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Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3β, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists., This work was supported by grants from the National Institutes of Health (National Cancer Institute NIH P01 CA171983 (T.P.L., M.K., M.C.C.), DOD-W81XWH-19-1-0213 (K.H.F.-W.), Spanish Ministry of Science and Innovation PID2020-113813RB-100 (G.F.), and the Brody Brothers Foundation (K.H.F.-W.), Kinston, NC.

The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)–dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8– or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (MlklFLAG/FLAG), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death.Casp8−/−MlklFLAG/FLAGmice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared toCasp8+/+mice. Studies in vitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 inCasp8−/−MlklFLAG/FLAGmice suppressed spontaneous MLKL phosphorylation in vivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.

The in vitro pharmacological profile of TD-5108, a novel, selective 5-HT(4) receptor agonist, was compared to that of clinically efficacious gastroprokinetic 5-HT(4) receptor agonists. TD-5108 produced an elevation of cyclic adenosine monophosphate in human embryonic kidney 293 cells expressing the human recombinant 5-HT(4(c)) (h5-HT(4(c))) receptor (pEC(50) = 8.3) and 5-HT(4) receptor-mediated relaxation of the rat esophagus (pEC(50) = 7.9) and contraction of the guinea pig colon (pEC(50) = 7.9). In all in vitro assays, TD-5108 was a high intrinsic activity agonist, unlike tegaserod, mosapride, and cisapride which, in the majority of test systems, had lower intrinsic activity. TD-5108 had high affinity (pK (i) = 7.7) and selectivity (> or =25-fold) for h5-HT(4(c)) receptors over other biogenic amine receptors. TD-5108 was >500-fold selective over other 5-HT receptors (including h5-HT(2B) and h5-HT(3A)) and, at 3 microM, had no effect on human ether-a-go-go-related gene K+ channels. In conclusion, TD-5108 is a selective 5-HT(4) receptor agonist in vitro. The high intrinsic activity and preferential binding of TD-5108 to 5-HT4 over other 5-HT receptors may result in an improved clinical profile for the treatment of gastrointestinal disorders of reduced motility.

The in vivo preclinical pharmacodynamic profile of TD-5108, a selective 5-HT4 receptor agonist with high intrinsic activity, was compared to that of the clinically studied gastrointestinal pro-kinetic agents, tegaserod, cisapride and mosapride. The activity of TD-5108 was evaluated in guinea pig colonic transit, rat oesophageal relaxation and dog gastrointestinal smooth muscle contractility models. Subcutaneous administration of TD-5108, tegaserod, cisapride and mosapride increased guinea pig colonic transit (rank order of potencies: TD-5108 > tegaserod > cisapride > mosapride). Following intravenous and intraduodenal dosing, TD-5108, tegaserod, cisapride and mosapride produced dose-dependent relaxation of the rat oesophagus. On a molar basis, TD-5108 was approximately twofold less potent than tegaserod following intravenous dosing but 6- or 86-fold more potent than cisapride or mosapride, respectively, and 9- or 18-fold more potent than tegaserod or cisapride, respectively, after intraduodenal administration. Orally dosed TD-5108 increased the contractility of the canine antrum, duodenum and jejunum with higher potency than tegaserod. The selective 5-HT4 receptor agonist, TD-5108, demonstrates robust in vivo activity in the guinea pig, rat and dog gastrointestinal tracts.

Early theories of tumor angiogenesis suggested that preexisting vessels surrounding the tumor were the principal source of the tumor vasculature but recent evidence suggests that endothelial progenitor cells (EPC) migrate from the marrow play an important role in developing the tumor blood supply. In a mouse model, in which the vascularization of a transplantable tumor was studied after bone marrow (BM) transplantation, we show that cells that express Tie-2, Sca-1, CD31 and CD45 function as both BM EPC and primitive hematopoietic stem cells. BM cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the endothelial lineage-specific Tie-2 promoter (Tie-2 /GFP) were used to reconstitute irradiated (12 Gy) wild-type mice. Five donor BM cell populations were studied: (1) whole BM; (2) Sca-1-enriched BMC; (3) GFP/Tie-2+, Sca-1+ BMC; (4) GFP/Tie-2-, Sca-1+ BMC and (5) Sca-1-depleted BMC. After 4 weeks, the mice were injected with Tg.AC tumor cells. Three weeks later, sections from the tumors were stained for CD31 and examined for Tie-2-driven GFP expression. BM-derived endothelial cells were found only in mice transplanted with bone marrow containing populations of Tie-2+, Sca-1+ cells. As few as 3500 of these cells were sufficient to radioprotect lethally irradiated mice. Thus, we conclude that a rare subset of BMC (approximately 4 x 10(-3)%) with the putative properties of hemangioblasts have an active Tie-2 promoter. Selection of Tie-2+Sca-1+ BMC enriches for marrow-derived EPCs that participate in tumor angiogenesis and cells that can provide hematopoietic reconstitution of marrow-ablated mice.

