History of science--spores.

Bacterial endospores were first studied 130 years ago by Cohn in 1876 and independently by Koch in the same year. Although spore dormancy and resistance have been much studied since then, questions still remain concerning the basic mechanisms and the kinetics of heat inactivation in particular. Likewise, the extreme dormancy and longevity of spores was recognized early on and later greatly extended but still evade complete understanding. Evidence has accumulated for the involvement of specific spore components such as calcium, dipicolinic acid, small acid soluble proteins in the core and peptidoglycan in the cortex. Involvement of physical factors too, such as the relative dehydration of the core, maybe in a high-viscosity state or even in a glassy state, has added to appreciation of the multicomponent nature of dormancy and resistance. Spore-former morphology formed the basis for early classification systems of sporeformers from about 1880 and consolidated in the mid-1900s, well prior to the use of modern genetic procedures. With respect to sporulation, groundbreaking sequence studies in the 1950s provided the basis for later elucidation of the genetic control widely relevant to many cell differentiation mechanisms. With respect to the breaking of dormancy (activation and germination), the elucidation of mechanisms began in the 1940s following the observations of Hills at Porton who identified specific amino acid and riboside 'germinants', and laid the basis for the later genetic analyses, the identification of germinant receptor genes and the elucidation of key germination reactions. The nonexponential nature of germination kinetics has thwarted the development of practical Tyndallization-like processing. So inactivation by heat remains the premier method of spore control, the basis of a huge worldwide industry, and still relying on the basic kinetics of inactivation of Clostridium botulinum spores, and the reasoning regarding safety first evolved by Bigelow et al. in 1920 and Esty and Meyer in 1922. 'Newer' processes such as treatment with ionizing radiation (first proposed in 1905) and high hydrostatic pressure (first proposed in 1899) may be introduced if consumer resistance and some remaining technical barriers could be overcome.

Membrane traffic in cytokinesis.

A crucial facet of mammalian cell division is the separation of two daughter cells by a process known as cytokinesis. An early event in cytokinesis is the formation of an actomyosis contractile ring, which functions like a purse string in the constriction of the forming furrow between the cells. Far less well characterized are the membrane-trafficking steps which deliver new membrane to the cell surface during the plasma membrane expansion known to accompany furrow formation. It is now clearly established that the plasma membrane at the cleavage furrow of mammalian cells has a distinct lipid and protein composition from the rest of the plasma membrane. This may reflect a requirement for both increased surface area during furrowing and for the co-ordinated delivery of intracellular signalling or membrane re-modelling activities to the correct spatial coordinates during cleavage. In this review, we discuss recent work within the area of membrane traffic and cytokinesis.

Insect renal tubules constitute a cell-autonomous immune system that protects the organism against bacterial infection.

Innate immunity is a widespread and important defence against microbial attack, which in insects is thought to originate mainly in the fat body. Here we demonstrate that the fluid-transporting Malpighian (renal) tubule of Drosophila melanogaster constitutes an autonomous immune-sensing tissue utilising the nitric oxide (NO) signalling pathway. Reverse transcriptase PCR (RT-PCR) shows that tubules express those genes encoding components of the Imd pathway. Furthermore, isolated tubules bind and respond to lipopolysaccharide (LPS), by upregulating anti-microbial peptide (diptericin) gene expression and increased bacterial killing. Excised, LPS-challenged tubules, as well as tubules from LPS-infected flies, display increased NO synthase (NOS) activity upon immune challenge. Targetted expression of a Drosophila NOS (dNOS) transgene to only principal cells of the tubule main segment using the GAL4/UAS system increases diptericin expression. In live flies, such targetted over-expression of dNOS to tubule principal cells confers increased survival of the whole animal upon E. coli challenge. Thus, we describe a novel role of Malpighian tubules in immune sensing and insect survival.

Reduced insulin-stimulated GLUT4 bioavailability in stroke-prone spontaneously hypertensive rats.

