Cytotoxicity, apoptosis, cellular uptake, cell cycle arrest, photocleavage, and antioxidant activity of 1, 10-phenanthroline ruthenium(II) complexes

Two new ruthenium(II) complexes, [Ru(phen)2(DNPIP)](ClO4)2 (1) and [Ru(phen)2(DAPIP)](ClO4)2 (2), were synthesized and characterized. The DNA-binding properties of these complexes were investigated using UV/vis absorption titration, viscosity measurements, thermal denaturation, and photoactivated cleavage. The DNA binding constants for complexes 1 and 2 are 2.63?±?0.25×10(5) M(-1) (s=2.45) and 1.51±0.18×10(5) M(-1) (s=1.34). The results indicated that these complexes interacted with DNA through the intercalative mode. The cytotoxicity in vitro of complexes 1 and 2 were assessed against BEL-7402, HepG-2, and MCF-7 cell lines by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was studied with the acridine orange/ethidium bromide staining method. The antiproliferative mechanism was explored with flow cytometry. Cellular uptake studies showed that complexes 1 and 2 can enter into the cytoplasm and accumulate in the nuclei. Cell cycle arrest and antioxidant activity were also investigated.     

Subcloning,expression, purification,and characterization of recombinant human leptin-binding domain

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.     

Contamination of Floodplain Soils along the Wupper River,Germany, with As,Co, Cu,Ni, Sb,and Zn and the Impact of Pre-definite Redox Variations on the Mobility of These Elements

This study demonstrates that floodplain soils of the River Wupper, Germany, are seriously contaminated with metal(loid)s. We used an automated biogeochemical microcosm system allowing controlled variation of redox potential (E_H) to assess the impact of pre-definite redox conditions on the dynamics of arsenic (As), cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), antimony (Sb), and zinc (Zn) in homogenized soil material taken from an acid floodplain soil. The concentrations of Co, Cu, Mn, Ni, Sb, and Zn in soil solution were low at low E_H, possibly due to the precipitation of metal sulfides, and increased with rising E_H, presumably caused by their association with dissolved organic carbon (DOC). A significant positive correlation between metal/DOC-ratio and E_H indicated that the binding of the metals to DOC shifted from stronger to weaker when E_H rose. Decreasing As concentrations with increasing E_H in soil solution indicated co-precipitation with Fe(hydr)oxides and/or oxidation of more soluble As(III) to less soluble As(V) during oxidation. The other studied elements seemed not to co-precipitate with newly formed Fe(hydr)oxides when E_H rose, possibly due to the prevailing low pH. In the future, the specific role of DOC and sulfur chemistry on metal(loid) dynamics should be elucidated more fully, and similar studies should be conducted with additional frequently flooded soils worldwide to verify these results.     

The effects of cryopreservation on the acrosome structure,enzyme activity,motility, and fertility of bovine,ovine, and goat sperm

The study was designed to investigate the effects of cryopreservation on bovine, ovine, and goat sperm motility, acrosome structure, enzyme activity, and fertilization ability. Percentage of sperm with hyaluronidase enzyme (HYD) activity was detected by a modified sodium hyaluronate-gelatin membrane. The N-α-benzoyl-DL-arginine-p-nitroanilide (BNPNA) method was used to assess the sperm acrosome enzyme (ACE). The mean percentage of sperm acrosome integrity dropped significantly (P < 0.01) after cryopreservation. The ACE activity of bovine sperm (100.48) was higher (P < 0.01) than that of ovine (57.88) or goat sperm (50.30), while the percentage of sperm with HYD activity of bovine (71.10%) and ovine (67.60%) sperm was higher than that of goat sperm (58.52%) after cryopreservation (P < 0.01). Sperm motility was positively correlated with the activity of the two acrosome enzymes before and after cryopreservation (P < 0.01). Cryopreservation had a negative effect on acrosomal morphology, motility, and acrosomal enzyme activity in their sperm. The fertilization ability of ovine and goat sperm decreased significantly after cryopreservation, but that of frozen bovine sperm did not differ significantly when compared with fresh sperm. There was no significant difference between ovine and goat sperm indices, except for percentage of sperm with HYD activity.     

