Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.     

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.     

Physiological significance of C-28 hydroxylation in the metabolism of 1alpha,25-dihydroxyvitamin D(2).

In our previous study, we indicated for the first time that C-28 hydroxylation plays a significant role in the metabolism of 1alpha, 25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] by identifying 1alpha,24(S),25,28-tetrahydroxyvitamin D(2) [1alpha,24(S),25, 28(OH)(4)D(2)] as a major renal metabolite of 1alpha,25(OH)(2)D(2) [G. S. Reddy and K-Y. Tserng Biochemistry 25, 5328-5336, 1986]. The present study was performed to establish the physiological significance of C-28 hydroxylation in the metabolism of 1alpha, 25(OH)(2)D(2). We perfused rat kidneys in vitro with 1alpha, 25(OH)(2)[26,27-(3)H]D(2) (5 x 10(-10)M) and demonstrated that 1alpha,24(R),25-trihydroxyvitamin D(2) [1alpha,24(R),25(OH)(3)D(2)] and 1alpha,24(S),25,28(OH)(4)D(2) are the only two major physiological metabolites of 1alpha,25(OH)(2)D(2). In the same perfusion experiments, we also noted that there is no conversion of 1alpha,25(OH)(2)D(2) into 1alpha,25,28-trihydroxyvitamin D(2 )[1alpha,25,28(OH)(3)D(2)]. Moreover, 1alpha,24(S),25,28(OH)(4)D(2) is not formed in the perfused rat kidney when synthetic 1alpha,25, 28(OH)(3)D(2) is used as the starting substrate. This finding indicates that C-28 hydroxylation of 1alpha,25(OH)(2)D(2) occurs only after 1alpha,25(OH)(2)D(2) is hydroxylated at C-24 position. At present the enzyme responsible for the C-28 hydroxylation of 1alpha, 24(R),25(OH)(3)D(2) in rat kidney is not known. Recently, it was found that 1alpha,25(OH)(2)D(3)-24-hydroxylase (CYP24) can hydroxylate carbons 23, 24, and 26 of various vitamin D(3) compounds. Thus, it may be speculated that CYP24 may also be responsible for the C-28 hydroxylation of 1alpha,24(R),25(OH)(3)D(2) to form 1alpha, 24(S),25,28(OH)(4)D(2). The biological activity of 1alpha,24(S),25, 28(OH)(4)D(2), determined by its ability to induce intestinal calcium transport and bone calcium resorption in the rat, was found to be almost negligible. Also, 1alpha,24(S),25,28(OH)(4)D(2) exhibited very low binding affinity toward bovine thymus vitamin D receptor. These studies firmly establish that C-28 hydroxylation is an important enzymatic reaction involved in the inactivation of 1alpha,25(OH)(2)D(2) in kidney under physiological conditions.     

Effect of arterial oxygenation on quadriceps fatigability during isolated muscle exercise

The effect of various levels of oxygenation on quadriceps muscle fatigability during isolated muscle exercise was assessed in six male subjects. Twitch force (Q(tw)) was assessed using supramaximal magnetic femoral nerve stimulation. In experiment 1, maximal voluntary contraction (MVC) and Q(tw) of resting quadriceps muscle were measured in normoxia [inspired O(2) fraction (Fi(O(2))) = 0.21, percent arterial O(2) saturation (Sp(O(2))) = 98.4%, estimated arterial O(2) content (Ca(O(2))) = 20.8 ml/dl], acute hypoxia (Fi(O(2)) = 0.11, Sp(O(2)) = 74.6%, Ca(O(2)) = 15.7 ml/dl), and acute hyperoxia (Fi(O(2)) = 1.0, Sp(O(2)) = 100%, Ca(O(2)) = 22.6 ml/dl). No significant differences were found for MVC and Q(tw) among the three Fi(O(2)) levels. In experiment 2, the subjects performed three sets of nine, intermittent, isometric, unilateral, submaximal quadriceps contractions (62% MVC followed by 1 MVC in each set) while breathing each Fi(O(2)). Q(tw) was assessed before and after exercise, and myoelectrical activity of the vastus lateralis was obtained during exercise. The percent reduction of twitch force (potentiated Q(tw)) in hypoxia (-27.0%) was significantly (P < 0.05) greater than in normoxia (-21.4%) and hyperoxia (-19.9%), as were the changes in intratwitch measures of contractile properties. The increase in integrated electromyogram over the course of the nine contractions in hypoxia (15.4%) was higher (P < 0.05) than in normoxia (7.2%) or hyperoxia (6.7%). These results demonstrate that quadriceps muscle fatigability during isolated muscle exercise is exacerbated in acute hypoxia, and these effects are independent of the relative exercise intensity.     

