The 2nd International Workshop on Clinical Pharmacology of HIV Therapy. April 2nd-4th 2001, Noordwijk, The Netherlands.

The 2nd International Workshop on Clinical Pharmacology of HIV Therapy was convened in Noordwijk, The Netherlands on April 2nd-4th, 2001. The purpose of this meeting was to present and discuss research related to antiretroviral drug interactions, drug distribution to sanctuary sites, pharmacokinetics, pharmacodynamics (i.e., the relationship between pharmacokinetics and efficacy, toxicity or drug resistance), inhibitory quotient (IQ) and therapeutic drug monitoring (TDM). There were 28 oral and 49 poster presentations accepted to this meeting. While most studies focused on protease inhibitors (PIs), insight into the pharmacology of non-nucleoside reverse transcriptase inhibitors (NNRTIs) was also gleaned. Herein, we highlight some of the research that was presented.

Immobilization of Pseudomonas cepacia lipase in a phyllosilicate sol-gel matrix: effectiveness as a biocatalyst.

A novel procedure is described for immobilizing a lipase from Pseudomonas cepacia (PS-30) within a phyllosilicate sol-gel matrix. The method is based on cross-linking a phyllosilicate clay with silicate polymers produced by the controlled hydrolysis of tetramethyl orthosilicate (TMOS). The activity of the phyllosilicate sol-gel-immobilized lipase was dependent upon the type of alkylammonium salt, inorganic catalyst and volume ratio of phyllosilicate clay to TMOS used. Lipase PS-30 immobilized in this way was more stable and had higher activity compared with the free lipase. Studies on the lipase-catalysed esterification of lauric acid with octan-1-ol in iso-octane showed that under controlled water activity conditions the phyllosilicate sol-gel-immobilized lipase had better activity compared with other supported lipase preparations. In addition, the phyllosilicate sol-gel-immobilized lipase was reusable for at least five esterification cycles without significant loss of activity.

Immobilized lipoxygenase in a packed-bed column bioreactor: continuous oxygenation of linoleic acid.

The continuous oxygenation of linoleic acid (LA) by immobilized lipoxygenase (LOX) was studied. Enzymatic oxidation was carried out in a recirculating packed column reactor using immobilized LOX as the stationary phase and LA as the substrate. The column, when packed with LOX immobilized in either a calcium alginate sol-gel matrix or a phyllosilicate sol-gel matrix, is equivalent to five continuous stirred tank reactors (CSTRs). The reactor cascade was calculated from the residence-time distribution for the reactor. Based on mass-balance calculations, a set of mathematical equations for predicting the concentration of oxygenated product generated in each CSTR was calculated. Product formation in the packed column reactor was simulated and results calculated with the model were compared with the experimental results. The data indicated that product yield (hydroperoxyoctadecadienoic acid, HPOD) increased asymptotically with reaction time. Experimentally, when the bioreactor was packed with calcium alginate sol-gel-immobilized LOX, an initial linear increase in HPOD production with time was observed, but reached a steady state. For the bioreactor packed with phyllosilicate sol-gel-immobilized LOX, initial HPOD production increased more rapidly but reached a lower steady-state concentration. From these data, a simple computer simulation model was developed to determine the process kinetics of this reactor design.

Characterization of soybean lipoxygenase immobilized in cross-linked phyllosilicates.

Lipoxygenase (LOX) is an enzyme that regioselectively introduces the hydroperoxide functionality into polyunsaturated fatty acids, such as linoleic acid (LA). Hydroperoxide derivatives of polyunsaturated fatty acids are of interest because they can serve as important intermediates in the synthesis of chemical and pharmaceutical compounds. In this study, LOX was immobilized in dispersed phyllosilicate layers that were cross-linked with silicate polymers formed by the hydrolysis of tetramethyl orthosilicates. The effects of substrate concentration, reaction temperature and solvent participation were studied on the oxidation of LA by LOX. The temperature optimum for the oxidation of LA by immobilized LOX was 25 degrees C and values of Km and Vmax for this reaction were 1.7 mM and 0.023 micromol/min respectively. Enzymic activity was stimulated by the addition of 10% (v/v) iso-octane to the reaction mixture. The immobilized LOX preparation showed a degree of substrate preference that demonstrated that 1,3-dilinolein was a better substrate than LA in the oxidation reaction, followed in order by 1-monolinolein, methyl oleate and trilinolein. In general, LOX immobilized in cross-linked phyllosilicates retained the physical and chemical characteristics of free LOX.

