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Biomedical subjects

J M Pratt

Publications and source records attributed to J M Pratt.

At least 19 recordsLinked to original sources
PubMed1999

Cytotoxicity of 2,2':6',2' '-terpyridineplatinum(II) complexes against human ovarian carcinoma.

2,2':6',2''-Terpyridineplatinum(II) complexes are shown to possess cytotoxicity against a number of human ovarian tumor cell lines. Many of the complexes show similar activity against cisplatin- and doxorubicin-resistant cell lines as the parental cells suggesting that there is little or no cross-resistance with cisplatin or doxorubicin. The cytotoxicity of bis[2,2':6',2''-terpyridineplatinum(II)] complexes is strongly dependent on the nature of the linker. Bis[2,2':6',2''-terpyridineplatinum(II)] complexes with a flexible linker at the 4'-position show poor cytotoxicity; by contrast bis[2,2':6',2''- terpyridineplatinum(II)] complexes with rigid and short linkers at platinum(II) are strikingly effective. Several of the compounds show greater cytotoxicity against human ovarian cell lines than carboplatin, the therapeutic agent currently advocated for the treatment of human ovarian cancers.

Antineoplastic Agents↗
PubMed1999

Cytotoxicity of (2,2':6',2''-terpyridine)platinum(II) complexes to Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei.

A range of (2,2':6',2''-terpyridine)platinum(II) complexes are shown to possess antiprotozoal activity in vitro against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei,the causative organisms of tropical diseases leishmaniasis and trypanosomiasis. The best compounds caused 100% and 78% inhibition of growth of the intracellular amastigote forms of L. donovani and T. cruzi, respectively, at a concentration of 1 microM and 100% inhibition of growth of the bloodstream trypomastigote forms of T. brucei at a concentration of 0.03 microM. The results obtained with complexes in which the fourth ligand to platinum(II) is capable of being substituted with a substitution inert hydroxyethanethiolate complex are compared. The ammine complexes show high antiprotozoal activity suggesting that the trans influence of the 2,2':6',2''-terpyridine ligand has a profound effect on the ease of displacement of the fourth ligand in (2,2':6',2'' -terpyridine)platinum(II) complexes, although nonbonded interaction between the ammine ligand and the 6 and 6' ' hydrogens probably also weakens the ligation to Pt(II).

Animals↗
PubMed1997

Barriers to school-based health care programs.

Although school-based health care programs (SBHCPs) provide affordable and accessible health care to children and adolescents and are known to improve school attendance, a variety of barriers affect their development. Focus groups were conducted in three schools in Louisiana to demonstrate how barriers can affect the initiation and development of SBHCPs. Each school-based program was in a different stage of development. Identifying potential barriers and developing strategies to overcome them can enhance already existing SBHCPs and make it easier for new programs to begin. The social worker serves as an important ally in the development of SBHCPs and is a necessary part of the school-based health care team.

Adolescent↗
PubMed1994

Analysis of the membrane-anchoring properties of the putative amphiphilic alpha-helical anchor at the C-terminus of Escherichia coli PBP 6.

Penicillin-binding protein (PBP) 6 is anchored to the periplasmic face of the Escherichia coli inner membrane. Analysis of the C-terminal 20 amino acids of PBP 6 implies the presence of a C-terminal amphiphilic alpha-helical anchor comparable to that of PBP 5. A C-terminal deletion of PBP 6 was constructed; it resulted in the release of the protein from the inner membrane into the periplasm, thus confirming that this region is essential for anchoring. Treatment of E. coli K12 membrane vesicles with various reagents was used to probe the membrane-binding characteristics of both PBP 5 and PBP 6. The results indicate that, although the strength of membrane anchoring of PBP 6 is weaker than that of PBP 5, both modes of anchoring involve a large hydrophobic element and have similar membrane-binding characteristics. This is in agreement with the hypothesis that both proteins exhibit the same novel method of anchoring.

Amino Acid Sequence↗
PubMed1994

Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins.