AIMS/HYPOTHESIS: Insulin-stimulated glucose transport is impaired in a genetic model of hypertension, the stroke-prone spontaneously hypertensive rat (SHRSP), yet the molecular mechanisms that underlie this defect in the animals remain unclear. METHODS: We examined the effects of insulin on the trafficking of the insulin-responsive glucose transporter GLUT4 to the plasma membrane in isolated adipocytes from SHRSP and normotensive control Wistar-Kyoto (WKY) rats. RESULTS: Treatment of isolated adipocytes with insulin resulted in trafficking of GLUT4 to the plasma membrane. There was no significant difference in the magnitude of insulin-stimulated GLUT4 trafficking from intracellular membranes to the plasma membrane between strains. In contrast, we demonstrated that there is a significant reduction in GLUT4 accessible to the glucose photolabel Bio-LC-ATB-BGPA at the plasma membrane of SHRSP adipocytes compared with control rats. CONCLUSIONS/INTERPRETATION: We propose that a large proportion of GLUT4 translocated to the plasma membrane in response to insulin is not able to bind substrate and catalyse transport in the SHRSP. Therefore, there is a reduction in bioavailable GLUT4 in SHRSP animals that is likely to account, at least in part, for the reduced insulin-stimulated glucose uptake.

Skeletal muscle of stroke-prone spontaneously hypertensive rats exhibits reduced insulin-stimulated glucose transport and elevated levels of caveolin and flotillin.

Insulin resistance is of major pathogenic importance in several common human disorders, but the underlying mechanisms are unknown. The stroke-prone spontaneously hypertensive (SHRSP) rat is a model of human insulin resistance and is characterized by reduced insulin-mediated glucose disposal and defective fatty acid metabolism in isolated adipocytes (Collison et al. [Diabetes 49:2222-2226, 2000]). In this study, we have examined skeletal muscle and cultured skeletal muscle myoblasts for defects in insulin action in the male SHRSP rat model compared with the normotensive, insulin-sensitive control strain, Wistar-Kyoto (WKY). We show that skeletal muscle from SHRSP animals exhibits a marked decrease in insulin-stimulated glucose transport compared with WKY animals (fold increase in response to insulin: 1.4 +/- 0.15 in SHRSP, 2.29 +/- 0.22 in WKY; n = 4, P = 0.02), but the stimulation of glucose transport in response to activation of AMP-activated protein kinase was similar between the two strains. Similar reductions in insulin-stimulated glucose transport were also evident in myoblast cultures from SHRSP compared with WKY cultures. These differences were not accounted for by a reduction in cellular GLUT4 content. Moreover, analysis of the levels and subcellular distribution of insulin receptor substrates 1 and 2, the p85alpha subunit of phosphatidylinositol 3'-kinase, and protein kinase B (PKB)/cAKT in skeletal muscle did not identify any differences between the two strains; the insulin-dependent activation of PKB/cAKT was not different between the two strains. However, the total cellular levels of caveolin and flotillin, proteins implicated in insulin signal transduction/compartmentalization, were markedly elevated in skeletal muscles from SHRSP compared with WKY animals. Increased cellular levels of the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins syntaxin 4 and vesicle-associated membrane protein (VAMP)-2 were also observed in the insulin-resistant SHRSP strain. Taken together, these data suggest that the insulin resistance observed in the SHRSP is manifest at the level of skeletal muscle, that muscle cell glucose transport exhibits a blunted response to insulin but unchanged responses to activation of AMP-activated protein kinase, that alterations in key molecules in both GLUT4 trafficking and insulin signal compartmentalization may underlie these defects in insulin action, and that the insulin resistance of these muscles appears to be of genetic origin rather than a paracrine or autocrine effect, since the insulin resistance is also observed in cultured myoblasts over several passages.

Low grade chronic inflammation in women with polycystic ovarian syndrome.