Heme-copper/dioxygen adduct formation relevant to cytochrome c oxidase: spectroscopic characterization of [(6L)FeIII-(O2 2−)-CuII]+

In the further development and understanding of heme-copper dioxygen reactivity relevant to cytochrome c oxidase O(2)-reduction chemistry, we describe a high-spin, five-coordinate dioxygen (peroxo) adduct of an iron(II)-copper(I) complex, [((6)L)Fe(II)Cu(I)](BArF(20)) (1), where (6)L is a tetraarylporphyrinate with a tethered tris(2-pyridylmethyl)amine chelate for copper. Reaction of 1 with O(2) in MeCN affords a remarkably stable [t(1/2) (rt; MeCN) approximately 60 min] adduct, [((6)L)Fe(III)-(O(2) (2-))-Cu(II)](+) (2) [EPR silent; lambda(max)=418 (Soret), 561 nm], formulated as a peroxo complex based on manometry (1:O(2)=1:1; spectrophotometric titration, -40 degrees C, MeCN), mass spectrometry {MALDI-TOF-MS: (16)O(2), m/z 1191 ([((6)L)Fe(III)-((16)O(2) (2-))-Cu(II)](+)); (18)O(2), m/z 1195}, and resonance Raman spectroscopy (nu((O-O))=788 cm(-1); Delta(16)O(2)/(18)O(2)=44 cm(-1); Delta(16)O(2)/(16/18)O(2)=22 cm(-1)). (1)H and (2)H NMR spectroscopy (-40 degrees C, MeCN) reveals that 2 is the first heme-copper peroxo complex which is high-spin, with downfield-shifted pyrrole resonances (delta(pyrrole)=75 ppm, s, br) and upfield shifted peaks at delta= -22, -35, and -40 ppm, similar to the pattern observed for the mu-oxo complex [((6)L)Fe(III)-O-Cu(II)](BAr(F)) (3) (known S=2 system, antiferromagnetically coupled high-spin Fe(III) and Cu(II)). The corresponding magnetic moment measurement (Evans method, CD(3)CN, -40 degrees C) also confirms the S=2 spin state, with mu(B)=4.9. Structural insights were obtained from X-ray absorption spectroscopy, showing Fe-O (1.83 A) and Cu-O (1.882 A) bonds, and an Fe...Cu distance of 3.35(2) A, suggestive of a mu-1,2-peroxo ligand present in 2. The reaction of 2 with cobaltocene gives 3, differing from the observed full reduction seen with other heme-Cu peroxo complexes. Finally, thermal decomposition of 2 yields 3, with concomitant release of 0.5 mol O(2) per mol 2, as confirmed quantitatively by an alkaline pyrogallol dioxygen scavenging solution.     

