Metabolism of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate in the neuronal and glial compartments of the adult rat brain as detected by [(13)C, (2)H] NMR spectroscopy

Ex vivo ?(13)C, (2)H? NMR spectroscopy allowed to estimate the relative sizes of neuronal and glial glutamate pools and the relative contributions of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate to the neuronal and glial tricarboxylic acid cycles of the adult rat brain. Rats were infused during 60 min in the right jugular vein with solutions containing (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose or (2-(13)C, 2-(2)H(3)) acetate only. At the end of the infusion the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by high resolution (13)C NMR spectroscopy (90.5 MHz). The relative sizes of the neuronal and glial glutamate pools and the contributions of acetyl-CoA molecules derived from (2-(13)C, (2)H(3)) acetate or (1-(13)C) glucose entering the tricarboxylic acid cycles of both compartments, could be determined by the analysis of (2)H-(13)C multiplets and (2)H induced isotopic shifts observed in the C4 carbon resonances of glutamate and glutamine. During the infusions with (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose, the glial glutamate pool contributed 9% of total cerebral glutamate being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (4%), (2-(13)C) acetyl-CoA (3%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (2%). The neuronal glutamate pool accounted for 91% of the total cerebral glutamate being mainly originated from (2-(13)C) acetyl-CoA (86%) and (2-(13)C, 2-(2)H) acetyl-CoA (5%). During the infusions of (2-(13)C, 2-(2)H(3)) acetate only, the glial glutamate pool contributed 73% of the cerebral glutamate, being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (36%), (2-(13)C, 2-(2)H) acetyl-CoA (27%) and (2-(13)C) acetyl-CoA (10%). The neuronal pool contributed 27% of cerebral glutamate being formed from (2-(13)C) acetyl-CoA (11%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (16%). These results illustrate the potential of ?(13)C, (2)H? NMR spectroscopy as a novel approach to investigate substrate selection and metabolic compartmentation in the adult mammalian brain.     

不同浓度Ni、Cu处理对骆驼蓬光合作用和叶绿素荧光特性的影响水

以骆驼蓬幼苗为材料,采用盆栽试验研究不同浓度(0、50、100、200、400 mg·kg-1)Ni、Cu处理对骆驼蓬叶片光合作用、叶绿素荧光特性及生长状况的影响.结果表明:随着Ni浓度的增加,骆驼蓬幼苗叶片的光合色素含量、净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)、PS Ⅱ最大光化学效率(Fv/Fm)、PS Ⅱ电子传递量子产率(φpsⅡ)、光化学猝灭系数(qp)及各项生长指标均呈显著下降趋势,而细胞间隙CO2浓度(Ci)和非光化学猝灭系数(qn)呈显著增加趋势,其中Pn的下降主要是由非气孔限制所致;骆驼蓬幼苗叶片的光合色素含量、Pn、Gs、Tr、Ci、Fv/Fm、φpsⅡ、qp及各项生长指标均在50 mg·kg-1Cu处理时达到峰值,叶绿素a和b、Pn、Gs、Tr、Ci、Fv/Fm及各项生长指标值在100 mg·kg-1Cu处理时仍微高于对照,而后随Cu浓度的增加,光合色素含量、Pn、Gs、Tr、Ci、Fv/Fm、φpsⅡ、qp及各项生长指标均呈下降趋势,qN呈增加趋势,其中Pn的下降主要是由气孔限制所致.     

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase

T4 RNA ligase catalyzes the synthesis of ATP beta,gamma-bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH(2)p) (100), clodronate (pCCl(2)p) (52), and etidronate (pC(OH)(CH(3))p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH(2)-CH(2)-NH(2))p), and alendronate (pC(OH)(CH(2)-CH(2)-CH(2)-NH(2))p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E-AMP in a concentration dependent manner. The K(m) values and the rate of synthesis (k(cat)) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26+/-0.05 mM (0.28+/-0.05 s(-1)); clodronate, 0.54+/-0.14 mM (0.29+/-0.05 s(-1)); and etidronate, 4.3+/-0.5 mM (0.028+/-0.013 s(-1)). In the presence of GTP, and ATP or AppCCl(2)p the relative rate of synthesis of adenosine 5',5'-P(1),P(4)-tetraphosphoguanosine (Ap(4)G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH(2)p) was a very poor substrate for the synthesis of Ap(4)G. To our knowledge this report describes, for the first time, the synthesis of ATP beta,gamma-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases.     

