A combined three-year and four-year medical program with modified admission requirements

More flexible admission requirements have made it possible to admit students to the first year of a program leading to the B.Med.Sc. degree at the end of two years and to the M.D. degree in four years, saving one to three years by means of earlier entry following high school. More advanced students may enter at the beginning of second year and complete a three-year M.D. program. Encouraging preliminary results have been obtained.     

Withdrawal from chronic phencyclidine produces a pentylenetetrazol-like discriminative stimulus.

Rats trained to discriminate the anxiogenic compound pentylenetetrazol (PTZ) from water were implanted with osmotic mini-pumps containing 7 mg/kg/day phencyclidine (PCP) or water. Rats were tested for generalization to PTZ 24 hours prior to pump removal, and 4 to 96 hours after pump removal. While the pumps were in place, rats did not generalize to PTZ. When the pumps were removed on day 10, rats in the water group did not generalize to PTZ, but 69% of the rats in the chronic PCP group responded on the PTZ lever at 4 and/or 24 hours after pump removal, suggesting that the PCP withdrawal state mimics the interoceptive cue produced by PTZ. This withdrawal phenomenon was repeatable, in that rats that generalized once to PTZ during PCP withdrawal, generalized a second time when the procedure was repeated. In addition, the phenomenon was dose-dependent, as rats that did not generalize to PTZ after 7 mg/kg/day PCP did generalize when the chronic dose of PCP was increased to 10 mg/kg/day. These findings suggest that there is an anxiogenic component of PCP withdrawal and that tolerance does not develop to this effect.     

Biodistribution, kinetics, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase in the murine model of mucopolysaccharidosis VII

Enzyme replacement therapy (ERT) has been shown to be effective at reducing the accumulation of undegraded substrates in lysosomal storage diseases. Most ERT studies have been performed with recombinant proteins that are mixtures of phosphorylated and non-phosphorylated enzyme. Because different cell types use different receptors to take up phosphorylated or non-phosphorylated enzyme, it is difficult to determine which form of enzyme contributed to the clinical response. Here we compare the uptake, distribution, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase (GUSB) in the MPS VII mouse. Highly phosphorylated murine GUSB was efficiently taken up by a wide range of tissues. In contrast, non-phosphorylated murine GUSB was taken up primarily by tissues of the reticuloendothelial (RE) system. Although the tissue distribution was different, the half-lives of both enzymes in any particular tissue were similar. Both preparations of enzyme were capable of preventing the accumulation of lysosomal storage in cell types they targeted. An important difference in clinical efficacy emerged in that phosphorylated GUSB was more efficient than non-phosphorylated enzyme at preventing the hearing loss associated with this disease. These data suggest that both forms of enzyme contribute to the clinical responses of ERT in MPS VII mice but that enzyme preparations containing phosphorylated GUSB are more broadly effective than non-phosphorylated enzyme.     

n-Alkane uptake and utilisation by Streptomyces strains

Streptomyces strains isolated from the Kuwait Burgan oil field were defined as S. griseoflavus, S. parvus, and S. plicatus utilised n-hexadecane, n-octadecane (purified fractions of mineral oil), kerosene, and crude oil as sole carbon and energy sources. The strains were incubated with n-alkanes and increase of the fatty acid content with chain length equivalent to the employed n-alkanes was observed. Signal transducing GTP-binding proteins (GBPs) play an important role in n-alkane uptake in streptomycetes. Specific activators of GBPs increased the uptake of hydrocarbons. Using the hydrophobic fluorescent dye diphenylhexatrien (DPH) as a probe, it was found that the microviscosity of the hydrophobic inner region of the cellular membrane is significantly lower in hydrocarbon utilisers than in non-utilisers. This difference probably reflects differences in the fatty acid composition of the strains. When cultures were grown in n-alkane containing media, electron microscopy revealed that the hydrocarbon utilisers showed less-electron dense areas as inclusions in the cytoplasm. Soil samples inoculated with Streptomyces strains eliminated hydrocarbons much faster than those not containing these strains, serving as control. When inorganic medium was supplied with n-hexadecane-1-¹⁴C as sole carbon and energy source, radioactive CO₂ was detected. Since streptomycetes have not been used until now for oil elimination, though they are known as abundant soil bacteria tolerating extreme conditions, their possible use for bioremediation of hydrocarbon contaminated soils is discussed.     

