Dynamic docking and electron transfer between myoglobin and cytochrome b 5

The interaction of trypsin-digested bovine cytochrome b(5) (cyt b(5)) with horse heart myoglobin (Mb) and the interprotein electron transfer (ET) between these redox partners have been studied to gain better understanding of ET processes between weakly bound protein partners. The bimolecular rate constant ( k(2)) for photo-induced ET between zinc-substituted Mb (ZnMb) and cyt b(5) decreases with increasing ionic strength, consistent with the predominantly electrostatic character of this complex. The formation of a protein-protein complex has been confirmed and the binding affinities of metMb and ZnMb for cyt b(5) have been measured by two techniques: (1)H NMR titrations at pH 6.0 give binding constants of K(a) approximately (1.0+/-0.1)x10(3) M(-1) for metMb and K(a) approximately (0.75+/-0.1)x10(3) M(-1) for ZnMb; isothermal calorimetry gives K(a) approximately (0.35+/-0.1)x10(3) M(-1) for ZnMb. Brownian dynamic (BD) simulations show that cyt b(5) binds over a broad surface of Mb that includes its heme edge. The experimental results are described in terms of a dynamic docking model which proposes that Mb binds cyt b(5) in a large ensemble of protein binding conformations, not one or a few dominant ones, but that only a small subset are ET reactive. Aided by the BD simulations, this model explains why k(2) decreases with increasing pH: increasing pH not only weakens the binding affinity but also reduces the number of binding conformations with high ET reactivity.     

Boron deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of citrus seedlings

Seedlings of sweet orange (Citrus sinensis) were fertilized for 14 weeks with boron (B)-free or B-sufficient (2.5 or 10muM H(3)BO(3)) nutrient solution every other day. Boron deficiency resulted in an overall inhibition of plant growth, with a reduction in root, stem and leaf dry weight (DW). Boron-starved leaves showed decreased CO(2) assimilation and stomatal conductance, but increased intercellular CO(2) concentrations. Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) and stromal fructose-1,6-bisphosphatase (FBPase) were lower in B-deficient leaves than in controls. Contents of glucose, fructose and starch were increased in B-deficient leaves while sucrose was decreased. Boron-deficient leaves displayed higher or similar superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) activities, while dehydroascorbate reductase (DHAR) and catalase (CAT) activities were lower. Expressed on a leaf area or protein basis, B-deficient leaves showed a higher ascorbate (AsA) concentration, but a similar AsA concentration on a DW basis. For reduced glutathione (GSH), we found a similar GSH concentration on a leaf area or protein basis and an even lower content on a DW basis. Superoxide anion (O(2)(-)) generation, malondialdehyde (MDA) concentration and electrolyte leakage were higher in B-deficient than in control leaves. In conclusion, CO(2) assimilation may be feedback-regulated by the excessive accumulation of starch and hexoses in B-deficient leaves via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes. Although B-deficient leaves remain high in activity of antioxidant enzymes, their antioxidant system as a whole does not provide sufficient protection from oxidative damage.     

Spectral and kinetic studies on eosinophil peroxidase compounds I and II and their reaction with ascorbate and tyrosine.

Eosinophil peroxidase, the major granule protein in eosinophils, is the least studied human peroxidase. Here, we have performed spectral and kinetic measurements to study the nature of eosinophil peroxidase intermediates, compounds I and II, and their reduction by the endogenous one-electron donors ascorbate and tyrosine using the sequential-mixing stopped-flow technique. We demonstrate that the peroxidase cycle of eosinophil peroxidase involves a ferryl/porphyrin radical compound I and a ferryl compound II. In the absence of electron donors, compound I is shown to be transformed to a species with a compound II-like spectrum. In the presence of ascorbate or tyrosine compound I is reduced to compound II with a second-order rate constant of (1.0+/-0.2)x10(6) M(-1) s(-1) and (3.5+/-0.2)x10(5) M(-1) s(-1), respectively (pH 7.0, 15 degrees C). Compound II is then reduced by ascorbate and tyrosine to native enzyme with a second-order rate constant of (6.7+/-0.06)x10(3) M(-1) s(-1) and (2.7+/-0.06)x10(4) M(-1) s(-1), respectively. This study revealed that eosinophil peroxidase compounds I and II are able to react with tyrosine and ascorbate via one-electron oxidations and therefore generate monodehydroascorbate and tyrosyl radicals. The relatively fast rates of the compound I reduction demonstrate that these reactions may take place in vivo and are physiologically relevant.     