Kinetic study of the quenching reaction of singlet oxygen by tea catechins in ethanol solution

A kinetic study of the quenching reaction of singlet oxygen ((1)O(2)) with catechins (catechin (CA), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG)) and related compounds (5-methoxyresorcinol (MR), 4-methylcatechol (MC), and n-propyl gallate (PG)) was performed in ethanol at 35 degrees C. MR, MC, and PG are considered to be a model of resorcinol (A)-, catechol (B)-, and gallate (G)-rings in catechins, respectively. The overall rate constants, k(Q) (= k(q) + k(r), physical quenching + chemical reaction), for the reaction of catechins with (1)O(2) increased in the order of PG < MR < MC < CA < EC < EGC < ECG < EGCG. In a comparison of the rate constants, the relationship between quenching rates and chemical structures is discussed. The catechins which have lower peak oxidation potentials, E(P), show higher reactivities. It was observed that the chemical reaction (k(r)) is almost negligible in the quenching reaction of (1)O(2) by catechins. The k(Q) values of EGCG (1.47 x 10(8) M(-1) s(-1)) and ECG (7.81 x 10(7)) were found to be larger than those of lipids (1.3 x 10(5)-1.9 x 10(5) M(-1) s(-1)), amino acids (<3.7 x 10(7)), and DNA (5.1 x 10(5)). Further, these values are similar to those (1.15 x 10(8)-2.06 x 10(8) M(-1) s(-1)) of alpha- and gamma-tocopherol, ubiquinol-10, and gamma-tocopherol hydroquinone (plastoquinol model). The result suggests that catechins may contribute to the protection of oxidative damage in biological systems, by quenching (1)O(2).     

Characterisation and biological activity of Glu3 amino acid substituted GIP receptor antagonists

Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release and glucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biological activity of several Glu(3)-substituted analogues of GIP namely; (Ala(3))GIP, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP. Only (Lys(3))GIP demonstrated moderately enhanced resistance to DPP-IV (p<0.05 to p<0.01) compared to native GIP. All analogues demonstrated a decreased potency in cAMP production (EC(50) 1.47 to 11.02 nM; p<0.01 to p<0.001) with (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated cAMP production (p<0.05). In BRIN-BD11 cells, (Lys(3))GIP, (Phe(3))GIP, (Trp(3))GIP and (Tyr(3))GIP did not stimulate insulin secretion with both (Lys(3))GIP and (Phe(3))GIP significantly inhibiting GIP-stimulated insulin secretion (p<0.05). Injection of each GIP analogue together with glucose in ob/ob mice significantly increased the glycaemic excursion compared to control (p<0.05 to p<0.001). This was associated with lack of significant insulin responses. (Ala(3))GIP, (Phe(3))GIP and (Tyr(3))GIP, when administered together with GIP, significantly reduced plasma insulin (p<0.05 to p<0.01) and impaired the glucose-lowering ability (p<0.05 to p<0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronounced than (Pro(3))GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala(3)), (Phe(3)), (Tyr(3)) or (Pro(3)) provides a new class of functional GIP receptor antagonists.     

Design of peptide oxytocin antagonists with strikingly higher affinities and selectivities for the human oxytocin receptor than atosiban.