Effectiveness of cross-linked phyllosilicates for intercalative immobilization of soybean lipoxygenase.

A novel procedure was developed to intercalate enzymes into dispersed phyllosilicates that were cross-linked with silicate polymers formed by the hydrolysis of tetramethyl orthosilicate (TMOS). Lipoxygenase (LOX) intercalated into cross-linked phyllosilicates exhibited high enzymatic activity. The enzyme-phyllosilicate composite prepared by this procedure had an improved pore network. Alkylamines were used to occupy the charge sites of the phyllosilicate, which increased the hydrophobicity of the phyllosilicate and reduced charge-charge interaction between LOX and the phyllosilicate. The amount of macropores and the enzymatic activity of the lipoxygenase-phyllosilicate composites increased with an increase in the ratio of trimethylammonium (TMA)-phyllosilicate to cross-linking reagent TMOS. LOX intercalatively immobilized into phyllosilicates displayed good storage stability and reusability at ambient temperature.

Kinetic analysis of proton transport by the vanadate-sensitive ATPase from maize root microsomes.

Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in Kl-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO(3) or KCl. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.

Factors associated with the instability of nitrate-insensitive proton transport by maize root microsomes.

Proton transport catalyzed by the nitrate-insensitive, vanadate-sensitive H(+)-ATPase in microsomes from maize (Zea mays L.) roots washed with 0.25 molar KI decreased as a function of time at 0 to 4 degrees C. The rate of proton transport was approximately one-half of that by freshly isolated microsomes after 6 to 18 hours of cold storage. The decrease in proton transport coincided with losses in membrane phosphatidylcholine and was not associated with a change in vanadate-sensitive ATP hydrolysis. A technique based on a protocol developed for the reconstitution of Neurospora crassa plasma membrane H(+)-ATPase (DS Perlin, K Kasamo, RJ Brooker, CW Slayman 1984 J Biol Chem 259: 7884-7892) was employed to restore proton transport activity to maize microsomes. These results indicated that the decline in proton transport by maize root membranes during cold storage was not due to degradation of the protein moiety of the H(+)-ATPase, but was due to the loss of phospholipids.

Sugars present in tobacco extracts.

The presence of fructose, glucose, sucrose, maltose, and isomaltose in commercial tobacco products was identified and quantitated. Gas-liquid chromatographic studies showed that these five types of sugar were present in the water-soluble extracts of pouch and plug chewing tobacco, yet only fructose and glucose were found in extracts of snuff and unprocessed natural tobaccos. The amount of sucrose present in pouch chewing tobacco was twice that in plug chewing tobacco. No detectable amount of sucrose was found in snuff or unprocessed natural tobaccos. The content of maltose and isomaltose was much less than the content of fructose, glucose, or sucrose. All unprocessed natural tobacco leaves studied as controls contained low amounts of fructose and glucose, and no detectable amounts of sucrose, maltose, or isomaltose. The larger amounts of fructose and glucose, and the additional sucrose, maltose, and isomaltose present in pouch and plug chewing tobaccos are probably added during the manufacturing process.

Studies of the human testis. X. Properties of human chorionic gonadotropin receptor in adult testis and relation to intratesticular testosterone concentration.

Receptors for [125I]hCG were found in adult human testis. The specific binding of [125I]hCG to testicular receptor is temperature dependent and is a saturable process with respect to added receptor protein and hormone. Scatchard analysis revealed a dissociation constant of 5.0 X 10(-10) M, and 6.2 fmol binding site/mg protein. Intact unlabeled hCG effectively inhibits the specific binding of [125I]hCG to human testicular receptors. For inhibition of binding of [125I]hCG, the alpha subunit has 3.0% of the potency of intact hCG and the beta subunit has 0.4% of the potency of intact hCG. Specific binding is pH dependent, with an optimum at pH 7.4. Brief exposure to extremes of pH causes irreversible damage to the receptors. Incubation with protease and trypsin results in an almost complete loss of binding activity, while ribonuclease, deoxyribonuclease, phospholipase C, or neuraminidase treatment does not significantly alter hormone-binding activity. Binding activity was found to be positively correlated to the concentration of intratesticular testosterone.

