Green fluorescent protein functions as a reporter for protein localization in Escherichia coli

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.     

Use of lipid lowering drugs for primary prevention of coronary heart disease: meta-analysis of randomised trials

ObjectiveTo summarise the effect of primary prevention with lipid lowering drugs on coronary heart disease events, coronary heart disease mortality, and all cause mortality.DesignMeta-analysis.IdentificationSystematic search of the Medline database from January 1994 to June 1999 for English language studies examining drug treatment for lipid disorders (use of the MeSH terms “hyperlipidemia” and “anticholesteremic agents,” keyword searches for individual drug names, and a search strategy for identifying randomised trials to capture relevant articles); identification of older studies through systematic reviews and hand search of bibliographies.ResultsFour studies met eligibility criteria. Drug treatment reduced the odds of a coronary heart disease event by 30% (summary odds ratio 0.70, 95% confidence interval 0.62 to 0.79) but not the odds of all cause mortality (0.94, 0.81 to 1.09). When statin drugs were considered alone, no substantial differences in results were found.ConclusionsTreatment with lipid lowering drugs lasting five to seven years reduces coronary heart disease events but not all cause mortality in people with no known cardiovascular disease.     

Functional requirement for SAP in 2B4-mediated activation of human natural killer cells as revealed by the X-linked lymphoproliferative syndrome

X-linked lymphoproliferative syndrome (XLP) is an immunodeficiency characterized by life-threatening infectious mononucleosis and EBV-induced B cell lymphoma. The gene mutated in XLP encodes SLAM (signaling lymphocytic activation molecule-associated protein)-associated protein (SAP), a small SH2 domain-containing protein. SAP associates with 2B4 and SLAM, activating receptors expressed by NK and T cells, and prevents recruitment of SH2 domain-containing protein tyrosine phosphatase-2 SHP-2) to the cytoplasmic domains of these receptors. The phenotype of XLP may therefore result from perturbed signaling through SAP-associating receptors. We have addressed the functional consequence of SAP deficiency on 2B4-mediated NK cell activation. Ligating 2B4 on normal human NK cells with anti-2B4 mAb or interaction with transfectants bearing the 2B4 ligand CD48 induced NK cell cytotoxicity. In contrast, ligation of 2B4 on NK cells from a SAP-deficient XLP patient failed to initiate cytotoxicity. Despite this, CD2 or CD16-induced cytotoxicity of SAP-deficient NK cells was similar to that of normal NK cells. Thus, selective impairment of 2B4-mediated NK cell activation may contribute to the immunopathology of XLP.     

Overwintering adaptations in the lettuce root aphid Pemphigus bursarius (L.)

Pemphigus bursarius (L.) is a host alternating root-feeding aphid with a proportion of the population overwintering as asexual hiemalis in the soil. These hiemalis must be sufficiently cold tolerant to survive at the temperatures they would experience in winter, and also be able to overcome a period of prolonged starvation brought about by the absence of secondary host plants. Cold tolerance experiments showed field collected hiemalis to be considerably more cold hardy than laboratory summer apterae, with an LTemp(50) of -13.1 degrees C compared with 2.3 degrees C. In a constant exposure at 0 degrees C some field collected hiemalis survived for 18 days, while no summer apterae survived more than 8 h. Hiemalis, collected from the field in winter and induced in the laboratory, had significantly higher levels of triglycerides, 12.8% fresh weight (39.9% dry wt.) and 11.4% fresh weight (43.7% dry wt.), respectively, compared with summer apterae with a value of 7.1% fresh weight (32.5% dry wt.). These two adaptations of increased cold tolerance and accumulation of energy reserves confirm that the hiemalis morph is adapted for overwintering and hence physiologically distinct from summer morphs, and in turn, contribute to the success of the asexual life cycle strategy in this species.     

Variation in cuticular hydrocarbons among strains of the Anopheles gambiae sensu stricto by analysis of cuticular hydrocarbons using gas liquid chromatography of larvae

Cuticular hydrocarbons of larvae of individual strains of the Anopheles gambiae sensu stricto were investigated using gas liquid chromatography. Biomedical discriminant analysis involving multivariate statistics suggests that there was clear hydrocarbon difference between the Gambian(G3), the Nigerian (16CSS and, its malathion resistant substrain, REFMA) and the Tanzanian (KWA) strains. The high degree of segregation (95%) in hydrocarbons among the four strains investigated indicates that further analysis is needed to enable understanding of hydrocarbon variation in samples of An. gambiae especially from areas where these populations co-exist.     

