Membrane enzymes: artifacts in Arrhenius plots due to temperature dependence of substrate-binding affinity.

For the membrane sodium-stimulated magnesium-adenosinetriphosphatase of Acholeplasma laidlawii B both the Vmax and Km values in the Michaelis equation very strongly with temperature. Simulations of Arrhenius plots show that an enzyme with a temperature-dependent Km can yield a variety of Arrhenius plot artifacts, most notably erroneous "breaks," if activity is assayed at a fixed substrate concentration.

Biophysical characterization of small molecule antiviral-loaded nanolipogels for HIV-1 chemoprophylaxis and topical mucosal application.

UNLABELLED: Nanocarriers are versatile vehicles for drug delivery, and emerging as platforms to formulate and deliver multiple classes of antiretroviral (ARV) drugs in a single system. Here we describe the fabrication of hydrogel-core and lipid-shell nanoparticles (nanolipogels) for the controlled loading and topical, vaginal delivery of maraviroc (MVC) and tenofovir disoproxil fumarate (TDF), two ARV drugs with different mechanisms of action that are used in the treatment of HIV. The nanolipogel platform was used to successfully formulate MVC and TDF, which produced ARV drug-loaded nanolipogels that were characterized for their physical properties and antiviral activity against HIV-1 BaL in cell culture. We also show that administration of these drug carriers topically to the vaginal mucosa in a murine model leads to antiviral activity against HIV-1 BaL in cervicovaginal lavages. Our results suggest that nanolipogel carriers are promising for the encapsulation and delivery of hydrophilic small molecule ARV drugs, and may expand the nanocarrier systems being investigated for HIV prevention or treatment. STATEMENT OF SIGNIFICANCE: Topical, mucosal intervention of HIV is a leading strategy in the efforts to curb the spread of viral infection. A significant research thrust in the field has been to characterize different dosage forms for formulation of physicochemically diverse antiretroviral drugs. Nanocarriers have been used to formulate and deliver small molecule and protein drugs for a range of applications, including ARV drugs for HIV treatment. The broad significance of our work includes evaluation of lipid-shell, hydrogel-core nanoparticles for formulation and topical, vaginal delivery of two water-soluble antiretroviral drugs. We have characterized these nanocarriers for their physical properties and their biological activity against HIV-1 infection in vitro, and demonstrated the ability to deliver drug-loaded nanocarriers in vivo.

Influence of membrane lipid fluidity on glucose and uridine facilitated diffusion in human erythrocytes.

A central question which must be resolved before acceptable molecular descriptions of facilitated diffusion systems can be provided is the nature of the spatial and functional relationships between the transport proteins and the membrane lipids. In the work reported here, this question was addressed by investigating the dependence of the rates of glucose and uridine facilitated diffusion in human erythrocytes on membrane lipid fluidity. Two approaches were used to alter the lipid fluidity: treatment with ether, an anesthetic, and the exchange of a synthetic 3-ketosteroid, cholest-4-en-3-one, for membrane chloesterol. Both of these treatments result in a significant increase in membrane lipid fluidity, as judged by the increase in the rates of passive diffusion of uridine through cell membranes and of glucose through membrane lipid bilayer vesicles. Ether produces no change in the Km of either transport process, a slight decrease in the V for glucose transport, and no significant change in the V for uridine transport. Replacement of membrane cholesterol by cholest-4-en-3-one reduces the V for glucose transport slightly, without altering the Km, and reduces both the Km and V for uridine transport. The absence of the expected increase in the V of facilitated diffusion with increasing membrane lipid fluidity observed here with human erythrocytes is not consistent with models for the transport process which feature movement of transport proteins which are in direct contact with the bulk lipids of the membrane.

Specific interaction of concanavalin A with glycolipid monolayers.

The effect of 131I-labelled concanavalin A on the surface pressure and surface radioactivity of monolayers formed from phospholipids and from natural and synthetic glycolipids has been studied. The lectin binds to and penetrates dipalmitoyl phosphatidylcholine monolayers at a surface pressure of 15 dynes/cm and this interaction is inhibited by the presence of alpha-methyl mannose in the subphase. At surface pressures of 25 dynes/cm or higher, concanavalin A will interact with monoglucosyl diglyceride or diglucosyl diglyceride from Acholeplasma laidlawii and with synthetic glycolipids containing 2 or 3 alpha 1 leads to 4-linked D-glucose residues in the headgroup, but not with phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or with the ganglioside II3NeuAc-GgOse4-Cer. The binding to the glycolipid sugar group and penetration of the hydrocarbon region seem to occur simultaneously, as the time courses for the development of surface pressure and surface radioactivity coincide.