The peptide oxytocin (OT) antagonist atosiban, approved for tocolytic use in Europe (under the tradename Tractocile), represents an important new therapeutic advance for the treatment of premature labor. This paper presents some new peptide OT antagonists which offer promise as superior tocolytics. The solid phase synthesis is reported of four pairs of L and D-2-naphthylalanine (L/D-2Nal) position-2 modified analogs of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly-NH(2),d(CH(2))(5)[Tyr(Me)(2),Thr(4)]OVT) (A); the Tyr-NH(2) (9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2) (9)]OVT (B); the Eda(9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)]OVT (C); and the retro COCH(2)Ph(4-0H)(10) modified analog of (C), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT (D). The eight new analogs of A-D are (1) desGly-NH(2),d(CH(2))(5)[D-2Nal(2),Thr(4)]OVT, (2) desGly-NH(2),d(CH(2))(5)[2-Nal(2),Thr(4)]OVT, (3) d(CH(2))(5)[D-2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (4) d(CH(2))(5)[2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (5) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)]OVT, (6) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)]OVT, (7) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT, (8) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-OH)(10)]OVT. Peptides 1-8 were evaluated for agonistic and antagonistic activities in in vitro and in vivo rat bioassays, in rat OT receptor (rOTR) binding assays and in human OT receptor (hOTR) and human vasopressin (VP) vasopressor (V(1a)) receptor (hV(1a)R) binding assays. Also reported are the hOTR and hV(1a)R affinity data for atosiban and for B. None of the eight peptides exhibit oxytocic or vasopressor agonism. Peptides 1-8 exhibit weak antidiuretic agonism (activities in the range 0.014-0.21 U/mg). Peptides 1-6 exhibit potent in vitro (no Mg(2+)) OT antagonism (anti-OT pA(2) values range from 7.63 to 8.08). Peptides 7 and 8 are weaker OT antagonists. Peptides 1-6 are all OT antagonists in vivo (estimated in vivo anti-OT pA(2) values in the range 6.94-7.23). Peptides 1-8 exhibit vasopressor antagonism, anti-V(1a) pA(2) values in the range 5.1-7.65. Peptides 1-8 exhibit high affinities for the rOTR (K(i) values = 0.3-7.8 nM). Peptides 1-4 and B exhibit surprisingly very high affinities for the hOTR; their K(i) values are 0.17, 0.29, 0.07, 0.14 and 0.59 nM, respectively. Peptides 1-4 and B exhibit respectively 449, 263, 1091, 546 and 129 times greater affinity for the hOTR than atosiban (K(i) = 76.4 nM). Peptides 1-4 exhibit high affinities for the hV(1a)R (K(i)s = 1.1 nM, 1.3 nM, 0.19 nM and 0.54 nM, all higher than the hV1(a)R affinities exhibited by atosiban (K(i) = 5.1 nM) and by B (K(i) = 5.26 nM). Because of their strikingly higher affinities for the hOTR than atosiban, peptides 1-4 and B exhibit gains in anti hOT/anti hV(1a) receptor selectivity compared with atosiban of 93, 64, 39, 56 and 127, respectively. These OT antagonists are thus promising candidates for development as potential new tocolytic agents.     

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)).     

Antigen-specific T cell factors: a fine analysis of specificity.

The specificity of T cell factors produced in presence of synthetic polypeptide antigens was studied. Factors prepared with either one of the three antigens: poly(Tyr,Glu)-poly(DLALa)--poly(Lys), (T,G)-A--L, poly(Phe,Glu)-poly(DLALa)--poly(Lys), (Phe,G)-A--L, and poly(His,Glu)-poly(DLALa)--poly(Lys), (H,G)-A--L, successfully cooperated with B cells for antibody production to the homologous as well as to the other two immunogens. Furthermore, the activity of a (T,G)-A--L-specific factor was removed after passage through immunoadsorbents built of Sepharose coupled to: (T,G)A--L, (Phe-G)-A--L and poly(Glu)-poly(DLAa)--poly(Lys), (G)-A--L, but not to poly (DLALa)--poly(LLys),A--L. No cross-reactivity was observed between (T,G)-A--L and poly(Tyr,Glu)-poly(Pro)--poly(Lys), (T,G)-Pro--L, at the level of T cell factors, as shown using the above approaches. These results lead to the conclusion that specificity of T cell factors, although not identical, is similar to that of antibodies.     