Prostacyclin and thromboxane synthesis by endometrial cancer and leiomyomas

To study the role of prostacyclin (PGI2) and thromboxane A2 (TxA2) in uterine tumors, pieces of endometrial cancer (n = 12) and leiomyomas (n = 12) were incubated in vitro, and the productions of 6-keto-prostaglandin F1a (6-keto-PGF1a, a hydration product of PGI2) and thromboxane B2 (TxB2, a hydration product of TxA2), measured by radioimmunoassay, were compared to those of corresponding healthy tissues. The production of 6-keto-PGF1a by endometrial cancer (20.8; 15.1-85.0 ng/mg protein/min, median and interquartile range), by healthy endometrium (25.5; 10.0-55.0), by healthy myometrium (34.9; 25.0-59.9) and by leiomyoma (20.3; 10.2-45.1) was similar. The production of TxB2 was increased by endometrial cancer (55.5; 10.5-155.2, p less than 0.02) in comparison with endometrium (9.8; 4.3-35.1), myometrium (3.8; 2.1-8.0) and leiomyoma (1.9; 1.0-3.8). The 6-keto-PGF1a/TxB2 ratio in endometrial cancer (0.9; 0.3-1.5) was smaller (p less than 0.02) than that in healthy endometrium (3.3; 1.9-4.8). Thus, TxA2 may be a factor in endometrial cancer.     

Lipolysis and lipid oxidation in cirrhosis and after liver transplantation

On the basis of the finding that plasma glycerol concentration is not controlled by clearance in healthy humans, it has been proposed that elevated plasma free fatty acid (FFA) and glycerol concentrations in cirrhotic subjects are caused by accelerated lipolysis. This proposal has not been validated. We infused 10 volunteers, 10 cirrhotic subjects, and 10 patients after orthotopic liver transplantation (OLT) with [1-(13)C]palmitate and [(2)H(5)]glycerol to compare fluxes (R(a)) and FFA oxidation. Cirrhotic subjects had higher plasma palmitate (52%) and glycerol (33%) concentrations than controls. Palmitate R(a) was faster (1.45+/-0.18 vs. 0.85+/-0.17 micromol x kg(-1) x min(-1)) but glycerol R(a) and clearance slower (1.20+/-0.09 vs. 1.90+/-0.24 micromol x kg(-1) x min(-1) and 21.2+/-1.2 vs. 44.7+/- 4.9 ml x kg(-) x h(-1), respectively) than in controls. After OLT, plasma palmitate and glycerol concentrations and palmitate R(a) did not differ, but glycerol R(a) (1.16+/-0.11 micromol x kg(-1) x min(-1)) and clearance (26.7+/-2.4 ml x kg(-1) x h(-1)) were slower than in controls. We conclude that 1) impaired reesterification, not accelerated lipolysis, elevates FFA in cirrhotic subjects; 2) normalized FFA after OLT masks impaired reesterification; and 3) plasma glycerol concentration poorly reflects lipolytic rate in cirrhosis and after OLT.     

