Yeast sequencing reports. Isolation and sequence analysis of a gene from the linear DNA plasmid pPAC1–2 of Pichia acaciae that shows similarity to a killer toxin gene of Kluyveromyces lactis

The toxin-encoding linear plasmid systems found in Pichia acaciae and Kluyveromyces lactis yeasts appear to be quite similar, both in function and structural organization. By Southern hybridization, a linear plasmid of P. acaciae, pPac1–2, was found to hybridize to the second open reading frame (ORF2) of K. lactis plasmid pGKL1, known to encode the α and β subunits of the K. lactis toxin. A 1·7 kbp segment of pPac1–2 DNA was cloned, sequenced and shown to contain four regions of strong homology to four similarly oriented regions of K. lactis ORF2. This 1·7 kbp fragment also contained an ORF of 1473 bp that could encode a protein of ～ 55·8 kDa. Like the α subunit gene of K. lactis ORF2, a very hydrophobic region occurs at the N-terminus, perhaps representing a signal sequence for transport out of the cell. Unlike K. lactis ORF2, however, the encoded polypeptide is much smaller and lacks a recognizable domain common to chitinases. The structure of a toxin that includes the translation product of this P. acaciae ORF would likely be quite different from that of the K. lactis toxin. Analysis of the upstream region of the P. acaciae ORF revealed an upstream conserved sequence identical to that found before ORFs 8 and 9 of pGKL2. A possible hairpin loop structure, as has been described for each of the four K. lactis pGKL1 ORFs, was found just upstream of the presumed start codon. The similarity of the promoter-like elements found in the linear plasmid genes of these diverse yeasts reinforces the idea of the existence of a unique, but highly conserved, expression system for these novel plasmids. The sequence has been deposited in the GenBank data library under Accession Number U02596.     

Defect Analysis of Barrier Height Inhomogeneity in Titanium 4H-SiC Schottky Barrier Diodes

Over 350 4H-SiC Schottky barrier diodes (SBDs) of varying size are characterized using current–voltage (I–V) measurements, with some also measured as a function of temperature. Devices display either a characteristic single-barrier height or atypical dual-barrier heights. Device yields are shown to decrease as device area increases. Molten KOH etching is used to highlight defects for analysis by optical microscopy and atomic force microscopy. The I–V characteristics are compared against the defect density. A positive correlation between effective barrier height and effective electrically active area of the SBDs is found. No correlation is found between threading dislocations and ideality factor or barrier height.     

Monitoring the sizes of denatured ensembles of staphylococcal nuclease proteins: implications regarding m values, intermediates, and thermodynamics

Fluorescence and size-exclusion chromatography (SEC) are used to monitor urea denaturation of wild-type staphylococcal nuclease (SN) as well as the m+ and m- mutants A69T and V66W, respectively. It is found that the SEC partition coefficient, 1/Kd, is directly proportional to the Stokes radii of proteins. From the Stokes radii, the denatured ensembles of the three proteins are found to be highly compact in the limit of low urea concentration and expand significantly with increasing urea concentration. The m values from fluorescence-detected denaturation of the SN proteins are generally considered to reflect the relative sizes of denatured ensembles. However, the rank order of m values of the SN proteins studied do not correspond to the rank order of denatured ensemble sizes detected by 1/Kd, suggesting that m values reflect more than just surface area increases on denaturation. SEC provides two complementary ways to demonstrate the existence of intermediates in urea denaturation and illustrates that V66W undergoes a three-state transition. Fluorescence-detected urea denaturations of A69T and wt SN do not correspond with 1/Kd-detected denaturation profiles, a result that would ordinarily mean that the transitions are non-two-state. However, this interpretation fails to recognize the rapidly changing size and thermodynamic character of the denatured ensembles of these proteins both within and outside of the transition zone. The implications of the changing sizes and thermodynamic character of the denatured ensembles for SN proteins are manifold, requiring a reconsideration of the thermodynamics of proteins whose denatured ensembles behave as those of SN proteins.     

Comparison of scotopic sensitivity in diurnal (Anas platyrhynchos) and crepuscular (Dendrocygna autumnalis) ducks.

Obtained scotopic visual adaptation curves from 4 mallard ducks. A curve of best fit was used to compare the mallards' mean adaptation curve to the curve previously reported for the black-bellied tree duck, a crepuscular species. The curves did not differ significantly in either their slopes or base levels (thresholds). The mallards' curve had a rod-cone "break" at approximately 25 min. This break was evident in the scotopic curve for pigeons but was absent from the black-bellied tree ducks' curve. Examination of retinal tissues indicated that the black-bellied tree ducks had significantly more rods and cones, and a larger rod-cone ratio than the mallards. The mallards' scotopic visual threshold was exceeded by the natural illumination present under several nocturnal conditions. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Reproducibility of the histologic diagnosis of pneumonia among a panel of four pathologists: analysis of a gold standard

