Thermal denaturation of Escherichia coli thioredoxin studied by hydrogen/deuterium exchange and electrospray ionization mass spectrometry: monitoring a two-state protein unfolding transition

Thermally denatured oxidized Escherichia coli thioredoxin (TRX) in 2% acetic acid was examined by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism. Conformational dynamics during thermal unfolding were probed by hydrogen/deuterium (H/D) exchange-in experiments. ESI-MS was used to determine the H/D ratios. TRX shows only a marginal change in negative ellipticity at 222 nm during thermal unfolding, but in the near-UV circular dichroism (240-350 nm) a clear transition is observed (Tm = 61 degrees C), and unfolding goes to completion. ESI mass spectra were recorded as a function of temperature, and the observed bimodal charge state distributions were analyzed assuming a two-state unfolding mechanism which allowed an estimation of the midpoint temperature, Tm = 64 degrees C. Under conditions at which the compact, folded conformational state is only marginally stable (80 degrees C, 2% acetic acid-d1), H/D exchange-in experiments in combination with ESI-MS resulted in mass spectra differing in the number of incorporated deuteriums which indicates the presence of two distinct populations of molecules after short incubation periods. As the exchange-in time increases, the population representing the unfolded state increases and the population which is protected against exchange decreases. The rate of conversion was used to estimate the rate constant of unfolding which was 2.1 +/- 0.2 min-1. The results presented here indicate that thermally denatured TRX under the conditions used may represent a collapsed unfolded state with properties often attributed to molten globule-like states, such as pronounced secondary structure but absence of rigid tertiary structure and, hence, lack of protection against H/D exchange.     

Aberrant methylation of p16INK4a and deletion of p15INK4b are frequent events in human esophageal cancer in Linxian, China

Esophageal transit scintigraphy seems to be a valid methodology to assess impaired esophageal motility in early stages of disease. The purpose of this study was to discriminate patients with primary Raynaud's phenomenon (RP) and patients with systemic sclerosis (SSc) from healthy subjects by esophageal scintigraphy with a semisolid meal. METHODS: We studied 32 patients with primary RP, 18 with SSc and 13 healthy subjects. Dysphagia, acid regurgitation and heartburn were scored. After an overnight fast, all subjects underwent esophageal scintigraphy, using a semisolid orally ingested bolus (10 mL apple puree) labeled with 99mTc-sulfur colloid. Esophageal transit and emptying time and integral value were evaluated with the subjects in the upright (sitting) and supine positions. Transit time was defined as the time from the entry of 50% of radioactivity into the upper esophagus until the clearance of 50% of the bolus from the whole esophagus. Emptying time was defined as the time from the entry of 50% of radioactivity into the upper esophagus, until the clearance of 100% of the bolus from the whole esophagus. Integral value was defined as the total counts under the time-activity curve normalized to the maximum. RESULTS: Esophageal transit and emptying time and integral value, evaluated in both positions, were significantly higher in patients with SSc than in healthy subjects and than in patients with RP. Moreover, patients with RP had all three parameters, assessed in supine position, significantly longer compared to healthy subjects. Clinical scores regarding dysphagia, acid regurgitation and heartburn were not significantly different between patients with RP and SSc. CONCLUSION: Esophageal transit and emptying time and integral value appear to be able to discriminate patients with primary RP from patients with SSc and patients with RP from healthy subjects, suggesting an early mild esophageal dysfunction in RP.     

A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Multiple symmetric lipomatosis in the Chinese: ultrasound, CT and MR imaging

We report four cases of multiple symmetric lipomatosis in the Chinese population. We believe that multiple symmetric lipomatosis in the Chinese is not uncommon and may be related to the increasing incidence of alcoholism. The ultrasound appearances of these lipomatous masses are presented for the first time. Heterogeneous echogenic masses with fine fibrous strands that insinuate around fascial planes, lymph nodes and vascular tissues are typical findings. Unlike diseases affecting the Western population, multiple symmetric lipomatosis in the Chinese appears to be limited to the head and neck.     

Developmental expression and location of IGF-I and IGF-II mRNA and protein in skeletal muscle

To investigate the role of IGF in muscle development in vivo, developmental expression and location of IGF-I and -II protein and mRNA were examined in fetal, postnatal, and adult skeletal muscle. Muscle tissue was collected from 30-, 44-, 59-, 68-, 75-, 89-, and 109-d porcine fetuses, 21-d neonatal pigs, and 6-mo-old (adult) pigs. Relative amounts of IGF-II mRNA peaked (P < .05) in 59-d fetal muscle and decreased thereafter. Inversely, muscle IGF-I expression increased (P < .05) to maximal levels around birth. For in situ hybridization, frozen muscle tissue sections (10 microm) were hybridized with a hydrolyzed form of the same riboprobes or incubated with polyclonal or monoclonal antibodies to IGF-I or -II, respectively. The majority of IGF-I and IGF-II mRNA was localized to developing muscle fibers, whereas little signal was found in the surrounding connective tissues. Immunofluorescent localization of IGF-I and -II confirmed that muscle IGF are present in developing muscle fibers. Collectively, these data show that IGF-I and -II are expressed and produced primarily in muscle cells within developing muscle tissue and support the hypothesis that IGF-I and -II modulate fetal muscle development.     

Involvement of interleukin-3 in delayed-type hypersensitivity

The in vivo functions of interleukin-3 (IL-3) were investigated by generating IL-3-deficient mice. Although hematopoiesis was unimpaired in homozygous mutant animals, contact hypersensitivity reactions were compromised. IL-3 was required for efficient priming of hapten-specific contact hypersensitivity responses, but was dispensable for T-cell-dependent sensitization to tumor cells. These findings reveal a critical role for IL-3 in some forms of delayed-type hypersensitivity.     

Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis

Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.     

Spray‐Deposited Al‐Si (Osprey CE) Alloys and their Properties

Osprey Metals Ltd. has developed a new family of lightweight, low expansion aluminium‐silicon alloys to suit electronic applications, containing between 30 and 70 wt.% aluminium. They are produced using the Osprey spray deposition process, which achieves homogeneous and isotropic properties. Their advantageous physical characteristics, combining low thermal expansion, high thermal conductivity and low density are particularly suited for packaging electronic circuitry. These CE (controlled expansion) alloys are easy to machine to tight tolerances using standard machine tools and they can be electroplated with gold, silver or nickel finishes without difficulty. This paper focuses on the measurement of mechanical (flexural strength) and thermo‐mechanical properties (CTE, thermal diffusivity and specific heat) of these alloys, carried out in collaboration with AMTT. The property measurements were consistent with those measured previously elsewhere, apart from the CTE values, which deviated significantly and were at variance with values predicted from a rule of mixtures. This discrepancy was resolved in a short follow‐up series of measurements carried out under conditions corresponding more closely to temperature equilibrium.