The effects of drug complexation on the stability and conformation of human serum albumin

We report different analytical methods used to study the effects of 3\'-azido-3\'-deoxythymidine, aspirin, taxol, cisplatin, atrazine, 2,4-dichlorophenoxyacetic, biogenic polyamines, chlorophyll, chlorophyllin, poly(ethylene glycol), vanadyl cation, vanadate anion, cobalt-hexamine cation, and As2O3, on the stability and secondary structure of human serum albumin (HSA) in aqueous solution, using capillary electrophoresis, Fourier transform infrared, ultraviolet visible, and circular dichroism (CD) spectroscopic methods. The concentrations of HSA used were 4% to 2% or 0.6 to 0.3 mM, while different ligand concentrations were 1 microM to 1 mM. Structural data showed drugs are mostly located along the polypeptide chains with both specific and nonspecific interactions. The stability of drug-protein complexes were in the order K(VO(2+)) 1.2 x 10(8) M(-1) > K(AZT) 1.9 x 10(6) M(-)1 > K(PEG) 4.1 x 10(5) M(-1) > K(atrazine) 3.5 x 10(4) M(-1) > K(chlorophyll) 2.9 x 10(4) M(-1) > K2,4-D 2.5 x 10(4) M-1 > K(spermine) 1.7 x 10(4) M(-1) > K(taxol) 1.43 x 10(4) M(-1) > K(Co(3+)) > 1.1 x 10(4) M(-1) > K(aspirin) 1.04 x 10(4)i(-1) > K(chlorophyllin) 7.0 x 10(3) M(-1) > K(VO(3)(-)) 6.0 x 103 M(-1) > K(spermidine) 5.4 x 10(3) M(-1) > K(putrescine) 3.9 x 10(3) M(-1) > K(As(2)O(3)) 2.2 x 10(3) M(-1)> K(cisplatin) 1.2 x 10(2) M(-1). The protein conformation was altered (infrared and CD results) with major reduction of alpha-helix from 60 to 55% (free HSA) to 49 to 40% and increase of beta-structure from 22 to 15% (free HSA) to 33 to 23% in the drug-protein complexes. The alterations of protein secondary structure are attributed to a partial unfolding of HSA on drug complexation.     

Organization of the telencephalon in the channel catfish,Ictalurus punctatus

The cytoarchitectonics of the telencephalon of the channel catfish, Ictalurus punctatus, are described as a basis for experimental analysis of telencephalic afferents and efferents. The olfactory bulb comprises: (1) an outer layer of olfactory nerve fibers, (2) a glomerular layer, (3) an external cell layer, (4) an inner fiber layer, and (5) an internal cell layer. The telencephalic hemispheres comprise the areas ventralis and dorsalis telencephali. The area ventralis consists of: (1) a precommissural, periventricular zone including nucleus 'nother (Vn), the ventral nucleus (Vv), and the dorsal nucleus (Vd); (2) a precommissural, migrated zone of central (Vc) and lateral (VI) nuclei; (3) a supracommissural nucleus (Vs); (4) a caudal commissural zone of postcommissural (Vp) and intermediate (Vi) nuclei; and (5) a preoptic area (PP). The area dorsalis comprises: (1) medial (DM), (2) dorsal (Dd), (3) lateral [DL, containing dorsal (DLd), ventral (DLv), and posterior (DLp) regions], (4) posterior (DP), and (5) central (DC-1, -2, -3) areas. Nucleus taeniae (NT) is transitional between areas dorsalis and ventralis.     

Regulation of TXB(2) and PGE(2) production by TGF-beta(1) in in vitro silica dust-exposed rat alveolar macrophage

We investigated the effect of transforming factor factor-beta(1) (TGF-beta(1)) on thromboxane B(2) (TXB(2)) and prostaglandin E(2) (PGE(2)) production in in vitro silica dust-exposed rat alveolar macrophages (AM). In the presence of 5 mug of anti-TGF-beta(1) antibodies, TXB(2) production decreased, but PGE(2) production increased. Addition of 2 ng of TGF-beta(1) to the culture medium potentiated TXB(2) production, but PGE(2) production apparently did not change. At 50 ng of TGF-beta(1), TXB(2) production decreased, and PGE(2) production varied. Our data suggest that in rat AM: (1) both endogenous and exogenous TGF-beta(1) regulate TXB(2) production; and (2) in the absence of endogenous TGF-beta(1) the liberation of PGE(2) increases; however, exogenous TGF-beta(1) does not have a regulatory effect on PGE(2).     

The factor IXa second epidermal growth factor (EGF2) domain mediates platelet binding and assembly of the factor X activating complex.