Inhibitor "double occupancy" in the Q(o) pocket of the chloroplast cytochrome b6f complex.

Electron paramagnetic resonance (EPR) spectra of the "Rieske" 2Fe-2S cluster revealed that two molecules of the inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) can bind to each monomer of the spinach cytochrome (cyt) b6f complex, both in isolated form and in intact thylakoid membranes. Binding to the high-affinity site, which accounts for the observed inhibitory effects, caused small shifts in the g(x) transition of the 2Fe-2S cluster EPR spectrum, similar to those induced by stigmatellin or 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether (DNP-INT). Occupancy of the low-affinity site was only observed after addition of superstoichiometric amounts of the inhibitor and was accompanied by the appearance of a g = 1.94 EPR signal. The shape of the equilibrium binding titration curve, the effects on the 2Fe-2S EPR spectrum, and the ability of the DBMIB binding to displace DNP-INT were consistent with two molecules of DBMIB binding at the Q(o) pocket, with the strongly binding species binding close to the 2Fe-2S cluster. Possible implications of these findings for so-called "double-occupancy" models for Q(o) site catalysis are discussed.     

Ligand-induced heme ruffling and bent no geometry in ultra-high-resolution structures of nitrophorin 4

The nitrophorins are a family of proteins that use ferric heme to transport nitric oxide (NO) from the salivary glands of blood-sucking insects to their victims, resulting in vasodilation and reduced blood coagulation. We have refined atomic resolution structures of nitrophorin 4 (NP4) from Rhodnius prolixus complexed with NO (1.08 A) and NH(3) (1.15 A), yielding a highly detailed picture of the iron coordination sphere. In NP4-NO, the NO nitrogen is coordinated to iron (Fe-N distance = 1.66 A) and is somewhat bent (Fe-N-O angle = 156 degrees ), with bending occurring in the same plane as the proximal histidine ring. The Fe(NO)(heme)(His) coordination geometry is unusual but consistent with an Fe(III) oxidation state that is stabilized by a highly ruffled heme. Heme ruffling occurs in both structures, apparently due to close contacts between the heme and leucines 123 and 133, but increases on binding NO even though the steric contacts have not changed. We also report the structure of NP4 in complexes with histamine (1.50 A) and imidazole (1.27 A). Unexpectedly, two mobile loops that rearrange to pack against the bound NO in NP4-NO, also rearrange in the NP4-imidazole complex. This conformational change is apparently driven by the nonpolar nature of the NO and imidazole (as bound) ligands. Taken together, the desolvation of the NO binding pocket through a change in protein conformation, and the bending of the NO moiety, possibly through protein-assisted heme ruffling, may lead to a nitrosyl-heme complex that is unusually resistant to autoreduction.     

Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI

The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.     

Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

Formation of reactive cyclopentenone compounds in vivo as products of the isoprostane pathway

Cyclopentenone prostaglandins A2 and J2 are reactive compounds that possess unique biological activities. However, the extent to which they are formed in vivo remains unclear. In this study, we explored whether D2/E2-isoprostanes undergo dehydration in vivo to form A2/J2-isoprostanes. Oxidation of arachidonic acid in vitro generated a series of compounds that were confirmed to be A2/J2-isoprostanes by mass spectrometric analyses. A2/J2-isoprostanes were detected in vivo esterified to lipids in livers from normal rats at a level of 5. 1 +/- 2.3 ng/g, and levels increased dramatically by a mean of 24-fold following administration of CCl4. An A2-isoprostane, 15-A2t-isoprostane, was obtained and found to readily undergo Michael addition with glutathione and to adduct covalently to protein. A2/J2-isoprostanes could not be detected in the circulation, even following CCl4 administration, which we hypothesized might be explained by rapid formation of adducts. This was supported by finding that essentially all the radioactivity excreted into the urine following infusion of radiolabeled 15-A2t-isoprostane into a human volunteer was in the form of a polar conjugate(s). These data identify a new class of reactive compounds that are produced in vivo as products of the isoprostane pathway that can exert biological effects relevant to the pathobiology of oxidant injury.