The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M(r) PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M(r) PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and 6 are DD-carboxypeptidases. All three low M(r) PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic alpha-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.

Amino Acid Sequence↗
PubMed1993

Membrane interaction of Escherichia coli penicillin binding protein 5 is modulated by the ectomembranous domain.

E. coli penicillin binding protein (PBP) 5 is anchored to the periplasmic face of the inner membrane by a C-terminal domain which is predicted to form an amphiphilic alpha-helix. Here we show that the presence of a substrate analogue, benzyl penicillin, causes the protein to be converted from a membrane bound urea inaccessible form to a urea extractable form. If the anchor region is fused to the periplasmic protein, beta-lactamase, the fusion protein becomes membrane bound but is unable to exhibit the changes in urea extractability which are observed with PBP5. We therefore conclude that although the C-terminus of PBP5 is sufficient to anchor the protein to the membrane surface the ectomembranous domain can affect the state of the anchor and in vivo changes in the state of anchoring may be related to enzyme activity.

Bacterial Proteins↗
PubMed1991

A coupled in vitro transcription-translation system for the exclusive synthesis of polypeptides expressed from the T7 promoter.

A coupled transcription-translation in vitro system has been developed in Escherichia coli specifically for the expression of genes under the exclusive control of the T7 promoter. This system consists of an E. coli crude extract (prepared from cells containing endogenous T7 RNA polymerase), rifampicin (an E. coli RNA polymerase inhibitor) and a labelled amino acid. When primed with a plasmid template encoding the target gene under exclusive control of the T7 promoter, this system has the capability to synthesize relatively large amounts of a unique, labelled polypeptide. This paper describes the characteristics and use of such a T7 RNA polymerase/T7-promoter specific in vitro system.

Bacterial Proteins↗
PubMed1991

Active transport of amino acids by gamma-glutamyl transpeptidase through Caco-2 cell monolayers.

The possible role of the gamma-glutamyl cycle in the transport of amino acids, using the Caco-2 cell monolayer as an in vitro model of the small intestine, has been investigated. The transport of [2-3H]glycine and [2-3H]glycylglycine through the Caco-2 monolayer has been shown to occur by two modes of action. Active transport is unidirectional from apical to basolateral region and is a carrier mediated system. The enzyme gamma-glutamyl transpeptidase seems to be involved in this process, since when the enzyme is inhibited, the active transport is also inhibited. However transport still takes place, and this occurs by a slower non-active process, which is bidirectional and is mediated by passive diffusion. The rate of transport of [2-3H]glycylglycine and [2-3H]glycine were 585 (+/- 24) and 287 (+/- 16) pmolcm-2min-1 respectively, while the non-active transport takes place at 87 (+/- 6) pmolcm-2min-1. Thus, amino acid translocation in Caco-2 cells is shown to occur by two methods, one of which involves the gamma-glutamyl cycle.

Amino Acids↗
PubMed1991

Molecular recognition in the binding of vitamin B12 by the cobalamin-specific Intrinsic Factor.

Equilibrium constants (given as log K/M-1) have been determined at pH 7.4 and 4 degrees C for binding by porcine Intrinsic Factor (B12-binding protein from the gut, specific for the 'cobalamin' series of Co corrinoids) of vitamin B12 or cyanocobalamin (10.5), cyanocobinamide, alpha-ribazole and alpha-ribazole-phosphate (main fragments produced by cleaving off the 'cobalamin' side-chain, all less than or equal to 3), and cyanocobinamide in the presence of greater than or equal to 10(-9) M ribazole (5.6 and independent of ribazole concentration), i.e. ribazole catalyses the binding of the cobinamide. It is proposed that the specificity of Intrinsic Factor for the cobalamins depends on the presence of the ribazole fragment in the cobalamin side-chain to promote an essential change in conformation before the corrinoid fragment can be bound.

Animals↗
PubMed1991

Analysis of the membrane organization of an Escherichia coli protein translocator, HlyB, a member of a large family of prokaryote and eukaryote surface transport proteins.