Thermotropic phase transitions of phosphatidylcholines with odd-numbered n-acyl chains.

Diacyl phosphatidylcholines with n-C13, -C15, -C17 and -C19 saturated acyl chains have been synthesized and their phase transitions in the presence of excess water monitored by differential thermal analysis. The C15-, C17- and C19-diacyl species show gel to liquid-crystalline transitions and pretransitions like those of the even-chain phosphatidylcholines. A plot of the main phase transition temperature, Tc, vs. acyl chain length is a smooth curve on which the Tc values of both odd- and even-chain species fall, while a similar plot for the pretransition temperature, Tp, shows significant alternation of Tp between odd- and even-chain species. Consideration of these results in terms of the physical basis of the odd-even alternation of phase transition temperatures in homologous series of paraffinic compounds suggests that the acyl chains of disaturated phosphatidylcholines are tilted with respect to the bilayer normal below Tp but become perpendicular to the bilayer surface above the pretransition temperature.

Inbreeding: one word, several meanings, much confusion.

Because conservation biologists must frequently deal with small populations, inbreeding (a frequent consequence of small population size) has played a central role in many genetic management programs. However, the word "inbreeding" has several, often contradictory meanings, and a failure to distinguish among these meanings has caused much misunderstanding on the role of inbreeding in genetic management. Three different biological meanings of inbreeding are discussed in this paper: (1) inbreeding as a measure of shared ancestry in the paternal and maternal lineages of an individual; (2) inbreeding as a measure of genetic drift in a finite population, and (3) inbreeding as a measure of system of mating in a reproducing population. The distinction and use of these different measures of inbreeding are discussed and illustrated with a worked example, the North American captive population of Speke's gazelle (Gazella spekei). It is shown that these different meanings of the word inbreeding must be kept separated, otherwise erroneous management recommendations and evaluations can occur. On the positive side, the different measures of inbreeding when used jointly can be a powerful management tool precisely because they measure different biological phenomena.

Diagnosis of congenital dislocated hips (CDH).

Radiology imaging, along with clinical orthopedic assessments, plays an important role in the diagnosis and treatment of Congenital Dislocated Hips. The validity of the radiologic examinations relies heavily on the examiner or technologist. This article explains some of these examinations and describes the benefits, disadvantages, and important facts to be aware of while performing them.

High substrate specificity factor ribulose bisphosphate carboxylase/oxygenase from eukaryotic marine algae and properties of recombinant cyanobacterial RubiSCO containing "algal" residue modifications.

Marine algae play an important role in removing carbon dioxide from the atmosphere. In this investigation, we have determined the substrate specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase from several marine chromophytic and rhodophytic algae. The enzymes were purified to homogeneity and all possessed significantly higher substrate specificity factors than the enzymes from terrestrial plants, green algae, or bacteria. There are substantial differences in the sequence in a helix 6 of the large subunit of these enzymes, which is intriguing since residues of this region had been previously shown to influence the ability of ribulose bisphosphate carboxylase to discriminate between CO2 and O2, presumably by influencing the adjacent flexible loop 6 region. Sequence divergence at this and other key regions might contribute to the substantial differences in the substrate specificity factor of the chromophyte/rhodophyte enzyme. Initial studies on probing the basis for the high substrate specificity factor employed single amino acid substitutions in the recombinant cyanobacterial ribulose bisphosphate carboxylase. Residues in the vicinity of loop 6 were changed to reflect the corresponding residues in the chromophyte/rhodophyte large subunit. Some changes in the substrate specificity factor were noted, as were alterations in other important kinetic parameters. Since marine algae show little evidence of photorespiratory metabolism, the high substrate specificity of ribulose bisphosphate carboxylase is consistent with the physiology of these organisms. The results of this study provide further evidence that the properties of this enzyme may evolve or change according to the environment in which the host organism is found.