Cyanogenic allosides and glucosides from Passiflora edulis and Carica papaya

Leaf and stem material of Passiflora edulis (Passifloraceae) contains the new cyanogenic glycosides (2R)-beta-D-allopyranosyloxy-2-phenylacetonitrile (1a) and (2S)-beta-D-allopyranosyloxy-2-phenylacetonitrile (1b), along with smaller amounts of (2R)-prunasin (2a), sambunigrin (2b), and the alloside of benzyl alcohol (4); the major cyanogens of the fruits are (2R)-prunasin (2a) and (2S)-sambunigrin (2b). The major cyanogenic glycoside of Carica papaya (Caricaceae) is 2a; only small amounts of 2b also are present. We were not able to confirm the presence of a cyclopentenoid cyanogenic glycoside, tetraphyllin B, in Carica papaya leaf and stem materials. In detailed 1H NMR studies of 1a/b and 2a/b, differences in higher order effects in glucosides and allosides proved to be valuable for assignment of structures in this series. The diagnostic chemical shifts of cyanogenic methine and anomeric protons in 1a/b are sensitive to anisotropic environmental effects. The assignment of C-2 stereochemistry of 1a/b was made in analogy to previous assignments in the glucoside series and was supported by GLC analysis of the TMS ethers.     

Synthesis and antitumoral activities of marine ent-chromazonarol and related compounds.

Efficient syntheses of ent-isozonarol (6a), ent-isozonarone (7a) and ent-chromazonarol (8) from (-)-sclareol (12) are described. 6a and 7a show a significative antitumoral activity.     

The column types of Neottia and Archineottia of the family Orchidaceae and their taxonomic and phylogenetic significance

The column is the most characteristic part of an orchid flower. It is considered to be formed by the union of stamens with a central style and stigma. In the Apostasieae, for example, the column is rather primitive in the stamens and style only partially united, whereas in the majority of higher orchids it becomes more advanced through a eomplete union of them into a single organ. Within the family, indeed, the column structure is greatly diversified and of great taxonomic significance. It is interesting to note that a great range of diversity of column structure is bund in Neottia (sensu lato), a small but widespread genus consisting of 14 species, about two thirds of which, however, are of local occurance and seem to be little known to many botanists. In some speeies of this genus we find a very primitive column structure which is quite unique in the family, while in the others it is much more complicated. In all, five types of their column structure can be distinguished as fol- lows: (1) column rather longer; anther erect with a short filament attached to the back of the column near the apex; stigma terminal; neither clinandrium nor rostellure; (f. 2, 4) (2) as the preceding, 'except for the stigma more or less curved foreward and filament longer; (f. 6, 8) (3) column rather longer with a clinandrium at its summit, upon which a sessile and incumbent anther sits; rostellum large, horizontally projecting out over the concave stigma situated in the front of the column; (f. 10, 13, 15, 17) (4) as the preceding, except, for the anther and rostellum almost erect, and the stigma more or less bilabiate; (f. 19,21) (5) column very short; anther and rostellum erect; stigma lamellate, erect; reflexed and almost clasping the rostellum. (f.,2g) In these .five types, with the exception of the first one in which the labellum (the median petal) is very similar to the lateral: petals, they all possess zygomorphic perianth with labellum bilobed or entire which is quite different from the two lateral petals. Here, we see a great change in the column structure from one form with stamen and style not fully united to another form in which they have been well fused. Speaking strictly, these are two sorts of entirely different column structure. The former one, represented by (1) and (2) as stated above, is, in fact, an incomplete or s very primitive column in having a terminal stigma and an erect stamen with its free filament attached to the back of the column; and the absence of clinandrium and rostellum. Furthermore, there exists on the back of the column a thick ridge with its upper end joined to the filament, with which it is of the same texture and appearance. In Neottia pantlingii (=Arohineottia pantlingii) the free filament is even rather longer than the ridge, (f. 6) while in the other three species (f. 2, 4, 8) they are shorter. It is in my opinion the lower part of the filament adnate to the compound style or column. This is another fact of interest perhaps not occuring in any other living orchids. On the other hand, the latter one, represented by (3), (4) and (5), is a more advanced column structure, in which a higher level of specialisation with well-developed clinandrium and rostellum is reached. The stigma becomes shallow depressed on the anterior side of the column, or sometimes in the form of somewhat a bilabiate lip projecting out before or under the long rostellum. This is apparently a complete column both in structure and function quite different from the former and, contrarily, much like that of Listera. Basing upon the facts just mentioned, we may subdivided Neottia (sensu lato) into two distinct genera, with two and three sections respectively. They are as follows: 1. Archineottia S. C. Chen, gen. nov. (1) Sect. Archineottia 1) A. gaudissartii (Hand.-Mzt.) S. C. Chen, comb. nov. (China) 2) A. microglottis (Duthie) S. C. Chen, comb. nov. (India) (2) Sect. Furciila S. C. Chen, sect. nov. 3) A. pantlingii (W. W. Smith) S. C. Chen, comb. nov. (Sikkim) 4) A. smithiana (Schltr.) S. C. Chen, comb. nov. (China) 2. Neottia Guett. (1) Sect. Listeroides S.C. Chen, sect. nov. 1) N. listeroides (L.) Rchb. f. (China, Sikkim, Kashmir) 2) N. camtschatea (L.) Rchb. f, (China, Soviet Union) 3) N. megalochila S. C. Chen, nom. nov. (China) 4) N. inayatii (I)uthie) Schltr. (Pakistan, Kashmir) 5) N. tenii Schltr; (China) (2) Sect. Neottia 6) N. papilligera Schltr. (Chinas: Japan, Korea, Soviet Union, Sikkim) 7) N. nidus-avis (L.) L. C. Rich. (Europe, Iran, Western Siberia) 8) N. brevilabris Tang et Wang: (China) (3) Sect. Hologlossa S. C. Chen, sect. nov. 9) N. acuminata Schltr. (China, Japan, Korea, Soviet Union, Sikkim) Inperfeetly known species: 10) N. ussuriensis (Kom. et Nevski) S6o (Soviet Union) Thus, the subtribe Neottiinae are composed of four genera, namely, Diplandrorchis, Archineottia, Neottia and Listera. The new genus Archineottia, as one of the most primitive genera in the family, is of great interest from a phylogenetic point of view. It shows dose similarity to Diplandrorchis and Neottia in habit, but sharply distinct from them in column structure. These genera, as indicated By some authors, also show affinity in some respects with the subtribe Limodorinae, especially to Tangtsinia and Sinorchis, the other two quite primitive genera in the family. There is, indeed, a great need of further study of these interesting or relic genera and this, I think, would go a long way towards solving the problems concerning the origin ofthe Orchidaceae.     

