GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.     

A method for isolation of sequences missing in one of two related genomes

We describe a novel technique for isolation of sequences that are present in one genome (tracer), but absent in another (driver). Tracer DNA, cleaved with Sau 3A and capped with a single stranded PCR adapter, is allowed to hybridize with an excess of sheared biotinylated driver; biotinylated DNA and its hybrids with the tracer are removed by phenol/chloroform extraction after incubation with streptavidin. After several rounds of subtraction the ends of self-annealed tracer molecules from the nonextractable fraction are filled-in with Tag polymerase and amplified, using the single stranded PCR adapter as a primer. The method has been applied to purification of fragments from a 2.9 kb plasmid added to E. coli DNA at equimolar quantity. Plasmid derived fragments (250-1000 bp), initially comprising 1/1400th part of tracer DNA, were purified to homogeneity after two rounds of subtraction followed by PCR.     

Characterisation of erythrocyte shapes and sizes by NMR diffusion-diffraction of water: correlations with electron micrographs

Irradiation of the mammalian foetus produces a broad spectrum of congenital abnormalities, growth retardations, developmental delays, and functional deficits, depending upon the dose and the specific gestational phase of irradiation. The developing brain is particularly susceptible to production of deleterious effects, with decreased brain size, behavioural alterations, and mental retardation having been documented. Supplementing the limited human data, rodent models have been extensively used to investigate the specific processes by which relatively low doses, with correspondingly minor cellular damage to the developing neocortex, can produce dramatic postnatal consequences in brain structure and function. The effects of a variety of physical (dose, linear energy transfer, dose rate, fractionation) and biological (species, strain, gestational age, time course post-irradiation) parameters have been examined in an attempt to provide much needed information on such critical aspects as dose response, threshold doses for effect, and extrapolation to human risk estimates. Various acute cellular responses (e.g. appearance of pyknotic cells and macrophages) observed in the developing neocortex 0-24 h after in utero irradiation can be associated with postnatal effects. Moreover, it is possible to correlate thinning of specific layers of the cerebral cortex with specific behavioural aberrations, allowing prediction of brain structural changes from functional alterations, and vice versa. Thus, it is possible to speculate as to the mechanisms and targets for extremely sensitive, radiation-induced cellular damage in the developing foetal brain, that will interfere with the orderly and precisely programmed development of the mammalian brain, leading finally to postnatal expression as delays in growth and development, perturbations in behaviour, and alterations in brain structure.     

Methyl tert-butyl ether (MTBE) contamination in private wells near gasoline stations in upstate New York

BACKGROUND: Auxiliary liver transplantation has several advantages over standard orthotopic liver transplantation. However, functional competition has been reported even in auxiliary partial orthotopic liver transplantation (APOLT). We evaluated herein the interaction in APOLT between the native liver and the graft in terms of portal blood flow and regeneration. The need for diversion of the portal blood flow to the graft was also assessed. METHODS: A total of 15 patients received APOLT from living donors. Portal blood flow to the native liver was preserved in 6 patients, and the portal vein to the native liver was preemptively transected at the time of transplantation in 9 patients. RESULTS: Of the patients with preservation of the portal blood flow to the native liver, two showed inadequate graft portal blood flow just after operation, and in the other three patients the graft portal blood flow decreased or the graft atrophied after deterioration of the graft function. In the patients with preemptive transection of the portal vein to the native liver, optimal graft portal blood flow was obtained, and the native liver, supplied only by arterial inflow, supported a small-for-size graft until the graft regenerated. The damage to the native liver was minimal. CONCLUSIONS: Functional competition may occur in APOLT with preservation of the portal blood flow to the native liver, whereas preemptive transection of the native liver portal vein is a safe procedure and effectively prevents the portal steal phenomenon.     