Amino acid substitutions in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that influence catalytic activity of the holoenzyme.

Four unique amino acid substitutions were introduced by site-directed mutagenesis into the third conserved region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Anacystis nidulans (Synechococcus sp., PCC6301), resulting in the formation of four mutant enzymes, I87V, R88K, G91V, and F92L. Wild-type and mutant proteins were purified after synthesis in Escherichia coli. These single amino acid substitutions do not appear to perturb intersubunit interactions or induce any gross conformational changes; purified mutant proteins are stable, for the most part like the wild-type holoenzyme, and exhibit nearly identical CD spectra. Three of the four mutants, however, are severely deficient in carboxylase activity, with kcat less than or equal to 35% of the wild-type enzyme. While the substrate specificity factors were the same for the mutant and wild-type enzymes, significant alterations in some kinetic parameters were observed, particularly in the Michaelis constants for CO2, O2, and RuBP. All four mutant proteins exhibited lower KCO2 values, ranging from 37 to 88% of the wild-type enzyme. Two of the mutants, in addition, exhibited significantly lower KRuBP values, and one mutant showed a substantial decrease in KO2. The effects of the single-site mutations in rbcS of this study strengthen the hypothesis that small subunits may not contribute directly to substrate specificity; however, individual residues of the small subunit substantially influence catalysis by large subunits.

A hybrid ribulosebisphosphate carboxylase/oxygenase enzyme exhibiting a substantial increase in substrate specificity factor.

Two hybrid ribulose-1,5 bisphosphate carboxylase/oxygenase (RubisCO) enzymes were constructed using RubisCO small subunit genes (rbcS) from two eucaryotic marine organisms, Cylindrotheca sp. N1 and Olisthodiscus luteus, cloned downstream of the RubisCO large subunit gene (rbcL) of the cyanobacterium Synechococcus PCC 6301. The expression products synthesized by Escherichia coli JM107 (pVTAC223 and pANOLI) were purified and examined by polyacrylamide gel electrophoresis and compared to the purified products generated by E. coli MV1190 (pBGL710), containing cyanobacterial rbcL and rbcS genes. Both Cylindrotheca and Olisthodiscus small subunits were able to assemble in vivo with the Synechococcus large subunit octamer to form heterologous hexadecameric L8S8 enzymes, the pVTAC223 and pANOLI hybrid enzymes, respectively. Like the Synechococcus RubisCO, the hybrid enzymes were rapidly activated by Mg2+ plus HCO3-, even in the presence of RuBP. The hybrid enzymes, however, were considerably more sensitive to the competitive inhibitor 6-phosphogluconate. Detailed kinetic analysis indicated that while the carboxylase activity of both chimeric enzymes was severely reduced, in the case of the pVTAC223 hybrid enzyme, the degree of partitioning between carboxylation and oxygenation was increased nearly 60% relative to the Synechococcus RubisCO. Other kinetic properties, including the Michaelis constants for the gaseous substrates and RuBP, were altered in the hybrid proteins. These studies also led to the finding that the substrate specificity factor of the Cylindrotheca RubisCO is unusually high.

Vascular distal ureteral obstruction.

The diagnosis, treatment and results of 6 patients with 7 obstructed distal ureters secondary to vascular compression are presented. Three ureters were treated by transection of the offending vessels and the remaining 4 required additional ureteroneocystostomy.

Factitious polymicrobial bacteremia: a case report.

A 23-year-old student nurse presented with polymicrobial bacteremia and symptoms suggestive of a renal abscess. After extensive evaluation, including an exploratory laparotomy, failed to reveal the source of sepsis, factitious illness owing to self-inoculation was suspected. In the absence of urologic, biliary or gastrointestinal obstruction self-inoculation must be considered in all patients with polymicrobial bacteremia.

Glucose transport in Acholeplasma laidlawii B: dependence on the fluidity and physical state of membrane lipids.