Electronic ground states of low-spin iron(III) porphyrinoids

Six-coordinate low-spin iron(III) porphyrinates adopt either common (d(xy))(2)(d(xz),d(yz))(3) or less common (d(xz),d(yz))(4)(d(xy))(1) ground state. In this review article, three major factors that affect the electronic ground state have been examined. They are (i) nature of the axial ligand, (ii) electronic effect of peripheral substituents, and (iii) deformation of porphyrin ring. On the basis of the (1)H NMR, (13)C NMR, and EPR data, it is now clear that (i) the axial ligands with low-lying pi* orbitals such as tert-butylisocyanide and 4-cyanopyridine, (ii) the electron donating groups at the meso-carbon atoms, and (iii) the ruffled deformation of porphyrin ring stabilize the (d(xz),d(yz))(4)(d(xy))(1) ground state. By manipulating these factors, we are able to prepare various low-spin iron(III) porphyrinates with unusual electronic structures such as bis(imidazole) complexes with the (d(xz),d(yz))(4)(d(xy))(1) ground state or bis(tert-butylisocyanide) complexes with the (d(xy))(2)(d(xz),d(yz))(3) ground state; bis(imidazole) and bis(tert-butylisocyanide) complexes usually adopt the (d(xy))(2)(d(xz),d(yz))(3) and (d(xz),d(yz))(4)(d(xy))(1) ground state, respectively.     

Synthesis, characterization and assessment of the cytotoxic properties of cis and trans-[Pd(L)(2)Cl(2)] complexes involving 6-benzylamino-9-isopropylpurine derivatives

A series of square-planar Pd(II) complexes of the composition cis-[Pd(L(n))(2)Cl(2)] {L(1)=2-chloro-6-benzylamino-9-isopropylpurine (1), L(2)=2-chloro-6-[(4-methoxybenzyl)amino]-9-isopropylpurine (2), L(3)=2-chloro-6-[(2-methoxybenzyl)amino]-9-isopropylpurine (3) and 2-[(chloropropyl)amino]-6-benzylamino-9-isopropylpurine (6)} has been synthesized by the reaction of PdCl(2) with L(n) in a 1:2 molar ratio. In contrast, the same reaction followed by recrystallization of the product from N,N'-dimethylformamide (DMF) leads to trans-[Pd(L(n))(2)Cl(2)] x nDMF {L(3), n=0 (4), n=1(4( *)DMF); L(4)=2-chloro-6-[(2,3-dimethoxybenzyl)-amino]-9-isopropylpurine, n=0 (5), n=1.5 (5( *)DMF). The compounds have been characterized by elemental analyses, conductivity measurements, electrospray mass spectra in the positive ion mode (ES+MS), FTIR, (1)H and (13)C NMR spectra, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Moreover, the complexes 2 and 6 have been also investigated by (15)N NMR spectroscopy. The molecular structures of L(5), {(H(2+)L(5))(Cl(-))(2)} x H(2)O, i.e. the protonated form of L(5), trans-[Pd(L(3))(2)Cl(2)] (4) and trans-[Pd(L(4))(2)Cl(2)] (5) have been determined by single crystal X-ray analysis. NMR data and X-ray structures revealed that the organic molecules are coordinated to Pd via N7 atom of a purine moiety. All the complexes and the corresponding ligands have been tested in vitro for their cytotoxicity against four human cancer cell lines: breast adenocarcinoma (MCF7), malignant melanoma (G361), chronic myelogenous leukaemia (K562) and osteogenic sarcoma (HOS). Promising in vitro cytotoxic effect has been found for cis-[Pd(L(2))(2)Cl(2)] (2), having the IC(50) values of 12, 10, 25, and 14 microM against MCF7, G361, K562, and HOS, respectively, and for trans-[Pd(L(3))(2)Cl(2)].DMF (4) with the IC(50) value of 15 microM against G361.     

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH?HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).     

Glycogen synthase kinase 3beta phosphorylates Alzheimer's disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanism

Phosphorylation of tau on S(396) was suggested to be a key step in the development of neurofibrillary pathology in Alzheimer's disease brain [Bramblett, G. T., Goedert, M., Jacks, R., Merrick, S. E., Trojanowski, J. Q., and Lee, V. M.-Y. (1993) Neuron 10, 1089-1099]. GSK3beta phosphorylates Ser(396) of tau in the brain by a mechanism which is not clear. In this study, when HEK-293 cells were cotransfected with tau and GSK3beta, GSK3beta co-immunoprecipitated with tau and phosphorylated tau on S(202), T(231), S(396), and S(400) but not on S(262), S(235), and S(404). Blocking phosphorylation on T(231), S(235), S(396), S(400), or S(404) did not prevent the subsequent phosphorylation on S(202) by GSK3beta. These data suggest that GSK3beta directly phosphorylates tau on S(202) (without requiring prephosphorylation). However, preventing phosphorylation on S(235), S(400), and S(404) prevented GSK3beta-dependent phosphorylation of T(231), S(396), and S(400), respectively. This indicates that phosphorylation of T(231), S(396), and S(400) by GSK3beta depends on a previous phosphorylation of S(235), S(400), and S(404), respectively. To examine S(396) phosphorylation, we analyzed phosphorylation of S(396), S(400), and S(404). Blocking phosphorylation of S(404) prevented the subsequent GSK3beta-dependent phosphorylation of both S(400) and S(396). When phosphorylation of S(404) was allowed but S(400) blocked, GSK3beta failed to phosphorylate S(396). Thus, GSK3beta phosphorylates S(396) by a two-step mechanism. In the first step, GSK3beta phosphorylates S(400) of previously S(404)-phosphorylated tau. This event primes tau for second-step phosphorylation of S(396) by GSK3beta. We conclude that GSK3beta phosphorylates tau directly at S(202) but requires the previous phosphorylation on S(235) to phosphorylate T(231). Phosphorylation of S(396), on the other hand, occurs sequentially. Once a priming kinase phosphorylates S(404), GSK3beta sequentially phosphorylates S(400) and then S(396).     

Experimental and natural weed host-virus relations

Weeds, as alternative hosts of plant viruses and nutrient plants of virus vectors play important role in virus ecology and epidemiology. The aim of our study was to discover new weed-virus relations. Therefore some weed species were mechanically inoculated with 28 viruses (strains or isolates) maintained in our glasshouse. Different weed species with and without visible symptoms were collected from agro-, water ecosystems and wastelands of Hungary between 1997 and 2003. Virus infections were evaluated by biotests, DAS ELISA serological methods, electronmicroscopy and immunosorbent electronmicroscopy (ISEM). Under glasshouse conditions Ambrosia artemisifolia was considered as a virophob species, showing resistance to all viruses listed above. A series of new artificial (Chenopodium album--SoMV (LH+SH)*, AMV (LH+SH); C. berlandieri--PVY(NTN) (LH), AMV (LH+SH), CMV (LH), SoMV (LH+SH), ObPV (LH+SH), ZYMV-10 (LH): C. ugandae--ObPV (LH), SoMV (L); C. glaucum--ObPV (LH), SoMV (L); Echinocystis lobata--PVX (L), ZYMV (LH+SH); Solanum nigrum--MYFV (LH+SH), PVY(N) (L), PVY(NTN) (LH+SH), SoMV (LH), TMV (SH), CMV (SH); S. dulcamara--CMV-U/246 (SH), PVY(NTN) (LH), SoMV-H (L), TMV-O (L); S. luteum--PVY(N) (SH), PVY(NTN) (LH+L), TMV(SH).) and natural (Asclepias syriaca--TMV, AMV, TSWV; Alisma plantago-aquatica--PVY, SoMV; Ambrosia artemisiifolia--CMV; Chenopodium album--CMV, PVS, PLRV; C. hybridum--CMV; Cirsium canum--CMV, PVM; Carex vulpina--CMV; Comium maculatum--PVY; Datura stramonium--PVA, PVX, PVS, PVM, CMV, TMV; Lysimachia vulgaris--ArMV, BNYVV, CMV, TMV; Lythrum salicaria--ArMV; Malva neglecta--CMV; Mercurialis annua--SoMV; Solanum nigrum--CMV, PVY, PVY(N); Solidago gigantea--CMV, RpRSV, BNYVV; Stenactis annua--PVM, PVA) weed--virus relations were detected. The epidemiological role of perennial hosts (A. syriaca, A. planlago aquatica, C. canurm, L. vulgaris, L. salicaria, S. gigantea) is especially high, because they can serve as infection sources as well as overwintering hosts of different plant viruses.     

On-line monitoring of intrinsic PEEP in ventilator-dependent patients.