Previously we have determined that residues 88-109 (but not Arg(94)) in the second epidermal growth factor (EGF2)-like domain of factor IXa (FIXa) are important for assembly of the factor X (FX) activating complex on phospholipid vesicles (Wilkinson, F. H., London, F. S., and Walsh, P. N. (2002) J. Biol. Chem. 277, 5725-5733). Here we report that these residues are important for platelet binding affinity, stoichiometry, and assembly of the FX activating complex. We prepared several chimeric FIXa proteins using homologous sequences from factor VII (FVII): FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), and FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127) and tested their ability to bind to thrombin-activated platelets. Binding affinities (K(d) values in 10(-9) m) for the proteins were as follows in the presence and absence of FVIIIa, respectively: FIXa(N) (0.55 +/- 0.06, 2.9 +/- 0.45), FIXa(WT) (0.80 +/- 0.08, 3.5 +/- 0.5), FIXa(loop1) (19 +/- 4.0, 27 +/- 5.0), FIXa(loop2) (35 +/- 9.0, 65 +/- 12.0), and FIXa(loop3) (1.1 +/- 0.09, 5.0 +/- 0.90). These K(d) values are in good agreement with K((d)(app)) values (in 10(-9) m) determined from the activation of FX (in the presence and absence of FVIIIa, respectively): FIXa(N) (0.46 +/- 0.05, 1.40 +/- 0.14), FIXa(WT) (0.72 +/- 0.08, 3.8 +/- 0.08), FIXa(loop1) (3.2 +/- 0.72, 14.0 +/- 1.60), FIXa(loop2) (18.4 +/- 1.60, 26.3 +/- 3.40), and FIXa(loop3) (0.7 +/- 0.05, 3.0 +/- 0.15). Moreover, the stoichiometry of binding (sites/platelet) showed an agreement with V(max) of FX activation and was reduced in those proteins that also showed a decreased platelet binding affinity. A peptide corresponding to the FIX EGF2 domain (Leu(84)-Val(128)) was an effective inhibitor of FIXa binding to platelets in both the presence (K(i) = 0.7 x 10(-6) m) and the absence (K(i) = 1.5 x 10(-6) m) of FVIIIa and FX. We conclude that residues 88-109 of the FIXa EGF2 domain mediate binding to platelets and assembly of the FX activating complex.ut not Ar     

Efficiency of the normal human diaphragm with hyperinflation.

We evaluated an index of diaphragm efficiency (Eff(di)), diaphragm power output (Wdi) relative to electrical activation, in five healthy adults during tidal breathing at usual end-expiratory lung volume (EELV) and diaphragm length (L(di ee)) and at shorter L(di ee) during hyperinflation with expiratory positive airway pressure (EPAP). Measurements were repeated with an inspiratory threshold (7.5 cmH(2)O) plus resistive (6.5 cmH(2)O.l(-1).s) load. Wdi was the product of mean inspiratory transdiaphragmatic pressure (DeltaPdi(mean)), diaphragm volume displacement measured fluoroscopically, and 1/inspiratory duration (Ti(-1)). Diaphragm activation, measured with esophageal electrodes, was quantified by computing root-mean-square values (RMS(di)). With EPAP, 1) EELV increased [mean r(2) = 0.91 (SD 0.01)]; 2) in four subjects, L(di ee) decreased [mean r(2) = 0.85 (SD 0.07)] and mean Eff(di) decreased 34% per 10% decrease in L(di ee) (P < 0.001); and 3) in one subject, gastric pressure at EELV increased two- to threefold, L(di ee) was unchanged or increased, and Eff(di) increased at two of four levels of EPAP (P < or = 0.006, ANOVA). Inspiratory loading increased Wdi (P = 0.003) and RMS(di) (P = 0.004) with no change in Eff(di) (P = 0.63) or its relationship with L(di ee). Eff(di) was more accurate in defining changes in L(di ee) [(true positives + true negatives)/total = 0.78 (SD 0.13)] than DeltaPdi(mean).RMS(di)(-1), RMS(di), or DeltaPdi(mean).Ti (all <0.7, P < or = 0.05, without load). Thus Eff(di) was principally a function of L(di ee) independent of inspiratory loading, behavior consistent with muscle force-length-velocity properties. We conclude that Eff(di), measured during tidal breathing and in the absence of expiratory muscle activity at EELV, is a valid and accurate measure of diaphragm contractile function.     

