Preventive Medicine and Public Health: Using Health Risk Appraisal in Clinical Practice

The Scientific Board of the California Medical Association presents the following inventory of items of progress in preventive medicine and public health. Each item, in the judgment of a panel of knowledgeable physicians, has recently become reasonably firmly established, both as to scientific fact and important clinical significance. The items are presented in simple epitome, and an authoritative reference, both to the item itself and to the subject as a whole, is generally given for those who may be unfamiliar with a particular item. The purpose is to assist busy practitioners, students, researchers, or scholars to stay abreast of these items of progress in preventive medicine and public health that have recently achieved a substantial degree of authoritative acceptance, whether in their own field of special interest or another.The items of progress listed below were selected by the Advisory Panel to the Section on Preventive Medicine and Public Health of the California Medical Association, and the summaries were prepared under its direction.     

Analysis of coiled-coil interactions between core proteins of the spindle pole body

The spindle pole body (SPB) is a multiprotein complex that organizes microtubules in yeast. Due to its large size and association with the nuclear membrane, little is known about its detailed structure. In particular, although many SPB components and some of the interactions between them have been identified, the molecular details of how most of these interactions occur are not known. The prevalence of predicted coiled-coil regions in SPB proteins suggests that some interactions may occur via coiled coils. Here this hypothesis is supported by biochemical characterization of isolated coiled-coil peptides derived from SPB proteins. Formation of four strongly self-associating coiled-coil complexes from Spc29, Spc42, and Spc72 was demonstrated by circular dichroism (CD) spectroscopy and a fluorescence resonance energy transfer (FRET) assay. Many weaker self- and heteroassociations were also detected by CD, FRET, and/or cross-linking. The thermal stabilities of nine candidate homooligomers were assessed; six unfolded cooperatively with melting temperatures ranging from <11 to >50 degrees C. Solution studies established that coiled-coil peptides derived from Spc42 and Spc72 form parallel dimers, and this was confirmed for Spc42 by a high-resolution crystal structure. These data contribute to a growing body of knowledge that will ultimately provide a detailed model of the SPB structure.     

Structure and mode of action of a mosquitocidal holotoxin

The crystal structure of the full mosquitocidal toxin from Bacillus sphaericus (MTX_holo) has been determined at 2.5 Å resolution by the molecular replacement method. The resulting structure revealed essentially the complete chain consisting of four ricin B-type domains curling around the catalytic domain in a hedgehog-like assembly. As the structure was virtually identical in three different crystal packings, it is probably not affected by packing contacts. The structure of MTX_holo explains earlier autoinhibition data. An analysis of published complexes comprising ricin B-type lectin domains and sugar molecules shows that the general construction principle applies to all four lectin domains of MTX_holo, indicating 12 putative sugar-binding sites. These sites are sequence-related to those of the cytotoxin pierisin from cabbage butterfly, which are known to bind glycolipids. It seems therefore likely that MTX_holo also binds glycolipids. The seven contact interfaces between the five domains are predominantly polar and not stronger than common crystal contacts so that in an appropriate environment, the multidomain structure would likely uncurl into a string of single domains. The structure of the isolated catalytic domain plus an extended linker was established earlier in three crystal packings, two of which showed a peculiar association around a 7-fold axis. The catalytic domain of the reported MTX_holo closely resembles all three published structures, except one with an appreciable deviation of the 40 N-terminal residues. A comparison of all structures suggests a possible scenario for the translocation of the toxin into the cytosol.     

Structure of 2,6-dihydroxypyridine 3-hydroxylase from a nicotine-degrading pathway

The enzyme 2,6-dihydroxypyridine-3-hydroxylase catalyzes the sixth step of the nicotine degradation pathway in Arthrobacter nicotinovorans. The enzyme was produced in Escherichia coli, purified and crystallized. The crystal structure was solved at 2.6 Å resolution, revealing a significant structural relationship with the family of FAD-dependent aromatic hydroxylases, but essentially no sequence homology. The structure was aligned with those of the established family members, showing that the FAD molecules are bound at virtually identical locations. The reported enzyme is a dimer like most other family members, but its dimerization contact differs from the others. The binding position of NAD(P)H to this enzyme family is not clear. Since the reported enzyme accepts only NADH for flavin reduction in contrast to the other established members using NADPH, we searched through the structural alignment and found an indication for the position of the 2′-phosphate of NADPH that is in general agreement with mutational studies on a related enzyme, but contradicts a crystal soaking experiment. Using a bound glycerol molecule and the known substrate positions of three related enzymes as a guide, the substrate 2,6-dihydroxypyridine was placed into the active center. The access to the binding site is discussed. The new active center geometry introduces constraints that render some reaction scenarios more likely than others. It suggests that flavin is reduced at its out-position and then drawn into its in-position, where it binds molecular oxygen. The geometry is consistent with the proposal that peroxy-flavin is protonated by the solvent to yield the electrophilic hydroperoxy-flavin. The substrate is activated by two buried histidines but there is no appropriate base to store the surplus proton of the hydroxylated carbon atom. The implications of this problem are discussed.     

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model

Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.     

Programmed drug-dependent ribosome stalling

The ribosome has the intrinsic capacity to monitor the sequence and structure of the nascent peptide. This fundamental property of the ribosome is often exploited in regulation of gene expression, in particular, for activation of expression of genes conferring resistance to ribosome-targeting antibiotics. Induction of expression of these genes is controlled by the programmed stalling of the ribosome at a regulatory open reading frame located upstream of the resistance cistron. Formation of the stalled translation complex depends on the presence of an antibiotic in the ribosome exit tunnel and the sequence of the nascent peptide. In this review, we summarize our current understanding of the molecular mechanisms of drug- and nascent peptide-dependent ribosome stalling.     

Association of Body Mass and Brain Activation during Gastric Distention: Implications for Obesity

Background

Gastric distention (GD), as it occurs during meal ingestion, signals a full stomach and it is one of the key mechanisms controlling food intake. Previous studies on GD showed lower activation of the amygdala for subjects with higher body mass index (BMI). Since obese subjects have dopaminergic deficits that correlate negatively with BMI and the amygdala is innervated by dopamine neurons, we hypothesized that BMI would correlate negatively with activation not just in the amygdala but also in other dopaminergic brain regions (midbrain and hypothalamus).

Methodology/Principal Findings

We used functional magnetic resonance imaging (fMRI) to evaluate brain activation during GD in 24 healthy subjects with BMI range of 20–39 kg/m². Using multiple regression and cross-correlation analyses based on a family-wise error corrected threshold P = 0.05, we show that during slow GD to maximum volumes of 500 ml and 700 ml subjects with increased BMI had increased activation in cerebellum and left posterior insula, and decreased activation of dopaminergic (amygdala, midbrain, hypothalamus, thalamus) and serotonergic (pons) brain regions and anterior insula, regions that were functionally interconnected with one another.

Conclusions

The negative correlation between BMI and BOLD responses to gastric distention in dopaminergic (midbrain, hypothalamus, amygdala, thalamus) and serotonergic (pons) brain regions is consistent with disruption of dopaminergic and serotonergic signaling in obesity. In contrast the positive correlation between BMI and BOLD responses in posterior insula and cerebellum suggests an opposing mechanism that promotes food intake in obese subjects that may underlie their ability to consume at once large food volumes despite increasing gastric distention.