Low grade chronic inflammation as reflected by increased C-reactive protein (CRP) concentrations independently predicts those at risk for coronary heart disease (CHD) and type 2 diabetes. Women with polycystic ovarian syndrome (PCOS) are insulin resistant and have increased risk for CHD and type 2 diabetes, but currently there are no data on markers of inflammation in women with PCOS. Seventeen women with PCOS (defined on the basis of elevated testosterone and oligomenorrhea) and 15 healthy women matched as a group for body mass index were recruited. Measurement of CRP concentrations was made using a highly sensitive assay. Insulin resistance was assessed using the hyperinsulinemic euglycemic clamp technique. The women with PCOS had significantly elevated CRP concentrations relative to controls (geometric means, 2.12 and 0.67 mg/L, respectively; P = 0.016). Log CRP correlated with body mass index in both PCOS and controls (r = 0.58; P < 0.05 and r = 0.78; P < 0.01, respectively) and inversely with insulin sensitivity (r = -0.57; P < 0.05 and r = -0.69; P < 0.01). Total testosterone did not correlate with log CRP in either group. On adjustment for body mass index and age, there remained a significant difference in log CRP between PCOS and controls (t = 2.13; P < 0.05). On further adjustment for insulin sensitivity, log CRP was no longer significantly different between groups (t = 1.51; P = 0.14). We conclude that women with PCOS have significantly increased CRP concentrations relative to women with normal menstrual rhythm and normal androgen levels. We propose low grade chronic inflammation as a novel mechanism contributing to increased risk of CHD and type 2 diabetes in these women.

SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis.

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts ( approximately 25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.

Regulation of glucose transport in aortic smooth muscle cells by cAMP and cGMP.

We have studied the ability of cGMP and cAMP to modulate platelet-derived growth factor (PDGF)-stimulated 2-deoxy-D-glucose (deGlc) transport in primary cultures of vascular smooth muscle cells (VMSC) from rat aorta. PDGF stimulated deGlc transport in a time- and concentration-dependent manner. 8-Bromo-cGMP and atrial natriuretic peptide(1-28) [ANP(1-28)] were found to reduce PDGF-stimulated deGlc transport without affecting basal (unstimulated) transport activity. In contrast, 8-bromo-cAMP and dibutyryl-cAMP stimulated basal deGlc transport 2-fold and were without effect on PDGF-stimulated deGlc transport. 8-Bromo-cGMP also inhibited 8-bromo-cAMP-stimulated deGlc transport. The stimulation of deGlc transport by PDGF was sensitive to the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase (MEK) inhibitor PD98059, and we show that ERK1/2 was activated by PDGF. Neither 8-bromo-cGMP nor ANP(1-28) inhibited PDGF-stimulated ERK activation, suggesting that the effects of cGMP and ANP(1-28) were not mediated by inhibition of this kinase. Our data also argue against a role for cGMP-dependent protein kinase in mediating the effects of cGMP or ANP(1-28). Collectively, our data suggest that in VSMC: (i) cGMP and cAMP have opposing effects on deGlc transport; (ii) PDGF and cAMP have common elements in the pathways by which they activate deGlc transport; and (iii) a common element may be the target of the cGMP-mediated inhibition of deGlc transport.

The synaptic vesicle protein, cysteine-string protein, is associated with the plasma membrane in 3T3-L1 adipocytes and interacts with syntaxin 4.

Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.

Symposium on 'nutritional effects of new processing technologies'. New processing technologies: an overview.

Most food-preservation techniques act by slowing down or completely inhibiting the growth of micro-organisms. Few techniques act by inactivating them. While heat remains the technique most extensively used for inactivation, there has been increasing interest recently in the development of alternative approaches in response to the desires of consumers for products which are less organoleptically and nutritionally damaged during processing and less reliant on additives than previously. The new approaches, therefore, mostly involve technologies that offer full or partial alternatives to heat for the inactivation of bacteria, yeasts and moulds. They include the application to foods of high hydrostatic pressure, high-voltage electric discharges, high-intensity laser and non-coherent light pulses, 'manothermosonication' (the combination of mild heating with ultrasonication and slightly-raised pressure), and high-magnetic-field pulses. In addition, a number of naturally-occurring antimicrobials, including lysozyme and low-molecular-weight products of micro-organisms are finding increasing use. High pressure is being used commercially to non-thermally pasteurize a number of foods, while the other physical procedures are in various stages of development and commercial evaluation. Possible nutritional consequences have so far been given little attention compared with microbiological ones.

The long term health consequences of polycystic ovary syndrome.

Women with polycystic ovary syndrome have both insulin resistance and beta cell dysfunction. Consequently, they are at increased risk of developing diabetes and cardiovascular disease. Women with polycystic ovary syndrome present to clinicians at a young age and as such offer a unique opportunity to identify insulin resistant patients at an early stage. This enables the modification of risk factors and diagnosis of diabetes before the onset of macro- and micro-vascular symptoms. Increased emphasis should thus be placed on long term risk management and diabetic screening with advice on smoking, exercise and, if appropriate, weight loss. Where possible drugs that exacerbate insulin resistance should be avoided and consideration should be given to the use of insulin sensitising agents, particularly in the obese.