Rat heparan sulphates. A study of the antithrombin-binding properties of heparan sulphate chains from rat adipose tissue, brain, carcase, heart, intestine, kidneys, liver, lungs, skin and spleen.

Adult male rats were given [35S]sulphate intraperitoneally. Heparan [35S]sulphate (HS) chains were recovered from adipose tissue, brain, carcase, heart, intestine, kidneys, liver, lungs, skin and spleen by digestion with Pronase, precipitation with cetylpyridinium chloride, digestion with chondroitin ABC lyase and DNAase and gradient elution from DEAE-Sephacel. Purity was confirmed by agarose-gel electrophoresis and degradation with HNO2. Fractionation by gradient elution from antithrombin-agarose indicated that the proportion of HS with high binding affinity for antithrombin (HA-HS) ranged from 4.7% (kidneys) to 21.5% (brain). On a mass basis the major sources of HA-HS were carcase, skin and intestine. HA-HS from intestine was arbitrarily divided into subfractions I-VI, with anticoagulant activities ranging from 1 to 60 units/mg [by amidolytic anti-(Factor IIa) assay] and from 4 to 98 units/mg [by amidolytic anti-(Factor Xa) assay], indicating that the antithrombin-binding-site densities of HA-HS chains covered a wide range, as shown previously for rat HA-heparin chains [Horner, Kusche, Lindahl & Peterson (1988) Biochem. J. 251, 141-145]. HA-HS subfractions II, IV and VI were mixed with samples of HA-[3H]heparin chains and rechromatographed on antithrombin-agarose. Affinity for matrix-bound antithrombin did not correlate with anticoagulant activity, e.g. HA-HS subfraction IV [38 anti-(Factor Xa) units/mg] was co-eluted with HA-heparin chains [127 anti-(Factor Xa) units/mg].     

A method for concurrent measurement of picomole quantities of acetylcholine, choline, dopamine, norepinephrine, serotonin, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, tryptophan, tyrosine, glycine, aspartate, glutamate, alanine, and gamma-aminobutyric acid in single tissue samples from different areas of rat central nervous system.

A rapid and sensitive method for separation and concurrent assay of 14 compounds at the picomole level in individual rat brain parts is described. The putative amino acid neurotransmitters (aspartate, γ-aminobutyric acid, glutamate, and glycine) are extracted from a 20–30 mg portion of the tissue with 5% TCA and assayed as their respective DNP-amino acid methyl ester derivatives by glc. Four other putative neurotransmitters (acetylcholine, dopamine, norepinephrine, and serotonin) and some of their precursors and metabolites (choline, tryptophan, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, and tyrosine) are extracted in 1n formic acid-acetone (v/v:15/85) from the remaining tissue. The lipids are removed with a heptane-chloroform (v/v:8/1) wash and the aqueous phase is dried at 37°C under N₂. The dried extract is dissolved in water (pH 4). With one portion of this solution, acetylcholine and choline are assayed using a radioenzymatic method whereas with the rest, dopamine, norepinephrine, serotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, tryptophan, and tyrosine are separated with three ion exchange resins arranged in tandem. These compounds are assayed fluorometrically with modified microadaptations of previously reported methods.     

