant improvement in BDI-II score (p=0.05 and p=0.04, respectively) and ADL value (p=0.04 and p=0.05, respectively). AE rates did not differ between groups.

Mutations in the rhodopsin gene (RHO) are suggested to be the most common cause of autosomal dominant retinitis pigmentosa (RP). However, the exact spectrum and frequency of RHO mutations in different forms of RP has not been well defined, especially in Chinese populations. In this study, direct cycle sequencing was used to analyze all five coding exons and adjacent intronic regions of RHO in 248 Chinese probands with different forms of non X-linked RP. Eight heterozygous nucleotide changes were detected, including two novel mutations (c.628G>T, p.Val210Phe; c.945C>G, p.Asn315Lys) and six known mutations (c.527C>T, p.Ser176Phe; c.568G>T, p.Asp190Tyr; c.768_770delCAT, p.Ile256del; c.1040C>T, p.Pro347Leu; c.310G>A, p.Val104Ile; c.895G>T, p.Ala299Ser), in 14 probands (nine isolated cases, three from autosomal dominant families, and three from autosomal recessive families). Of the eight mutations, seven were missense changes and one was a small deletion. Six may be pathogenic mutations, and two others (c.310G>A, c.895G>T) may not be causative on their own. The p.Ala299Ser change was present in six of the 248 probands with RP but only in one of 384 normal controls, while the p.Val104Ile was present in two probands but none of the 384 controls. Our results demonstrate an overview of the spectrum and frequency of RP RHO mutations in a Chinese population and emphasize that RHO mutations in isolated RP are not uncommon.

Genetic counselling endeavours to be nondirective. However, the availability of prenatal diagnosis may direct clients towards accepting and using these methods. It is time to investigate the attitudes of clients in order to monitor the psychological and social effects of new genetic techniques. As prenatal diagnosis was possible for choroideremia (C), but not for retinitis pigmentosa (RP) in 1988-89, we used a questionnaire to compare the attitudes of C and RP patients, their relatives and C carriers to prenatal diagnosis. The response rate was low (35%) and no significant differences between RP and C groups came to light. However, C carriers accepted prenatal diagnosis and also selective abortion more easily, but, on the other hand, they showed more uncertainty than did the other groups. This indicates that the availability of prenatal diagnosis may confuse those concerned. In general, about 60% of all the respondents had a positive attitude to the prenatal diagnosis of RP or choroideremia, though only about 30% would use if for abortion. Over 80% of all the respondents wanted to know the opinion of the genetic counsellor.

The value of reversed-phase high-performance liquid chromatography (RP-HPLC) and the field of proteomics would be greatly enhanced by accurate prediction of retention times of peptides of known composition. The present study investigates the hydrophilicity/hydrophobicity of amino acid side-chains at the N- and C-termini of peptides while varying the functional end-groups at the termini. We substituted all 20 naturally occurring amino acids at the N- and C-termini of a model peptide sequence, where the functional end-groups were N(alpha)-acetyl-X- and N(alpha)-amino-X- at the N-terminus and -X-C(alpha)-carboxyl and -X-C(alpha)-amide at the C-terminus. Amino acid coefficients were subsequently derived from the RP-HPLC retention behaviour of these peptides and compared to each other as well as to coefficients determined in the centre of the peptide chain (internal coefficients). Coefficients generated from residues substituted at the C-terminus differed most (between the -X-C(alpha)-carboxyl and -X-C(alpha)-amide peptide series) for hydrophobic side-chains. A similar result was seen for the N(alpha)-acetyl-X- and N(alpha)-amino-X- peptide series, where the largest differences in coefficient values were observed for hydrophobic side-chains. Coefficients derived from substitutions at the C-terminus for hydrophobic amino acids were dramatically different compared to internal coefficients for hydrophobic side-chains, ranging from 17.1 min for Trp to 4.8 min for Cys. In contrast, coefficients derived from substitutions at the N-terminus showed relatively small differences from the internal coefficients. Subsequent prediction of peptide retention time, within an error of just 0.4 min, was achieved by a predictive algorithm using a combination of internal coefficients and coefficients for the C-terminal residues. For prediction of peptide retention time, the sum of the coefficients must include internal and terminal coefficients.