脂蛋白(a)与动脉粥样硬化及脑梗死的研究进展

脂蛋白(a)是由载脂蛋白(a)和低密度脂蛋白构成的。脂蛋白(a)不随血浆中同型半胱氨酸、载脂蛋白A、高密度脂蛋白的变化而变化,是一种相对独立的脂蛋白。脂蛋白(a)的合成过程主要是在肝脏中完成的,脂蛋白(a)可以抑制NO介导的血管舒张,破坏血管壁中促凝与抗凝因子的平衡,参与动脉粥样硬化斑块的形成。动脉粥样硬化是脑梗死最基本的病因之一,在临床工作中我们应当重视脂蛋白(a)与脑梗死的关系,我们可以通过测定血清中脂蛋白(a)的水平来预测脑梗死的患病风险,尤其能对病变损害程度重,病变累及范围广的脑梗死的发生提出预警。高水平的血浆脂蛋白(a)是脑梗死及动脉粥样硬化的独立危险因素,本文就脂蛋白(a)与动脉粥样硬化及脑梗死研究中的进展做一综述,为脑梗死的预防提供参考和研究依据。     

Relationships between the molecular structure and moisture-absorption and moisture-retention abilities of carboxymethyl chitosan. II. Effect of degree of deacetylation and carboxymethylation

