The syntaxin Tlg1p mediates trafficking of chitin synthase III to polarized growth sites in yeast

Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers. We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p. Remarkably, growth is possible in the absence of all four proteins. In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64. However, although Pep12p is required for the ligand-induced internalization of the alpha factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not. In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p. In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme. Pep12p is neither necessary nor sufficient for this process. We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.     

Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli

DNA topoisomerase VI from the hyperthermophilic archaeon Sulfolobus shibatae is the prototype of a novel family of type II DNA topoisomerases that share little sequence similarity with other type II enzymes, including bacterial and eukaryal type II DNA topoisomerases and archaeal DNA gyrases. DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity. The native enzyme is a heterotetramer composed of two subunits, A and B, with apparent molecular masses of 47 and 60 kDa, respectively. Here wereport the overexpression in Escherichia coli and the purification of each subunit. The A subunit exhibits clusters of arginines encoded by rare codons in E.coli . The expression of this protein thus requires the co-expression of the minor E.coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in E.coli was obtained from inclusion bodies after denaturation and renaturation. The B subunit was overexpressed in E.coli and purified in soluble form. When purified B subunit was added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase were reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from S.shibatae . It catalyzes transient double-strand cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA. This cleavage is detected only in the presence of both subunits and in the presence of ATP or its non-hydrolyzable analog AMPPNP.     

Cervical lymphadenopathy revealing differentiated thyroid cancer. Review of the literature and therapeutic approach]

Well-differentiated thyroid cancer (i.e. papillary microcarcinoma) may be revealed by a clinically palpable cervical node, especially in young patients. Primary tumor specificity (small-sized, superior location, invasion, multifocality) and characteristics of cervical node involvement (with synchronous central and lateral multiple node spread) require appropriate treatment by a skilled surgeon. Contrary to the primary tumor treatment (total thyroidectomy), surgical management of the cervical node basins remains controversial. Only a special knowledge of the specificity of this disease and cervical lymphatic anatomy will ensure a good prognosis for the patient.     

Monte Carlo simulation of a miniature, radiosurgery x-ray tube using the ITS 3.0 coupled electron-photon transport code

A miniature, interstitial x-ray generator has recently been developed and is currently undergoing clinical trials for the treatment of brain tumors. The maximum photon energy from this x-ray tube is 50 keV, although most of the initial testing has been carried out at 40 keV. Dose rates of up to 2 Gy/min in a water phantom at a distance of 10 mm from the tube tip are produced. In this paper we describe the modeling and simulation of x-ray production from this device using the ITS 3.0 Monte Carlo code. Verification of the simulation of x-ray production in the device was carried out by comparing predictions of spatial photon distribution, energy spectrum, and dose versus depth in water with experimentally obtained measurements. Agreement between the simulated results and experimental measurements was fairly good when comparing the angular distribution of photons emitted from the x-ray tube and very good when comparing dose rate versus depth in a water phantom. Discrepancies observed when comparing the calculated and measured estimates of characteristic line radiation were reduced by incorporation of a modification to the ITS code. Possible causes of the remaining discrepancy in bremsstrahlung intensity are discussed.     

Targeting peptide nucleic acid-protein conjugates to structural features within duplex DNA

URF13 is the product of a mitochondrial-encoded gene (T-urf13) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility of cms-T maize to the fungal pathogens Bipolaris maydis race T and Phyllosticata maydis. Mitochondria isolated from cms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed in Escherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and can contribute to pore formation. Chemical cross-linking of URF13 was used to demonstrate the existence of URF13 oligomers in cms-T mitochondria and E. coli cells. The ability of the carboxylate-specific reagent, N,N'-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor in cms-T mitochondria.     

Psychosocial characteristics of pregnant and nonpregnant HIV-seropositive women

Thirty-three HIV-positive women, 12 of whom were pregnant, participated in semistructured interviews to define areas of psychosocial need. Eighty-eight percent of the subjects reported current unemployment. A history of substance abuse was reported by 82 percent, suicide attempts by 52 percent, and sexual problems by 43 percent. Approximately 30 percent reported elevated levels of depressive symptoms on standardized symptom inventories. The pregnant women appeared psychologically healthier than the nonpregnant group. HIV-positive women face multiple psychosocial stressors and may experience significant psychological distress.     

Assessing obesity: classification and epidemiology

Obesity is generally defined as a body mass index (BMI) of 30 kg/m2 and higher. Overweight is defined as a BMI between 25 and 30 kg/m2. The prevalence varies considerably between countries, and between regions within countries. It is estimated that more than half of adults aged 35-65 living in Europe are either overweight or obese. Overweight is more common among men than among women but obesity is more common among women. The prevalence of obesity in Europe is probably in the order of 10-20% in men and 15-25% in adult women. In most European countries who have reliable data on time-trends the prevalence of obesity seems to be increasing. In most European countries, obesity is usually inversely associated with socio-economic status, particularly among women. New classifications of overweight may be based on cut-off points for simple anthropometric measures which reflects both total adiposity as well as abdominal fatness.