Although it has been known for decades that arsenic forms fat-soluble arsenic compounds, only recent attempts to identify the compounds have been successful by using a combination of fractionation and elemental and molecular mass spectrometry. Here we show that arsenolipids can directly be identified and quantified in biological extracts using reversed-phase high-performance liquid chromatography (RP-HPLC) simultaneously online-coupled to high-resolution inductively coupled plasma mass spectrometry (ICPMS) and high-resolution electrospray mass spectrometry (ES-MS) without having a lipophilic arsenic standard available. Using a methanol gradient for the separation made it necessary to use a gradient-dependent arsenic response factor for the quantification of the fat-soluble arsenic species in the extract. The response factor was obtained by using the ICPMS signal of known concentration of arsenic. The arsenic response was used to determine species-specific response factors for the different arsenic species. The retention time for the arsenic species was utilized to mine the ES-MS data for accurate mass and their tandem mass spectrometry (MS/MS) fragmentation pattern to give information of molecular formula and structure information. The majority of arsenolipids, found in the hexane phase of fish meal from capelin ( Mallotus villosus ) was in the form of three dimethylarsinoyl hydrocarbons (C(23)H(38)AsO, C(17)H(38)AsO, C(19)H(42)AsO) with minor amounts of dimethylarsinoyl fatty acids (C(17)H(36)AsO(3), C(23)H(38)AsO(3), C(24)H(38)AsO(3)). One of the dimethylarsinoyl fatty acids (C(24)H(38)AsO(3)), with an even number of carbon in the fatty acid chain, was identified for the first time in this work. This molecular formula is unusual and in contrast to all previously identified arsenic-containing fatty acids with odd numbers of carbon.

In this paper, we characterize the polarization impedance behavior of several common metals in diluted NaCl solution operated at low current densities. The objective was to provide a useful reference for those wishing to calculate the electrode polarization impedance in diluted NaCl solutions. Serial equivalent resistance (R) and capacitance (C) for silver, aluminum, gold, platinum, and medical stainless-steel were measured as a function of frequency (10(-2)-10(3) Hz) and NaCl concentration (2.4-77.0 mmol/L). The ratio of electrode polarization impedance with respect to the bulk resistance was calculated and plotted against concentration for each metal. Such a ratio shows the effect of the electrode polarization contribution as a function of electrolyte concentration when the bulk resistance of the solution changes. All metals showed a decrease of serial resistance Rp and capacitance Cp as a function of frequency. The medical stainless-steel electrode showed largest impedance values at lower frequencies compared to the other electrodes, and was concentration independent at all frequencies. Aluminum had smallest polarization impedance at low frequencies. Pure gold and platinum behaved similar with the exception that the serial resistance for gold showed a lower value at higher frequencies.

We investigated the effects of the vasoconstrictor endothelin-1 (ET-1) on the whole-cell ATP-sensitive K+ (KATP) currents of smooth muscle cells that were isolated enzymatically from rabbit coronary artery (CASMCs) and pulmonary artery (PASMCs). The size of the KATP current did not differ significantly between CASMCs and PASMCs. ET-1 reduced the KATP current in a concentration-dependent manner, and this inhibition was greater in PASMCs than in CASMCs (half-inhibition values of 12.20 nM and 1.98 nM in CASMCs and PASMCs, respectively). However, the level of inhibition induced by other vasoconstrictors (angiotensin II, norepinephrine, and serotonin) were not significantly different between CASMCs and PASMCs. Pretreatment with the protein kinase C (PKC) inhibitors staurosporine (100 nM) and GF 109203X (1 microM) prevented ET-1-induced inhibition of the KATP current in both arterial smooth muscle cell preparations. The PKC activators phorbol-12,13-dibutyrate (PDBu) and 1-olelyl-2-acetyl-sn-glycerol (OAG) reduced the KATP current in dose-dependent manner. Although the numbers of ET receptors were not significantly different between the 2 arterial smooth muscle cell preparations, the effects of PDBu and OAG were greater on PASMCs. ET-1-induced inhibition of the KATP current was unaffected by the PKA inhibitor Rp-cAMPs (100 microM) and PKA inhibitory peptide (5 microM).