Carboxymethyl chitosans (CM-chitosan) of various degrees of deacetylation (DD 28-95%) and substitution (DS 0.15-1.21) were successfully prepared from N-acetylchitosans in NaOH of varying concentrations. Infrared spectroscopy (IR), elemental analysis, potentiometric titration, 13C NMR, X-ray diffraction and gel-permeation chromatographic (GPC) techniques were used to characterize their molecular structures. The moisture-absorption (R(a)) and -retention (R(h)) abilities of CM-chitosan are closely related to the DD and DS values. Under conditions of high relative humidity, the maximum R(a) and R(h) were obtained at DD values of about 50%, and when the DD value deviated from 50%, R(a) and R(h) decreased. Under dry conditions, when the DD value was 50%, the R(h) was the lowest. With the DS value increasing, R(a) and R(h) increased. However, further increase of the DS value above 1.0 reduced the increasing tendency of R(a) and R(h), and even some decreases in R(a) and R(h) were observed. Intermolecular hydrogen bonds play a very important role in moisture-absorption and retention ability of CM-chitosan.     

Synthesis and binding affinities of methylvesamicol analogs for the acetylcholine transporter and sigma receptor

We synthesized methylvesamicol analogs 13-16 and investigated the binding characteristics of 2-[4-phenylpiperidino]cyclohexanol (vesamicol) and methylvesamicol analogs 13-16, with a methyl group introduced into the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (-)-o-methylvesamicol [(-)-OMV] (13) (Ki=6.7 nM), as well as (-)-vesamicol (Ki=4.4 nM), had a high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (Ki=3.0 nM), as well as SA4503 (Ki=4.4 nM), reported as a sigma-1 mapping agent for positron emission tomography (PET), had a high affinity for the sigma-1 receptor. The binding affinity of (+)-PMV (16) for the sigma-1 receptor (Ki=3.0 nM) was about 13 times higher than that for the sigma-2 (sigma-2) receptor (Ki=40.7 nM). (+)-PMV (16) (Ki=199 nM) had a much lower affinity for VAChT than SA4503 (Ki=50.2 nM) and haloperidol (Ki=41.4 nM). These results showed that the binding characteristics of (-)-OMV (13) to VAChT were similar to those of (-)-vesamicol and that (+)-PMV (16) bound to the sigma-1 receptor with high affinity. In conclusion, (-)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a methyl group for labeling with 11C, may become not only a new VAChT ligand and a new type of sigma receptor ligand, respectively, but may also become a new target compound of VAChT and the sigma-1 receptor radioligand for PET, respectively.     

Model complexes with naturally occurring ligands (salicylglycine and imidazol) and the biometals copper and cobalt

Two new complexes [Cu(Imz)(4)Cl(2)][Cu(Imz)(4)Cl] (2)(2-OH-Hip)(2) (1) and [Co(2-OH-Hip)(Imz)(3)].H(2)O (2) (with Imz=Imidazol and 2-OH-Hip=2-hydroxyhippuric acid) were prepared and characterized. The molecular structures and the solution and solid state behavior of the complexes were investigated. Complex 1 crystallizes in the monoclinic space group P2(1)/c with a=16.880(1), b=8.046(1), c=24.683(1) A, beta=107.88(1) degrees, and Z=2, while complex 2 crystallizes in the orthorhombic space group Pbca with a=11.712(2), b=15.741(4), c=22.254(4) A, and Z=8. The [Cu(Imz)(4)Cl(2)][Cu(Imz)(4)Cl](2)(2-OH-Hip)(2) solid consists in two distinct monomeric Cu(II) complexes: one of them is neutral octahedral [Cu(Imz)(4)Cl(2)] and the other, charged square basis pyramida [Cu(Imz)(4)Cl](+). The 2-hydroxyhippuric acid, which here acts as a counter ion, is deprotonated at its carboxylic group. Cobalt(III) ion in [Co(2-OH-Hip)(Imz)(3)].H(2)O is at the center of an octahedral environment, coordinated to three Imidazol ligands and to a triply deprotonated 2-hydroxyhippuric acid molecule acting as a tridentate ligand. Aqueous solution equilibrium of the quaternary system Cu(2+)/2-OH-Hip/Imz/H(+) was studied by potentiometric titrations.     