Biosynthetic studies on the alpha-glucosidase inhibitor acarbose: the chemical synthesis of isotopically labeled 2-epi-5-epi-valiolone analogs

2-epi-5-epi-valiolone is a cyclization product of the C(7) sugar phosphate, sedoheptulose 7-phosphate, involved in the biosynthesis of the aminocyclitol moieties of acarbose, validamycin, and pyralomicin. As part of our investigation into the pathway from 2-epi-5-epi-valiolone to the valienamine moiety of acarbose, we prepared 1-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-6], 5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-17], 1-epi-2-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-12] and 2-epi-5-epi-(6-(2)H(2))valiolamine [(6-(2)H(2))-11]. Compounds (6-(2)H(2))-6 and (6-(2)H(2))-17 were synthesized from 2,3,4,6-tetra-O-benzyl-D-glucopyranose in 10 and seven steps, respectively, whereas (6-(2)H(2))-12 and (6-(2)H(2))-11 were synthesized from 2,3,4,6-tetra-O-benzyl-D-mannopyranose in eight and 10 steps, respectively.     

H(2)O(2) opens BK(Ca) channels via the PLA(2)-arachidonic acid signaling cascade in coronary artery smooth muscle

H(2)O(2) is a reactive oxygen species that contracts or relaxes vascular smooth muscle, but the molecular basis of these effects remains obscure. We previously demonstrated that H(2)O(2) opens the large-conductance, calcium- and voltage-activated (BK(Ca)) potassium channel of coronary myocytes (2) and now report physiological and biochemical evidence that the effect of H(2)O(2) on coronary smooth muscle involves the phospholipase A(2) (PLA(2))/arachidonic acid (AA) signaling cascades. H(2)O(2) stimulation of BK(Ca) channel activity was inhibited by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA(2). Furthermore, H(2)O(2) stimulated release of [(3)H]AA from coronary myocytes, and exogenous AA mimicked the effect of H(2)O(2) on BK(Ca) channels. Inhibitors of protein kinase C activity attenuated the effect of H(2)O(2) on BK(Ca) channels, [(3)H]AA release, or intact coronary arteries. In addition, the effect of H(2)O(2) or AA on BK(Ca) channels was inhibited by blockers of lipoxygenase metabolism. In contrast, inhibitors of cyclooxygenase or cytochrome P-450 had no effect. We propose that H(2)O(2) relaxes coronary arteries by stimulating BK(Ca) channels via the PLA(2)/AA signaling cascade and that lipoxygenase metabolites mediate this response.     

Inhibition of pyrophosphatase activity of mouse duodenal alkaline phosphatase by magnesium ions

Duodenal alkaline phosphatase of juvenile (11-day-old) mice, like other non-specific alkaline phosphatases, has the ability to hydrolyse PP(i). When a constant Mg(2+)/PP(i) concentration ratio is maintained, plots of velocity as a function of PP(i) concentration are consistent with Michaelis-Menten kinetics. Mg(2+) activates pyrophosphate hydrolysis and maximal activity is obtained at a constant Mg(2+)/PP(i) concentration ratio of 0.66. At higher ratios there is strong inhibition. At constant concentrations of Mg(2+) and increasing concentrations of PP(i), the velocity-substrate (PP(i)) concentration plots show sigmoidal dependence. By assuming that the true substrate is MgP(2)O(7) (2-) complex, and using complexity constants, the concentrations of free Mg(2+), Mg(2)P(2)O(7) and MgP(2)O(7) (2-) were calculated in assay mixtures ranging in PP(i) concentration from 0.1 to 2.5mm and in total Mg(2+) concentration from 0.6 to 2.6mm. From these data, the concentrations of added Mg(2+) and PP(i) in the assay mixtures were selected so that the velocity could be measured (1) at three fixed concentrations of free Mg(2+) ions with varied concentrations of MgP(2)O(7) (2-) and (2) at four fixed concentrations of Mg(2)P(2)O(7) with varied concentrations of MgP(2)O(7) (2-). Lineweaver-Burk and Hill plots from these data showed that the inhibition is caused by free Mg(2+) ions, of a mixed type and consistent with Michaelis-Menten kinetics. The sigmoidal dependence observed between velocity and PP(i) concentration at constant concentration of total Mg(2+) is therefore not due to allosteric inhibition. It is due to a combined effect of (1) inhibition by free Mg(2+) ions, (2) depletion of the true substrate, MgP(2)O(7) (2-), owing to the formation of Mg(2)P(2)O(7) and (3) the manner in which the concentrations of these three molecular or ionic species change when PP(i) concentration is increased maintaining the total Mg(2+) concentration constant.     