1. Intrathecal (i.t.) injections of the (tachykinin) NK1 receptor agonist, substance P methyl ester (SPME; 20 pmol), or the NK2 receptor agonist, neurokinin A (NKA; 20 pmol), substantially decreased the cutaneous mechanical threshold and markedly enhanced the touch-evoked response of posterior biceps femoris-semitendinosus (PBF-ST) spinal flexor motoneurones in decerebrate-spinal rats. This cutaneous mechanical reflex allodynia was prevented by pretreatment with the NK1 antagonist RP 67580 (2.28 nmol, i.t.) and the NK2 antagonist MEN 10376 (0.7 nmol, i.t.), respectively. 2. Electrical stimulation of the sural nerve at C fibre strength or cutaneous application of the irritant, mustard oil, produced prolonged cutaneous mechanical allodynia in PBF-ST motoneurones (15 min and>> 1 h, respectively). Pretreatment with RP 67580 but not MEN 10376 prevented this, but when RP 67580 was administered 25 min after the application of mustard oil, the established hypersensitivity of the flexor motor reflex was not reversed. The enantiomer of RP 67580, RP 68651 was without effect. 3. Injection of bradykinin (60 microM, 80 microliters) into the gastrocnemius muscle increased the cutaneous mechanical hypersensitivity of PBF-ST flexor motoneurones for 40-50 min. MEN 10376, but not RP 67580, prevented this, but only when administered prior to the bradykinin injection. 4. These results suggest that the induction, but not the maintenance, of cutaneous mechanical allodynia in flexor motoneurones is NK receptor dependent, with cutaneous C fibre conditioning inputs acting via NK1 and muscle C fibre conditioning inputs via NK2 receptor subtypes.

We compared the effectiveness in changing human immunodeficiency virus (HIV) risk behavior of two different approaches to acquired immunodeficiency syndrome (AIDS) education given by residential drug abuse treatment programs differing in their planned duration. Two randomized controlled trials compared (a) a 6-month with a 12-month therapeutic community (TC) program, and (b) a 6-month with a 3-month relapse prevention (RP) program. Three composite variables assessing HIV risk (a) drug injection, (b) sexual partners, and (c) condom use-were measured for the 3 months prior to admission and follow-up. The TC program comprised a four-part AIDS information intervention. The RP program comprised a 21- or 42-session small-group program in the principles of RP, 5 skills-building AIDS education sessions, and 6 other health education sessions. Four hundred ninety-five clients were enrolled in the study and completed a follow-up interview within 6 months of exit (79% of those enrolled). Clients in the RP program reduced their drug injection and condom use risk. Female clients in the TC program reduced their condom use risk. There were no differential effects on risk behavior change of either planned duration (randomization assignment) or program type (RP versus TC). Thus, differences in the treatment programs, including AIDS education components, had no apparent effect on HIV risk behavior change. The contribution of residential drug abuse treatment programs to AIDS prevention remains unproved.

As life expectancy continually increases, it is imperative to identify determinants of survival to the extreme end of the lifespan and more importantly to identify factors that increase the chance of survival free of major morbidities. As such, the current study assessed 45 common disease factors as predictors of survival and morbidity-free survival to age 85 years. Within the Rotterdam Study, a population-based cohort, we evaluated morbidity-free participants who were able to attain age 85 within the study duration (n = 2,008). Risk factors were assessed at baseline (1990-1993), and mortality and morbidities were then collected continuously until mortality or the occurrence of their 85th birthday (average time of 7.9 years). Risk factors included demographic and lifestyle variables, health and morbidity indicators and physiological makers. Major morbidities examined included dementia, cancer, cerebrovascular accident, heart failure and myocardial infarction. Logistic regression analyses demonstrated that many of the variables were independently predictive for survival and for morbidity-free ageing to 85 years. These included being female, absence of left ventricular abnormalities, stable body weight, unimpaired instrumental activities of daily living, lower C-RP levels and higher levels of femoral neck bone mineral density and albumin. Relative to non-survival, predictors were stronger for morbidity-free survival than for total survival or survival with morbidity. This suggests that lifespan and healthy survival to older age can be relatively well predicted. Understanding predictors of a long and healthy lifespan is vital for developing primary and secondary preventions to help improve the quality of life of older adults and for reducing the financial burden of the rapidly escalating ageing population.