Cd36 and molecular mechanisms of insulin resistance in the stroke-prone spontaneously hypertensive rat.

Insulin resistance is of pathogenic importance in several common human disorders including type 2 diabetes, hypertension, obesity and hyperlipidemia, but the underlying mechanisms are unknown. The spontaneously hypertensive rat (SHR) is a model of these human insulin resistance syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridemia, and hypertension map to a single region on rat chromosome 4. Genetic analysis of an SHR derived from a National Institutes of Health colony led to the identification of a causative mutation in the SHR Cd36. We have investigated glucose and fatty acid metabolism in the stroke-prone SHR (SHRSP). We demonstrate defects in insulin action on 2-deoxy-D-glucose transport (SHRSP 3.3 +/- 1.5 vs. 21.0 +/- 7.4 pmol x min(-1) x [20 microl packed cells](-1), SHRSP vs. WKY, respectively, P = 0.01) and inhibition of catecholamine-stimulated lipolysis (P < 0.05 at all concentrations of insulin) in adipocytes isolated from SHRSP. In contrast, basal levels of catecholamine-stimulated nonesterified free fatty acid (NEFA) release and plasma levels of NEFA are similar in SHRSP and WKY. These results are in agreement with the data on the SHR.4 congenic strain, which suggested that the QTL containing Cd36 mutations accounted for the entire defect in basal catecholamine action but only for approximately 40% of the SHR defect in insulin action. In the SHR, both abnormalities appear consequent of defective Cd36 expression. Because Cd36 sequence and expression are apparently normal in SHRSP, it is likely that the molecular mechanism for defective insulin action in this strain is caused by a gene(s) different than Cd36.

Sex hormones induce insulin resistance in 3T3-L1 adipocytes by reducing cellular content of IRS proteins.

AIM/HYPOTHESIS: Numerous studies have suggested a relation between sex hormones and insulin sensitivity but the ability of sex hormones to directly influence insulin action in peripheral tissues has not been investigated. METHODS: We have examined the effects of estriol, estradiol and estrone on insulin action in cultured 3T3-L1 adipocytes, a useful model of adipocytes. RESULTS: Treatment of these cells with each of these sex hormones resulted in a statistically significant reduction in the ability of insulin to stimulate glucose transport independently of a reduction in total cellular GLUT-4 content. This diminished ability of insulin to stimulate glucose transport was accompanied by a reduction in the total cellular content of insulin receptor substrates -1 and -2 and the p85alpha subunit of phosphatidylinositol 3'-kinase. By contrast, cellular content of protein kinase B was unchanged by hormone treatment but the magnitude of insulin-stimulated kinase activity was statistically significantly reduced after incubation with each of the sex hormones tested. We have further shown that treatment of 3T3-L1 adipocytes with these hormones alters the subcellular distribution of insulin receptor substrate proteins such that the particulate and soluble pools of these proteins were differentially affected by hormone treatment. CONCLUSION/INTERPRETATION: These data show that sex hormones can directly induce a state of insulin resistance in 3T3-L1 adipocytes in culture. The mechanism of this defect seems to be at least in part due to decreased cellular content and altered subcellular distribution of insulin receptor substrate proteins which in turn results in a reduction in proximal insulin-stimulated signalling cascades.

Long-term insulin treatment of 3T3-L1 adipocytes results in mis-targeting of GLUT4: implications for insulin-stimulated glucose transport.

