Genetic diversity and proviral DNA load in different neural compartments of HIV-1 subtype C infection

In India, the low prevalence of HIV-associated dementia (HAD) in the Human immunodeficiency virus type 1 (HIV-1) subtype C infection is quite paradoxical given the high-rate of macrophage infiltration into the brain. Whether the direct viral burden in individual brain compartments could be associated with the variability of the neurologic manifestations is controversial. To understand this paradox, we examined the proviral DNA load in nine different brain regions and three different peripheral tissues derived from ten human subjects at autopsy. Using a highly sensitive TaqMan probe-based real-time PCR, we determined the proviral load in multiple samples processed in parallel from each site. Unlike previously published reports, the present analysis identified uniform proviral distribution among the brain compartments examined without preferential accumulation of the DNA in any one of them. The overall viral DNA burden in the brain tissues was very low, approximately 1 viral integration per 1000 cells or less. In a subset of the tissue samples tested, the HIV DNA mostly existed in a free unintegrated form. The V3-V5 envelope sequences, demonstrated a brain-specific compartmentalization in four of the ten subjects and a phylogenetic overlap between the neural and non-neural compartments in three other subjects. The envelope sequences phylogenetically belonged to subtype C and the majority of them were R5 tropic. To the best of our knowledge, the present study represents the first analysis of the proviral burden in subtype C postmortem human brain tissues. Future studies should determine the presence of the viral antigens, the viral transcripts, and the proviral DNA, in parallel, in different brain compartments to shed more light on the significance of the viral burden on neurologic consequences of HIV infection.

Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.

Relative sensitivity of the corpus luteum of different days of pregnancy to LH-deprivation in the rat and hamster

Deprivation of endogenous LH by LH antiserum (LH A/S) in 6-day pregnant rats did not affect the luteal or serum progesterone within 24 h. LH A/S treatment on day 7 or 8 of pregnancy, however, caused a 70 and 92% reduction in luteal progesterone, respectively, within 24 h. Serum levels of progesterone showed a similar reduction. In the case of pregnant hamster, unlike the rat, there was a significant decrease in progesterone in the serum, luteal and non-luteal compartments whether the A/S was administered on day 4, 5 or 6. There was more than a 10-fold increase in the luteal cholesterol esters within 24 h whether the A/S was given on day 6, 7 or 8 of pregnancy in the rat. Rat corpora lutea of days 6 and 8 of pregnancy reacted in a like manner to LH-deprivation, showing an increased utilization of [U-14C]glucose to form 14CO2 in vitro. In the rat, LH (25 μg NIH-S19) administration in vivo either on day 6 or day 8 of pregnancy, caused within 2 h an increase in serum and non-luteal progesterone, but luteal progesterone was unchanged. On the other hand, LH administration to hamsters on day 8 of pregnancy caused an increase in progesterone levels in serum, luteal and non-luteal tissue. Incubation of corpora lutea isolated from untreated 6- and 8-day pregnant rats with LH brought about an increase in progesterone secretion into the medium in both cases. The results show that, even though LH-deprivation does not apparently affect progesterone concentration in the corpus luteum of 6-day pregnant rats, it does affect other metabolic parameters such as glucose utilization and cholesterol turnover, suggesting that the corpus luteum of early pregnancy exhibits a continuous dependency on LH for the maintainence of metabolic functions.

Mitochondrial localization of functional ferrochelatase from Plasmodium falciparum

In the malarial parasite, enzymes of heme-biosynthetic pathway are distributed in different cellular compartments. The site of localization of ferrochelatase in the malarial parasite is crucial, since it will decide the ultimate site of heme synthesis. Earlier results have differed in terms of localization, being the mitochondrion or apicoplast and the functional enzyme has not been cloned, expressed and characterized. The present study reveals that Plasmodium falciparum ferrochelatase (PfFC) gene encodes multiple transcripts of which the one encoding the full length functional protein (PfFC) has been cloned and the recombinant protein over-expressed and purified from E. coli cells. The enzyme shows maximum activity with iron, while zinc is a poor substrate. Immunofluorescence studies with antibodies to functional ferrochelatase reveal that the native enzyme is localized to the mitochondrion of the parasite indicating that this organelle is the ultimate site of heme synthesis.