Haemolysin B (HlyB) is essential for secretion of the 107 x 10(3) Mr haemolysin A protein from Escherichia coli and is a member of a family of highly conserved, apparently ATP-dependent surface proteins in many organisms. We have shown in this study that both HlyB and HlyD fractionate primarily with the cytoplasmic membrane of E. coli and are accessible to proteases after removal of the outer membrane. We have measured experimentally the topological organization of HlyB within the membrane by construction of fusions to beta-lactamase as a reporter. The predicted folding of HlyB, with a minimum of six transmembrane segments, does not always coincide with regions of highest average hydrophobicity. This suggests that HlyB may have a novel organization within the bilayer. From our data and comparative sequence analysis, we have been able to predict very similar topological models for the other members of the HlyB family.

Amino Acid Sequence↗
PubMed1990

pH-induced insertion of the amphiphilic alpha-helical anchor of Escherichia coli penicillin-binding protein 5.

By treating vesicles prepared from Escherichia coli K12 with various reagents, we have investigated the mechanism by which penicillin-binding protein 5 anchors to the inner membrane. The results indicate that there are two forms of anchoring; one which is inaccessible to urea and probably inserted into the bilayer and one which is accessible. Association of the accessible form with the membrane seems to involve significant hydrophobic interaction and this form is triggered to undergo reversible 'insertion' by a decrease in pH.

Bacterial Proteins↗
PubMed1988

Oxygen activation and ligand binding by pure heme-octapeptide microperoxidase-8 (MP-8).

The rapid, efficient preparation of pure microperoxidase-8 (MP-8) is described. Ligand binding studies confirm that MP-8 is monomeric in alkaline solution. It is shown that the monomeric MP-8 activates oxygen in a similar manner to that already reported for alkaline hemin, establishing the octapeptide as a possible second generation model for the oxygen activation/insertion reactions of the cytochrome P-450.

Animals↗
PubMed1988

Analysis of the membrane-binding domain of penicillin-binding protein 5 of Escherichia coli.

Internal deletions close to the C-terminus of the Escherichia coli penicillin binding protein 5 (PBP5, DacA) have defined the C-terminal 18 residues of the protein as essential for membrane binding. This C-terminal sequence is capable of forming a strongly amphiphilic alpha-helix. In this paper we show that the PBP5 amphiphilic helix is able to anchor the periplasmic TEM-beta-lactamase to the inner membrane. In addition, we have demonstrated that mature PBP5 (lacking the N-terminal signal sequence) possesses the ability to bind to the membrane from a soluble form of the protein, showing that translocation across the membrane is unnecessary for anchoring to be established.

Amino Acid Sequence↗
PubMed1987

Hemes and hemoproteins. 3. The reaction of microperoxidase-8 with cyanide: comparison with aquocobalamin and hemoproteins.

The monomeric heme octapeptide from cytochrome c, microperoxidase-8, (MP-8), coordinates CN- with log K = 7.55 +/- 0.04 at 25 degrees C in 20% (v/v) aqueous methanol. Log K values are independent of pH between 6 and 9. A spectrophotometric titration of cyanoMP-8 between pH 5.5 and 13.8 gave a single pKa greater than or equal to 13.5 ascribed to ionization of the proximal His ligand. A study of the kinetics of the reaction of MP-8 with cyanide between pH 5.5 and 12, at 25 degrees C and mu = 0.1, indicates that formation of cyanoMP-8 occurs via three routes: attack of CN- on Fe(III) (k1 = 6.0 +/- 0.3 X 10(5) M-1 sec-1); attack of HCN on Fe(III) (k2 = 4.8 +/- 2.0 X 10(3) M-1 sec-1), followed by deprotonation and isomerization to form the C-bound species; and displacement of OH- by CN- when the proximal His ligand is ionized (k5 = 1.8 +/- 0.1 X 10(5) M-1 sec-1). These results are compared with available data for the reaction of cyanide with aquocobalamin and with various hemoproteins.

