The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5

The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K(+), which can be blocked by the anion channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organism's principal K(+) transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K(+) ((86)Rb(+)) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5-7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.     

Microhomology-dependent end joining and repair of transposon-induced DNA hairpins by host factors in Saccharomyces cerevisiae

The maize, cut-and-paste transposon Ac/Ds is mobile in Saccharomyces cerevisiae, and DNA sequences of repair products provide strong genetic evidence that hairpin intermediates form in host DNA during this transposition, similar to those formed for V(D)J coding joints in vertebrates. Both DNA strands must be broken for Ac/Ds to excise, suggesting that double-strand break (DSB) repair pathways should be involved in repair of excision sites. In the absence of homologous template, as expected, Ac excisions are repaired by nonhomologous end joining (NHEJ) that can involve microhomologies close to the broken ends. However, unlike repair of endonuclease-induced DSBs, repair of Ac excisions in the presence of homologous template occurs by gene conversion only about half the time, the remainder being NHEJ events. Analysis of transposition in mutant yeast suggests roles for the Mre11/Rad50 complex, SAE2, NEJ1, and the Ku complex in repair of excision sites. Separation-of-function alleles of MRE11 suggest that its endonuclease function is more important in this repair than either its exonuclease or Rad50-binding properties. In addition, the interstrand cross-link repair gene PSO2 plays a role in end joining hairpin ends that is not seen in repair of linearized plasmids and may be involved in positioning transposase cleavage at the transposon ends.     

Preparation and immunological studies of protein conjugates of N -acylneuraminic acids

The overexpression of N -acetylneuraminic acid (Neu5Ac) is closely correlated with malignant transformations. Thus, Neu5Ac is an important target in the design of cancer vaccines. To study the influence of chemical modifications of Neu5Ac on its immunological properties, the alpha-allyl glycosides of five differently N -acylated neuraminic acid derivatives were prepared. Following selective ozonolysis of their allyl group to form an aldehyde functionality, they were coupled to keyhole limpet hemocyanin (KLH) via reductive amination. Resultant glycoconjugates were studied in C57BL/6 mice. The N -propionyl, N - iso- butanoyl and N -phenylacetyl derivatives of neuraminic acid provoked robust immune responses of various antibody isotypes, including IgM, IgG1, IgG2a and IgG3, whereas N -trifluoropropionylneuraminic acid and natural Neu5Ac were essentially nonimmunogenic. Moreover, the N -phenylacetyl and N - iso- butanoyl derivatives mainly induced IgG responses that are desirable for antitumor applications. These results raise the promise of formulating effective glycoconjugate cancer vaccines via derivatizing sialic acid residues of sialooligosaccharides.     

Identification of angiotensin I in a cyclostome, Lampetra fluviatilis

Angiotensin I (ANG I) was isolated from incubates of plasma and kidney extracts of the river lamprey, Lampetra fluviatilis, using eel vasopressor activity as an assay during purification. Its sequence was Asn-Arg-Val-Tyr-Val-His-Pro-Phe-Thr-Leu as determined by the sequence analysis and mass spectrometry. The sequence was confirmed by identity of the elution profile with the synthetic peptide in two different reverse-phase columns of high-performance liquid chromatography. Lamprey ANG I produced dorsal-aortic pressor responses in L. fluviatilis but the rise was very small in comparison to that produced by angiotensin II. Angiotensin III produced an even bigger increase. It was not possible to demonstrate a difference in response to Asn(1) (lamprey) ANG I and Asp(1) (human) ANG I. The present study directly demonstrated the presence and biological activity of the renin-angiotensin system in the most primitive extant vertebrates, the cyclostomes. Thus the renin-angiotensin system is a phylogenetically old hormonal system that is present throughout the vertebrates.     

Do P2X purinergic receptors regulate skeletal muscle blood flow during exercise?

Although there is evidence that sympathetic nerves release ATP as a neurotransmitter to produce vasoconstriction via P2X purinergic receptors, the role of these receptors in the regulation of blood flow to exercising skeletal muscle has yet to be determined. We hypothesized that there is tonic P2X receptor-mediated vasoconstriction in exercising skeletal muscle. To test this hypothesis, the effect of P2X receptor blockade on skeletal muscle blood flow was examined in six exercising mongrel dogs. P2X receptor antagonism was accomplished with pyridoxal-phosphate-6-azophenyl-2'4'-disulfonic acid (PPADs). Animals were instrumented chronically with flow probes on the external iliac arteries of both hindlimbs and a catheter in one femoral artery. PPADs (40 mg) was infused as a bolus into the femoral artery catheter during steady-state exercise at 6 miles/h. Intra-arterial infusion of PPADs increased iliac blood flow from 542 +/- 55 to 677 +/- 69 ml/min (P < 0.05) and iliac vascular conductance from 5.17 +/- 0.62 to 6.53 +/- 0.80 ml.min(-1).mmHg(-1). The PPADs infusion did not affect blood flow in the contralateral iliac artery. These data support the hypothesis that P2X purinergic receptors produce vasoconstriction in exercising skeletal muscle.     