High-light stress and photoprotection in Umbilicaria antarctica monitored by chlorophyll fluorescence imaging and changes in zeaxanthin and glutathione

The effect of high light on spatial distribution of chlorophyll (Chl) fluorescence parameters over a lichen thallus (Umbilicaria antarctica) was investigated by imaging of Chl fluorescence parameters before and after exposure to high light (1500 micro mol m (-2) s (-1), 30 min at 5 degrees C). False colour images of F (V)/F (M) and Phi (II) distribution, taken over thallus with 0.1 mm (2) resolution, showed that maximum F (V)/F (M) and Phi (II) values were located close to the thallus centre. Minimum values were typical for thallus margins. After exposure to high light, a differential response of F (V)/F (M) and Phi (II) was found. The marginal thallus part exhibited a loss of photosynthetic activity, manifested as a lack of Chl fluorescence signal, and close-to-centre parts showed a different extent of F (V)/F (M) and Phi (II) decrease. Subsequent recovery in the dark led to a gradual return of F (V)/F (M) and Phi (II) to their initial values. Fast (30 min) and slow (1 - 22 h) phase of recovery were distinguished, suggesting a sufficient capacity of photoprotective mechanisms in U. antarctica to cope with low-temperature photoinhibition. Glutathione and xanthophyll cycle pigments were analyzed by HPLC. High light led to an increase in oxidized glutathione (GSSG), and a conversion of violaxanthin to zeaxanthin, expressed as their de-epoxidation state (DEPS). The responses of GSSG and DEPS were reversible during subsequent recovery in the dark. GSSG and DEPS were highly correlated to non-photochemical quenching (NPQ), indicating involvement of these antioxidants in the resistance of U. antarctica to high-light stress. Heterogeneity of Chl fluorescence parameters over the thallus and differential response to high light are discussed in relation to thallus anatomy and intrathalline distribution of the symbiotic alga Trebouxia sp.     

Folding and unfolding of gammaTIM monomers and dimers

Kinetic simulations of the folding and unfolding of triosephosphate isomerase (TIM) from yeast were conducted using a single monomer gammaTIM polypeptide chain that folds as a monomer and two gammaTIM chains that fold to the native dimer structure. The basic protein model used was a minimalist Gō model using the native structure to determine attractive energies in the protein chain. For each simulation type--monomer unfolding, monomer refolding, dimer unfolding, and dimer refolding--thirty simulations were conducted, successfully capturing each reaction in full. Analysis of the simulations demonstrates four main conclusions. First, all four simulation types have a similar "folding order", i.e., they have similar structures in intermediate stages of folding between the unfolded and folded state. Second, despite this similarity, different intermediate stages are more or less populated in the four different simulations, with 1), no intermediates populated in monomer unfolding; 2), two intermediates populated with beta(2)-beta(4) and beta(1)-beta(5) regions folded in monomer refolding; 3), two intermediates populated with beta(2)-beta(3) and beta(2)-beta(4) regions folded in dimer unfolding; and 4), two intermediates populated with beta(1)-beta(5) and beta(1)-beta(5) + beta(6) + beta(7) + beta(8) regions folded in dimer refolding. Third, simulations demonstrate that dimer binding and unbinding can occur early in the folding process before complete monomer-chain folding. Fourth, excellent agreement is found between the simulations and MPAX (misincorporation proton alkyl exchange) experiments. In total, this agreement demonstrates that the computational Gō model is accurate for gammaTIM and that the energy landscape of gammaTIM appears funneled to the native state.     