Measurement of the intrinsic positive end-expiratory pressure (PEEP(i)) is important in planning the management of ventilated patients. Here, a new recursive least squares method for on-line monitoring of PEEP(i) is proposed for mechanically ventilated patients. The procedure is based on the first-order model of respiratory mechanics applied to experimental measurements obtained from eight ventilator-dependent patients ventilated with four different ventilatory modes. The model PEEP(i) (PEEP(i,mod)) was recursively constructed on an inspiration-by-inspiration basis. The results were compared with two well-established techniques to assess PEEP(i): end-expiratory occlusion to measure static PEEP(i) (PEEP(i, st)) and change in airway pressure preceding the onset of inspiratory airflow to measure dynamic PEEP(i) (PEEP(i,dyn)). PEEP(i, mod) was significantly correlated with both PEEP(i,dyn) (r = 0.77) and PEEP(i,st) (r = 0.90). PEEP(i,mod) (5.6 +/- 3.4 cmH(2)O) was systematically >PEEP(i,dyn) and PEEP(i,st) (2.7 +/- 1.9 and 8.1 +/- 5.5 cmH(2)O, respectively), in all the models without external PEEP. Focusing on the five patients with chronic obstructive pulmonary disease, PEEP(i,mod) was significantly correlated with PEEP(i,st) (r = 0.71), whereas PEEP(i,dyn) (r = 0.22) was not. When PEEP was set 5 cmH(2)O above PEEP(i,st), all the methods correctly estimated total PEEP, i.e., 11.8 +/- 5.3, 12.5 +/- 5.0, and 12.0 +/- 4.7 cmH(2)O for PEEP(i,mod), PEEP(i,st), and PEEP(i,dyn), respectively, and were highly correlated (0.97-0.99). We interpreted PEEP(i,mod) as the lower bound of PEEP(i,st) and concluded that our method is suitable for on-line monitoring of PEEP(i) in mechanically ventilated patients.     

Primary structure of ten galactosides formed by transglycosylation during lactose hydrolysis by Bifidobacterium bifidum

Oligosaccharides formed by a transgalactosylation reaction during lactose hydrolysis with Bifidobacterium bifidum were separated into eight fractions by gel-permeation chromatography and their structures studies determined by trimethylsilylation analysis, methylation analysis, f.a.b.-m.s., g.l.c.-m.s. and enzymic hydrolysis as beta-D-Galp-(1----3)-D-Glc, beta-D-Galp-(1----6)-D-Glc, beta-D-Galp-(1----6)-D-Gal, beta-D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, beta-D-Galp-(1----6)[beta-D-Galp-(1----4)]-D-Glc, beta-D-Galp-(1----2)[beta-D-Galp-(1----6)]-D-Glc, beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Ga lp- (1----4)-D-Glc, beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-DGalp-(1----3)-beta -D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, and beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Ga lp-(1----3)-beta-D-G-alp-(1----3) beta-D-Galp-(1----4)-D-Glc.     

Methane from cattle waste: Effects of temperature, hydraulic retention time, and influent substrate concentration on kinetic parameter (k)

The effects of temperature (35 and 55 degrees C), influent volatile solids (VS) concentration (S(0) = 43, 64, 82, 100, and 128 kg VS/m(3)) and hydraulic retention time (HRT = 4, 5, 8, 10, 15, and 25 days) on methane (CH(4)) production from cattle waste were evaluated using 3-dm(3) laboratoryscale fermentors. The highest CH(4) production rate achieved was 6.11 m(3) CH(4) m(-3) fermentor day(-1) at 55 degrees C, four days HRT, and S(0) = 100 kg VS/m(3). Batch fermentations showed an ultimate CH(4) yield (B(0)) of 0.42 m(3) CH(4)/kg VS fed. The maximum loading rates for unstressed fermentation were 7 kg VS m(-3) day(-1) at 35 degrees C and 20 kg VS m(-3) day(-1) at 55 degrees C. The kinetic parameter (K, an increasing K indicates inhibition of fermentation) increased exponentially as S(0) increased, and was described by: K = 0.8 + 0.0016 e(0.06S(0) ). Temperature had no significant effect on K for S(0) between 40 and 100 kg VS/m(3). The above equation predicted published K values for cattle waste within a mean standard error of 7%.     