Cytolysis and piecemeal degranulation as distinct modes of activation of airway mucosal eosinophils

In contrast to the protective effect of chronic caloric restriction on tumor development, we have shown that fasting sustained tumor initiation in rat liver by a noninitiating dose of diethylnitrosamine. Here we investigated whether fasting had a similar favorable effect on initiation in the colorectal mucosa in 80 male F344 rats. Animals fasted for 4 days were given a single s.c. dose of azoxymethane (AOM) (20 mg/kg) on the first day of re-feeding, and rates of kinetic proliferative parameters, and development of the pre-neoplastic lesions such as aberrant crypt foci (ACF), were evaluated. Starvation before AOM treatment enhanced the growth of ACF, as shown by the significantly higher crypt multiplicity of fasted/re-fed rats as compared with fully fed rats (3.97 +/- 0.50 vs. 2.64 +/- 0.20, p < or = 0.025). This difference was associated with perturbations in cell death and cell proliferation. Fasting induced apoptosis and depressed cell division, while re-feeding had opposite effects, resulting in a higher percentage of S-phase cells at the time of AOM injection and 2 days thereafter. Starvation-induced apoptosis may represent the mitogenic stimulus to an increase in the number of cells susceptible to AOM damage, and may favor its fixation, leading to enhanced growth of ACF. Our data therefore suggest that fasting/re-feeding enhances colon cancer.     

A histomorphological study in age related changes in the elastic fiber system of the basilar artery

The aim of the study was to quantify the distribution of elastic fibers within the wall of the basilar artery. Three age groups were studied: 1) 0-1 yr.; 2) 20-50 yr. 3) > 50 yr. A histomorphometrical study was undertaken using an estimation of the linear density of the components of the fibrous elastic tissue system: the full length in the proximal and distal segments of the basilar artery was evaluated. Our results showed that: The elastic tissue of the basilar artery is not evenly distributed throughout the tunica media. Compared to the 0 to 1-year age group, mature elastic, elauninic and oxytalan fibers decreased with age. In all age groups the linear density of the mature elastic fibers was more evident in the proximal than in the distal segment of the artery.     

Increased incidence of congenital malformations in children with transient thyroid-stimulating hormone elevation on neonatal screening

Huntington's disease is an autosomal-dominant inherited progressive neurodegenerative disease associated with an expanded trinucleotide repeat (CAG) sequence on the short arm of chromosome 4. The disease is considered rare in Africans. We report five black South African families of different ethnic origin with proven expansions typical of Huntington's disease and discuss the possible origins of the disease in Africa.     

Fourier transform Raman spectroscopy of polyacrylamide gels (PAGs) for radiation dosimetry

Polyacrylamide gels (PAGs) are used for magnetic resonance imaging radiation dosimetry. Fourier transform (FT) Raman spectroscopy studies were undertaken to investigate cross-linking changes during the copolymerization of polyacrylamide gels in the spectral range of 200-3500 cm(-1). Vibrational bands of 1285 cm(-1) and 1256 cm(-1) were assigned to acrylamide and bis-acrylamide single CH2 deltaCH2 binding modes. Bands were found to decrease in amplitude with increasing absorbed radiation dose as a result of copolymerization. Principal component regression was performed on FT-Raman spectra of PAG samples irradiated to 50 Gy. Two components were found to be sufficient to account for 98.7% of the variance in the data. Cross validation was used to establish the absorbed radiation dose of an unknown PAG sample from the FT-Raman spectra. The calculated correlation coefficient between measured and predictive samples was 0.997 with a standard error of estimate of 0.976 and a standard error of prediction of 1.140. Results demonstrate the potential of FT-Raman spectroscopy for ionizing radiation dosimetry using polyacrylamide gels.     

Spontaneous intracranial hypotension: report of four cases and review of the literature

Spontaneous intracranial hypotension is an unusual syndrome of postural headache and low cerebrospinal fluid pressure without an established cause. We present four cases, analyze those previously reported in the literature, examine the MRI, CT, angiographic and cisternographic finding and discuss the clinical picture, proposed pathophysiologic mechanisms and potential treatment.