The prevalence of Raynaud's phenomenon (RP) was studied in patients treated with cisplatin, vinblastine, and bleomycin. Thirty-two patients with germ cell cancer were followed for a median of 78 months (range, 49 to 106 months). All had obtained complete remission during treatment; none had relapsed at last follow-up examination. All blood samples, including serum magnesium, were normal. The arterial vasoconstrictor response to cold in the finger was measured by cuff- and strain-gauge techniques at 30, 15, and 10 degrees C. Blood pressure (BP) was measured auscultatorily, using a 12-cm broad cuff on the ipsilateral upper arm. Fourteen patients (44%) had developed anamnestic RP, and all showed an exaggerated response to cold; arterial closure was provoked in nine patients. Eighteen patients (56%) were without finger symptoms. These patients had an exaggerated response to cold in comparison with controls, and in two patients, RP was provoked. None of the patients had an increased systolic pressure gradient from arm to finger when compared with a control group. Thus, an exaggerated cold response was found to be a prolonged vasospastic side effect not only in patients with RP, but also in patients without finger symptoms.

BACKGROUND

Atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory and cytoprotective potential. The aim of this study was to characterize induction of heme oxygenase (HO)-1 by ANP in human umbilical vein endothelial cells (HUVEC).

METHODS

HUVEC were treated with ANP, 8-bromo-cyclic GMP (cGMP), or cANF in the presence or absence of various inhibitors. HO-1 was determined by Western blot and RT-PCR, c-jun N-terminal kinase (JNK) and ERK by the use of phospho-specific antibodies. Activator protein (AP)-1 activation was assessed by gelshift assay. Reporter gene assays were performed using native or mutated AP-1 binding sites of the HO-1 promoter. TNF-alpha-induced cell death was investigated by Hoechst staining, fluorescence-activated cell sorting analysis, caspase-3-measurement, and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide test.

RESULTS

ANP (10(-9)-10(-6) mol/liter) induced the expression of HO-1 protein and mRNA. Induction was mediated via the guanylate-cyclase-coupled receptor because 8-Br-cGMP mimicked the effect of ANP, whereas the clearance receptor agonist cANF did not induce HO-1. Endogenously produced cGMP also induced HO-1 because phosphodiesterase inhibition markedly elevated HO-1. The lack of effect of the cGMP-dependent protein kinase inhibitor 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-pCT-cGMPS) suggested no involvement for this cGMP effector pathway in the signal transduction. ANP lead to activation of the transcription factor AP-1, and subsequently of JNK, as well as of ERK. Cotreatment of the cells with U0126 or SP600125, as well as reporter gene assays revealed the involvement of AP-1/JNK activation in HO-1 induction. Abrogation of HO-1 induction by PD-98059 showed also a role for ERK. Treatment of HUVEC with ANP did not protect from TNF-alpha-induced apoptosis.

CONCLUSIONS

This work characterizes the induction of HO-1 by ANP in HUVEC, which is shown to be mediated via JNK/AP-1 and ERK pathways. ANP-induced HO-1 does not confer protection against TNF-alpha-induced apoptosis.

Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70-80%), NMDA-induced inward currents were inhibited by the adenosine A2A receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the A2A receptor selective antagonist 8-(3-chlorostyryl)caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP-beta-S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14-24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca2+ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, A2A receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca2+ pathway rather than the adenylate cyclase/PKA pathway.

Increased renal pelvic pressure or bradykinin increases afferent renal nerve activity (ARNA) via PGE(2)-induced release of substance P. Protein kinase C (PKC) activation increases ARNA, and PKC inhibition blocks the ARNA response to bradykinin. We now examined whether bradykinin mediates the ARNA response to increased renal pelvic pressure by activating PKC. In anesthetized rats, the ARNA responses to increased renal pelvic pressure were blocked by renal pelvic perfusion with the bradykinin B(2)-receptor antagonist HOE 140 and the PKC inhibitor calphostin C by 76 +/- 8% (P < 0.02) and 81 +/- 5% (P < 0.01), respectively. Renal pelvic perfusion with 4beta-phorbol 12,13-dibutyrate (PDBu) to activate PKC increased ARNA 27 +/- 4% and renal pelvic release of PGE(2) from 500 +/- 59 to 1, 113 +/- 183 pg/min and substance P from 10 +/- 2 to 30 +/- 2 pg/min (all P < 0.01). Indomethacin abolished the increases in substance P release and ARNA. The PDBu-mediated increase in ARNA was also abolished by the substance P-receptor antagonist RP 67580. We conclude that bradykinin contributes to the activation of renal pelvic mechanosensitive neurons by activating PKC. PKC increases ARNA via a PGE(2)-induced release of substance P.

