Characterization of microdialysis acidification for capillary isoelectric focusing-microelectrospray ionization mass spectrometry

A microdialysis junction, based on a microdialysis membrane connecting a separation capillary and a short, sharply tapered microelectrospray emitter capillary, is demonstrated for on-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization mass spectrometry (ESI-MS). The microdialysis junction provides the necessary electrical connection across the dialysis membrane for defining the electric fields needed for the CIEF separation and the electrospray process. Additionally, postseparation acidification of focused protein zones eluted from the CIEF capillary is achieved using the microdialysis junction while separation efficiency and resolution is maintained. A microelectrospray emitter produces a stable electrospray of protein analytes without the need for a makeup liquid flow and eliminates any subsequent sample dilution and reduction in MS sensitivity. The microdialysis junction is advantageous over the coaxial liquid sheath interface as evidenced by the simplicity in operation procedures, the enhancement in detection sensitivity, and the linear correlation between protein migration time and isoelectric point in CIEF-ESI-MS.     

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.     

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.     

Lepromatous uveitis diagnosed by iris biopsy

Ocular leprosy is rarely seen in developed countries. We report the long-term follow-up of a patient with bilateral uveitis, glaucoma, and keratitis. Skin, iris and aqueous humor biopsies disclosed abundant Wade-Fite-positive organisms consistent with Mycobacterium leprae. Leprosy must be considered in the differential diagnosis of keratitis and uveitis.     

Alpha-2 adrenoceptor subtype causing nitric oxide-mediated vascular relaxation in rats

The alpha-2 adrenoceptor subtype and its signal transduction pathway mediating vascular relaxation in rats were studied in vitro using rings of superior mesenteric arteries. Removal of endothelium or incubation with NG-nitro-L-arginine completely blocked relaxant responses to UK14,304, suggesting endothelium-derived nitric oxide mediates relaxation. The order of potency for full (F) or partial (P) agonists causing relaxation was guanabenz (P) > UK14,304 (F) > clonidine (P) > epinephrine (F) > norepinephrine (F). Affinities (Ka) of alpha-2 adrenoceptor subtype-selective drugs for blocking relaxation were obtained in side-by-side experiments comparing rat mesenteric arteries with pig coronary arteries. Relaxation of pig coronary arteries is known to be mediated by the alpha-2A adrenoceptor subtype. Ka values in nM for rauwolscine (19), WB-4101 (265), SKF-104078 (197), spiroxatrine (128), and prazosin (1531) for blocking relaxation in rat arteries were consistent with their affinities for binding at the alpha-2D adrenoceptor subtype. Ka values for rauwolscine and WB-4101, drugs distinguishing the alpha-2D from the alpha-2A adrenoceptor subtype, were significantly higher in blocking relaxation of rat arteries compared with pig arteries, suggesting the alpha-2D adrenoceptor subtype mediates NO-induced relaxation in rat arteries. We used forskolin to oppose alpha-2 adrenoceptor-mediated inhibition of cAMP formation by directly stimulating cAMP formation in endothelium. Forskolin did not affect the relaxant response to UK14,304, suggesting that cAMP is not involved in the coupling of alpha-2 adrenoceptors to nitric oxide-induced vascular relaxation.     

Suppression of human prostate carcinoma metastases in severe combined immunodeficient mice by interleukin 2 immunocytokine therapy

Immunocytokines are antibody-cytokine fusion proteins that combine the unique targeting ability of antibodies with the multifunctional activities of cytokines to activate effector cells in the tumor microenvironment. Here, we demonstrate the therapeutic efficacy of a tumor-specific immunocytokine, huKS1/4-IL2, which effectively inhibited growth and dissemination of lung and bone marrow metastases of human prostate carcinoma in severe combined immunodeficient mice. This antitumor effect was specific and highly effective, irrespective of reconstitution of these mice with human lymphokine-activated killer cells. Survival times of mice treated with huKS1/4-IL2 were increased 4-fold as compared with animals treated with a mixture of the corresponding antibody and recombinant human interleukin-2 (rhIL2). A persistent antitumor response after treatment with the huKS1/4-IL2 immunocytokine in B, T, and natural killer cell-deficient severe combined immuodeficient-BEIGE mice, depleted of granulocytes, implies a major role for macrophages in this treatment effect. Our data demonstrate that immunocytokine-directed interleukin-2 therapy to tumor sites is an immunotherapeutic approach with potent effects against disseminated metastases of human prostate carcinoma and suggest that this treatment could be effective in an adjuvant setting for patients with minimal residual disease.     

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.     

Role of hope in academic and sport achievement

Hope is the sum of goal thoughts as tapped by pathways and agency. Pathways reflect the perceived capability to produce goal routes; agency reflects the perception that one can initiate action along these pathways. Using trait and state hope scales, studies explored hope in college student athletes. In Study 1, male and female athletes were higher in trait hope than nonathletes; moreover, hope significantly predicted semester grade averages beyond cumulative grade point average and overall self-worth. In Study 2, with female cross-country athletes, trait hope predicted athletic outcomes; further, weekly state hope tended to predict athletic outcomes beyond dispositional hope, training, and self-esteem, confidence, and mood. In Study 3, with female track athletes, dispositional hope significantly predicted athletic outcomes beyond variance related to athletic abilities and affectivity; moreover, athletes had higher hope than nonathletes.