Animals↗
PubMed1987

Hemes and hemoproteins. 5: Kinetics of the peroxidatic activity of microperoxidase-8: model for the peroxidase enzymes.

The peroxidatic activity of the heme octapeptide from cytochrome c, microperoxidase-8 (MP-8), was assayed at 25 degrees C under conditions where formation of Compound I is rate limiting. In the pH range 6-9, the reaction rate increased linearly with a slope close to unity. The active form of the substrate is the hydroperoxide anion, HO2-, and an extrapolated second-order rate constant was obtained for the reaction of aquoMP-8 with HO2- of 3.7 X 10(8) M-1 sec-1, which is close to the second-order rate constants reported for reaction of the peroxidase enzymes with H2O2. Comparison with published data shows that the Fe3+ ion of MP-8 reacts as expected with simple anions, electrons, and HO2-, while the analogous reactions of the enzymes all show a requirement for one H+. We conclude that the peroxidase enzymes activate H2O2 under physiological conditions through a pH-independent, H+-coupled binding of the required H2O2-. The peroxidase activity of MP-8 can be increased more than tenfold by the presence of the guanidinium ion, which is ascribed to formation of the ion-pair GuaH+HO2-; this suggests a role for the invariant distal Arg in the enzymes.

Animals↗
PubMed1987

An 18 amino acid amphiphilic helix forms the membrane-anchoring domain of the Escherichia coli penicillin-binding protein 5.

Small (10 residue) C-terminal deletions of PBP5 cause release of this inner membrane protein into the periplasm, indicating disruption of the membrane binding domain. To define the extent of the membrane anchoring domain, oligonucleotide-directed mutagenesis was used to introduce both single amino acid changes and novel restriction sites into the DNA, allowing subsequent construction of precise internal deletions. The 10 C-terminal amino acid residues possess very weak membrane anchoring potential. By extending the sequence to 18 residues membrane binding equivalent to that of authentic PBP5 was achieved. A proline substitution in this region, breaking a potential alpha-helix, also disrupts the membrane binding domain. These results are discussed with respect to the amphiphilicity of the C-terminal sequence when arranged in an alpha-helix.

Amino Acid Sequence↗
PubMed1986

Intermediates in the assembly of the TonA polypeptide into the outer membrane of Escherichia coli K12.

The tonA gene of Escherichia coli K12 was cloned into a multicopy plasmid, leading to substantial overproduction of the corresponding 78,000 Mr polypeptide in the outer membrane. The approximate size of the tonA gene and its direction of transcription were established by Tn1000 mutagenesis. A family of tonA deletions was constructed in vitro by Bal31 exonuclease digestion, followed by splicing of an "oligo stop" sequence to each 3' terminus in order to ensure prompt termination of translation of the truncated polypeptides in vivo. All these polypeptides proved to be extremely unstable in exponentially growing cultures but were relatively stable in maxicells. Under these conditions the truncated polypeptides, unlike wild-type TonA, fractionated with the Sarkosyl-soluble fraction of the cell envelope, indicating that these proteins are blocked in assembly as inner membrane (translocation) intermediates or as outer membrane (maturation) intermediates unable to form Sarkosyl-resistant complexes. We have also examined the kinetics of assembly of wild-type TonA into the outer membrane and the results indicate that, following cleavage of the N-terminal signal peptide, the protein passes through an apparently membrane-free intermediate form and only appears in the outer membrane after a delay of at least 50 seconds, following the completion of synthesis. From these results, we propose that the assembly of TonA involves translocation (with concomitant cleavage of the signal sequence) directly into the periplasm, followed by partitioning into the outer membrane. We further propose that the C terminus of TonA is essential for final maturation in the outer membrane in Sarkosyl-resistant form but that the C-terminal half of the molecule probably does not contain any topogenic sequences required for partitioning to the outer membrane.

Bacterial Outer Membrane Proteins↗