Vasoactive intestinal peptide-camptothecin conjugates inhibit the proliferation of breast cancer cells

The effects of vasoactive intestinal peptide-camptothecin (VIP-CPT) conjugates were investigated on breast cancer cells and cells transfected with VIP receptors (R). (Ala(2,8,9,19,24.25.27), Nle(17), Lys(28))VIP, (A-NL-K)VIP, was synthesized and Lys(28) was coupled to a linker, N-methyl-amino-ethyl-glycine, L2, which formed a carbamate bond with CPT. The resulting (A-NL-K)VIP-L2-CPT was cytotoxic for MCF7 breast cancer cells, which have VPAC(1)-R, with IC(50) values of 380 and 90 nM using the MTT and clonogenic assays, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-VIP to 3T3 cells transfected with VPAC(1)-R with IC(50) values of 1.9, 56 and 126 nM, respectively. In contrast, (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-Ro25-1553 to 3T3 cells transfected with VPAC(2)-R with IC(50) values of 3.9, 3162 and 2690 nM, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT caused increased cAMP after addition to MCF7 cells. (125)I-(A-NL-K)VIP-L2-CPT was internalized by MCF7 cells at 37 degrees C but not 4 degrees C. These results indicate that (A-NL-K)VIP-L2-CPT is a VPAC(1)-R agonist which is cytotoxic for breast cancer cells.     

Comparative binding properties of metallobleomycins with DNA 10-mers.

Properties of the interaction of bleomycin (Blm) and metallobleomycins [M = Zn, Cu(II), Fe(III), and HO(2)-Co(III)] with site-specific and nonspecific DNA oligomers, d(GGAAGCTTCC)(2) (I) and d(GGAAATTTCC)(2) (II), respectively, were investigated. With both 10-mers association constants increased in the series Blm A(2), ZnBlm A(2), Cu(II)Blm A(2), Fe(III)Blm A(2), and HO(2)-Co(III)Blm A(2). Generally, the metallobleomycins were bound with a modestly higher affinity to I. One-dimensional (1)H NMR spectra of the imino proton region of I in the presence of this series of compounds revealed that Blm and Zn- and CuBlm bind in fast exchange on the NMR time scale, while the Fe and Co complexes bind in slow exchange. Blm, ZnBlm, and Cu(II)Blm caused little perturbation of the UV circular dichroism spectrum of I or II. In contrast, Fe(III)Blm and HO(2)-Co(III)Blm induced hypochromic effects in the CD spectrum of I and altered the spectrum of II to a smaller extent. On the basis of these results, the DNA binding structures and properties of Blm A(2), ZnBlm A(2), and CuBlm A(2) differ substantially from those of Fe(III)Blm A(2) and HO(2)-Co(III)Blm A(2).     

Synthesis, crystal structures and cytotoxicities of some transition metal complexes with N-[2-{(pyridin-2-ylmethylidene)amino}ethyl]acetamide

The synthesis and spectroscopic (IR, (1)H and (13)C NMR) characterization of new complexes of Pt(II), Pd(II), Cu(II), and Hg(II) with the Schiff base ligand MeCONHCH(2)CH(2)N=CHPy (L) (Py=pyridine) are reported, together with studies on the cytotoxicities of these complexes, L and [ReBr(CO)(3)(L)] against human leukemia (MOLT-4), breast cancer (MCF-7) and Chang Liver (non-cancerous) cells. The crystal structures of [Pt(L)Cl(2)] (2), [Cu(L)Cl(2)] (4) and [Hg(L)Cl(2)](2) (5) are also reported. Of the complexes studied, [Cu(L)Cl(2)] (4) was identified as the most cytotoxic active derivative against cells of neoplastic origin (MOLT-4, and MCF-7), while having low toxicity on cells of benign origin (Chang Liver).