Potentially orally bioavailable prodrugs of the antiretroviral agent 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) were evaluated. Alkyl methyl carbamates were synthesized by alkylation of PMPA with the corresponding alkyl chloromethyl carbonate and N-alkyl chloromethyl carbamate reagents. The prodrugs were evaluated for in vitro antiviral activity in addition to chemical and enzymic stability. The inhibition of human immunodeficiency virus type 1 (HIV-1) strain IIIB replication in MT-2 cells by the carbonate prodrugs was found to be 2.5-500-fold increased compared to PMPA. The alkyl methyl carbonates, except t-butyl methyl carbonate, had reasonable chemical stability at pH 2.2 and 7.4, but were rapidly converted to the corresponding monoester of PMPA in the presence of dog plasma. The alkyl methyl carbamate prodrugs such as N-t-butyl methyl carbamate were found to have high stability in vitro. Based on its chemical stability and good oral bioavailability, bis(POC)PMPA (isopropyl methyl carbonate) was chos...

Literature on the properties, forms and regulation of cytochrome P450 (CYP) in digestive gland of Mytilus sp., including studies indicating the existence of an organic contaminant-inducible CYP1A-like protein, are briefly reviewed. Laboratory and field studies show increases in digestive gland microsomal CYP1A-immunopositive protein levels and/or benzo[a]pyrene hydroxylase (BPH) activity (i.e. metabolism of benzo[a]pyrene to phenols) with exposure of Mytilus sp. to certain polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls. In order to examine further the relationship between these two parameters, M. edulis were collected 25 and 130 days after the release of oil following the grounding of the tanker 'Sea Empress' in South Wales, UK (15 February 1996); and M. galloprovincialis were sampled from sites in south-western France and south-eastern Spain during a cruise aboard the IFREMER Research Vessel 'L'Europe' (2-18 August 1996). In both studies, sites with higher levels of CYP1A-immunopositive protein showed higher levels of BPH activities. Positive correlations were observed between the two measurements - R=0.65 (M. edulis) and 0.68 (M. galloprovincialis), and both fitted linear regression models (P0.05). The CYP1A-immunopositive protein levels and BPH activities tended to be highest at sites with greatest PAH body burden for the Mediterranean study. It is concluded that development of the CYP1A-like protein into a robust biomarker of exposure to organic contaminants will depend upon sequencing of the gene/protein and the subsequent production of Mytilus-specific cDNA and antibody probes. Such probes will then allow a full characterization of the enzyme's properties and gene regulation.

The aim of this study was to compare the effect of the selective noradrenergic reuptake inhibitor desipramine and methylscopolamine bromide, a parasympatholytic agent, on late luteal phase dysphoric disorder (LLPDD), or premenstrual syndrome (PMS), as defined in DSM-III-R. Fourteen patients with PMS were assessed both retrospectively and prospectively during premenstrual cycles. All met the DSM-III-R criteria for LLPDD. They received 3 months treatment each with both desipramine and methylscopolamine in random order using a double-blind cross-over design. PMS symptomatology was significantly reduced after treatment with desipramine and methylscopolamine bromide compared with baseline, but there was no significant difference between them in efficacy. There is evidence from this study that both anticholinergic and noradrenergic pathways may be involved in the genesis of PMS.

Two aldehyde dehydrogenases involved in the degradation of toluene and xylenes, namely, benzaldehyde dehydrogenase and 2-hydroxymuconic semialdehyde dehydrogenase, are encoded by the xylC and xylG genes, respectively, on TOL plasmid pWW0 of Pseudomonas putida. The nucleotide sequence of xylC was determined in this study. A protein exhibiting benzaldehyde dehydrogenase activity had been purified from cells of P. putida (pWW0) (J. P. Shaw and S. Harayama, Eur. J. Biochem. 191:705-714, 1990); however, the amino-terminal sequence of this protein does not correspond to that predicted from the xylC sequence but does correspond to that predicted from the xylG sequence. The protein purified in the earlier work was therefore 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product). This conclusion was confirmed by the fact that this protein oxidized 2-hydroxymuconic semialdehyde (kcat/Km = 1.6 x 10(6) s-1 M-1) more efficiently than benzaldehyde (kcat/Km = 3.2 x 10(4) s-1 M-1). The xylC product, the genuine benzaldehyde dehydrogenase, was purified from extracts of P. putida (pWW0-161 delta rylG) which does not synthesize 2-hydroxymuconic semialdehyde dehydrogenase. The amino-terminal sequence of the purified protein corresponds to the amino-terminal sequence deduced from the xylC sequence. This enzyme efficiently oxidized benzaldehyde (kcat/Km = 1.7 x 10(7) s-1 M-1) and its analogs but did not oxidize 2-hydroxymuconic semialdehyde or its analogs.

The pterygospinous and pterygoalar foramina are illustrated photographically. To confirm the incidence of the foramina 454 skulls from various racial groups were examined. A survey of the literature shows that the pterygospinous foramen is present in about 10% of individuals and the pterygoalar foramen in about 1%. The possibility that either foramen may trap, compress, or cause traction of anomalous cutaneous branches of the mandibular division of the trigeminal nerve, or occlude blood vessels supplying the trigeminal ganglion is discussed in relation to trigeminal neuralgia. © 1993 Wiley-Liss, Inc.

