Sequence Determinants of GLUT1 Oligomerization: ANALYSIS BY HOMOLOGY-SCANNING MUTAGENESIS*

The human blood-brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes, where the GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in human embryonic kidney 293 (HEK293) cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8, and 11 also contribute to a less abundant heterocomplex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6- and 10-nm Stokes radius particles, which correspond to GLUT1 dimers and tetramers, respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6- and 10-nm Stokes radius particles, whereas GLUT3 resolves as a 6-nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6- and 10-nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6- and 4-nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.     

Historical and recent evidence for close relationships among Rickettsia parkeri,R. conorii,R. africae,and R. sibirica: Implications for rickettsial taxonomy

Rickettsia parkeri, a member of the spotted fever group rickettsias, was first described in 1939 and was thought to be non‐pathogenic until recently, when it was found to cause a spotted fever‐like illness in humans and areas of necrosis (eschars) at the sites of tick bites. Accordingly, there is currently much interest in this emerging pathogen. In this study, all published articles concerning R. parkeri were reviewed and analyzed for evidence of relatedness among this agent and other spotted fever group (SFG) rickettsiae which also produce similar clinical syndromes and/or eschars, including R. conorii, R. africae, and R. sibirica. A synthesis of the historical (antigenic) and recent (molecular) data supporting a phylogenetic sub‐grouping of these SFG organisms is presented and comments are offered about the taxonomy of rickettsial organisms in general, and R. parkeri in particular.     

The effect of pregnancy and foaling on intravaginal pressure in pony mares

Pressures were recorded monthly at two sites in the vagina of each of five pregnant and five nonpregnant pony mares; pressures in five mares were also recorded weekly after foaling. The developing pregnancy did not influence pressure, and after foaling the integrity of the seal formed by the caudal reproductive tract was rapidly restored.     

Maturation-related changes in mass and elemental contents of secretory granules as measured by electron-microprobe

Summary The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P<0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.     

The use of catheter-tipped pressure transducers for chronic measurement of genital tract pressures in the ewe. I. Implantation technique, catheter performance and data analysis

A computerised system for chronic in vivo monitoring of genital tract pressure in the ewe is described. Solid-state, catheter-tipped pressure transducers were surgically implanted in the uterine tube (ampulla), ipsilateral uterine horn and abdomen of five non-pregnant parous ewes. The catheters were connected to a portable computer which recorded the pressure at each location once per second and was programmed to analyse the data as mean pressure and area under the contraction curve over given periods. The catheters remained implanted for up to 129 days and 252 daily recordings were made. Analysis of spontaneous activity showed little variation in mean pressure of work done over four successive ten-minute periods. Although the amplitude of uterine contractions varied quite dramatically, chiefly in relation to variation in the plasma progesterone concentration, ampullary activity was unaffected by such changes. Genital tract pressures did not appear to be influenced by abdominal events. The catheters provoked minimal tissue reaction, and it is suggested that the system is suitable for chronic in vivo recording of genital tract pressure in the ewe.     

The use of catheter-tipped pressure transducers for chronic measurement of genital tract pressures in the ewe. II. Effects of oxytocin and prostaglandin F(2)alpha

Chronic catheterisation of the uterus, ampulla, and abdomen was performed in five ewes using solid-state, catheter-tipped pressure transducers. The catheters remained in place for up to 129 d, allowing in vivo studies of the effects of oxytocin and prostaglandin F(2)alpha (PGF(2)alpha). These agents did not produce any measurable increase in abdominal pressure. Intravenous (i.v.) oxytocin elicited a rapid increase in work done by both the uterus and ampulla. Intramuscular (i.m.) PGF(2)alpha produced a delayed uterine response but little change in the ampulla; i.v. PGF(2)alpha produced a rapid response at both sites. Low plasma progesterone concentrations (< 0.5 ng/ml) were associated with a greater uterine and ampullary response to oxytocin and with an enhanced uterine response to PGF(2)alpha. However, the uterine tube response to intravenous PGF(2)alpha was greatest when plasma progesterone concentrations were high.     

THE VALIDITY OF CENSUSING BLACK RHINOCEROS POPULATIONS FROM THE AIR

The efficacy of censusing black rhinoceros ( Diceros bicornis Linnaeus) populations from the air was tested. Repeated aerial counts were made of the black rhinoceros population centered on Qlduvai Gorge, Tanzania, where the number of rhinoceros on the ground is known accurately. Even under the most ideal conditions only 50% of the population was detected by observers in an aircraft, and factors accounting for this are considered. It is concluded that the light aircraft is of limited value in providing estimates of black rhinoceros populations, and estimates based purely on aerial counts are subject to considerable variation.