Quantitation of the involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC genes in the repair of X-ray-induced DNA double-strand breaks in Escherichia coli

Isogenic Escherichia coli strains carrying single DNA-repair mutations were compared for their capacity for (i) the repair of X-ray-induced DNA double-strand breaks (DSB) as measured using neutral sucrose gradients; (ii) medium-dependent resistance, i.e., a recA-dependent X-ray survival phenomenon that correlates closely with the capacity for repairing DSB; and (iii) the growth medium-dependent, recA-dependent repair of X-ray-induced DNA single-strand breaks (SSB) as measured using alkaline sucrose gradients (about 80% of these SSB are actually parts of DSB). These three capacities were measured to quantitate more accurately the involvement of the various genes in the repair of DSB over a wide dose range. The mutations tested were grouped into five classes according to their effect on the repair of X-ray-induced DSB: (I) the recA, recB, recC, and lexA mutants were completely deficient; (II) the radB and recN mutants were about 90% deficient; (III) the recF and recJ mutants were about 70% deficient; (IV) the radA and uvrD mutants were about 30% deficient; and (V) the umuC mutant resembled the wild-type strains in its capacity for the repair of DSB.     

Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues,Cell Lines,FFPE Tissues,Aspirates, Lavages,Effusions, Plasma,Serum, and Urine

DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.     

Purification,reconstitution, and characterization of Na(+)/serine symporter,SstT, of Escherichia coli

A gene encoding Na(+)/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacteriol. 180, 6749-6752]. In an attempt to overproduce the protein and purify it, we first constructed a plasmid pTSTH in which the modified sstT gene (sstT gene with 8 successive codons for His at the 3'-terminus) is located downstream from the trc promoter. Upon induction by IPTG, the His-tagged SstT protein was overproduced (about 15% of total membrane proteins), and showed activity as high as the wild type SstT. The His-tagged SstT was solubilized with octylglucoside and purified to homogeneity using a nickel nitrilotriacetic acid (Ni(2+)-NTA) affinity resin. The N-terminal sequence (20 amino acid residues) of the purified protein showed that the sequence was identical to that deduced from the DNA sequence of the sstT gene and that the initiation methionine was excised. The purified His-tagged SstT was reconstituted into liposomes by the detergent dilution method. Reconstituted proteoliposomes mediated the transport of serine driven by an artificially imposed electrochemical Na(+) gradient. The K(m) and the V(max) values for serine transport with the proteoliposomes were 0.82 microM and 0.37 nmol/min/mg protein, respectively. Serine transport was inhibited by L-threonine, but not by other amino acids. The purified protein was stable for at least 6 months at -80 degrees C.     

Sleep, sleepiness, fatigue, and performance of 12-hour-shift nurses

Nurses working 12-h shifts complain of fatigue and insufficient/poor-quality sleep. Objectively measured sleep times have not been often reported. This study describes sleep, sleepiness, fatigue, and neurobehavioral performance over three consecutive 12-h (day and night) shifts for hospital registered nurses. Sleep (actigraphy), sleepiness (Karolinska Sleepiness Scale [KSS]), and vigilance (Performance Vigilance Task [PVT]), were measured serially in 80 registered nurses (RNs). Occupational fatigue (Occupational Fatigue Exhaustion Recovery Scale [OFER]) was assessed at baseline. Sleep was short (mean 5.5?h) between shifts, with little difference between day shift (5.7?h) and night shift (5.4?h). Sleepiness scores were low overall (3 on a 1-9 scale, with higher score indicating greater sleepiness), with 45% of nurses having high level of sleepiness (score >?7) on at least one shift. Nurses were progressively sleepier each shift, and night nurses were sleepier toward the end of the shift compared to the beginning. There was extensive caffeine use, presumably to preserve or improve alertness. Fatigue was high in one-third of nurses, with intershift fatigue (not feeling recovered from previous shift at the start of the next shift) being most prominent. There were no statistically significant differences in mean reaction time between day/night shift, consecutive work shift, and time into shift. Lapsing was traitlike, with rare (39% of sample), moderate (53%), and frequent (8%) lapsers. Nurses accrue a considerable sleep debt while working successive 12-h shifts with accompanying fatigue and sleepiness. Certain nurses appear more vulnerable to sleep loss than others, as measured by attention lapses.     

