Alpha 2-macroglobulin and proliferative retinopathy in type 1 diabetes

Serum alpha 2-macroglobulin (alpha 2m) and total glycosylated haemoglobin (HbA1) concentrations were measured in 110 insulin dependent Type 1 diabetics with minimal or no fundoscopic retinopathy, referred to as non-retinopaths, and in 52 proliferative retinopaths. Proteinuria was recorded in 8 (7%) non-retinopaths and 29 (56%) retinopaths and was accompanied by elevated alpha 2m concentrations in both groups of diabetics but only significantly so in the non-retinopaths. Diabetics without proteinuria showed a significant correlation between alpha 2m concentration and duration of diabetes, HbA1 and age (being higher at extremes of age). Alpha 2m concentrations were significantly higher in retinopaths than in non-retinopaths without proteinuria when allowance was made for the influence of age and duration of diabetes on alpha 2m. This difference may be attributed to the higher HbA, levels found in retinopaths than in non-retinopaths and was no longer evident when account was taken of the prevailing HbA1 concentration in individual patients.     

An analysis of an ordinary differential equation model for a two-species predator-prey system with harvesting and stocking

An analysis is presented for a model of a two-species predator-prey system where each species can be harvested or stocked. Using methods from bifurcation theory the qualitative nature of the steady-state solutions is examined. The effect of harvesting and stocking rates and the prey carrying capacity is examined in detail.     

The Development of Diet-Induced Obesity and Glucose Intolerance in C57Bl/6 Mice on a High-Fat Diet Consists of Distinct Phases

High–fat (HF) diet-induced obesity and insulin insensitivity are associated with inflammation, particularly in white adipose tissue (WAT). However, insulin insensitivity is apparent within days of HF feeding when gains in adiposity and changes in markers of inflammation are relatively minor. To investigate further the effects of HF diet, C57Bl/6J mice were fed either a low (LF) or HF diet for 3 days to 16 weeks, or fed the HF-diet matched to the caloric intake of the LF diet (PF) for 3 days or 1 week, with the time course of glucose tolerance and inflammatory gene expression measured in liver, muscle and WAT. HF fed mice gained adiposity and liver lipid steadily over 16 weeks, but developed glucose intolerance, assessed by intraperitoneal glucose tolerance tests (IPGTT), in two phases. The first phase, after 3 days, resulted in a 50% increase in area under the curve (AUC) for HF and PF mice, which improved to 30% after 1 week and remained stable until 12 weeks. Between 12 and 16 weeks the difference in AUC increased to 60%, when gene markers of inflammation appeared in WAT and muscle but not in liver. Plasma proteomics were used to reveal an acute phase response at day 3. Data from PF mice reveals that glucose intolerance and the acute phase response are the result of the HF composition of the diet and increased caloric intake respectively. Thus, the initial increase in glucose intolerance due to a HF diet occurs concurrently with an acute phase response but these effects are caused by different properties of the diet. The second increase in glucose intolerance occurs between 12 - 16 weeks of HF diet and is correlated with WAT and muscle inflammation. Between these times glucose tolerance remains stable and markers of inflammation are undetectable.     

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.     

Synthesis and assembly of the cytochrome b-f complex in higher plants

The cytochrome b-f complex is composed of four polypeptide subunits, three of which, cytochrome f, cytochrome b-563 and subunit IV, are encoded in chloroplast DNA and synthesised within the chloroplast, and the fourth, the Rieske FeS protein, is encoded in nuclear DNA and synthesised in the cytoplasm. The assembly of the cytochrome b-f complex therefore requires the interaction of subunits encoded by different genomes. A key role for the nuclear-encoded Rieske FeS protein in the assembly of the complex is suggested by a study of cytochrome b-f complex mutants. The assembly of individual subunits of the complex may be regulated by the availability of prosthetic groups. The genes for the chloroplast-encoded subunits and cDNA clones for the Rieske FeS protein have been isolated and characterised. Cytochrome f and the Rieske FeS protein are synthesised initially with N-terminal presequences required for their correct assembly within the chloroplast. The deduced amino acid sequences of the four subunits have been used to suggest models for the arrangement of the polypeptides in the thylakoid membrane.     

Structural studies of mutants of the lysozyme of bacteriophage T4. The temperature-sensitive mutant protein Thr157----Ile

To understand the roles of individual amino acids in the folding and stability of globular proteins, a systematic structural analysis of mutants of the lysozyme of bacteriophage T4 has been undertaken. The isolation, characterization, crystallographic refinement and structural analysis of a temperature-sensitive lysozyme in which threonine 157 is replaced by isoleucine is reported here. This mutation reduces the temperature of the midpoint of the reversible thermal denaturation transition by 11 deg.C at pH 2.0. Electron density maps showing differences between the wild-type and mutant X-ray crystal structures have obvious features corresponding to the substitution of threonine 157 by isoleucine. There is little difference electron density in the remainder of the molecule, indicating that the structural changes are localized to the site of the mutation. High-resolution crystallographic refinement of the mutant lysozyme structure confirms that it is very similar to wild-type lysozyme. The largest conformational differences are in the gamma-carbon of residue 157 and in the side-chain of Asp159, which shift 1.0 A and 1.1 A, respectively. In the wild-type enzyme, the gamma-hydroxyl group of Thr157 participates in a network of hydrogen bonds. Substitution of Thr157 with an isoleucine disrupts this set of hydrogen bonds. A water molecule bound in the vicinity of Thr155 partially restores the hydrogen bond network in the mutant structure, but the buried main-chain amide of Asp159 is not near a hydrogen bond acceptor. This unsatisfied hydrogen-bonding potential is the most obvious reason for the reduction in stability of the temperature-sensitive mutant protein.     

Genetic Studies of Recombining DNA in Pneumococcal Transformation

The results of genetic fine structure experiments, performed on the amiA locus of Pneumococcus are summarized. The peculiar feature of transformation genetics is that a given donor marker mutation transforms with an efficiency characteristic of the mutated site. In spite of this difficulty, mapping procedures have been devised and quantitative recombination studies performed. It is concluded from these studies that transformation, in this locus, is the consequence of frequent, and essentially random exchanges occurring between donor DNA and the chromosomal DNA of the recipient cell. The average length of uninterrupted donor DNA polynucleotide strand which could be inserted into the chromosome of a transformed cell is estimated, from genetic data, to be probably not greater than 3·10⁵ daltons (for a double-stranded insertion). It is proposed, on the basis of genetic evidence, that following essentially random exchanges between donor DNA and recipient chromosome, a revision process, specific for certain types of mutated sites, occurs. The revision process appears to remove preferentially donor DNA sequences from the primary recombinant structure, and allow repair along the chromosomal template, leading to low efficiency in the genetic integration of these sites. A mechanism for this "destruction-choice" process is presented, and evidence in support of this mechanism discussed.