Location of Pathogenic Bacteria during Persistent Infections: Insights from an Analysis Using Game Theory

Bacterial persistent infections are responsible for a significant amount of the human morbidity and mortality. Unlike acute bacterial infections, it is very difficult to treat persistent bacterial infections (e.g. tuberculosis). Knowledge about the location of pathogenic bacteria during persistent infection will help to treat such conditions by designing novel drugs which can reach such locations. In this study, events of bacterial persistent infections were analyzed using game theory. A game was defined where the pathogen and the host are the two players with a conflict of interest. Criteria for the establishment of Nash equilibrium were calculated for this game. This theoretical model, which is very simple and heuristic, predicts that during persistent infections pathogenic bacteria stay in both intracellular and extracellular compartments of the host. The result of this study implies that a bacterium should be able to survive in both intracellular and extracellular compartments of the host in order to cause persistent infections. This explains why persistent infections are more often caused by intracellular pathogens like Mycobacterium and Salmonella. Moreover, this prediction is in consistence with the results of previous experimental studies.

Specificity for the Exchange of Phospholipids Through Polymyxin B Mediated Intermembrane Molecular Contacts

Structural specificity for the direct vesicle−vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (PxB) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O. G., & Jain, M. K. (1996) Biochemistry 35, 299−308], intermembrane molecular contacts between anionic vesicles are formed by a small number of PxB molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of PxB to vesicles, formation of stable vesicle−vesicle contacts, and exchange of phospholipids. Polycationic PxB binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through PxB contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. PxB does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange in covesicles is not mediated by PxB. Vesicles of dianionic phospholipids, like phosphatidic acid, bind PxB; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of PxB at the interface. PxB in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that PxB at the interface can exist in two forms depending on the lipid/PxB ratio. Additional studies show that stable PxB-mediated vesicle−vesicle contacts may be structurally and functionally distinct from “stalks”, the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis and trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundations of interfacial equilibria and catalysis.

Protoporphyrinogen IX oxidase from Plasmodium falciparum is anaerobic and is localized to the mitochondrion

Earlier studies in this laboratory had shown that the malarial parasite can synthesize heme de novo and inhibition of the pathway leads to death of the parasite. It has been proposed that the pathway for the biosynthesis of heme in Plasmodium falciparum is unique involving three different cellular compartments, namely mitochondrion, apicoplast and cytosol. Experimental evidences are now available for the functionality and localization of all the enzymes of this pathway, except protoporphyrinogen IX oxidase (PfPPO), the penultimate enzyme. In the present study. PfPPO has been cloned, expressed and shown to be localized to the mitochondrion by immunofluorescence microscopy. Interestingly, the enzyme has been found to be active only under anaerobic conditions and is dependent on electron transport chain (ETC) acceptors for its activity. The native enzyme present in the parasite is inhibited by the ETC inhibitors, atovaquone and antimycin. Atovaquone, a well known inhibitor of parasite dihydroorotate dehydrogenase, dependent on the ETC, inhibits synthesis of heme as well in P. falciparum culture. A model is proposed to explain the ETC dependence of both the pyrimidine and heme-biosynthetic pathways in P. falciparum. (C) 2010 Elsevier B.V. All rights reserved.

Chaperone expression profiles correlate with distinct physiological states of Plasmodium falciparum in malaria patients