AIMS/HYPOTHESIS: Insulin stimulates glucose transport in adipose and muscle tissue by the translocation of a specialised pool of intracellular GLUT4-containing vesicles to the cell surface. It is well established that defective insulin-stimulated GLUT4 translocation is associated with insulin resistance. Long-term insulin treatment (500 nmol/l for 24 h) of 3T3-L1 adipocytes has previously been shown to decrease cellular GLUT4 content and reduce insulin-stimulated GLUT4 translocation. Here, we test the hypothesis that the insulin resistance observed after long-term insulin treatment arises by the selective loss of GLUT4 from a specific intracellular compartment. METHODS: Using iodixanol gradient centrifugation we have separated intracellular GLUT4 containing membranes into two distinct populations corresponding to recycling endosomes and a distinct intracellular compartment which probably represents GLUT4 storage vesicles (GSVs). RESULTS: A short-term insulin stimulation reduced the content of GLUT4 in the GSV fraction (51 +/- 3.5%) with only a modest decrease from the endosomal fraction (23 +/- 2.6%). Long-term insulin treatment decreased cellular GLUT4 content by about 40% and diminished the ability of a short-term insulin challenge to promote GLUT4 translocation. We further show that this depletion of cellular GLUT4 is selectively from the GSV fraction (68 +/- 7% decrease compared to untreated cells). CONCLUSIONS/INTERPRETATION: Such data argue that long-term insulin treatment results in the mis-targeting of GLUT4 such that it no longer accesses the GSV compartment. These data imply that defective targeting of GLUT4 away from the GSV compartment plays an important role in the aetiology of insulin resistance.

5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes.

Incubation of skeletal muscle with 5-aminoimidazole-4carboxamide ribonucleoside (AICAR), a compound that activates 5'-AMP-activated protein kinase (AMPK), has been demonstrated to stimulate glucose transport and GLUT4 translocation to the plasma membrane. In this study, we characterized the AMPK cascade in 3T3-L1 adipocytes and the response of glucose transport to incubation with AICAR. Both isoforms of the catalytic alpha-subunit of AMPK are expressed in 3T3-L1 adipocytes, in which AICAR stimulated AMPK activity in a time- and dose-dependent fashion. AICAR stimulated 2-deoxy-D-glucose transport twofold and reduced insulin-stimulated uptake to 62% of the control transport rate dose-dependently, closely correlating with the activation of AMPK. AICAR also inhibited insulin-stimulated GLUT4 translocation, assessed using the plasma membrane lawn assay. The effects of AICAR on insulin-stimulated glucose transport are not mediated by either adenosine receptors or nitric oxide synthase and are mediated downstream of phosphatidylinositol 3'-kinase stimulation. We propose that in contrast to skeletal muscle, in which AMPK stimulation promotes glucose transport to provide ATP as a fuel, AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes, inhibiting triacylglycerol synthesis, to conserve ATP under conditions of cellular stress. Investigation of the mode of action of AICAR and AMPK may, therefore, give insight into the mechanism of insulin action.

Identification of further important residues within the Glut4 carboxy-terminal tail which regulate subcellular trafficking.

The insulin-responsive glucose transporter, Glut4, exhibits a unique subcellular distribution such that in the absence of insulin >95% of the protein is stored within intracellular membranes. In response to insulin, Glut4 exhibits a large mobilisation to the plasma membrane. Studies of the amino acid motifs which regulate the unique trafficking of Glut4 have identified several key residues within the soluble cytoplasmic N- and C-terminal domains of Glut4. Of particular note is a Leu-498Leu-499 motif within the C-terminal domain that has been proposed to regulate both internalisation from the plasma membrane and sorting to an insulin-sensitive compartment. In this study, we have examined the role of the adjacent amino acids (Glu-491, Gln-492 and Glu-493) by their sequential replacement with Ala. Our results are consistent with the notion that Glu-491 and Glu-493 play an important role in the sub-endosomal trafficking of Glut4, as substitution of these residues with Ala results in increased levels of these proteins at the cell surface, reduced insulin-stimulated translocation and increased susceptibility to endosomal ablation. These residues, together with other identified sequences within the C-terminus of Glut4, are likely to be crucial targeting elements that regulate Glut4 subcellular distribution.

Effects of insulin on intracellular GLUT4 vesicles in adipocytes: evidence for a secretory mode of regulation.