Impaired monocyte maturation in response to CpG oligodeoxynucleotide is related to viral RNA levels in human immunodeficiency virus disease and is at least partially mediated by deficiencies in alpha/beta interferon responsiveness and production

The biological activity of CpG oligodeoxynucleotide 2216 (ODN2216), a Toll-like receptor 9 agonist, was investigated with monocytes from human immunodeficiency virus (HIV)-negative and HIV-positive (HIV+) donors. Exposure of peripheral blood mononuclear cells to CpG ODN2216 led to decreased expression of the monocyte marker CD14 and increased expression of the dendritic cell marker CD83, as well as increased expression of HLA-DR, CD40, CD80, and CD86 among the monocytes. Several features of the CpG ODN-induced maturation were diminished in monocytes from HIV+ donors, and these deficiencies were related to increased viremia but not to CD4 cell counts. Alpha interferon (IFN-alpha) was implicated as at least a partial mediator of the CpG ODN-induced monocyte maturation. Reduced production of IFN-alpha in response to CpG ODN and reduced frequencies of plasmacytoid dendritic cells, the principal IFN-alpha-producing cell type in peripheral blood, were observed in peripheral blood mononuclear cells from HIV+ donors. These deficiencies also were related to levels of plasma HIV RNA. Responses of monocytes from HIV+ donors to direct stimulation with IFN-alpha also were partially impaired. Thus, reduced production of IFN-alpha and reduced IFN-alpha responsiveness may contribute to diminished functional responses to CpG ODN in HIV disease. Application of CpG ODNs in HIV disease for adjuvant or immunoregulatory purposes may be particularly useful for HIV+ donors without high-level viremia.     

The sulfogalactose moiety of sulfoglycosphingolipids serves as a mimic of tyrosine phosphate in many recognition processes. Prediction and demonstration of Src homology 2 domain/sulfogalactose binding

Multiple ligand co-recognition of 3'-sulfogalactosylceramide (SGC) and sulfotyrosine initiated the comparison of SGC and sulfotyrosine and, subsequently, phosphotyrosine (pY) binding. SGC is a receptor for ligands involved in cell adhesion/microbial pathology. pY forms a Src homology domain 2 recognition motif in intracellular signaling. Using hsp70, anti-SGC, and anti-pY antibodies, ligand binding is retained following phosphate/sulfate and tyrosine/galactose substitution in SGC and sulfate/phosphate exchange in pY. Remarkable lipid-dependent binding to phosphatidylethanolamine-conjugated sulfotyrosine suggests "microenvironmental" modulation of sulfotyrosine-containing receptors, similar to glycosphingolipids. Based on an aryl substrate-bound co-crystal of arylsulfatase A, a sulfogalactose and phosphotyrosine esterase, modeling provides a solvation basis for co-recognition. c-Src/Src homology domain 2:SGC/phosphogalactosylceramide binding confirms our hypothesis, heralding a carbohydrate-based approach to regulation of phosphotyrosine-mediated recognition.     

An implantable telemetry device to measure intra-articular tibial forces

Tibial forces are important because they determine polyethylene wear, stress distribution in the implant, and stress transfer to underlying bone. Theoretic estimates of tibiofemoral forces have varied between three and six times the body weight depending on the mathematical models used and the type of activity analyzed. An implantable telemetry system was therefore developed to directly measure tibiofemoral compressive forces. This system was tested in a cadaver knee in a dynamic knee rig. A total knee tibial arthroplasty prosthesis was instrumented with four force transducers located at the four corners of the tibial tray. These transducers measured the total compressive forces on the tibial tray and the location of the center of pressure. A microprocessor performed analog-to-digital signal conversion and performed pulse code modulation of a surface acoustic wave radio frequency oscillator. This signal was then transmitted through a single pin hermetic feed-through tantalum wire antenna located at the tip of the stem. The radio frequency signal was received by an external antenna connected to a receiver and to a computer for data acquisition. The prosthesis was powered by external coil induction. The tibial transducer accurately measured both the magnitude and the location of precisely applied external loads. Successful transmission of the radio frequency signal up to a range of 3m was achieved through cadaveric bone, bone cement, and soft tissue. Reasonable accuracy was obtained in measuring loads applied through a polyethylene insert. The implant was also able to detect unicondylar loading with liftoff.     

Mouse endothelial cells cross-present lymphocyte-derived antigen on class I MHC via a TAP1- and proteasome-dependent pathway

In vivo studies suggest that vascular endothelial cells (ECs) can acquire and cross-present exogenous Ag on MHC-I but the cellular mechanisms underlying this observation remain unknown. We tested whether primary female mouse aortic ECs could cross-present exogenous male Ag to the T cell hybridoma, MHH, specific for HYUty plus D(b). MHC-I-deficient male spleen cells provided a source of male Ag that could not directly stimulate the MHH cells. Addition of male but not female MHC-I-deficient spleen cells to wild-type syngeneic female EC induced MHH stimulation, demonstrating EC cross-presentation. Lactacystin treatment of the donor male MHC-I-deficient spleen cells, to inhibit proteasome function, markedly enhanced EC cross-presentation showing that the process is most efficient for intact proteins rather than degraded peptide fragments. Additional experiments revealed that this EC Ag-processing pathway is both proteasome and TAP1 dependent. These studies demonstrate that cultured murine aortic ECs can process and present MHC-I-restricted Ag derived from a separate, live cell, and they offer insight into the molecular requirements involved in this EC Ag presentation process. Through this pathway, ECs expressing cross-presented peptides can participate in the effector phase of T cell-mediated inflammatory responses such as autoimmunity, anti-tumor immunity, and transplant rejection.