Role of the inositol 1,4,5-trisphosphate receptor in Ca(2+) feedback inhibition of calcium release-activated calcium current (I(crac)).

We examined the activation and regulation of calcium release-activated calcium current (I(crac)) in RBL-1 cells in response to various Ca(2+) store-depleting agents. With [Ca(2+)](i) strongly buffered to 100 nM, I(crac) was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP(3)), and two metabolically stable IP(3) receptor agonists, adenophostin A and L-alpha-glycerophospho-D-myoinositol-4,5-bisphosphate (GPIP(2)). With minimal [Ca(2+)](i) buffering, with [Ca(2+)](i) free to fluctuate I(crac) was activated by ionomycin, thapsigargin, and by the potent IP(3) receptor agonist, adenophostin A, but not by GPIP(2) or IP(3) itself. Likewise, when [Ca(2+)](i) was strongly buffered to 500 nM, ionomycin, thapsigargin, and adenophostin A did and GPIP(2) and IP(3) did not activate detectable I(crac). However, with minimal [Ca(2+)](i) buffering, or with [Ca(2+)](i) buffered to 500 nM, GPIP(2) was able to fully activate detectable I(crac) if uptake of Ca(2+) intracellular stores was first inhibited. Our findings suggest that when IP(3) activates the IP(3) receptor, the resulting influx of Ca(2+) quickly inactivates the receptor, and Ca(2+) is re-accumulated at sites that regulate I(crac). Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca(2+) pools following activation by different IP(3) receptor agonists indicates that the critical regulatory pool of Ca(2+) may be very small in comparison to the total IP(3)-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP(3) receptors located on discrete IP(3)-sensitive Ca(2+) pools in calcium feedback regulation of I(crac) and capacitative calcium entry.     

Chemical weed management in wheat at higher altitudes

To evaluate the effect of different herbicides for controlling weeds in wheat (variety Fakhr-i-Sarhad),at higher attitude, an experiment was conducted at Agriculture Research Station, Chitral during Rabi season 2003-04, using Randomized Complete Block Design, keeping four replications. The experiment, sown in November comprised of eight treatments, viz; seven herbicides and a weedy check. Each treatment consisted of 5 rows each 30 cm apart and 5 m long thus giving a total size of 5 m x 1.5 m. The herbicides used included; terbutryn + triasulfuron at 0.16 kg, 2,4-D at 0.7 kg, fenoxaprop-P-ethyl at 0.93 kg, clodinafop at 0.05 kg, bromoxynil + MCPA at 0.49 kg, carfentrazon-ethyl at 0.02 kg and isoproturon at 1.0 kg a.i ha(-1). The data were recorded on weed kill efficiency (%), fresh weed biomass (kg ha(-1)), plant height (cm), spike length (cm), number of tillers m(-2), number of grains spike(-1), thousand grains weight (g), biological yield (kg ha(-1)), grain yield (kg ha(-1)) and harvest index (%). The data recorded on weed kill efficiency, weed biomass (kg ha1), grains yield (kg ha(-1)) and harvest index (%) were significantly affected by the different herbicidal treatments. Statistically isoproturon treatment exhibited the best performance, with maximum weed kill efficiency (48.26%) and minimum fresh weed biomass (433.3 kg ha(-1)) as compared to weedy check (6 %) and (1102 kg ha(-1)), respectively. Similarly, the spike length (8.34 cm), number of tillers (427 m(-2)), number of grains spike(-1) (38.0), thousand grains weight (39.85 g), biological yield (8475 kg ha(-1)), grain yield (2530 kg ha(-1)) and harvest index (31.3%) were the highest in isoproturon treatments as compared to weedy check having (7.64 cm), (356 m(-2)), (34.1), (37.12 g), (6858 kg), (1913 kg ha(-1)) and (27%), respectively.     