Background: Molecular chaperones have been shown to be important in the growth of the malaria parasite Plasmodium falciparum and inhibition of chaperone function by pharmacological agents has been shown to abrogate parasite growth. A recent study has demonstrated that clinical isolates of the parasite have distinct physiological states, one of which resembles environmental stress response showing up-regulation of specific molecular chaperones. Methods: Chaperone networks operational in the distinct physiological clusters in clinical malaria parasites were constructed using cytoscape by utilizing their clinical expression profiles. Results: Molecular chaperones show distinct profiles in the previously defined physiologically distinct states. Further, expression profiles of the chaperones from different cellular compartments correlate with specific patient clusters. While cluster 1 parasites, representing a starvation response, show up-regulation of organellar chaperones, cluster 2 parasites, which resemble active growth based on glycolysis, show up-regulation of cytoplasmic chaperones. Interestingly, cytoplasmic Hsp90 and its co-chaperones, previously implicated as drug targets in malaria, cluster in the same group. Detailed analysis of chaperone expression in the patient cluster 2 reveals up-regulation of the entire Hsp90-dependent pro-survival circuitries. In addition, cluster 2 also shows up-regulation of Plasmodium export element (PEXEL)-containing Hsp40s thought to have regulatory and host remodeling roles in the infected erythrocyte. Conclusion: In all, this study demonstrates an intimate involvement of parasite-encoded chaperones, PfHsp90 in particular, in defining pathogenesis of malaria.

Characterization of coproporphyrinogen III oxidase in Plasmodium falciparum cytosol

A unique hybrid pathway has been proposed for de novo heme biosynthesis in Plasmodium falciparum involving three different compartments of the parasite, namely mitochondrion, apicoplast and cytosol. While parasite mitochondrion and apicoplast have been shown to harbor key enzymes of the pathway, there has been no experimental evidence for the involvement of parasite cytosol in heme biosynthesis. In this study, a recombinant P. falciparum coproporphyrinogen III oxidase (rPfCPO) was produced in E. coli and confirmed to be active under aerobic conditions. rPfCPO behaved as a monomer of 61 kDa molecular mass in gel filtration analysis. Immunofluorescence studies using antibodies to rPfCPO suggested that the enzyme was present in the parasite cytosol. These results were confirmed by detection of enzyme activity only in the parasite soluble fraction. Western blot analysis with anti-rPfCPO antibodies also revealed a 58 kDa protein only in this fraction and not in the membrane fraction. The cytosolic presence of PfCPO provides evidence for a hybrid heme-biosynthetic pathway in the malarial parasite. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Vesicle and Stable Monolayer Formation from Simple ``Click'' Chemistry Adducts in Water

Click chemistry has been successfully extended into the field of molecular design of novel amphiphatic adducts. After their syntheses and characterizations, we have studied their aggregation properties in aqueous medium. Each of these adducts forms stable suspensions in water. These suspensions have been characterized by dynamic light scattering (DLS) studies and transmission electron microscopy (TEM). The presence of inner aqueous compartments in such aggregates has been demonstrated using dye (methylene blue) entrapment studies. These aggregates have been further characterized using X-ray diffraction (XRD), which indicates the existence of bilayer structures in them. Therefore, the resulting aggregates could be described as vesicles. The temperature-induced order-to-disorder transitions of the vesicular aggregates and the accompanying changes in their packing and hydration have been examined using high-sensitivity differential scanning calorimetry, fluorescence anisotropy, and generalized polarization measurements using appropriate membrane-soluble probe, 1,6-diphenylhexatriene, and Paldan, respectively. The findings of these studies are consistent with each other in terms of the apparent phase transition temperatures. Langmuir monolayer studies confirmed that these click adducts also form stable monolayers on buffered aqueous subphase at the air-water interface.

In vivo and in vitro studies on the differential role of luteinizing hormone and follicle-stimulating hormone in regulating follicular function in the bonnet monkey (Macaca radiata) using specific gonadotropin antibodies

Although a distinct need for FSH in the regulation of follicular maturation in the primate is well recognized, it is not clear how FSH controls the functionality of different cellular compartments of the follicle. It is also not evident whether there is a requirement for LH in follicular maturation in the primate. In the first part of the present study, female bonnet monkeys were administered a well-characterized ovine (o) LH antiserum to neutralize endogenous monkey LH for different periods during the follicular phase, and the effect on the overall follicular maturation process was assessed by analyzing serum estrogen (E) and progesterone (P) profiles. Neither continuous LH deprivation from Day 8 of the cycle nor deprivation of LH on any one day between Days 6 and 10 had a significant effect on serum E and P profiles and the follicular maturation process. The period for which the antiserum was effective was dependent upon the dose injected; 1 ml of the antiserum given on Day 8 blocked ovulation but not follicular maturation. To assess the effect of deprivation of LH/FSH at the cellular level, animals were deprived in vivo of LH (on Days 8 and 9 of the cycle) or of FSH (on Day 6 of the cycle) by injection of highly characterized hCG and ovine (o) FSH antisera, respectively; the in vitro responsiveness of granulosa and thecal cells isolated on Day 10 from these animals was then determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase relations and gibbs energies in the system Mn-Rh-O

