Solution structures of antimalarial drug-heme complexes

Paramagnetic metal centers [such as Fe(III) found within ferriprotoporphyrin IX heme (FPIX)] exert through space effects on the relaxation rate of nearby proton spins that depend critically on the metal-proton distance. We have measured these effects for all protons of several antimalarial drugs that bind to FPIX by systematically varying the drug:heme molar ratio in high field NMR experiments. These measurements allow us to determine precise FPIX Fe-drug H distances for the solution structures of noncovalent complexes formed between FPIX mu-oxo dimers and the antimalarial drugs chloroquine (CQ), quinine (QN), and quinidine (QD). Using these distances, we then performed distance restraint calculations to determine the lowest-energy solution structures of these complexes. Structures were solved for neutral, monoprotic (+1), and diprotic (+2) forms of the drugs. Analysis of these structures allows us to visualize for the first time the stereospecific differences between QN and QD binding to FPIX and the differences in populations of QN and QD solution structures upon changes in digestive vacuolar pH for drug resistant malarial parasites [Dzekunov, S. M., et al. (2000) Mol. Biochem. Parasitol. 110, 107-124]. The data indicate a previously unrecognized key role for the CQ aliphatic chain in stabilizing FPIX-CQ complexes, and suggest how lengthening or shortening the chain might perturb stability. We also define FPIX:drug stoichiometries of 2:1 for the complexes formed at physiological FPIX concentrations, in contrast to the 4:1 and 5:1 stoichiometries previously determined at higher FPIX concentrations [Dorn, A., et al. (1998) Biochem. Pharmacol. 55, 727-736]. These atomic resolution antimalarial drug-heme structures should help elucidate how these drugs inhibit formation of hemozoin during metabolism of heme within the malarial parasite Plasmodium falciparum and assist ongoing development of strategies for circumventing antimalarial drug resistance.     

In vivo evidence for interferon-gamma-mediated homeostatic mechanisms in small intestine of the NHE3 Na+/H+ exchanger knockout model of congenital diarrhea

Mice lacking NHE3, the major absorptive Na(+)/H(+) exchanger in the intestine, are the only animal model of congenital diarrhea. To identify molecular changes underlying compensatory mechanisms activated in chronic diarrheas, cDNA microarrays and Northern blot analyses were used to compare global mRNA expression patterns in small intestine of NHE3-deficient and wild-type mice. Among the genes identified were members of the RegIII family of growth factors, which may contribute to the increased absorptive area, and a large number of interferon-gamma-responsive genes. The latter finding is of particular interest, since interferon-gamma has been shown to regulate ion transporter activities in intestinal epithelial cells. Serum interferon-gamma was elevated 5-fold in NHE3-deficient mice; however, there was no evidence of inflammation, and unlike conditions such as inflammatory bowel disease, levels of other cytokines were unchanged. In addition, quantitative PCR analysis showed that up-regulation of interferon-gamma mRNA was localized to the small intestine and did not occur in the colon, spleen, or kidney. These in vivo data suggest that elevated interferon-gamma, produced by gut-associated lymphoid tissue in the small intestine, is part of a homeostatic mechanism that is activated in response to the intestinal absorptive defect in order to regulate the fluidity of the intestinal tract.     

Selective interleukin-12 synthesis defect in 12/15-lipoxygenase-deficient macrophages associated with reduced atherosclerosis in a mouse model of familial hypercholesterolemia

Targeted gene disruption or overexpression of 12/15-lipoxygenase in mice on the genetic background of apolipoprotein E or low density lipoprotein-receptor (LDL-R) deficiency has implicated 12/15-lipoxygenase in atherogenesis. The data support indirectly a role for 12/15-lipoxygenase in the oxidative modification of low density lipoprotein. In this study we set out to explore other potential mechanisms for 12/15-lipoxygenase in atherosclerosis using apolipoprotein B mRNA editing catalytic polypeptide-1/LDL-R double-deficient mice, a model highly related to the human condition of familial hypercholesterolemia. 12/15-Lipoxygenase deficiency in this strain led to approximately 50% decrease in aortic lesions in male and female mice at 8 months on a chow diet in the absence of cholesterol differences. While studying 12/15-lipoxygenase-deficient macrophages in culture, we discovered a remarkable selective defect (75-90% decrease) in interleukin-12 production but not in tumor necrosis factor-alpha or nitric oxide release, in response to lipopolysaccharide in the presence or absence of interferon-gamma priming. The lipopolysaccharide/interferon-gamma response was associated with a 33-50% decrease in nuclear interferon consensus sequence-binding protein, which is consistent with interferon consensus sequence-binding protein containing protein complex-dependent regulation of the interleukin-12 p40 gene. The decrease in interleukin-12 production was recapitulated in vivo in mouse aortas of the triple knockout group and was reflected in a marked decrease in interferon-gamma expression. The data provide support for a novel mechanism linking the 12/15-lipoxygenase pathway to a known immunomodulatory Th1 cytokine in atherogenesis.     