An androgen binding protein in the testicular cytosol of human testis. Comparison with human plasma testosterone-estrogen binding globulin.

After removal of plasma contamination, an androgen binding protein (hABP) was detected in a 105,000-g supernate of human testicular homogenate. The physicochemical properties of hABP have been compared with a similar androgen binding protein in human plasma, testosterone-estrogen binding globulin (TeBG). hABP had high affinity (K(d) = 7.8 nM) and low capacity (0.27 pmol/mg protein) for 5alpha-dihydrotestosterone (DHT). Binding affinity of human TeBG for DHT was greater (K(d) = 0.66 nM, binding capacity 0.68 pmol/mg protein). On the basis of sedimentation rates and Einstein Stokes radii of hABP and TeBG, the mol wt of the two proteins were similar in the range of 87,000-92,000. The ligand specificities of hABP and TeBG were the same. The binding of [(3)H]DHT to hABP and TeBG were reversible processes at 0 degrees C. The half-lives for the dissociation of [(3)H]DHT from hABP and TeBG were 100-120 min and 67-70 min, respectively. Heat sensitivity of hABP and TeBG were similar. hABP had a sharp pH binding curve with an optimum at 8, whereas TeBG had a stable pH optimum between 6.5 and 9. hABP and TeBG were eluted from an ion exchange column at 100 mM and 80 mM sodium chloride concentrations, respectively. Concanavalin A and ricin Sepharose affinity chromatography showed that TeBG is bound to the columns nearly quantitatively, whereas hABP is bound to the columns only partially. Differences between hABP and TeBG, plus the reduction of plasma contamination as marked by albumin, suggest that the human mature testis contains an androgen binding protein separate from that circulating in plasma.

The role of a dolichol-oligosaccharide as an intermediate in glycoprotein biosynthesis.

Incubation of mouse myeloma microsomes with GDP-[(14)C]mannose results in the biosynthesis of [(14)C]mannose phosphoryl dolichol [Baynes, J. W., Hsu, A.-F. & Heath, E. C. (1973) J. Biol. Chem. 248, 5693-5704] and a [(14)C]mannose- and N-acetylglucosamine (GlcNAc)-containing oligosaccharide derivative of dolichol. Thus, [(14)C]mannose phosphoryl dolichol and [(14)C]mannose-labeled oligosaccharide pyrophosphoryl dolichol were isolated from incubation mixtures by solubilization in 2% (w/v) Triton X-100 and the lipids were separated from small molecules by gel filtration fractionation. After removal of radioactive protein from the preparation, the two lipid derivatives were separated quantitatively by fractionation on a concanavalin A-Sepharose column; [(14)C]mannose phosphoryl dolichol was not retained by the affinity resin but [(14)C]mannose-oligosaccharide pyrophosphoryl dolichol adsorbed to the gel and was eluted with alpha-methylmannoside.[(14)C]Mannose-oligosaccharide pyrophosphoryl dolichol appeared to be homogeneous when fractionated on DEAE-cellulose and in several thin-layer chromatographic systems. Treatment of [(14)C]mannose oligosaccharide pyrophosphoryl dolichol with 10% (w/v) NH(4)OH at 100 degrees for 1 hr resulted in the formation of a water-soluble radioactive oligosaccharide phosphate which was isolated and characterized as [Man](5) --> [GlcNAc --> GlcNAc --> P. Incubation of [(14)C]mannose-oligosaccharide pyrophosphoryl dolichol with myeloma microsomal preparations results in the transfer, presumably, of the entire oligosaccharide to endogenous protein. Kinetic studies indicate that the dolichol derivatives serve as intermediates in the glycosylation of protein as follows: [Formula: see text]