Synthesis of alkyl and aryl esters of N-protected beta-homoamino acids from N-protected alpha-aminodiazoketones

A simple and concomitant esterification method for the synthesis of methyl, ethyl, t-butyl, benzyl, and 9-fluorenylmethyl esters of Fmoc-/Boc-/Z-beta-homoamino acids employing Fmoc-/Boc-/Z-alpha-aminodiazoketones by Wolff rearrangement is described. The method offers good yield with purity.     

Toxoplasma gondii cyclic GMP-dependent kinase: chemotherapeutic targeting of an essential parasite protein kinase

The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) has in vivo activity against the apicomplexan parasites Toxoplasma gondii and Eimeria tenella in animal models. The presumptive molecular target of this compound in E. tenella is cyclic GMP-dependent protein kinase (PKG). Native PKG purified from T. gondii has kinetic and pharmacologic properties similar to those of the E. tenella homologue, and both have been functionally expressed as recombinant proteins in T. gondii. Computer modeling of parasite PKG was used to predict catalytic site amino acid residues that interact with compound 1. The recombinant laboratory-generated mutants T. gondii PKG T761Q or T761M and the analogous E. tenella T770 alleles have reduced binding affinity for, and are not inhibited by, compound 1. By all other criteria, PKG with this class of catalytic site substitution is indistinguishable from wild-type enzyme. A genetic disruption of T. gondii PKG can only be achieved if a complementing copy of PKG is provided in trans, arguing that PKG is an essential protein. Strains of T. gondii, disrupted at the genomic PKG locus and dependent upon the T. gondii T761-substituted PKGs, are as virulent as wild type in mice. However, unlike mice infected with wild-type T. gondii that are cured by compound 1, mice infected with the laboratory-generated strains of T. gondii do not respond to treatment. We conclude that PKG represents the primary molecular target responsible for the antiparasitic efficacy of compound 1.     

Inhibition of vascular endothelial growth factor/vascular permeability factor action blocks estrogen-induced uterine edema and implantation in rodents

Estrogen induces a rapid increase in microvascular permeability in the rodent uterus, leading to stromal edema and a marked increase in uterine wet weight. This edema is believed to create an environment optimal for the growth and remodeling of the endometrium in preparation for implantation and pregnancy. Increased endometrial microvascular permeability also occurs in conjunction with implantation. Estrogen-induced uterine edema is immediately preceded by an increase in the expression of vascular endothelial growth factor (VEGF), a potent stimulator of microvascular permeability. The objective of this study was to determine to what degree immunoneutralization of VEGF would interfere with a) estradiol-induced uterine edema and b) pregnancy. In the first set of experiments, immature female rats were injected with either VEGF antiserum or normal rabbit serum (NRS) prior to 17beta-estradiol treatment. Rats treated with estradiol alone showed a 57% increase in uterine wet weight at 6 h compared with controls. Injection of 200 or 300 micro l of VEGF antiserum reduced the response to only 20% and 10% above controls, respectively. In the second set of experiments, young adult female mice were treated with 100 micro l of either VEGF antiserum or NRS at 1200 h on the fourth day after mating. NRS-treated mice had normal pregnancies. VEGF antiserum, however, completely blocked pregnancy. When VEGF antiserum-treated females were examined on Day 5 for the presence of implantation sites, none were found. These results show that a) VEGF is the major mediator of estrogen-induced increase in uterine vascular permeability and b) VEGF-induced edema is absolutely essential for implantation to take place.     

Importance of the conserved Walker B glutamate residues, 556 and 1201, for the completion of the catalytic cycle of ATP hydrolysis by human P-glycoprotein (ABCB1)

The human MDR1 (ABCB1) gene product, P-glycoprotein (Pgp), functions as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. In this study, we assessed the role of conserved glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [alpha-(32)P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [alpha-(32)P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [alpha-(32)P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [alpha-(32)P]-8-azidoADP trapping. Interestingly, the double mutants E556/1201Q and E556/1201A trapped [alpha-(32)P]-8-azidoADP even in the absence of Vi, and the occluded nucleotide was not released after incubation at 37 degrees C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of the wild-type protein trapped in the presence of Vi (Pgp x [alpha-(32)P]-8-azidoADP xVi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. In aggregate, these data suggest that E556 and E1201 residues in the Walker B domains may not be critical as catalytic carboxylates for the cleavage of the bond between the gamma-P and the beta-P of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle.     

Antimicrobial constituents from the rhizomes of Rheum emodi

The bioassay-guided chemical examination of the rhizomes of R. emodi resulted in the isolation of two new oxanthrone esters, revandchinone-1, revandchinone-2, a new anthraquinone ether revandchinone-3 and a new oxanthrone ether, revandchinone-4. Their structures were established based on spectroscopic and degradative evidence. Occurrence of oxanthrone ether is reported for the first time. The anti bacterial and anti fungal activity of the isolates is studied.     

An immuno-affinity method for the purification of mannose 6-phosphate receptor proteins

In a recent study, we have developed an ELISA method to quantify the mannose 6-phosphate receptor (MPR) proteins [J. Biochem. Biophys. Methods 52 (2002) 111]. In the present study, we have used the goat MPR 300 antibody and peptide specific antibodies to human MPR 46 to develop simple and efficient immuno-affinity matrices, which can be used to purify the MPR proteins from goat liver in a single step. The identity of the immuno-affinity purified receptors is confirmed by their molecular masses as well as by their immunoreactivity.     

Is type 2 diabetes mellitus a vascular disease (atheroscleropathy) with hyperglycemia a late manifestation? The role of NOS, NO, and redox stress.

Background

The impressive correlation between cardiovascular disease and glucose metabolism alterations has raised the likelihood that atherosclerosis and type 2 diabetes may share common antecedents. Inflammation is emerging as a conceivable etiologic mechanism for both. Interleukins are regulatory proteins with ability to accelerate or inhibit inflammatory processes.

Presentation of the hypothesis

A novel interleukins classification is described, based on their role in diabetes and atherosclerosis, hypothesizing that each interleukin (IL) acts on both diseases in the same direction – regardless if harmful, favorable or neutral.

Testing the hypothesis

The 29 known interleukins were clustered into three groups: noxious (the "bad", 8 members), comprising IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-17 and IL-18; protective (the "good", 5 members), comprising IL-4, IL-10, IL-11, IL-12 and IL-13; and "aloof", comprising IL-5, IL-9, IL-14, IL-16 and IL-19 through IL-29 (15 members). Each group presented converging effects on both diseases. IL-3 was reluctant to clustering.

Implications

These observations imply that 1) favorable effects of a given IL on either diabetes or atherosclerosis predicts similar effects on the other; 2) equally, harmful IL effects on one disease can be extrapolated to the other; and 3) absence of influence of a given IL on one of these diseases forecasts lack of effects on the other. These facts further support the unifying etiologic theory of both ailments, emphasizing the importance of a cardiovascular diabetologic approach to interleukins for future research. Pharmacologic targeting of these cytokines might provide an effective means to simultaneously control both atherosclerosis and diabetes.