Primary cultures of prepubertal rat Sertoli cells secrete two major tissue inhibitors of metalloproteinases: TIMP-1 (M(r) 28K) and TIMP-2 (M(r) 21 K). FSH stimulated Sertoli cell TIMP-1 and TIMP-2 activity in a time- and dose-dependent manner and also stimulated TIMP-1 and TIMP-2 protein and messenger RNA levels. These effects were mimicked by the cAMP analog, 8-bromo-cAMP, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The protein kinase C activating phorbol ester phorbol myristate acetate (TPA) stimulated TIMP-1 but not TIMP-2 activity and messenger RNA levels. Cycloheximide and actinomycin-D inhibited basal TIMP-1 and TIMP-2 activity and inhibited the ability of FSH, 8-bromo-cAMP, and TPA to stimulate TIMP activity. The protein kinase A (PKA) inhibitor AMP Rp isomer did not affect basal TIMP-1 and TIMP-2 activity or TPA-stimulated TIMP-1 activity. However, the PKA inhibitor markedly reduced FSH and 3-isobutyl-1-methylxanthine stimulation of TIMP-1 and TIMP-2 activity. FSH, 8-bromo-cAMP, and TPA stimuli induced DNA binding complexes capable of binding to a TIMP-1 AP-1 site consensus sequence oligonucleotide. The AP-1 site binding complex(es) induced by all three treatments reacted with antibodies directed broadly against fos and jun protooncogene families and against the specific family members c-fos, junB, and junD but not c-jun proteins. Constitutive cAMP response element binding activity capable of binding an artificial cAMP response element binding site oligonucleotide was demonstrated in Sertoli cell nuclear extracts. This activity was minimally modulated by FSH, 8-bromo-cAMP, or TPA treatment. In summary, Sertoli cells secrete TIMP-1 and TIMP-2 that can be coordinately up-regulated by FSH through a cAMP, PKA-dependent pathway. a convergence of TPA, FSH, and cAMP mediated signals in prepubertal Sertoli cells may occur with the induction of specific AP-1 site binding complex(es) containing jun and fos proteins. Our data suggest that FSH stimulation of TIMP-2 expression may be regulated independently to that of TIMP-1. We propose that the ability of FSH to stimulate Sertoli cell TIMP activity suggests a central role for this hormone in the control of extracellular matrix turnover during testicular development at the level of metalloproteinase inhibition.

Complex biological samples, such as urine, contain a very large number of endogenous metabolites reflecting the metabolic state of an organism. Metabolite patterns can provide a comprehensive signature of the physiological state of an organism as well as insights into specific biochemical processes. Although the metabolites excreted in urine are commonly highly polar, the samples are generally analyzed using reversed-phase liquid chromatography mass spectrometry (RP-LC/MS). In Part 1 of this work, a method for detecting highly polar metabolites by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) is described as a complement to RP-LC/ESI-MS. In addition, in an accompanying paper (Part 2), different multivariate approaches to extracting information from the resulting complex data are described to enable metabolic fingerprints to be obtained. The coverage of the method for the screening of as many metabolites as possible is highly improved by analyzing the urine samples using both a C(18) column and a ZIC-HILIC column. The latter was found to be a good alternative when analyzing highly polar compounds, e.g., hydroxyproline and creatinine, to columns typically used for reversed-phase liquid chromatography.

Hemoglobin, α-chain, β-chain and fragmented hemoglobin of Crocodylus siamensis demonstrated both antibacterial and antioxidant activities. Antibacterial and antioxidant properties of the hemoglobin did not depend on the heme structure but could result from the compositions of amino acid residues and structures present in their primary structure. Furthermore, thirteen purified active peptides were obtained by RP-HPLC analyses, corresponding to fragments in the α-globin chain and the β-globin chain which are mostly located at the N-terminal and C-terminal parts. These active peptides operate on the bacterial cell membrane. The globin chains of Crocodylus siamensis showed similar amino acids to the sequences of Crocodylus niloticus. The novel amino acid substitutions of α-chain and β-chain are not associated with the heme binding site or the bicarbonate ion binding site, but could be important through their interactions with membranes of bacteria.