Interactions of isopenicillin N synthase with cyclopropyl-containing substrate analogues reveal new mechanistic insight

Isopenicillin N synthase (IPNS), a non-heme iron oxidase central to penicillin and cephalosporin biosynthesis, catalyzes an energetically demanding chemical transformation to produce isopenicillin N from the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV). We describe the synthesis of two cyclopropyl-containing tripeptide analogues, delta-(l-alpha-aminoadipoyl)-l-cysteinyl-beta-methyl-d-cyclopropylglycine and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-cyclopropylglycine, designed as probes for the mechanism of IPNS. We have solved the X-ray crystal structures of these substrates in complex with IPNS and propose a revised mechanism for the IPNS-mediated turnover of these compounds. Relative to the previously determined IPNS-Fe(II)-ACV structure, key differences exist in substrate orientation and water occupancy, which allow for an explanation of the differences in reactivity of these substrates.     

Antimicrobial peptide-lipid binding interactions and binding selectivity

Surface pressure measurements, external reflection-Fourier transform infrared spectroscopy, and neutron reflectivity have been used to investigate the lipid-binding behavior of three antimicrobial peptides: melittin, magainin II, and cecropin P1. As expected, all three cationic peptides were shown to interact more strongly with the anionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-(phosphor-rac-(1-glycerol)) (DPPG), compared to the zwitterionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-phosphocholine (DPPC). All three peptides have been shown to penetrate DPPC lipid layers by surface pressure, and this was confirmed for the melittin-DPPC interaction by neutron reflectivity measurements. Adsorption of peptide was, however, minimal, with a maximum of 0.4 mg m(-2) seen for melittin adsorption compared to 2.1 mg m(-2) for adsorption to DPPG (from 0.7 microM solution). The mode of binding to DPPG was shown to depend on the distribution of basic residues within the peptide alpha-helix, although in all cases adsorption below the lipid layer was shown to dominate over insertion within the layer. Melittin adsorption to DPPG altered the lipid layer structure observed through changes in the external reflection-Fourier transform infrared lipid spectra and neutron reflectivity. This lipid disruption was not observed for magainin or cecropin. In addition, melittin binding to both lipids was shown to be 50% greater than for either magainin or cecropin. Adsorption to the bare air-water interface was also investigated and surface activity followed the trend melittin>magainin>cecropin. External reflection-Fourier transform infrared amide spectra revealed that melittin adopted a helical structure only in the presence of lipid, whereas magainin and cecropin adopted helical structure also at an air-water interface. This behavior has been related to the different charge distributions on the peptide amino acid sequences.     

A large genomic island allows Neisseria meningitidis to utilize propionic acid,with implications for colonization of the human nasopharynx

Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid‐generating bacteria. Data from classical microbiological and sequence‐based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp‐containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.     

A model of care for familial hypercholesterolaemia: key role for clinical biochemistry

Familial hypercholesterolaemia (FH) is a dominantly inherited disorder present from birth that causes marked elevation in plasma low-density lipoprotein (LDL) cholesterol concentrations and premature coronary heart disease. There are at least 45,000 people with FH in Australia and New Zealand, but most remain unrecognised and those diagnosed remain inadequately treated. To bridge this gap in coronary prevention the FH Australasia Network has developed a model of care for FH. An executive summary of the model of care is presented, with a commentary on its recommendations and the key role of the clinical biochemistry laboratory.     

A putative role for apelin in the etiology of obesity

Apelin, the endogenous ligand of the G protein-coupled APJ receptor has been shown to promote tumor angiogenesis. However, the effect of apelin on inducing angiogenesis in adipose tissue has not been investigated. In this review, we propose a putative role for apelin in promoting angiogenesis in adipose tissue. We further propose that targeting adipose tissue vasculature by blocking apelin signaling with anti-apelin antibodies will lead not only to inhibition of angiogenesis in adipose tissue but also to decreased adiposity.     

Trophy heads from Nawinpukio, Perú: physical and chemical analysis of Huarpa-era modified human remains

Rescue excavations at the site of Nawinpukio in Perú's Ayacucho Valley exposed a cache of fragmented skulls dating to the Huarpa-era, about AD 400-700. Physical analysis of these remains revealed that they belonged to individuals of both sexes and a range of ages (MNI = 8), and that four crania had been modified through drilling, cutting, and scraping. The occipital and parietal bones of one cranium had been modified to form a shallow basin. Carbon stable isotope analysis of these remains revealed that five individuals had isotopic signatures consistent with maize consumption and one individual exhibited a carbon isotope value indicative of a C(3) plant based diet. Such a nonmaize diet distinguishes this individual from all other prehistoric humans analyzed from the Ayacucho Valley and is consistent with an origin a different ecozone of the valley. On the basis of their physical properties it is argued that these remains represent trophies obtained during raiding. Drawing on the formal properties of the specimens as well as ethnographic and archaeological analogies, it is suggested that the cranial basin served as a vessel for liquid.     

Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes

Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = -0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism.     

Maternal and cord plasma cytokine and chemokine profile in pregnancies complicated by asthma

The mechanisms contributing to worsening of asthma during pregnancy have not been well characterized. Both asthma and pregnancy are conditions associated with a skewing of the immune response from T helper (Th) 1 toward a Th2 response. We hypothesise that worsening of asthma during pregnancy may be due to an enhanced production of circulating proinflammatory cytokines and chemokines and this may be modified by the use of inhaled glucocorticoid treatment. Peripheral blood was collected from asthmatic (n = 35) and control non-asthmatic patients (n = 13) in the third trimester (30–37 weeks) of pregnancy. Fetal blood was collected from the umbilical vein of the placenta after delivery from normal (n = 24) and pregnancies complicated by asthma (n = 24). Plasma samples were assayed for IL-6, -8, eotaxin and RANTES using conventional ELISA. In addition, a range of Th1 and Th2 cytokines measured using Luminex system. There were no significant differences in the levels of maternal IL-6, IL-8, eotaxin and RANTES between asthmatics and nonasthmatics. The results of this study suggest that the presence of asthma does not result in an enhanced circulation of Th2 related cytokines and chemokines during the third trimester of pregnancy. Furthermore peripheral blood cytokine concentrations appear unaffected by inhaled glucocorticoid treatment. Cord plasma eotaxin concentrations were increased in pregnancies complicated by asthma, compared with control. This is the first study to show increased eotaxin production in the feto-placental unit of asthmatic pregnancies and may be one mechanism by which allergy susceptibility is increased in the offspring of asthmatic women.     

A laminar flow model of aerosol survival of epidemic and non-epidemic strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolated from people with cystic fibrosis

Background  

Cystic fibrosis (CF) is an inherited multi-system disorder characterised by chronic airway infection with pathogens such as Pseudomonas aeruginosa.     

Nutritional skewing of conceptus sex in sheep: effects of a maternal diet enriched in rumen-protected polyunsaturated fatty acids (PUFA)

Background  

Evolutionary theory suggests that in polygynous mammalian species females in better body condition should produce more sons than daughters. Few controlled studies have however tested this hypothesis and controversy exists as to whether body condition score or maternal diet is in fact the determining factor of offspring sex. Here, we examined whether maternal diet, specifically increased n-6 polyunsaturated fatty acid (PUFA) intake, of ewes with a constant body condition score around the time of conception influenced sex ratio.