STUDY OBJECTIVE: To establish a histologic diagnosis of pneumonia by consensus of a panel of pathologists, to test the interobserver and intraobserver variation in the histologic diagnosis of pneumonia, to compare the diagnostic accuracy of diagnosing pneumonia with and without preselected histologic criteria, and to establish more specific histologic criteria for the diagnosis of pneumonia. METHODS: The study group consisted of 39 patients who died after a mean of 14 days of mechanical ventilation. A postmortem open lung biopsy was performed on all patients. The tissue was reviewed independently by four pathologists who categorized the slides from each patient as showing or not showing pneumonia. Interobserver variation was calculated using the kappa statistic. Six months following the initial evaluation, the same slides were resubmitted to one of the pathologists for reevaluation to look for intraobserver error. Finally, the slides were reviewed and categorized by the criteria of Johanson et al into no pneumonia, mild, moderate, or severe bronchopneumonia. A comparison was made of the patients selected as demonstrating histologic pneumonia by each of the examinations. RESULTS: The reliability coefficient (kappa) measuring agreement among the four pathologists was good at 0.916. However, the prevalence of pneumonia as determined by each of the four pathologists varied; pathologist A, 15 of 39 (38%); pathologist B, 12 of 39 (31%); pathologist C, 9 of 39 (23%); and pathologist D, 7 of 39 (18%). Resubmitting the same slides to the same pathologist 6 months later resulted in reclassification of 2 of 39 patients. Using the histologic criteria of Johanson and colleagues, 14 patients were selected as having pneumonia compared with only nine patients selected by consensus of three of four pathologists. CONCLUSIONS: Recognition of histologic pneumonia varies among pathologists. The preselected criteria of Johanson and colleagues detected histologic pneumonia in eight of nine patients picked by consensus of pathologists, but six additional patients classified as "no histologic pneumonia" by the consensus of pathologists were judged to have histologic pneumonia by these criteria. The results established the necessity for standardization of histologic criteria for studies using biopsy as the gold standard for bacterial pneumonia. An atlas showing the criteria used in our selection was developed.     

Osmolyte-driven contraction of a random coil protein

The Stokes radius characteristics of reduced and carboxamidated ribonuclease A (RCAM RNase) were determined for transfer of this "random coil" protein from water to 1 M concentrations of the naturally occurring protecting osmolytes trimethylamine N-oxide, sarcosine, sucrose, and proline and the nonprotecting osmolyte urea. The denatured ensemble of RCAM RNase expands in urea and contracts in protecting osmolytes to extents proportional to the transfer Gibbs energy of the protein from water to osmolyte. This proportionality suggests that the sum of the transfer Gibbs energies of individual parts of the protein is responsible for the dimensional changes in the denatured ensemble. The dominant term in the transfer Gibbs energy of RCAM RNase from water to protecting osmolytes is the unfavorable interaction of the osmolyte with the peptide backbone, whereas the favorable interaction of urea with the backbone dominates in RCAM RNase transfer to urea. The side chains collectively favor transfer to the osmolytes, with some protecting osmolytes solubilizing hydrophobic side chains as well as urea does, a result suggesting there is nothing special about the ability of urea to solubilize hydrophobic groups. Protecting osmolytes stabilize proteins by raising the chemical potential of the denatured ensemble, and the uniform thermodynamic force acting on the peptide backbone causes the collateral effect of contracting the denatured ensemble. The contraction decreases the conformational entropy of the denatured state while increasing the density of hydrophobic groups, two effects that also contribute to the ability of protecting osmolytes to force proteins to fold.     

Forcing thermodynamically unfolded proteins to fold

A growing number of biologically important proteins have been identified as fully unfolded or partially disordered. Thus, an intriguing question is whether such proteins can be forced to fold by adding solutes found in the cells of some organisms. Nature has not ignored the powerful effect that the solution can have on protein stability and has developed the strategy of using specific solutes (called organic osmolytes) to maintain the structure and function cellular proteins in organisms exposed to denaturing environmental stresses (Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., and Somero, G. N. (1982) Science 217, 1214-1222). Here, we illustrate the extraordinary capability of one such osmolyte, trimethylamine N-oxide (TMAO), to force two thermodynamically unfolded proteins to fold to native-like species having significant functional activity. In one of these examples, TMAO is shown to increase the population of native state relative to the denatured ensemble by nearly five orders of magnitude. The ability of TMAO to force thermodynamically unstable proteins to fold presents an opportunity for structure determination and functional studies of an important emerging class of proteins that have little or no structure without the presence of TMAO.     

Behavioral predictors of blood pressure variation in hypertensives and normotensives.

Examined the relative impact of activity, posture, location, social involvement, and tension on the 24-hr blood pressure (BP) variability of 21 normotensives, 18 borderline hypertensives, and 18 sustained essential hypertensives. Within each diagnostic group, activity accounted for more variance in BP variability than any other behavioral dimension. For each behavioral dimension, the magnitude of the relationship with BP was greater for the normotensives than for both hypertensive groups. Variation due to individuals was a better predictor of BP variability for the 2 hypertensive groups than for the normotensive group. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Syk tyrosine kinase required for mouse viability and B-cell development

The Syk cytoplasmic protein-tyrosine kinase has two amino-terminal SH2 domains and a carboxy-terminal catalytic domain. Syk, and its close relative ZAP-70, are apparently pivotal in coupling antigen- and Fc-receptors to downstream signalling events. Syk associates with activated Fc receptors, the T cell receptor complex and the B-cell antigen-receptor complex (BCR) in immature and mature B lymphocytes. On receptor activation, the tandem SH2 domains of Syk bind dual phosphotyrosine sites in the conserved ITAM motifs of receptor signalling chains, such as the immunoglobulin alpha and beta-chains of the BCR, leading to Syk activation. Here we have investigated Syk function in vivo by generating a mouse strain with a targeted mutation in the syk gene. Homozygous syk mutants suffered severe haemorrhaging as embryos and died perinatally, indicating that Syk has a critical role in maintaining vascular integrity or in wound healing during embryogenesis. Analysis of syk-/- lymphoid cells showed that the syk mutation impaired the differentiation of B-lineage cells, apparently by disrupting signalling from the pre-BCR complex and thereby preventing the clonal expansion, and further maturation, of pre-B cells.