Postnatal development of northern fulmar chicks, Fulmarus glacialis

The slow growth and large fat stores characteristic of many pelagic seabird chicks were generally assumed to reflect infrequent and unpredictable food provisioning by parents. Much less attention has been focused on the importance of intrinsic physiological processes in shaping patterns of development. In this study, we examined postnatal growth and changes in water content of different organs in fulmar chicks, Fulmarus glacialis, from Fair Isle, United Kingdom. After correcting for body size, mass growth rate was as high as in inshore-feeding species, which did not support the notion of an external constraint on growth imposed by the unpredictability of pelagic prey. Pectoral muscles and plumage grew more rapidly than other tissues. Pectorals also had a high water index, probably indicating slower maturation compared with leg muscles, which need to generate heat earlier on to free adults from brooding requirements. Lean dry mass of liver, kidney, and gut decreased markedly toward fledging, presumably because of high energetic costs of maintaining large metabolic machinery in older chicks and analogous to the situation in adult waders before migration. These results suggest that the general pattern of development of fulmars may be linked to changes in resource allocation as chicks grow and possibly a compromise at the tissue level between cell division and the attainment of mature function.     

The isolated hepatocyte preparation: 30 years on

A method for the preparation of intact rat hepatocytes in high yield was first described in 1969. The procedure involved digestion of hepatic tissue by perfusion of the liver with crude collagenase; later, purified collagenase without other enzymic additions was shown to be ineffective. Recently it has been discovered that the combination of purified collagenase plus elastase is superior to crude collagenase in that it consistently provides high yields of undamaged hepatocytes. The isolated hepatocyte preparation has proved particularly useful for the study of mechanisms responsible for long-range interactions within the cell. These can be studied over prolonged time courses and in the presence of graded concentrations of specific inhibitors. Studies of this kind have demonstrated a close relationship between cytoplasmic metabolic flows and mitochondrial forces and have also revealed that the cytoplasmic and mitochondrial free NAD-linked redox potentials are maintained by energy-dependent reactions.     

Increases in leptin levels, sympathetic drive, and weight gain in obstructive sleep apnea

Patients with obstructive sleep apnea (OSA) are frequently obese and are predisposed to weight gain. They also have heightened sympathetic drive. We reasoned that noradrenergic activation of beta(3)-receptors on adipocytes would inhibit leptin production, predisposing to obesity in sleep apnea. We therefore tested the hypothesis that obesity and predisposition to weight gain in OSA are associated with low levels of plasma leptin. We prospectively studied 32 male patients (43 +/- 2 yr) with OSA who were newly diagnosed and never treated and who were free of any other diseases. Control measurements were obtained from 32 similarly obese closely matched male subjects (38 +/- 2 yr). Leptin levels were 13.7 +/- 1.3 and 9.2 +/- 1.2 ng/ml in patients with OSA and controls, respectively (P = 0.02). Weight gain over the year before diagnosis was 5.2 +/- 1.7 and 0.5 +/- 0.9 kg in sleep apnea patients and similarly obese control subjects, respectively (P = 0.04). Muscle sympathetic activity was 46 +/- 4 and 30 +/- 4 bursts/min in patients with OSA (n = 16) and control subjects (n = 18), respectively (P = 0.01). Plasma leptin levels are elevated in newly diagnosed otherwise healthy patients with untreated sleep apnea beyond the levels seen in similarly obese control subjects without sleep apnea. Higher leptin levels in OSA, independent of body fat content, suggest that OSA is associated with resistance to the weight-reducing effects of leptin.     

Effects of lipid interaction on the lysine microenvironments in apolipoprotein E

Lysines in apolipoprotein (apo) E are key factors in the binding of apoE to the low density lipoprotein receptor, and high affinity binding requires that apoE be associated with lipid. To gain insight into this effect, we examined the microenvironments of the eight lysines in the 22-kDa fragment of apoE3 (residues 1-191) in the lipid-free and lipid-associated states. As shown by (1)H,(13)C heteronuclear single quantum coherence nuclear magnetic resonance, lysine resonances in the lipid-free fragment were poorly resolved over a wide pH range, whereas in apoE3.dimyristoyl phosphatidylcholine (DMPC) discs, the lysine microenvironments and protein conformation were significantly altered. Sequence-specific assignments of the lysine resonances in the spectrum of the lipidated 22-kDa fragment were made. In the lipid-free protein, six lysines could be resolved, and all had pK(a) values above 10. In apoE3.DMPC complexes, however, all eight lysines were resolved, and the pK(a) values were 9.2-11.1. Lys-143 and Lys-146, both in the receptor binding region in helix 4, had unusually low pK(a) values of 9.5 and 9.2, respectively, likely as a result of local increases in positive electrostatic potential with lipid association. Shift reagent experiments with potassium ferricyanide showed that Lys-143 and Lys-146 were much more accessible to the ferricyanide anion in the apoE3.DMPC complex than in the lipid-free state. The angle of the nonpolar face of helix 4 is smaller than the angles of helices 1, 2, and 3, suggesting that helix 4 cannot penetrate as deeply into the DMPC acyl chains at the edge of the complex and that its polar face protrudes from the edge of the disc. This increased exposure and the greater positive electrostatic potential created by interaction with DMPC may explain why lipid association is required for high affinity binding of apoE to the low density lipoprotein receptor.