Sleep,Sleepiness, Fatigue,and Performance of 12-Hour-Shift Nurses

Nurses working 12-h shifts complain of fatigue and insufficient/poor-quality sleep. Objectively measured sleep times have not been often reported. This study describes sleep, sleepiness, fatigue, and neurobehavioral performance over three consecutive 12-h (day and night) shifts for hospital registered nurses. Sleep (actigraphy), sleepiness (Karolinska Sleepiness Scale [KSS]), and vigilance (Performance Vigilance Task [PVT]), were measured serially in 80 registered nurses (RNs). Occupational fatigue (Occupational Fatigue Exhaustion Recovery Scale [OFER]) was assessed at baseline. Sleep was short (mean 5.5?h) between shifts, with little difference between day shift (5.7?h) and night shift (5.4?h). Sleepiness scores were low overall (3 on a 1–9 scale, with higher score indicating greater sleepiness), with 45% of nurses having high level of sleepiness (score ?>?7) on at least one shift. Nurses were progressively sleepier each shift, and night nurses were sleepier toward the end of the shift compared to the beginning. There was extensive caffeine use, presumably to preserve or improve alertness. Fatigue was high in one-third of nurses, with intershift fatigue (not feeling recovered from previous shift at the start of the next shift) being most prominent. There were no statistically significant differences in mean reaction time between day/night shift, consecutive work shift, and time into shift. Lapsing was traitlike, with rare (39% of sample), moderate (53%), and frequent (8%) lapsers. Nurses accrue a considerable sleep debt while working successive 12-h shifts with accompanying fatigue and sleepiness. Certain nurses appear more vulnerable to sleep loss than others, as measured by attention lapses. (Author correspondence: jgeiger@son.umaryland.edu)     

Anti-plasmodial action of de novo-designed, cationic, lysine-branched, amphipathic, helical peptides

ABSTRACT: BACKGROUND: A lack of vaccine and rampant drug resistance demands new anti-malarials. METHODS: In vitro blood stage anti-plasmodial properties of several de novo-designed, chemically synthesized, cationic, amphipathic, helical, antibiotic peptides were examined against Plasmodium falciparum using SYBR Green assay. Mechanistic details of anti-plasmodial action were examined by optical/fluorescence microscopy and FACS analysis. RESULTS: Unlike the monomeric decapeptides {(Ac-GXRKXHKXWA-NH2) (X = F,DeltaF) (Fm, DeltaFm IC50 >100 muM)}, the lysine-branched,dimeric versions showed far greater potency {IC50 (muM) Fd 1.5 , DeltaFd 1.39}. The more helical and proteolytically stable DeltaFd was studied for mechanistic details. DeltaFq, a K-K2 dendrimer of DeltaFm and (DeltaFm)2 a linear dimer of DeltaFm showed IC50 (muM) of 0.25 and 2.4 respectively. The healthy/infected red cell selectivity indices were >35 (DeltaFd), >20 (DeltaFm)2 and 10 (DeltaFq). FITC-DeltaFd showed rapid and selective accumulation in parasitized red cells. Overlaying DAPI and FITC florescence suggested that DeltaFd binds DNA. Trophozoites and schizonts incubated with DeltaFd (2.5 muM) egressed anomalously and Band-3 immunostaining revealed them not to be associated with RBC membrane. Prematurely egressed merozoites from peptide-treated cultures were found to be invasion incompetent. CONCLUSION: Good selectivity (>35), good resistance index (1.1) and low cytotoxicity indicate the promise of DeltaFd against malaria.     

Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V, and IX with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes; the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V, and the tumor-associated, transmembrane hCA IX, with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate; such as chlorate, perchlorate, bromate, iodate, periodate, silicate, bismuthate, vanadate, molybdate, and wolframate is reported. Apparently, the geometry of the inhibitor (tetrahedral or trigonal) does not influence its binding to the Zn(II) ion of the enzyme active site, but the nature of the central element is the most important factor influencing potency. Isozymes hCA I and II are best inhibited by chlorate, perchlorate, and silicate, together with the anions structurally related to sulfate, sulfamate, and sulfamidate, but sulfate itself is a weak inhibitor (inhibition constant of 74 mM against hCA I and 183 mM against hCA II). Molybdate is a very weak hCA I inhibitor (K(I) of 914 mM) but it interacts with hCA II (K(I) of 27.5mM). Isozyme IV is well inhibited by sulfate (K(I) of 9 mM), sulfamate, and sulfamidate (in the low micromolar range), but not by perchlorate (K(I) of 767 mM). The mitochondrial isozyme V has the lowest affinity for sulfate (K(I) of 680 mM) and carbonate (K(I) of 95 mM) among all the investigated isozymes, suggesting on one hand its possible participation in metabolon(s) with sulfate anion exchanger(s), and on the other hand an evolutionary adaptation to working at higher pH values (around 8.5 in mitochondria) where rather high amounts of carbonate in equilibrium with bicarbonate may be present. Metasilicate, isosteric to carbonate, is also about a 10 times weaker inhibitor of this isozyme as compared to other CAs investigated here (K(I) of 28.2 mM). Surprisingly, the tumor-associated isozyme IX is resistant to sulfate inhibition (K(I) of 154 mM) but has affinity in the low micromolar range for carbonate, sulfamate, and sulfamidate (K(I) in the range of 8.6-9.6 microM). This constitutes another proof that this isozyme best works at acidic pH values present in tumors, being inhibited substantially at higher pH values when more carbonate may be present. Bromate and chlorate are quite weak CA IX inhibitors (K(I) s of 147-274 mM).     

Floristic description and ecological characteristics of the plants of Pashat Valley,Pak-Afghan border,district Bajaur,Pakistan

This study was conducted during 2018–19 to evaluate the floristic composition and ecological characteristics of vegetation of Pashat valley, Bajaur. Floristic list of the valley contains 385 species distributed in 291 genera and 102 families. The dicotyledons having 83 families with 311 species and monocotyledons with 12 families contains 61 species. Pteridophytes having 05 families with 11 species and gymnosperm with a single family of two species. The leading family was Asteraceae with 42 species (10.90%) followed by Poaceae with 39 species (10.10%), Papilionaceae (Fabaceae) with 24 species (6.23%), Lamiaceae with 20 species (5. 19%) and Rosaceae with 15 species (3.90%). Therophytes 180 species (46.80%), hemicryptophytes 53 species (13.80%), nanophanerophytes 51 species (13.20%) and geophytes 35 species (9.09%) were the dominant life form groups. Leaf size class was dominated by microphylls (138 species, 35.84%) followed by nanophylls (124 species, 32.21%) and mesophylls (89 species, 23.12%). Habit wise the vegetation was mostly herbs (289 species) followed by shrubs (54 species), trees (38 species) and lianas (4 species). According to flowering phenology, most plants (62.30%) bloomed in spring followed by in summer (34%), in autumn (1.82%), in the whole year (1.30%) and in winter (0.52%). Based on chorological affinities, most of the species were bioregional in distribution (170 species, 44.15%) followed by mono-regional (157 species, 40.78%), pluri-regional (43 species, 11.17%) and cosmopolitan (15 species, 3.89%). The ecological characteristics like life form and leaf size spectra of the vegetation have a strong correlation with the prevailing environmental conditions such as altitude, slope, precipitation and temperature.     

Effects of calcium channel blocker, mibefradil, and potassium channel opener, pinacidil, on the contractile response of mid-pregnant goat myometrium

