Isolation,structural characterization,and immunological evaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl beta-(1-->6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide-protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of beta-(1-->6)-linked glucosamine containing 45-60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis.     

The actin-regulating kinase Prk1p negatively regulates Scd5p,a suppressor of clathrin deficiency,in actin organization and endocytosis

Endocytosis is a dynamic process requiring a network of interacting proteins that assemble and disassemble during cargo capture and vesicle formation. A major mechanism for regulation of this process involves the reversible phosphorylation of endocytic factors. Recently, members of a new kinase family, the Ark/Prk kinases, which include mammalian AAK1 and GAK as well as yeast Prk1p, Ark1p, and Akl1p, were shown to regulate components of the endocytic machinery. These include animal AP-1/AP-2 mu chains and yeast Pan1p (Eps15-like), Sla1p, and epsins, but other potential targets are likely. SCD5, an essential yeast gene, was identified as a suppressor of clathrin deficiency. We also showed that Scd5p is required for normal cortical actin organization and endocytosis, possibly as a targeting subunit for protein phosphatase type 1 (PP1). Scd5p contains a central triple repeat (3R) motif related to a known Prk1p consensus phosphorylation site L/IxxQxTG, except that Q is replaced by T. In this study we demonstrate that the Scd5p 3R sequence is phosphorylated by Prk1p to negatively regulate Scd5p. Furthermore, we show that Prk1p, Ark1p, and Akl1p have different substrate specificities and play distinct roles in actin organization and endocytosis.     

Cross-bridge mechanisms underlying the history-dependent properties of muscle spindles and stretch reflexes

The effects of prior movement on the force responses of skeletal muscle are compared with the effects of movement history on the changes in firing rate of muscle spindle receptors. Prior release results in the linearization of the mechanical properties of skeletal muscles, which can be provisionally explained by cross-bridge models of muscular contraction. The history-dependence of responses of muscle spindle receptors in unanesthetized decerebrate preparations appears to result from the kinetics of cycling and noncycling cross-bridges. The results of this comparison indicate that the integration of mechanical properties of muscle and spindle receptor promotes stiffness regulation.     

Keratinocyte growth factor-2 production in an ompT-deficient Escherichia coli K-12 mutant

A variant form of Keratinocyte growth factor-2 (KGF-2) spanning amino acids A63-S208 was produced in the Escherichia coli K-12 host W3110. When the protein was purified using a standard process, the first six N-terminal amino acids were rapidly and specifically removed from the protein. This cleavage resulted in a truncated KGF-2 species (S69-S208). To circumvent this problem, guanidine-HCl was used to inhibit the putative proteolytic activity. This modified process resulted in a massive loss of protein product due to precipitation, in addition to the cost and corrosiveness of guanidine-HCl. To develop an economically feasible, scaleable, and robust process for KGF-2 production, we were tasked with identifying the protease(s) responsible for the N-terminal degradation. Experimental evidence revealed that OmpT (outer membrane protein T) was the primary protease involved in the N-terminal cleavage of the A63-S208 KGF-2 protein. Moreover, the OmpT-mediated cleavage occurred at a novel site (Arg-Ser). From this work, we show that production of the A63-S208 form of KGF-2 in an ompT-deleted E. coli host nearly abolished the N-terminal cleavage issue.     

Regulation of a wheat actin-depolymerizing factor during cold acclimation

We have previously shown that the wheat (Triticum aestivum) TaADF gene expression level is correlated with the plants capacity to tolerate freezing. Sequence analysis revealed that this gene encodes a protein homologous to members of the actin-depolymerizing factor (ADF)/cofilin family. We report here on the characterization of the recombinant TaADF protein. Assays for ADF activity showed that TaADF is capable of sequestering actin, preventing nucleotide exchange, and inducing actin depolymerization. In vitro phosphorylation studies showed that TaADF is a substrate for a wheat 52-kD kinase. The activity of this kinase is modulated by low temperature during the acclimation period. Western-blot analyses revealed that TaADF is expressed only in cold-acclimated Gramineae species and that the accumulation level is much higher in the freezing-tolerant wheat cultivars compared with the less tolerant ones. This accumulation was found to be regulated by a factor(s) encoded by a gene(s) located on chromosome 5A, the chromosome most often found to be associated with cold hardiness. The induction of an active ADF during cold acclimation and the correlation with an increased freezing tolerance suggest that the protein may be required for the cytoskeletal rearrangements that may occur upon low temperature exposure. These remodelings might be important for the enhancement of freezing tolerance.     

