Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-[Pt(NH3)2(d(GpG]], built into a specific site in a viral genome

A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-[Pt(NH3)2(d(GpG]] intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II). The duplex dodecamer d(AGAAGGCCTAGA).d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site. A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant. The dodecamer d(TCTAGGCCTTCT) was synthesized by the solid-phase phosphotriester method and platinated by reaction with cis-[Pt(NH3)2(H2O)2]2+ (yield 39%). Characterization by pH-dependent 1H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines. The platinated oligonucleotide was phosphorylated in the presence of [gamma-32P]ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome. Approximately 80% of the gapped duplexes incorporated a dodecanucleotide in the ligation reaction. Of these, approximately half did so with the dodecanucleotide covalently joined to the genome at both 5' and 3' termini. The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII. The cis-[Pt(NH3)2(d(GpG]] cross-link completely inhibited StuI cleavage, which was fully restored following incubation of the platinated genome with cyanide to remove platinum as [Pt(CN)4]2-. Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-[Pt(NH3)2(d(GpG]] cross-link induces localized weakening of the DNA double helix. In addition, double- and single-stranded genomes, in which the cis-[Pt(NH3)2(d(GpG]] cross-link resides specifically in the plus strand, were constructed. Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes. In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-[Pt(NH3)2(d(GpG]] cross-link is lethal to the single-stranded bacteriophage.     

Cytokine Balance in Human Malaria: Does Plasmodium vivax Elicit More Inflammatory Responses than Plasmodium falciparum?

Background

The mechanisms by which humans regulate pro- and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum.

Methodology/Principal Findings

We measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF)-α receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n = 85), P. falciparum (n = 30), or both species (n = 12), and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL)-10, which correlated positively with parasite density, and elevated IL-10/TNF-α, IL-10/interferon (IFN)-γ, IL-10/IL-6 and sTNFRII/TNF-α ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-α receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species.

Conclusions

Controlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction of regulatory cytokines may be a critical mechanism protecting vivax malaria patients from severe clinical complications.     

Daily rhythms of leukocytes populations,biochemical and enzymatic blood parameters in a carnivorous freshwater catfish (Lophiosilurus alexandri)

The aim of this study was to investigate the daily rhythms of hematological, biochemical and enzymatic parameters of the blood of a nocturnal model of fish (Lophiosilurus alexandri) bred in the laboratory (F1). Thirty-six juveniles were stocked in six tanks of a recirculation aquaculture system for 20 days. The fish were exposed to a light:dark cycle of 12:12 h and were fed 1% of biomass twice a day with commercial diet. The daily rhythms of hematological, biochemical and enzymatic parameters were then measured at six sampling times “zeitgeber time = ZT” at four-hour intervals under light:dark 12:12 h (lights on = ZT0, at 8.00 a.m). No differences were observed to alkaline phosphatase, glucose, cortisol, aspartate aminotransferase, superoxide dismutase, total protein and hematocrit (p > 0.05). However, white blood cell count, Lymphocytes (LYN), Neutrophils (NEU), Eosinophil and Neutrophils to Lymphocytes ratio were significant different between sample times (p < 0.05). Also, a significant difference in alanine transaminase was observed, with a peak of production at nighttime. In contrast, glutathione peroxidase peaked at 8:00. Uric acid, magnesium and Calcium (Ca⁺⁺) showed statistically significant differences (p < 0.05). A significant difference was observed (p < 0.05), with a peak of albumin at 08:00 and triglycerides at 12:00, while cholesterol was low (p < 0.05) at 08:00 and higher from 12:00 to 04:00. Cosinor analysis revealed also rhythmicity to SOD, UA, Mg and Ca⁺⁺, ALB and CHO (p < 0.05). In conclusion, the time of day must be considered a key factor when using blood parameters as biomarkers for disease, health and welfare in the L. alexandri aquaculture.     

Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus

The influenza virus M2 proton-selective ion channel activity facilitates virus uncoating, a process that occurs in the acidic environment of the endosome. The M2 channel causes acidification of the interior of the virus particle, which results in viral protein-protein dissociation. The M2 protein is a homotetramer that contains in its aqueous pore a histidine residue (His-37) that acts as a selectivity filter and a tryptophan residue (Trp-41) that acts as a channel gate. Substitution of His-37 modifies M2 ion channel properties drastically. However, the results of such experiments are difficult to interpret because substitution of His-37 could cause gross structural changes to the channel pore. We described here experiments in which partial or, in some cases, full rescue of specific M2 ion channel properties of His-37 substitution mutants was achieved by addition of imidazole to the bathing medium. Chemical rescue was demonstrated for three histidine substitution mutant ion channels (M2-H37G, M2-H37S, and M2-H37T) and for two double mutants in which the Trp-41 channel gate was also mutated (H37G/W41Y and H37G/W41A). Currents of the M2-H37G mutant ion channel were inhibited by Cu(II), which has been shown to coordinate with His-37 in the wild-type channel. Chemical rescue was very specific for imidazole. Buffer molecules that were neutral when protonated (4-morpholineethanesulfonic acid and 3-morpholino-2-hydroxypropanesulfonic acid) did not rescue ion channel activity of the M2-H37G mutant ion channel, but 1-methylimidazole did provide partial rescue of function. These results were consistent with a model for proton transport through the pore of the wild-type channel in which the imidazole side chain of His-37 acted as an intermediate proton acceptor/donor group.     