The stereochemistry at position 2 of the title compound, C 14 H 17 BrO 4 , has been confirmed as S.

The substrate-specificities of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, encoded by TOL plasmid pWW0 of Pseudomonas putida mt-2, were determined. The rates of benzyl alcohol dehydrogenase-catalysed oxidation of substituted benzyl alcohols and reduction of substituted benzaldehydes were independent of the electronic nature of the substituents at positions 3 and 4. Substitutions at position 2 of benzyl alcohol affected the reactivity of benzyl alcohol dehydrogenase: the velocity of the benzyl alcohol dehydrogenase-catalysed oxidation was lower for compounds possessing electron-withdrawing substitutions. In the reverse reaction of benzyl alcohol dehydrogenase, none of the substitutions tested influenced the apparent kcat. values. The rates of benzaldehyde dehydrogenase-catalysed oxidation of substituted benzaldehydes were influenced by the electronic nature of the substitutions: electron-withdrawing groups at positions 3 and 4 favoured the oxidation of benzaldehydes. Substitution at position 2 of benzaldehyde greatly diminished the benzaldehyde dehydrogenase-catalysed oxidation. Substitution at position 2 with electron-donating groups essentially abolished reactivity, and only substitutions that were strongly electron-withdrawing, such as nitro and fluoro groups, permitted enzyme-catalysed oxidation. The influence of the electronic nature and the position of substitutions on the aromatic ring of the substrate on the velocity of the catalysed reactions provided some indications concerning the transition state during the oxidation of the substrates, and on the rate-limiting steps of the enzymes. Pseudomonas putida mt-2 containing TOL plasmid pWW0 cannot grow on toluene derivatives substituted at position 2, nor can it grow on 2-substituted benzyl alcohols or aldehydes. One of the reasons for this may be the substrate-specificities of the benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase.

A series of copolymers and blends based on polybutylene were characterized using 13C NMR and thermal analysis. Optical microscopy techniques were used to measure crystallization rates and examine their morphology, and this behavior was related to their composition. As expected, the highest melting and crystallization temperatures, and the fastest crystallization rates were obtained for the polybutylene homopolymer. Copolymerized ethylene randomly distributed along the chain, even at a level of 1%, significantly reduced the melting temperature and crystallization rate. Increasing the ethylene content to 5% results in a polybutylene which crystallizes with difficulty to produce poorly defined spherulites. Four blends based on a polybutylene containing 1% ethylene were studied. A dispersed crystalline phase could be detected in all cases. Two of the blends contained 6-7% ethylene but differed in molar mass. Their melting and crystallization behavior was similar although the spherulite growth rate fo...

Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia. To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF. The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM. Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF. Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine. Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h. Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies. These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors.

Xylene monooxygenase, encoded by the TOL plasmid of Pseudomonas putida, catalyzes the oxidation of toluene and xylenes and consists of two different subunits encoded by xylA and xylM. In this study, the complete nucleotide sequences of these genes were determined and the amino acid sequences of the xylA and xylM products were deduced. The XylM sequence had a 25% homology with alkane hydroxylase, which catalyzes the omega-hydroxylation of fatty acids and the terminal hydroxylation of alkanes. The sequence of the first 90 amino acids of XylA exhibited a strong similarity to the sequence of chloroplast-type ferredoxins, whereas the rest of the XylA sequence resembled that of ferredoxin-NADP+ reductases. Based on this information, the structure and function of xylene monooxygenase were deduced. XylM may be a catalytic component for the hydroxylation of the carbon side chain of toluene and xylenes and, as is the alkane hydroxylase protein, may be a membrane-bound protein containing ferrous ion as a prosthetic group. XylA may have two domains consisting of an N-terminal region similar to chloroplast-type ferredoxins and a C-terminal region similar to ferredoxin-NADP+ reductases. The ferredoxin portion of XylA may contain a [2Fe-2S] cluster and reduce the oxidized form of the XylM hydroxylase. The activity determined by the C-terminal region of the XylA sequence may be the reduction of the oxidized form of ferredoxin by concomitant oxidation of NADH.

The title compound, C 17 H 14 O 5 , was obtained as a byproduct from a procedure to regioselectively hydroxylate homochiral acid derivatives. Structural analysis confirmed its identity and showed it to be achiral.