The structures of oligosaccharides excreted by sheep with swainsonine toxicosis

Eleven oligosaccharides were purified form the urine of sheep with swainsonine toxicosis induced by the feeding of Astragalus lentiginosus. Oligosaccharides were extracted by charcoal adsorption, chromatographed on Bio-Gel P-2, and partially fractionated by preparative-layer chromatography. Separation into individual compounds was completed by semi-preparative high pressure liquid chromatography. Structures were determined by a combination of high pressure liquid chromatography and exo- and endo- glycosidase action, methanolysis followed by gas-liquid chromatography, methylation analysis, and high resolution nuclear magnetic resonance spectroscopy. Two homologous series of oligosaccharides were identified: (a) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----2)-alpha-D-Manp(1----3)-[alpha-D-Manp+ ++-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----3)-[alpha- D-Manp-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc (minor series); (b) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-alpha-D-Manp-(1----6)-beta-D-Manp -(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----6)-alpha-D-Manp-(1----6)-beta-D-Manp++ +-(1----4)-beta-D-GlcpNAc - (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)-[alpha-D-Manp -(1----3)]-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)- [alpha-D-Manp-(1----3)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D- GlcpNAc (major series).     

Solution chemistry and cytotoxic properties of novel organogold(III) compounds

The solution behaviour of some novel organogold(III) compounds was investigated, and their cytotoxic properties evaluated against a few human tumour cell lines (A2780/S, A2780/R, MCF7, HT29 and A549). Specifically, the following compounds were considered: [Au(bipy(dmb)-H)(2,6-xylidine-H)][PF(6)] (AuXyl) and [Au(bipy(dmb)-H)(p-toluidine-H)][PF(6)] (AuTol) (in which bipy(dmb)=6-(1,1-dimethylbenzyl)-2,2'-bipyridine), [Au(py(dmb)-H)(AcO)(2)] (AuPyAcO) (in which py(dmb)=2-(1,1-dimethylbenzyl)-pyridine) and [Au(pz(Ph)-H)Cl(3)]K (AuPzCl) (in which pz(Ph)=1-phenylpyrazole). The solution chemistry of these compounds, under physiological-like conditions, was investigated through UV-vis absorption and (1)H NMR spectroscopies. Significant cytotoxic effects in vitro were observed in selected cases.     

Isoprenoid phenol and quinone precursors of ubiquinones and dihydroubiquinones [ubiquinones(H2)] in fungi

1. Ten moulds and two yeasts were analysed for the presence of 2-polyprenylphenols, 2-polyprenyl(H(2))phenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-2-polyprenyl(H(2))phenols, 6-methoxy-2-polyprenyl-1,4-benzoquinones, 6-methoxy-2-polyprenyl(H(2))-1,4-benzoquinones, 5-demethoxyubiquinones, 5-demethoxyubiquinones(H(2)), ubiquinones and ubiquinones(H(2)). 2. The organisms were found to be of three types: (a) those that contained only ubiquinones (Aspergillus fumigatus and Penicillium brevi-compactum) or ubiquinones(H(2)) (Alternaria solani, Claviceps purpurae and Penicillium stipitatum); (b) those that contained 5-demethoxyubiquinones and ubiquinones (Agaricus campestris, Aspergillus niger, Phycomyces blakesleeanus, Rhodotorula glutinis and Saccharomyces cerevisiae) or 5-demethoxyubiquinones(H(2)) and ubiquinones(H(2)) (Aspergillus quadrilineatus and Neurospora crassa); (c) one that contained 2-decaprenyl(H(2))phenol, 6-methoxy-2-decaprenyl(H(2))phenol, 6-methoxy-2-decaprenyl(X-H(2))-1,4-benzoquinone, 5-demethoxyubiquinone-10(X-H(2)) and ubiquinones(H(2)) (Aspergillus flavus). 3. Studies were made on the biosynthesis of ubiquinones and ubiquinones(H(2)) by Asp. flavus, Phyc. blakesleeanus and S. cerevisiae. These provided evidence that in Phyc. blakesleeanus 5-demethoxyubiquinone-9 is a precursor of ubiquinone-9 and that in S. cerevisiae 5-demethoxyubiquinone-6 is a precursor of ubiquinone-6. In addition they yielded results that may be interpreted as providing evidence that in Asp. flavus 6-methoxy-2-decaprenyl(X-H(2))-1,4-benzoquinone and 5-demethoxyubiquinone-10(X-H(2)) are precursors of ubiquinone-10(X-H(2)).     