The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro

The UspA1 and UspA2 proteins of Moraxella catarrhalis are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of uspA1 and uspA2 mutants of M. catarrhalis strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113-3119, 1998). In the present study, isogenic uspA1, uspA2, and uspA1 uspA2 mutations were constructed in three additional M. catarrhalis strains: 012E, TTA37, and 046E. The uspA1 mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the uspA1 gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the uspA2 gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the uspA2 genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this "hybrid" protein was designated uspA2H. PCR-based analysis revealed that approximately 20% of M. catarrhalis strains apparently possess a uspA2H gene instead of a uspA2 gene. The M. catarrhalis uspA1, uspA2, and uspA2H genes were cloned and expressed in Haemophilus influenzae cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.     

Oxygen dependency and precision of cytochrome oxidase signal from full spectral NIRS of the piglet brain

Oxidation changes of the copper A (Cu(A)) center of cytochrome oxidase in the brain were measured during brief anoxic swings at both normocapnia and hypercapnia (arterial PCO(2) approximately 55 mmHg). Hypercapnia increased total hemoglobin from 37.5 +/- 9.1 to 50.8 +/- 12.9 micromol/l (means +/- SD; n = 7), increased mean cerebral saturation (Smc(O(2))) from 65 +/- 4 to 77 +/- 3%, and oxidized Cu(A) by 0.43 +/- 0.23 micromol/l. During the onset of anoxia, there were no significant changes in the Cu(A) oxidation state until Smc(O(2)) had fallen to 43 +/- 5 and 21 +/- 6% at normocapnia and hypercapnia, respectively, and the maximum reduction during anoxia was not significantly different at hypercapnia (1.49 +/- 0.40 micromol/l) compared with normocapnia (1.53 +/- 0.44 micromol/l). Residuals of the least squares fitting algorithm used to convert near-infrared spectra to concentrations are presented and shown to be small compared with the component of attenuation attributed to the Cu(A) signal. From these observations, we conclude that there is minimal interference between the hemoglobin and Cu(A) signals in this model, the Cu(A) oxidation state is independent of cerebral oxygenation at normoxia, and the oxidation after hypercapnia is not the result of increased cerebral oxygenation.     

Abnormal exhaled ethane concentrations in scleroderma.

Scleroderma (systemic sclerosis) is a chronic multisystem autoimmune disease in which oxidative stress is suspected to play a role in the pathophysiology. Therefore, it was postulated that patients with scleroderma would have abnormally high breath ethane concentrations, which is a volatile product of free-radical-mediated lipid peroxidation, compared with a group of controls. There was a significant difference (p<0.05) between the mean exhaled ethane concentration of 5.27 pmol ml(-1) CO(2) (SEM=0.76) in the scleroderma patients (n=36) versus the mean exhaled concentration of 2.72 pmol ml(-1) CO(2) (SEM=0.71) in a group of healthy controls (n=21). Within the scleroderma group, those subjects taking a calcium channel blocker had lower ethane concentrations compared with patients who were not taking these drugs (p=0.05). There was a significant inverse association between lung diffusion capacity for carbon monoxide (per cent of predicted) and ethane concentration (b=-2.8, p=0.026, CI=-5.2 to -0.35). These data support the presence of increased oxidative stress among patients with scleroderma that is detected by measuring breath ethane concentrations.     

AGE STRUCTURE AND GROWTH OF THE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) FROM STRANDINGS IN THE MISSISSIPPI SOUND REGION OF THE NORTH-CENTRAL GULF OF MEXICO FROM 1986 TO 2003

Despite their high abundance and wide distribution, little is known about the historical or current growth and age structure of coastal bottlenose dolphins ( Tursiops truncatus ) in the north-central Gulf of Mexico. Between 1986 and 2003, teeth were collected from bottlenose dolphins stranded on the mainland coast of Mississippi and the adjacent barrier islands. Bottlenose dolphin strandings occurred year round, peaking in March and April. Neonate strandings also peaked during these 2 mo. Age estimates were obtained from 111 animals by reading the growth layer groups in the dentine layer of the teeth. The ages ranged from <1 yr to 30 yr of age. The two-stage Laird–Gompertz growth model was fitted to the total length and age data. On the basis of this model, the asymptotic lengths were estimated at 250 cm for females and 255 cm for males. The length at birth estimates were 98–103 cm for females and 100–107 cm for males. These lengths are similar to those of bottlenose dolphin populations from other Gulf of Mexico areas and from the North Atlantic Ocean along the southeastern United States.     

Thermal liquid biopsy for monitoring melanoma patients under surveillance during treatment: A pilot study

Background

Differential Scanning Calorimetry (DSC) is a technique traditionally used to study thermally induced macromolecular transitions, and it has recently been proposed as a novel approach for diagnosis and monitoring of several diseases. We report a pilot study applying Thermal Liquid Biopsy (TLB, DSC thermograms of plasma samples) as a new clinical approach for diagnostic assessment of melanoma patients.