Formoterol is a beta2-adrenoceptor agonist marketed as a racemic mixture of the active (R; R)- and inactive (S; S)-enantiomers (rac-formoterol). The drug produces prolonged bronchodilation by inhalation but there is significant interpatient variability in duration of effect. Previous work has shown that in humans formoterol is metabolized by conjugation with glucuronic acid but little is known about the stereoselectivity of this reaction. The aim of the present study was to investigate the glucuronidation of formoterol enantiomers in vitro by human liver microsomes.The kinetics of formation of formoterol glucuronides during incubation of racemate and of single formoterol enantiomers with human liver microsomes (n=9) was characterized by chiral h.p.l.c. assay.The kinetics of glucuronidation of the two formoterol enantiomers obeyed the Michaelis-Menten equation. Glucuronidation of formoterol was stereoselective and occurred more than two times faster for (S; S)-formoterol than for (R; R)-formoterol. In incubations with single formoterol enantiomers, the median (n=9) Km values for (R; R)-glucuronide and (S; S)-glucuronide were 827.6 and 840.4 microm, respectively, and the median V max values were 2625 and 4304 pmol min-1 mg-1, respectively. Corresponding values determined in incubations with rac-formoterol were 357.2 and 312.1 microm and 1435 and 2086 pmol min-1 mg-1 for (R; R)- and (S; S)-glucuronide, respectively. Interindividual variation was large with the ratio of V max/Km (S; S/R; R) ranging from 0.57 to 6.90 for incubations with rac-formoterol.Our study demonstrates that glucuronidation of formoterol by human liver microsomes is stereoselective and subject to high interindividual variability. These findings suggest that clearance of formoterol in humans is subject to variable stereoselectivity which could explain the variation in duration of bronchodilation produced by inhaled formoterol in patients with asthma.

The chemical, enzymatic, and biological stabilities and physical properties of a series of salicylate and aryl ester prodrugs of the antiviral agent, cyclic HPMPC, were evaluated to support the selection of a lead compound for clinical development.Chemical stabilities of the prodrugs in buffered solutions at 37 degrees C were determined. Stability was also studied in the presence of porcine liver carboxyesterases (PLCE) at pH 7.4 and 25 degrees C. Tissue stabilities were examined in both human and dog intestinal homogenates, plasmas and liver homogenates. Prodrug and product concentrations were determined by reverse phase HPLC.Chemical degradation of the prodrugs resulted in the formation of both cyclic HPMPC and the corresponding HPMPC monoester. Chemical stability was dependent on the orientation of the exo-cyclic ligand; the equatorial isomers were 5.4- to 9.4-fold more reactive than the axial isomers. In the presence of PLCE, the salicylate prodrugs cleaved exclusively to give cyclic HPMPC and not the HPMPC monoester. In plasma, but not intestinal or liver homogenates, the salicylate esters of cyclic HPMPC cleaved readily with a rate dependent on the chain length of the alkyl ester substituent.The carboxylate function on the salicylate prodrugs of cyclic HPMPC provides an additional handle to chemically modify the lipophilicity, solubility and the biological reactivity of the prodrug. In tissue and enzymatic studies, the major degradation product is cyclic HPMPC. The salicylate ester prodrugs are attractive drug candidates for further in vivo evaluation.

In July 1996, because of concern regarding the possible transmission of Creutzfeldt-Jakob disease, the province of Quebec stopped the reuse of percutaneous transluminal coronary angioplasty (PTCA) catheters. Prior to this time, PTCA balloon catheters were used a maximum of four times and guide catheters a maximum of two times in the cardiac catheterization laboratory at our institution. After this time, only new catheters were used. In order to examine the effects of catheter reuse on duration of PTCA procedures and clinical outcomes, we compared 53 consecutive patients undergoing PTCA prior to 21 July 1996 with 54 consecutive patients undergoing PTCA after that time. A total of 81 men and 26 women underwent PTCA (average age, 64 +/- 12 years). There were no significant differences between the single-use and reuse groups with respect to baseline characteristics. There were also no significant differences in the numbers of PTCA catheters used (97 vs. 103, P = NS) or angiographic success rates (88% vs. 83%, P = NS). There was a trend for total procedure time and fluoroscopy time to be slightly longer for single-use compared with reuse cases (49.2 vs. 45.7 min and 19.7 vs. 16.8 min, respectively; P = NS for both comparisons). However, after controlling for case severity and the use of stents, there were no significant differences in total procedure time or fluoroscopy time between the two groups. We found little evidence to suggest that the reuse of PTCA catheters is associated with longer total procedure time or fluoroscopy time. We conclude that if catheter reuse is not found to be associated with infectious disease transmission, its widespread use should be considered. Cathet. Cardiovasc. Intervent. 48:54-60, 1999.

A rapid and sensitive chiral high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of salmeterol and its principal human metabolite alpha-hydroxysalmeterol is described. The two pairs of enantiomers were resolved on a chiral-cellobiohydrolase column and detected by electrochemical detection at +700 mV. Standard curves were linear over the concentration range 0.1 to 4.0 microM for alpha-hydroxysalmeterol enantiomers and 2.5 to 40.0 microM for salmeterol enantiomers. Intra- and inter-day coefficients of variation were10%. The method was applied to a study of human hepatic metabolism in vitro which showed that microsomal metabolism of salmeterol to alpha-hydroxysalmeterol is not stereoselective.