Vascular volumes and hematology in male and female runners and cyclists

To examine the hypothesis that foot-strike hemolysis alters vascular volumes and selected hematological properties is trained athletes, we have measured total blood volume (TBV), red cell volume (RCV) and plasma volume (PV) in cyclists (n = 21) and runners (n = 17) and compared them to those of untrained controls (n = 20). TBV (ml x kg(-1)) was calculated as the sum of RCV (ml x kg(-1)) and PV (ml x kg(-1)) obtained using 51Cr and 125I-labelled albumin, respectively. Hematological assessment was carried out using a Coulter counter. Peak aerobic power (VO2peak) was measured during progressive exercise to fatigue using both cycle and treadmill ergometry. RCV was 15% higher (P < 0.05) in male cyclists [35.4 (1.0), mean (SE); n = 12] and runners [35.3 (0.98); n = 9] compared to the controls [30.7 (0.92); n = 12]. Similar differences existed between the female cyclists [28.2 (2.1); n = 9] and runners [28.4 (1.0); n = 8] compared to the untrained controls [24.9 (1.4); n = 8]. For the male athletes, PV was between 19% (cyclists) and 28% (runners) higher (P < 0.05) in the trained athletes compared to the untrained controls. The differences in PV between the female groups were not significant. Although the males had a higher (P < 0.05) TBV, RCV and PV than the females, no differences between cyclists and runners were found for either gender. Mean cell volume was not different between the athletic groups. VO2peak (ml x kg(-1) x min(-1)) was higher (P < 0.05) in both male [68.4 (1.5)] and female [54.8 (2.1)] runners when compared to the untrained males [47.1 (1.0)] and females [40.5 (2.1)]. Although differences existed between the genders in VO2peak for both cyclists and runners, no differences were found between the athletic groups within a gender. Since the vascular volumes were not different between cyclists and runners for either the males or females, foot-strike hemolysis would not appear to have an effect on that parameter. The significant correlations (P < 0.05) found between VO2peak and RCV (r = 0.64 and 0.64) and TBV (r = 0.82 and 0.63) for the males and females, respectively, suggests a role for the vascular system in realizing a high aerobic power.     

Viral abundance and genome size distribution in the sediment and water column of marine and freshwater ecosystems

The size distribution of viral DNA in natural samples was investigated in a number of marine, brackish and freshwater environments by means of pulsed field gel electrophoresis (PFGE). The method was modified to work with both water and sediment samples, with an estimated detection limit for individual virus genome size groups of 1-2 x 10(4) virus-like particles (VLP) mL(-1) water and 2-4 x 10(5) VLP cm(-3) sediment in the original samples. Variations in the composition and distribution of dominant virus genome sizes were analyzed within and between different habitats that covered a range in viral density from 0.4 x 10(7) VLP mL(-1) (sea water) to 300 x 10(7) VLP cm(-3) (lake sediment). The PFGE community fingerprints showed a number of cross-system similarities in the genome size distribution with a general dominance of genomes in the 30-48, 50-70 and 145-200 kb size fractions, and with many of the specific genome sizes detected in all the investigated habitats. However, large differences in community fingerprints were also observed between the investigated sites, and some virus genome sizes were found only in specific biotopes (e.g. lake water), in specific ecosystems (e.g. a particular lake) or even in specific microhabitats (e.g. a particular sediment stratum).     

Integrin alphaMbeta2 orchestrates and accelerates plasminogen activation and fibrinolysis by neutrophils