The present study was undertaken to investigate the in vitro influence of mibefradil, a calcium channel blocker, and pinacidil, a potassium channel opener, on pregnant goat myometrial spontaneous rhythmic contractility and contractions induced with the agonist, oxytocin. Longitudinal strips from the distal region of uterus, collected from goats at midgestation, were mounted in an organ bath for recording isometric contractions. Mibefradil (10(-8)-10(-4) M) or pinacidil (10(-10)-10(-4) M), added cumulatively to the bath at an increment of 1 log unit, caused concentration-dependent inhibition of the spontaneous rhythmic contractions of isolated uterine strips. The rhythmic contraction was, respectively, abolished at 100 and 10 microM concentrations of mibefradil and pinacidil. In a concentration-dependent manner, mibefradil (1 and 10 microM) antagonized the contractions elicited with oxytocin (10(-5)-10(-2) IU). Pretreatment of uterine strips with glibenclamide (10 microM), a selective KATP channel blocker, caused a rightward shift of the concentration-response curve of pinacidil with a concomitant decrease in its pD2 value. Pinacidil (0.3, 1 and 3 microM), in a concentration-related manner, antagonized the oxytocin (10(-5)-10(-2) IU)-induced contractile response. The inhibition of spontaneous rhythmic contractions and antagonism of oxytocin-induced contraction by mibefradil in the pregnant goat myometrium may be related to the antagonism of voltage-dependent Ca2+ channels, while by pinacidil suggests that KATP channel could be a therapeutic target for tocolysis.     

Mechanisms of the vasorelaxant effect of 1, 5-dihydroxy-2, 3-dimethoxy-xanthone, an active metabolite of 1-hydroxy-2, 3, 5-trimethoxy-xanthone isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery

1, 5-Dihydroxy-2, 3-dimethoxy-xanthone (HM-5) is one of the naturally-occurring xanthones of a Tibetan medicinal herb Halenia elliptica. Recently, it has been shown that HM-5 is one of the phase I metabolites of 1-hydroxy-2, 3, 5-trimethoxy-xanthone (HM-1), the major active component of H. elliptica with potent vasorelaxant actions. This study investigated the vasorelaxant effect of HM-5 and its mechanism(s). HM-5 (0.35-21.9 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 4.40+/-1.08 microM. Unlike HM-1, the effect of HM-5 was endothelial-independent such that removal of the endothelium did not affect its vasodilator potency. Nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME, 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM) did not affect the vasodilatory effects of HM-5, thus confirming the non-involvement of endothelium related mechanisms. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-5 was inhibited by a potassium channel blocker, TEA (10 mM), and 4-aminopyridine (4-AP, a K(v) blocker; 1 mM) but not by other K+ channel blockers such as iberiotoxin (100 nM), barium chloride (100 microM) and glibenclamide (10 microM). The involvement of Ca2+ channel was studied in artery rings pre-incubated with Ca2+-free buffer (intact endothelium or endothelium-denuded) and primed with 1 microM 5-HT or 60 mM KCl prior to the addition of CaCl2 to elicit contraction. In the 5-HT-primed preparations, HM-5 (34.7 microM) significantly inhibited the CaCl(2)-induced vasoconstriction (89.9% inhibition in intact endothelium artery rings; 83.3% inhibition in endothelium-denuded rings). In the KCl-primed preparations, HM-5 (34.7 microM) produced a 34% inhibition in endothelium-denuded rings. The same concentration of HM-5 inhibited (by 62.3%) the contractile response to 10 microM phorbol 12, 13-diacetate (PDA), a protein kinase C activator, in Ca2+-free solutions. Taken together, this study showed that the mechanisms of the vasorelaxant effects of HM-5 were distinctly different from those of its parent drug HM-1. The vasorelaxant effect of HM-5 was mediated through opening of potassium channel (4-AP) and altering intracellular calcium by partial inhibition of Ca2+ influx through L-type voltage-operated Ca2+ channels and intracellular Ca2+ stores.     

Effects of two-locus combinations, using the roux, lavender, and beige mutations, on plumage color of Japanese quail

The interactions between the effects of three plumage color mutations taken two-by-two (sex-linked recessive roux, autosomal recessive lavender, and autosomal dominant beige) were studied in Japanese quail by producing a total of 121 F(1) and 1118 F(2) quail from the three pure stocks. Three new plumage colors were obtained in F(2) quail: roux-diluted beige, cream, and lavender-diluted beige. Two of them, roux-diluted beige (from the roux and beige mutations) and cream (from the roux and lavender mutations) corresponded to double homozygotes or hemizygous birds, and could therefore be used to tag a quail line. On the other hand, an F(3) from F(2) birds with lavender-diluted beige plumage was necessary to show that quail with this plumage color were homozygous for the lavender mutation, but were either homozygous or heterozygous for the beige gene. In all three F(2)s, observed segregation of plumage colors fit simple two-locus Mendelian inheritance.     