Background: Helicobacter pylori seroprevalence increases with age in adult life but spontaneous reversion may occur in childhood and adolescence. Aims: To determine the seroprevalence of H. pylori in a longitudinal study of New Zealanders at ages 11 and 21. Methods: Serum from members of the Dunedin Multidisciplinary Health and Development Study (DMHDS) at age 11 (n=561; 303 males, 258 females) and 21 (n=785; 413 males, 372 females) was tested for H. pylori antibodies. A large proportion of those tested at age 11 was retested at age 21 (n=465; 262 males, 203 females). Serological status was examined in terms of gender, socioeconomic status (SES) and self-reported use of antibiotics. Results: The seroprevalence of H. pylori decreased by 38% from 6.6% (37/561) at age 11 to 4.1% (32/785) at age 21. Seroprevalence at age 11 was not associated with gender or SES. For those tested at both ages, the drop in seroprevalence from 6.7% to 4.1% was statistically significant (t=2.57, p

To report on the changing use of slow acting antirheumatic drugs in the treatment of rheumatoid arthritis by contrasting prescribing patterns in 1990 and 1995.Data were extracted from the case notes of 103 outpatients with rheumatoid arthritis. Results were compared with those obtained in 1990 in a survey of 81 patients using identical methods.There was a significant increase in the use of methotrexate between 1990 and 1995, and a marked decrease in the use of auranofin. A new feature was the use of drugs in combination. Methotrexate was the most effective agent and auranofin least effective (p = 0.02). The agent with the highest average toxicity score was D-penicillamine. The long term tolerability of methotrexate was superior, with a median time for remaining on therapy 6.4 times longer than that of the other slow acting antirheumatic drugs (p = 0.01).Our results suggest that identified trends in the altered use of slow acting antirheumatic drugs for treatment of rheumatoid arthritis are rationally based on the increased use of the most effective agents and decreased use of those with greater toxicity and lesser efficacy.

A series of prodrugs designed to enhance the oral bioavailability of the antiretroviral agent 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA; 1) have been synthesized, including a bis-(acyloxymethyl) ester 2 and a series of bis-(alkoxycarbonyloxymethyl) esters 3-9. The in vitro biological stability and in vivo pharmacokinetics of these prodrugs were evaluated to support selection of a prodrug candidate for clinical evaluation.The in vitro biological stability of the prodrugs was examined in dog tissues (intestinal homogenate, plasma and liver homogenate). The apparent half-lives were determined based on the disappearance of prodrug using reverse-phase HPLC with UV detection. Oral bioavailability of PMPA from each prodrug was determined in fasted beagle dogs. Concentrations of PMPA in plasma were determined by HPLC following fluorescence derivatization. Data for prodrugs were compared to historical data for intravenous PMPA.All prodrug were rapidly hydrolyzed in dog plasma and tissues (t1/260 min). In fasted beagle dogs, bis-[(pivaloyloxy)methyl] PMPA (bis-POM PMPA) 2 had the highest oral bioavailability as PMPA (37.8 +/- 5.1%). The oral bioavailabilities of PMPA from bis-(alkoxycarbonyloxymethyl) esters ranged from 16.0% to 30.7% and PMPA was the major metabolite formed.There was a correlation between oral bioavailability and intestinal stability of bis-(alkoxycarbonyloxymethyl) ester prodrugs (r2 = 0.96). Lipophilicity (log P) was not a good predictor of oral bioavailability. The most labile prodrugs in dog intestinal homogenates, bis-(n-butyloxycarbonyloxymethyl) PMPA 5 and bis-(neo-pentyloxy-carbonyloxymethyl) PMPA 8 (t1/25 min) had the lowest oral bioavailabilities. Based on good oral bioavailability (30.1%), chemical and intestinal stability bis-(isopropyloxycarbonyloxymethyl) PMPA (bis-POC PMPA) 4 was selected as a candidate for clinical evaluation.

The oral bioavailability of PMEA [9-[2-(phosphonomethoxy)ethyl]adenine; adefovir) has been determined in rats from three bisester prodrugs of PMEA: bis-(pivaloyloxymethyl) PMEA (bis-POM PMEA), bis-(phenyl) PMEA, and bis-(o-ethoxyphenyl) PMEA. The prodrugs were each administered to 9 male rats as solutions in PEG 400 at a dose of 10 mg-equivalent of PMEA per kg. Plasma samples were obtained over the course of 12 hr and concentrations of PMEA were determined by fluorescence derivatization and analysis by HPLC. Concentrations of PMEA observed in plasma following oral administration of PMEA prodrugs were compared with levels observed for intravenous PMEA. The observed oral bioavailabilities of PMEA from bis-POM PMEA, bis-(phenyl) PMEA, and bis-(o-ethoxyphenyl) PMEA were 38.2%, 2.46%, and 40.1%, respectively. PMEA was the only metabolite formed after oral administration of bis-POM PMEA. Three metabolites were detected after oral administration of either bis-(phenyl) PMEA or bis-(o-ethoxyphenyl) PMEA to rats: PMEA, the corresponding monoester, and 2-adenylacetic acid. The major metabolite of bis-(phenyl) PMEA was 2-adenylacetic acid following oral administration. 2-Adenylacetic acid appears to have been formed from the intact prodrugs by a P450 mediated oxidation of the ethyl side chain.