Molecular confinement of human amylin in lipidic nanoparticles

Amylin is a pancreatic hormone involved in the regulation of glucose metabolism and homeostasis. Restoration of the post-prandial and basal levels of human amylin in diabetic individuals is a key in controlling glycemia, controlling glucagon, reducing the insulin dose and increasing satiety, among other physiologic functions. Human amylin has a high propensity to aggregate. We have addressed this issue by designing a liposomal human amylin formulation. Nanoparticles of multilamellar liposomes comprising human amylin were obtained with 53% encapsulation efficiency. The in vitro kinetic release assay shows a biphasic profile. The stabilization of the lipidic nanoparticle against freeze-drying was achieved by using mannitol as a cryoprotectant, as evidenced by morphological characterization. The effectiveness of the human amylin entrapped in lipidic nanoparticles was tested by the measurement of its pharmacological effect in vivo after subcutaneous administration in mice. Collectively these results demonstrate the compatibility of human amylin with the lipidic interface as an effective pharmaceutical delivery system.     

Microsite determinants of variability in seedling and cutting establishment in tropical forest restoration plantations

Plantations are frequently established on abandoned pasture lands to speed forest recovery. This strategy requires matching a tree species mix with the prevailing microenvironmental conditions. In four degraded pastures of the Mexican Lacandon rainforest, we planted 2,400 trees of 6 species (Guazuma ulmifolia, Inga vera, Ochroma pyramidale, Trichospermum mexicanum, Bursera simaruba, and Spondias mombin) to (1) test survival, initial growth, and establishment costs; (2) evaluate whether vegetative cuttings outperform direct seeding or transplants of nursery‐raised seedlings; (3) determine tree response to herbaceous dominance and soil compaction; and (4) scrutinize the results' consistency across sites and sampling scales of tree–microenvironment interactions (individual tree vs. averaged plot responses). After 2 years, overall survival and growth rates were high for 2 of 3 nursery‐raised species. Contrary to expectations, all seedlings outperformed the cuttings while direct seeding resulted in a cost‐effective option of intermediate efficacy. The impact of soil resistance to root penetration on tree biomass accumulation was species dependent while bulk density was not relevant. Soil‐covering, herbaceous vegetation accelerated growth in 3 of 4 tested species during the dry season. At this initial stage of forest restoration in abandoned pastures, Guazuma and Trichospermum were the most restoration‐effective species. Costs can be reduced by using direct‐seeding Inga and avoiding weeding during the dry season. Finally, our results demonstrate how species selection trials can be misleading due to site variations in tree response and to sampling scales that fail to account for small‐scale environmental heterogeneity. We recommend ways to improve the design of restoration trials.     

Suppressor analysis of a histone defect identifies a new function for the hda1 complex in chromosome segregation

Histones are essential for the compaction of DNA into chromatin and therefore participate in all chromosomal functions. Specific mutations in HTA1, one of the two Saccharomyces cerevisiae genes encoding histone H2A, have been previously shown to cause chromosome segregation defects, including an increase in ploidy associated with altered pericentromeric chromatin structure, suggesting a role for histone H2A in kinetochore function. To identify proteins that may interact with histone H2A in the control of ploidy and chromosome segregation, we performed a genetic screen for suppressors of the increase-in-ploidy phenotype associated with one of the H2A mutations. We identified five genes, HHT1, MKS1, HDA1, HDA2, and HDA3, four of which encode proteins directly connected to chromatin function: histone H3 and each of the three subunits of the Hda1 histone deacetylase complex. Our results show that Hda3 has functions distinct from Hda2 and Hda1 and that it is required for normal chromosome segregation and cell cycle progression. In addition, HDA3 shows genetic interactions with kinetochore components, emphasizing a role in centromere function, and all three Hda proteins show association with centromeric DNA. These findings suggest that the Hda1 deacetylase complex affects histone function at the centromere and that Hda3 has a distinctive participation in chromosome segregation. Moreover, these suppressors provide the basis for future studies regarding histone function in chromosome segregation.