The facilitative glucose transporter, GLUT4 undergoes insulin-dependent movement to the cell surface in adipocytes. The magnitude of the insulin effect is much greater for GLUT4 than other recycling proteins such as the CD-MPR. In the present study we have studied the colocalisation of these proteins in adipocytes in an effort to explain this selective insulin-dependent recruitment of GLUT4. Using immunofluorescence microscopy or immuno-EM on 3T3-L1 adipocytes we find that there is considerable colocalisation between these proteins particularly within the area of the TGN. However, the distribution of CD-MPR was not significantly effected by insulin. The insulin-dependent recruitment of GLUT4 was concomitant with a selective decrease in GLUT4 labelling of cytoplasmic vesicles whereas the amount of GLUT4 in the TGN region (approx. 50% of total GLUT4) was relatively unaffected. To explore the possibility that the cytoplasmic GLUT4(+) vesicles represent an intracellular insulin-responsive storage compartment we performed quantitative immuno-EM on whole mounts of intracellular vesicles isolated from basal and insulin-stimulated adipocytes. These studies revealed that: (1) GLUT4 and CD-MPR were concentrated in small (30-200 nm) vesicles at a labelling density of 1-20+ gold particles/vesicle; (2) there was significant overlap between both proteins in that 70% of the total GLUT4 pool colocalised with CD-MPR; (3) a significant amount of GLUT4 (approx. 50% of total) was found in a subpopulation of vesicles that contained as little as 5% of the total CD-MPR pool; (4) the GLUT4(+)/CD-MPR(-) vesicles were highly insulin-responsive, and (5) the total number of GLUT4(+) vesicles, but not CD-MPR(+) vesicles, decreased by approx. 30% in response to insulin treatment. These data are consistent with a model in which GLUT4 is selectively sorted into a vesicular compartment in adipocytes that is recruited to the plasma membrane by insulin stimulation.

The cytosolic C-terminus of the glucose transporter GLUT4 contains an acidic cluster endosomal targeting motif distal to the dileucine signal.

The insulin-responsive glucose transporter GLUT4 is targeted to a post-endocytic compartment in adipocytes, from where it moves to the cell surface in response to insulin. Previous studies have identified two cytosolic targeting motifs that regulate the intracellular sequestration of this protein: FQQI(5-8) in the N-terminus and LL(489,490) (one-letter amino acid notation) in the C-terminus. In the present study we show that a GLUT4 chimaera in which the C-terminal 12 amino acids in GLUT4 have been replaced with the same region from human GLUT3 is constitutively targeted to the plasma membrane when expressed in 3T3-L1 adipocytes. To further dissect this domain it was divided into three regions, each of which was mutated en bloc to alanine residues. Analysis of these constructs revealed that the targeting information is contained within the residues TELEYLGP(498-505). Using the transferrin-horseradish peroxidase endosomal ablation technique in 3T3-L1 adipocytes, we show that mutants in which this C-terminal domain has been disrupted are more sensitive to chemical ablation than wild-type GLUT4. These data indicate that GLUT4 contains a targeting signal in its C-terminus, distal to the dileucine motif, that regulates its sorting into a post-endosomal compartment. Similar membrane-distal, acidic-cluster-based motifs are found in the cytosolic tails of the insulin-responsive aminopeptidase IRAP (insulin-regulated aminopeptidase) and the proprotein convertase PC6B, indicating that this type of motif may play an important role in the endosomal sequestration of a number of different proteins.

Preservation: past, present and future.

Foods deteriorate in quality due to a wide range of reactions including some that are physical, some that are chemical, some enzymic and some microbiological. The various forms of spoilage and food poisoning caused by micro-organisms are preventable to a large degree by a number of preservation techniques, most of which act by preventing or slowing microbial growth. These include freezing, chilling, drying, curing, conserving, vacuum packing, modified atmosphere packing, acidifying, fermenting, and adding preservatives. In contrast, a smaller number of techniques act by inactivating micro-organisms, predominantly heating (pasteurization and sterilization). Complementary techniques restrict access of micro-organisms to food products, e.g. aseptic processing and packaging. New and 'emerging' preservation techniques include more that act by inactivation. They include the application of ionizing radiation, high hydrostatic pressure, high voltage electric discharges, high intensity light, ultrasonication in combination with heat and slightly raised pressure ('manothermosonication'), and the addition to foods of bacteriolytic enzymes, bacteriocins, and other naturally-occurring antimicrobials. Major trends, reacting to consumers' needs, are towards the use of procedures that deliver food products that are less 'heavily' preserved, higher quality, more convenient, more 'natural', freer from additives, nutritionally healthier, and still with high assurance of microbiological safety.