Phase relations in the system Mn-Rh-O are established at 1273 K by equilibrating different compositions either in evacuated quartz ampules or in pure oxygen at a pressure of 1.01 x 10(5) Pa. The quenched samples are examined by optical microscopy, X-ray diffraction, and energy-dispersive X-ray analysis (EDAX). The alloys and intermetallics in the binary Mn-Rh system are found to be in equilibrium with MnO. There is only one ternary compound, MnRh2O4, with normal spinel structure in the system. The compound Mn3O4 has a tetragonal structure at 1273 K. A solid solution is formed between MnRh2O4 and Mn3O4. The solid solution has the cubic structure over a large range of composition and coexists with metallic rhodium. The partial pressure of oxygen corresponding to this two-phase equilibrium is measured as a function of the composition of the spinel solid solution and temperature. A new solid-state cell, with three separate electrode compartments, is designed to measure accurately the chemical potential of oxygen in the two-phase mixture, Rh + Mn3-2xRh2xO4, which has 1 degree of freedom at constant temperature. From the electromotive force (emf), thermodynamic mixing properties of the Mn3O4-MnRh2O4 solid solution and Gibbs energy of formation of MnRh2O4 are deduced. The activities exhibit negative deviations from Raoult's law for most of the composition range, except near Mn3O4, where a two-phase region exists. In the cubic phase, the entropy of mixing of the two Rh3+ and Mn3+ ions on the octahedral site of the spinel is ideal, and the enthalpy of mixing is positive and symmetric with respect to composition. For the formation of the spinel (sp) from component oxides with rock salt (rs) and orthorhombic (orth) structures according to the reaction, MnO (rs) + Rh2O3 (orth) --> MnRh2O4 (sp), DELTAG-degrees = -49,680 + 1.56T (+/-500) J mol-1. The oxygen potentials corresponding to MnO + Mn3O4 and Rh + Rh2O3 equilibria are also obtained from potentiometric measurements on galvanic cells incorporating yttria-stabilized zirconia as the solid electrolyte. From these results, an oxygen potential diagram for the ternary system is developed.

Primary Sequence That Determines the Functional Overlap between Mitochondrial Heat Shock Protein 70 Ssc1 and Ssc3 of Saccharomyces cerevisiae

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can ``make or break'' mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.

Thermal Lipid Order−Disorder Transitions in Mixtures of Cationic Cholesteryl Lipid Analogues and Dipalmitoyl Phosphatidylcholine Membranes

Two types of cationic cholesteryl amphiphiles, one where the headgroup is attached to the steroid by an ester linkage and the second by an ether linkage, were synthesized. A third type of cholesteryl lipid bearing an oligoethylene glycol segment was also prepared. Each of these synthetic lipids generated vesicle-like aggregates with closed inner aqueous compartments from their aqueous suspensions. We examined their interaction with L-α-dipalmitoyl phosphatidylcholine (DPPC) membranes using fluorescence anisotropy, transmission electron microscopy (TEM), and differential scanning calorimetry (DSC). When included in membranes, the synthetic cholesteryl lipids were found to quench the chain motion of the acyl chains of DPPC. This suggests that these cationic cholesteryl derivatives act as filler molecules despite modification at the headgroup level from the molecular structure of natural cholesterol. Careful analyses of DSC and fluorescence anisotropy data suggest that the nature of perturbation induced by each of these cationic cholesterol derivatives is dependent on the details of their molecular structure and provides significant information on the nature of interaction of these derivatives with phospholipid molecules. In general, amphiphiles that support structured water at the interfacial region tend to rigidify the fluid phase more than others. Importantly, these cholesteryl amphiphiles behave less like cholesterol in that their incorporation in DPPC not only abolishes the phase transition but also depresses the phase transition temperature.