The pharmacokinetics, distribution, and metabolism of [14C]cidofovir and its cyclic analog {[2-14C]-1-[((S)-2-hydroxy-2-oxo-1,4, 2-dioxaphosphorinan-5-yl)methyl]cytosine; [14C]cyclic HPMPC} were compared after administration to Sprague-Dawley rats. After intravenous (iv) administration of [14C]cidofovir (3 or 5 mg/kg) or [14C]cyclic HPMPC (5 mg/kg), plasma concentrations of total radioactivity declined in a multiexponential manner for both drugs with terminal half-lives of 7-11 hr. No metabolites of cidofovir were observed in plasma. However, iv [14C]cyclic HPMPC was converted in vivo to cidofovir (3.9% of the AUC of total radioactivity). The corrected half-life of cyclic HPMPC was 0.53 +/- 0.05 hr. The clearance of iv cidofovir (0.60-0.79 liter/hr/kg) was significantly less than that of cyclic HPMPC (1.10 +/- 0.12 liter/hr/kg). Cyclic HPMPC was more efficiently secreted by the kidney than cidofovir. The steady-state volume of distribution of cyclic HPMPC (0.32 +/- 0.5 liter/kg) was substantially less than that of cidofovir (0.9-3.0 liter/ kg). Concentrations in kidney at 24 hr after iv administration of 5 mg/kg were approximately 20-fold higher for cidofovir (6.6 micrograms-eq/g) than for cyclic HPMPC, whereas levels of radioactivity in the remaining tissues were similar for both drugs. This is consistent with the improved therapeutic index observed for cyclic HPMPC in animals. After iv administration of [14C]cidofovir (3 mg/kg), concentrations in most tissues declined with half-lives12 hr, presumably reflecting the long intracellular half-life of phosphorylated drug. Kidneys and liver of animals given iv [14C]cidofovir (5 mg/kg) contained unchanged cidofovir (35-65%), metabolite I (attributed to cidovofir phosphocholine) (29-60%), and a peak coeluting with cyclic HPMPC on three different HPLC systems (metabolite II) (5-7%). Kidneys, liver, and lung of animals given iv [14C]cyclic HPMPC contained cyclic HPMPC (1-6%), cidofovir (44-50%), and metabolite I (44-54%). The subcutaneous bioavailability of cidofovir was 91.5%. The subcutaneous and oral bioavailabilities of cyclic HPMPC were 82.7% and 3.50 +/- 1.07%, respectively, based on total radioactivity.

To determine the pharmacokinetics of GS-522, an oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, after constant infusion and bolus administration in the cynomolgus monkey.Using a stability indicating HPLC method, the GS-522 plasma concentration versus time data were obtained after constant infusion (0.1, 0.3, 0.5 mg/kg/min) and bolus administration (11.25 and 22.5 mg/kg). Plasma data after bolus administration was fit to a three-compartment model.The half-lives for the alpha and beta phases were 1.4 and 5.4 min, respectively. Steady state GS-522 concentrations were reached within 10 minutes after initiation of constant infusions. Termination of infusions resulted in a rapid elimination of GS-522 with an average elimination half-life equal to 1.5 min. The Vss calculated from both the constant infusion and bolus data approximated the blood volume of the monkey. Substitution of the phosphodiester backbone at the 3' end of GS-522 with two phosphorothioate linkages did not substantially effect the elimination half-life upon termination of infusion.These data in conjunction with published biodistribution data suggest that oligodeoxynucleotides are rapidly cleared from plasma by tissue uptake and that little efflux back into blood takes place. Additionally, strategies designed to increase oligodeoxynucleotide resistance to exonucleases will not dramatically increase plasma half-lives.

To determine the degradation rates and pathways of GS-522, a potent oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, in serum and plasma.A stability-indicating, anion-exchange HPLC method was developed and used to determine concentrations of GS-522 and metabolites.In monkey plasma at 2 microM or below, the degradation of GS-522 can be fit to a first-order exponential with a kpobs approximately 0.01 min-1. At 3 microM and above the degradation process deviates from a monoexponential decay profile. An initial fast degradation process is followed by a slower phase with an observed rate constant equal to that observed at 2 microM and below. In monkey serum, the KM and Vmax are 8.4 microM and 0.87 microM min-1, respectively.The kinetics are consistent with an equilibrium binding of GS-522 to prothrombin in plasma (Kd = 50 nM) which saturates at GS-522 concentrations2 microM. Compared to a scrambled sequence (GGTGGTGGTTGTGGT), with no defined tertiary structure, GS-522 is 4-fold more stable in serum. The metabolic profile in plasma is consistent with a 3'-exonuclease catalyzed hydrolysis of GS-522.

The Small eye (Sey) gene in the mouse is lethal in the homozygous state. It is located on chromosome 2, is a mutation in the Pax-6 gene, and is genetically homologous with the human aniridia 2 (AN2) gene mutation. Numerous studies over the last few years, using genetic and molecular biological approaches, have investigated both the location of the gene as well as its possible mode of action. In the homozygous state, the primary defect appears to be limited to the failure of differentiation of the presumptive lens and nasal placodes. Such mice therefore display a characteristic phenotype; they possess neither eyes nor any nasal derivatives. Their heterozygous (Sey/+) and normal (+/+) littermates may be distinguished before birth only by a detailed examination of their eyes. Few detailed morphological/histological studies have been undertaken to date in the Sey/Sey embryos and newborn, and in the present study we describe a variety of craniofacial abnormalities that have not previously been reported. We observed, with one exception, delayed closure of the palate, and the presence in 80% of mice of an abnormal complement of upper incisor teeth, so that 35% possessed 1 supernumerary tooth while 45% possessed 2 supernumerary teeth. In these mice, a total of either 3 or 4, rather than the normal complement of 2, upper incisor teeth were present. Possibly the most unexpected finding, however, was the presence of a median cartilaginous rod-like structure which protruded between the 2 maxillae to give the Alizarin red S and Alcian blue-stained 'cleared' skulls of the newborn mice a characteristic 'unicorn-like' appearance. While this structure appeared to be a rostral extension of the chondrocranium, its exact derivation is unclear.