Mechanisms of the vasorelaxant effect of 1-hydroxy-2, 3, 5-trimethoxy-xanthone, isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery

1-Hydroxy-2, 3, 5-trimethoxyxanthone (HM-1) is a xanthone isolated from Halenia elliptica, a Tibetan medicinal herb. HM-1 (0.33-42.1 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 1.67+/-0.27 microM. Removal of the endothelium significantly affected the vasodilator potency of HM-1, resulting in 46% decrease in E(max) value. The endothelium-dependent effects of HM-1 was confirmed when its vasorelaxant effect was inhibited after addition of nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (100 microM) or the soluble guanylate cyclase inhibitor 1H-[1, 2, 4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM). Atropine (100 nM), flurbiprofen (10 microM), propranolol (100 microM), pyrilamine (10 microM), cimetidine (10 microM) and SQ22536 (100 microM) had no effect on the vasorelaxant activity of HM-1 indicated the non-involvement of other receptor/enzyme systems. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-1 was unaffected by potassium channel blockers such as tetraethylammonium (10 mM), iberiotoxin (100 nM), barium chloride (100 microM) and 4-aminopyridine (1 mM). The involvement of Ca(2+) channel in 5-HT-primed artery ring preparations incubated with Ca(2+)-free buffer was confirmed when HM-1 (9.93 microM) partially abolished the CaCl(2)-induced vasoconstriction (87% inhibition in intact-endothelium artery rings; 50% inhibition in endothelium-denuded rings). In the KCl-primed preparations incubated with Ca(2+)-free buffer, HM-1 (9.93 microM) produced a 27.3% inhibition in endothelium-denuded rings. HM-1 (3.31-33.1 microM) had minimal relaxant effects (14.4%-20.3%) on the contractile response generated by 10 microM phorbol 12,13-diacetate (PDA) in Ca(2+)-free solutions, suggesting minimal effects on intracellular Ca(2+) mechanisms. These findings suggest the vasodilator action of HM-1 involved both an endothelium-dependent mechanism involving NO and an endothelium-independent mechanism by inhibiting Ca(2+) influx through L-type voltage-operated Ca(2+) channels; a minor contribution to the effects of HM-1 may be related to inhibition of the protein kinase C-mediated release of intracellular Ca(2+) stores.     

Interaction of ceruloplasmin, lactoferrin, and myeloperoxidase

When lactoferrin (LF) and myeloperoxidase (MPO) are added to ceruloplasmin (CP), a CP-LF-MPO triple complex forms. The complex is formed under physiological conditions, but also in the course of SDS-free PAGE. Polyclonal antibodies to both LF and MPO displace the respective proteins from the CP-LF-MPO complex. Similar replacement is performed by a PACAP38 fragment (amino acids 29-38) and protamine that bind to CP. Interaction of LF and MPO with CP-Sepharose is blocked at ionic strength above 0.3 M NaCl and at pH below 4.1 (LF) and 3.9 (MPO). Two peptides (amino acids 50-109 and 929-1012) were isolated by affinity chromatography from a preparation of CP after its spontaneous proteolytic cleavage. These peptides are able to displace CP from its complexes with LF and MPO. Both human and canine MPO could form a complex when mixed with CP from seven mammalian species. Upon intravenous injection of human MPO into rats, the rat CP-human MPO complex could be detected in plasma. Patients with inflammation were examined and CP-LF, CP-MPO, and CP-LF-MPO complexes were revealed in 80 samples of blood serum and in nine exudates from purulent foci. These complexes were also found in 45 samples of serum and pleural fluid obtained from patients with pleurisies of various etiology.