Genomic evidence for the absence of a functional cholesteryl ester transfer protein gene in mice and rats

Mice and rats are naturally deficient in cholesteryl ester transfer protein (CETP) activity, although the reason behind the deficiency in activity is unknown. A search of mouse genome databases revealed sequences resembling 7 of the 16 human exons. However, these sequences could not code for a functional CETP. Analysis of the rat genome using Southern blotting revealed sequences complementary to human CETP cDNA, but RNase protection assays were unable to detect any Cetp gene expression in liver, adipose, or muscle. A search of rat whole-genome shotgun databases revealed exon-like sequences that would be unable to code for a functional CETP. An Ap3s1 pseudogene lay immediately upstream of the CETP-like sequences in mouse, but was nearly identical to the functional gene and unlikely to have been inserted prior to mouse-rat divergence. In contrast, a deletion leading to a nonsense codon was found in the exon 11-like sequences of both rat and mouse and not in any other species. Thus, the lack of CETP activity in both the mouse and the rat is most likely due to an evolutionary event that occurred before these species diverged and not to altered regulation of the gene or function of the gene product.     

Characterization of the targeting signal of the Arabidopsis 22-kD integral peroxisomal membrane protein

Using a combination of in vivo and in vitro assays, we characterized the sorting pathway and molecular targeting signal for the Arabidopsis 22-kD peroxisome membrane protein (PMP22), an integral component of the membrane of all peroxisomes in the mature plant. We show that nascent PMP22 is sorted directly from the cytosol to peroxisomes and that it is inserted into the peroxisomal boundary membrane with its N- and C-termini facing the cytosol. This direct sorting of PMP22 to peroxisomes contrasts with the indirect sorting reported previously for cottonseed (Gossypium hirsutum) ascorbate peroxidase, an integral PMP that sorts to peroxisomes via a subdomain of the endoplasmic reticulum. Thus, at least two different sorting pathways for PMPs exist in plant cells. At least four distinct regions within the N-terminal one-half of PMP22, including a positively charged domain present in most peroxisomal integral membrane-destined proteins, functions in a cooperative manner in efficient peroxisomal targeting and insertion. In addition, targeting with high fidelity to peroxisomes requires all four membrane-spanning domains in PMP22. Together, these results illustrate that the PMP22 membrane peroxisomal targeting signal is complex and that different elements within the signal may be responsible for mediating unique aspects of PMP22 biogenesis, including maintaining the solubility before membrane insertion, targeting to peroxisomes, and ensuring proper assembly in the peroxisomal boundary membrane.     

Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.     

Visualizing genetic influences on human brain functions

, in this issue of Cell, integrate genetics and functional brain imaging by showing that variation in the human brain-derived neurotrophic factor (BDNF) gene is associated with variation in episodic memory ability and in hippocampal neurochemistry and function.     

Dynamics of bacterial and fungal communities on decaying salt marsh grass

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated microbial community. Here we describe the bacterial and fungal assemblages associated with two decomposition stages of Spartina alterniflora detritus in a productive southeastern U.S. salt marsh. 16S rRNA genes and 18S-to-28S internal transcribed spacer (ITS) regions were used to target the bacterial and ascomycete fungal communities, respectively, based on DNA sequence analysis of isolates and environmental clones and by using community fingerprinting based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Seven major bacterial taxa (six affiliated with the alpha-Proteobacteria and one with the Cytophagales) and four major fungal taxa were identified over five sample dates spanning 13 months. Fungal terminal restriction fragments (T-RFs) were informative at the species level; however, bacterial T-RFs frequently comprised a number of related genera. Amplicon abundances indicated that the salt marsh saprophyte communities have little-to-moderate variability spatially or with decomposition stage, but considerable variability temporally. However, the temporal variability could not be readily explained by either successional shifts or simple relationships with environmental factors. Significant correlations in abundance (both positive and negative) were found among dominant fungal and bacterial taxa that possibly indicate ecological interactions between decomposer organisms. Most associations involved one of four microbial taxa: two groups of bacteria affiliated with the alpha-Proteobacteria and two ascomycete fungi (Phaeosphaeria spartinicola and environmental isolate "4clt").     

Population Reduction and Social Disorganization in Alouatta pigra Following a Hurricane

The opportunity to study the effects of a powerful hurricane on monkey populations, diet, and behavior via pre- and post-hurricane data was presented when hurricane Iris virtually destroyed the forest along Monkey River in southern Belize on October 8, 2001, including a 52-ha area where black howlers have been subjects since 1999. Before the hurricane, 8 social groups, averaging 6.37 members, had been stable in both group composition and range for q 2 years. The hurricane, which levelled much of the forest, resulted in the complete loss of the forest canopy. The trees that remained standing lost most or all branches and were 100% defoliated. The monkey population in the study area was reduced by 42% and survivers experienced a period of extended social disorganization involving transient individuals, high numbers of solitary monkeys, and small fragmentary social groups. The period of disorganization lasted 12 weeks, after which the number of solitaries reduced and stability of the large groups increased. Within the study area, 5 social groups have been more or less stable since ca. week 15; however, home ranges had yet to stabilize at week 35. The social and ranging effects are similar to what has been described for translocated primates. Post-hurricane diet was limited to fruit and leaves remaining in the deadfall for the first 2 weeks and to new leaves and leaf buds for many weeks after that. Normal fruit consumption in April and May was prevented by the failure of surviving trees to produce fruit. With the loss of forest canopy there has been increased use of low foliage and ground travel, and with the reduction in population density there has been a reduction in vocalization frequency.