Because prolonged survival of heart transplant recipients is expected with the current immunosuppressive treatment, the functional capacity of these long-term survivors is of interest. Previous exercise studies showed no objective improvement in exercise tolerance several years after transplantation, but the extent to which chronotropic incompetence and allograft diastolic dysfunction observed early after transplantation may improve over time has not been defined.Thirteen untrained heart transplant recipients without symptoms, between 27 and 70 months after transplantation, and 13 age-matched sedentary normal controls underwent maximal upright bicycle exercise testing with simultaneous hemodynamic, radionuclide, and expired gas measurements.Systolic function as measured by ejection fraction was supranormal at rest in the transplant group and normalized with exercise. Despite their maximal exercise effort, transplant recipients had a 60% reduction in their exercise capacity compared with nontransplant recipients. Peak oxygen consumption was similarly reduced by 52%. Cardiac output response to exercise was 43% lower in the transplant group because of a 78% reduction in heart rate reserve and an 18% reduction in maximal stroke volume. Ventricular volumes were similarly reduced after transplantation, but filling pressures remained normal, indicating allograft diastolic dysfunction. Despite the significantly reduced maximal cardiac output, maximal arteriovenous oxygen difference was 25% lower in the transplant recipients, suggesting a peripheral deficit in oxygen handling.Therefore, patients, 2 to 6 years after transplantation, continue to have a significant reduction in exercise tolerance as a result of a combination of severe chronotropic incompetence, limited stroke volume reserve caused by a reduced ventricular size and allograft diastolic dysfunction, and an abnormality in peripheral oxygen delivery or use. Efforts aimed at improving these factors may further enhance the functional capacity of these long-term survivors of heart transplantation.

The bioavailability of PMEA from three oral formulations of the prodrug bis(POM)-PMEA has been evaluated in fasted male cynomolgus monkeys. The formulations examined included a hydroxypropyl-beta-cyclodextrin (HPBCD) complex, a PEG based cosolvent solution, and an aqeous suspension. Oral formulations containing 3H-bis(POM)-PMEA were compared to intravenous 3H-PMEA at 10.9 mg-eq/kg in a crossover study in four monkeys, with a 7 day washout period. No intact bis(POM)-PMEA or monoester were detected in plasma. Bioavailabilities of PMEA from the prodrug were 24.7 +/- 6.5%, 27.3 +/- 12.3% and 22.2 +/- 15.6% for the HPBCD complex, PEG solution and aqueous suspension, respectively. The oral bioavailability of PMEA from bis(POM)-PMEA was not limited by dissolution rate of the prodrug. Data for the PEG cosolvent solution and suspension indicate that the prodrug could potentially be formulated as a soft gelatin capsule or a tablet.

BACKGROUND Functional capacity and quality of life are subjectively improved after cardiac transplantation. However, the objective improvement in exercise tolerance after transplantation has been disappointing. The extent to which allograft diastolic dysfunction contributes to this exercise intolerance has not been defined. METHODS AND RESULTS Thirty cardiac transplant recipients between 3 and 16 months after transplantation and 30 age-matched normal control subjects underwent maximal symptom-limited graded upright bicycle exercise testing with simultaneous radionuclide angiography, invasive hemodynamic monitoring, and breath-by-breath gas analysis. Mean blood pressure was higher in the transplant group at supine rest (112.1 versus 97.7 mm Hg), normalized with upright posture, and became lower than normal at peak exercise (121.1 versus 133.2 mm Hg). Systolic function as measured by ejection fraction was normal in both groups. However, the cardiac transplant recipients had significantly lower exercise tolerance, achieving a mean maximal work rate of 390 kilopond-meters per minute (kpm/min), compared with 825 kpm/min in the normal subjects. Peak oxygen consumption was 12.3 mL.min-1.kg-1 in the transplant group, 46% lower than the normal group's value of 22.9 mL.min-1.kg-1. The transplant patients had a resting tachycardia (94 beats per minute) and a 79% reduction in exercise heart rate reserve compared with normal. Despite this chronotropic incompetence, stroke index response to exercise was consistently lower after transplantation, accounting for a 41% reduction in cardiac index at maximal exercise. The lower stroke index was accompanied by a 32% lower end-diastolic volume index at rest and a 14% lower end-diastolic volume index at peak exercise. Despite the smaller ventricular volumes after transplantation, pulmonary capillary wedge pressure was 35% higher than normal at supine rest and 50% higher at maximal exercise. Right atrial and mean pulmonary arterial pressures were similarly elevated. The ratio of pulmonary capillary wedge pressure to end-diastolic volume index was significantly higher during the postural change and exercise, suggesting allograft diastolic dysfunction. Arteriovenous oxygen difference was similar between groups at rest and with submaximal exercise but was 24% lower at maximal exercise in the transplant group, suggesting an abnormality in peripheral oxygen uptake or utilization. CONCLUSIONS Exercise tolerance is severely limited during the first 16 months after cardiac transplantation despite preservation of allograft left ventricular systolic function. This intolerance is due to an inadequate cardiac index response from a combination of chronotropic incompetence and diastolic dysfunction limiting the appropriate compensatory use of the Starling mechanism. In addition, there is a peripheral abnormality in oxygen transport or utilization that may partially reflect the effects of deconditioning.