Immunohistological investigations of PAS-negative globular intracisternal hyalin in human liver biopsy specimens

Eight liver biopsy specimens from five patients with PAS-negative intracisternal hyalin were investigated by immunofluorescence for: (1) immunoglobulins (Ig) G, A, M, D, E; (2) light chains (kappa and lambda); (3) complement components C1q, C4, C3c, C5, C9; (4) C1-inactivator; (5) C3-activator; (6) alpha 1-antitrypsin; (7) alpha 1-antichymotrypsin; (8) plasminogen; (9) fibrinogen; (10) fibrinogen breakdown products D and E; (11) fibronectin; (12) prealbumin; (13) albumin; (14) betalipoprotein; (15) apolipoprotein; (16) alpha 1- and alpha 2-glycoprotein; (17) cholinesterase; (18) ceruloplasmin; (19) haemopexin; (20) myoglobin; (21) placenta lactogen; (22) transferrin; (23) actin; (24) myosin; (25) cathepsin D; and (26) hepatitis B surface and core antigens (HBsAg and HBcAg). The globules reacted significantly with antisera against C3c (three patients), C4 (three patients), C3-activator (one patient) and fibrinogen (two patients). The cause of the protein accumulation is not clear. Serial studies indicate the possibility of a disturbance of protein secretion and an as yet unidentified immune complex disorder.     

Volume and compressibility changes accompanying thermally-induced native-to-unfolded and molten globule-to-unfolded transitions of cytochrome c: a high pressure study

We have measured the transition temperatures, T(M), and van't Hoff enthalpies, DeltaH(M), of the thermally induced native-to-unfolded (N-to-U) and molten globule-to-unfolded (MG-to-U) transitions of cytochrome c at pressures between 50 and 2200 bar. We have used the pressure dependence of T(M) to evaluate the changes in volume, Delta(v), accompanying each protein transition event as a function of temperature and pressure. From analysis of the temperature and pressure dependences of Delta(v), we have additionally calculated the changes in expansibility, Delta(e), and isothermal compressibility, Delta(k)(T), associated with the thermally induced conformational transitions of cytochrome c. Specifically, if extrapolated to 25 degrees C, the native-to-unfolded (N-to-U) transition is accompanied by changes in volume, Delta(v), expansibility, Delta(e), and isothermal compressibility, Delta(k)(T), of -(5 +/- 3) x 10(-3) cm(3) g(-1), (1.8 +/- 0.3) x 10(-4) cm(3) g(-1) K(-1), and approximately 0 cm(3) g(-1) bar(-1), respectively. The molten globule-to-unfolded (MG-to-U) transition is accompanied by changes in volume, Delta(v), and isothermal compressibility, Delta(k)(T), of -(2.9 +/- 0.3) x 10(-3) cm(3) g(-1) at 40 degrees C and -(1.9 +/- 0.3) x 10(-6) cm(3) g(-1) bar(-1) at 35 degrees C, respectively. By comparing the volumetric properties of the N-to-U and N-to-MG transitions of cytochrome c, we have estimated the properties of the native-to-molten globule (N-to-MG) transition. For the latter transition, the changes in volume, Delta(v), and isothermal compressibility, Delta(k)(T), are approximately 0 cm(3) g(-1) at 40 degrees C and 1.9 cm(3) g(-1) bar(-1) at 35 degrees C, respectively. Our estimate for the change in expansibility, Delta(e), upon the N-to-MG is negative and equal to -(5 +/- 3) x 10(-4) cm(3) g(-1) K(-1). This finding contrasts with the results of previous studies all of which report positive changes in expansibility associated with protein denaturation. In general, our volumetric data permit us to assess the combined effect of temperature and pressure on the stability of various conformational states of cytochrome c.