Plasmin, the pivotal thrombolytic enzyme, is generated on the surface of many cell types, where urokinase receptor (uPAR)-bound urokinase (uPA) activates cell-bound plasminogen (Plg). It has been reported that neutrophils mediate endogenous thrombolysis involving a uPA-dependent mechanism, and we previously demonstrated that both uPAR and integrin alpha(M)beta(2) recognize uPA to control cell migration and adhesion. In the present study, we report that the alpha(M)beta(2) regulates neutrophil-dependent fibrinolysis. Phorbol 12-myristate 13-acetate (PMA)-stimulated but not resting neutrophils dissolved fibrin clots, and this activity was not only uPA- and Plg-dependent but also alpha(M)beta(2)-dependent. Purified alpha(M)beta(2) directly bound uPA (K(d) = 40 nm) and Plg (K(d) = 1 microm) in a dose-dependent and saturable manner. In Plg activation assays, addition of purified alpha(M)beta(2), but not a control protein, to a single chain uPA (sc-uPA)/Plg mixture, decreased the K(m) from 2 to 0.1 microm, thereby augmenting the overall reaction efficiency by 50-fold. The binding of sc-uPA to alpha(M)beta(2) was critical for the alpha(M)beta(2)-mediated enhancement of plasmin (Plm) generation, because this effect was lost when WT-sc-uPA was replaced with a kringle-less mutant (DeltaK-sc-uPA), which does not bind to alpha(M)beta(2). Plm inactivation by alpha(2)-antiplasmin was significantly delayed when Plm was preincubated with purified, soluble alpha(M)beta(2). When Plg was added to PMA-stimulated neutrophils, both uPA and Plg were co-immunoprecipitated with alpha(M)beta(2.) Thus, assembly of Plg and uPA on integrin alpha(M)beta(2) regulates Plm activity and, thereby, plays a crucial role in neutrophil-mediated thrombolysis.     

Neuropharmacology and Stereochemistry of Dopamine Receptor Agonist and Antagonist Enantiomeric Pairs

Abstract

Neuropharmacological evaluation of the R and S isomers of 11-hydroxy-N-n-propylnoraporphine (11-OH-NPa) supports the impression that the 11-OH group in aporphines (analogous to the meta hydroxyl of dopamine, DA) is sufficient to confer high affinity and activity at DA receptors. As in the case of the catechol congeners, (R)-apomorphine (APO) and (R)-N-n-propylnorapomorphine (NPA), (R)-11-OH-NPa is a potent DA agonist while, like (S)-NPA, (S)-11-OH-NPa is a DA antagonist. Thus, (R) and (S)-11-OH-NPa are an additional pair of compounds in which one enantiomer is a DA agonist and the other an antagonist. Other analogous pairs are the enantiomers of 3-(3-hydroxy-N-n-propylnoraporphine (3-PPP), and cis-1-methyl-5-hydroxy-N-n-propylamino) tetralin (5-OH-MDAT). All contain a meta hydroxyphenyl, an N-n-propyl, and a phenethylamine moiety which can be superimposed in a consistent way to discriminate the DA agonists from the antagonists, with the key feature in this discrimination being the direction of the ammonium hydrogen. An energy penalty must be incurred by 3-PPP to assume the required conformations and it may account     

Comparative circular dichroism and fluorescence studies of oligodeoxyribonucleotide and oligodeoxyribonucleoside methylphosphonate pyrimidine strands in duplex and triplex formation

An analogue of the homopyrimidine oligodeoxyribonucleotide d(CT)8 has been synthesized. This analogue, d(CT)8 contains nonionic methylphosphonate internucleoside linkages. The pH-dependent conformational transitions of d(CT)8 have been studied and its ability to form duplexes and triplexes with the normal homopurine oligonucleotide d(AG)8 has also been investigated as a function of pH. Circular dichroism spectroscopy and ethidium bromide fluorescence enhancement have been used to monitor pH-dependent conformational transitions driven by the protonation of cytosine residues, and the different behavior of d(CT)8 and d(CT)8 has been compared. It was possible to form self-associated complexes by using either d(CT)8 or d(CT)8, and both compounds combined with d(AG)8 to form duplex or triplex DNA. At neutral pH, the CD spectrum of d(AG)8.d(CT)8 duplex was quite different from the CD spectrum of d(AG)8.d(CT)8 duplex, reflecting most likely a difference in conformation. The duplex to triplex transition characteristic of this DNA sequence occurred at a lower pH when d(CT)8 was substituted for d(CT)8; however, at pH 4.2, triplex containing d(CT)8 was similar in conformation to triplex containing d(CT)8. Several of these observations can be related to the alterations in electrostatic and steric interactions that occur when the negatively charged phosphodiester backbone of d(CT)8 is replaced with a nonionic methylphosphonate backbone.