The nucleotide sequence of the structural gene for benzyl alcohol dehydrogenase encoded by TOL plasmid pWWO of Pseudomonas putida has been determined. Benzyl alcohol dehydrogenase is a member of the long-chain zinc alcohol dehydrogenase family and, like other alcohol dehydrogenases of this family, contains two zinc atoms per subunit. Benzyl alcohol dehydrogenase, while sharing 31% identical residues with horse liver alcohol dehydrogenase, contains several amino acid substitutions near the active site, some of which may be responsible for the substrate specificity of benzyl alcohol dehydrogenase, which oxidizes exclusively aromatic substrates. Benzyl alcohol dehydrogenase also notably lacks the His51 residue of horse liver alcohol dehydrogenase. Contrary to the results obtained with a mutant human liver alcohol dehydrogenase lacking this residue, the concentration and pKa of solvent proton acceptors had no effect on the catalytic efficiency of benzyl alcohol dehydrogenase. The electronic nature of substituents on the aromatic ring of the substrate influenced the kcat of the enzyme in low concentrations of external proton acceptor, but not in high concentrations. Product inhibition studies demonstrated that benzyl alcohol dehydrogenase followed a general Ordered Bi Bi kinetic mechanism in low proton acceptor conditions, while following a Theorell-Chance kinetic mechanism at high proton acceptor conditions.

The xylene monooxygenase system encoded by the TOL plasmid pWW0 of Pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. In this study, the electron-transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH:cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non-haem iron and 2 mol/mol of acid-labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe-2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH:acceptor reductase was capable of reducing either one- or two-electron acceptors, such as horse heart cytochrome c or 2,6-dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 microM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro-R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe-2S] center occurred with a midpoint redox potential of -171 mV; and the reduction of FAD to FAD. (semiquinone form), with a calculated midpoint redox potential of -244 mV. The reduction of FAD. to FAD.. (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of -297 mV. The [2Fe-2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe-2S]-depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH --FAD --[2Fe-2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6-dichloroindophenol at a reduced rate.

Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia. To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF. The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM. Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF. Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine. Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h. Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies. These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors.

The stereochemistry at position 2 of the title compound, C 14 H 17 BrO 4 , has been confirmed as S.

The stereochemistries at positions 2 and 3 of the title compound, C 10 H 10 BrClO 2 , have been confirmed by X-ray crystal structural analysis. The halogen atoms show an antiperiplanar arrangement

In Hemiphractus fang–like teeth are ankylosed to the premaxilla, maxilla and prevomer, and bony odontoids are found on the dentary, angular and palatine bones. The odontoids are small, but a larger pair at the front of the lower jaw project upwards and backwards into the mouth and fit into a diastema between the anterior premaxillary teeth when the mouth is closed. The teeth are unipartite and monocuspid, and each consists of a strongly recurved and elongated cone of orthodentine, capped at the tip by a thin layer of enamel. The inner circumpulpal layer of the dentine is tubular, but no tubules are present in the outer pallial layer. During tooth development, dentine is formed before the enamel matrix is produced, and the tooth germs lie horizontally beneath the ventral surface of each dentigerous bone. On eruption, the tooth germs migrate horizontally and become ankylosed to the outer edge of the jaw bone by a layer of cellular cementum. During tooth replacement, the vast majority of the dentine of each tooth, and the cementum at the tooth base, are resorbed by osteoclasts. It is not clear whether the tips of the teeth are shed or not.

A pterygomaxillary ligament, which runs postero-inferiorly from the posterior end of the maxilla to the pterygoid, is described macroscopically and microscopically in the aquatic anuran amphibian Xenopus laevis (Daudin). The quadratojugal is absent in this species, and consequently the short maxilla has no posterior articulation and is potentially mobile. However, the pterygomaxillary ligament limits upward and lateral displacement of the maxilla when pressure is applied upwards during closure of the lower jaw. The ligament absorbs forces applied to the posterior end of the maxilla and permits their dissipation via the pterygoid to the neurocranium.

SUMMARY A model based on the concept of the circle of influence of a plant is used to estimate the relationship between yield and area/plant for tobacco crops with gaps in stand. The model predicts the average yield of plants in any configuration that includes missing plants and is used to predict the economic consequences of loss in stand from pest or other damage.