The uptake of D-glucose by Acholeplasma laidlawii B occurs via a mediated transport process, as shown by the following observations: (i) glucose permeates A. laidlawii B cells at a rate at least 100 times greater than would be expected if its entry occurred only by simple passive diffusion; (ii) the apparent activation energy for glucose uptake in A. laidlawii is significantly lower than that expected and observed for the passive permeation of this sugar; (iii) glucose uptake appears to be a saturable process; (iv) glucose uptake can be completely inhibited by low concentrations of phloretin and phlorizin; and (v) glucose uptake is markedly inhibited at temperatures above 45 C, whereas the passive entry of erythritol continues to increase logarithmically until at least 60 C. The metabolism of D-glucose by this organism is rapid and, at low glucose concentrations, the intracellular radioactivity derived from D-[14-C]glucose is at any given time a reflection of the net effect of glucose transport, glucose metabolism, and loss from the cell of radioactive metabolic products. Care must thus be taken when attempting to determine the rate of glucose transport by measuring the accumulation by the cells of the total radioactivity derived from D-[14-C]glucose. The rate of uptake of D-glucose by A. laidlawii B cells is markedly dependent on the fatty acid composition and cholesterol content of the plasma membrane and exhibits a direct dependence on the fluidity of the membrane lipids as measured by their reversible, thermotropic gel to liquie-crystalline phase transition temperatures. In contrast to the transport rates, the apparent activation energy for glucose uptake above the phase transition temperature is not dependent on membrane lipid composition. At the temperature range within the membrane lipid phase transition region, the apparent activation energy of glucose uptake is different from the activation energy observed at temperatures above the phase transition. This may reflect the superimposed operation within the phase transition region of more than one temperature-dependent process.

Catalytic properties of recombinant octameric, hexadecameric, and heterologous cyanobacterial/bacterial ribulose- 1,5-bisphosphate carboxylase/oxygenase.

The recent isolation of a catalytically competent recombinant octameric core of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans (Synechococcus) (B. Lee and F. R. Tabita, 1990, Biochemistry 29, 9352-9357) has provided a useful system for examining the properties of this enzyme in the absence of small subunits. Unlike most sources of hexadecameric ribulose bisphosphate carboxylase, the nonactivated Anacystis holoenzyme was not inhibited markedly by preincubation with ribulose 1,5-bisphosphate. This was also true for the Anacystis octameric core and a heterologous recombinant enzyme that comprised large subunits from Anacystis and small subunits from the bacterium Alcaligenes eutrophus, suggesting that substrate-mediated inactivation is not influenced by small subunits. In addition, the CO2/O2 specificity factor was not affected by the source of the small subunits incorporated into the structure of the hexadecameric protein, in agreement with previous in vitro heterologous reconstitution studies. The activated octameric Anacystis enzyme, however, was significantly more sensitive to inhibition by the phosphorylated effector 6-phosphogluconate than were the hexadecameric Alcaligenes and Anacystis enzymes and the heterologous Anacystis-Alcaligenes hybrid.

HK/LK polymorphism and its genetic determination in Speke's gazelle.

Concentrations of potassium (K) and sodium (Na) were determined in the erythrocytes of 28 members of an interbreeding herd of Speke's gazelle. The distribution of K concentrations and Na/K concentration ratios suggested the presence of the high-K/low-K (HK/LK) polymorphism known in erythrocytes of domestic bovids. The pedigree of the herd of gazelles is known completely, permitting examination of the inheritance of HK/LK polymorphism by overlaying the distribution of phenotypes on the pedigree. Statistical analyses clearly indicate a strong genetic component in the phenotypic variability that is entirely consistent with a single autosomal locus, two-allele mode of inheritance, with the LK allele being dominant. This is the first demonstration of HK/LK polymorphism in a wild bovid species. The evidence indicates that HK/LK polymorphism is of considerable evolutionary age, is of monophyletic origin, and is maintained by selection.

DNA fingerprinting in Speke's gazelle: a test for genetic distinctness, and the correlation between relatedness and similarity.

In the absence of pedigree information, the determination of genetic distinctness of populations can only be made by genetic methods. Using DNA fingerprinting on the North American captive herd of Speke's gazelle Gazella spekei, we were able to address two hypotheses. First, two new individuals were found to have come from a genetically distinct population (P = 0.008, permutation test), and represent potential new founders to be added to the population. Secondly, genetic similarity was not significantly correlated with relatedness under extreme inbreeding and very close relationship (coefficient of relationship range 0.304-0.717).