Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes.

Kv1.3 potassium channels maintain the membrane potential of effector memory (T(EM)) T cells that are important mediators of multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5), containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin, is a potent and selective blocker of these channels. However, a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability, ShK-192, contains a nonhydrolyzable phosphotyrosine surrogate, a methionine isostere, and a C-terminal amide. ShK-192 shows the same overall fold as ShK, and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys(411) of the channel. ShK-192 blocks Kv1.3 with an IC(50) of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 microg/kg, approximately 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24, 48, and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T(EM) cells and suppresses delayed type hypersensitivity when administered at 10 or 100 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells.

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

The research nurse role in a clinic-based oncology research setting.

Public demand and government support for evaluating new cancer drugs and methods in a more timely manner have significantly affected clinical research programs. To meet these demands, it is critical to have research personnel with an appropriate skill mix to ensure that clinical trials are conducted safely and effectively, while scientific integrity is maintained. This article describes the development and integration of the research nurse role within an oncology research program in a large outpatient oncology clinic. Methods for evaluating the research nurse role included literature review, analysis of job descriptions, and dialogue with research staff, oncology staff, and a clinical nurse specialist. A Review of the Nurse Practice Act of Wisconsin and the Oncology Nursing Society standards provided license and practice standards. Similarities and differences between the roles of the research nurse and the chemotherapy nurse are analyzed. Complementary roles and functions are identified, and areas of role overlap are defined. This report expands the limited current literature regarding this subject. The findings provide the reader with a framework for evaluating the roles of registered nurse personnel in a clinical setting. Although each institution has unique characteristics or research needs, the method used to address the reported program is transferable.

Attainment of maximal exercise criteria in boys and men.

BACKGROUND: This study tested the hypothesis that the occurrence of a VO(2) plateau at maximal exercise would be greater in men versus boys. Secondary indicators of maximal effort also were examined. METHODS: Sixteen boys (10.7+/-0.6 yrs) and 21 men (22.5+/-2.0 yrs) performed a graded exercise test on a treadmill at a constant speed of 8.04 km x hr(-1) with 2.5% increments in elevation. The men also performed a second test at 11.26 km x hr(-1) with similar increases in slope. RESULTS: At 8.04 km x hr(-1) , VO(2) max was 52.3+/-6.0 and 52.5+/-5.1 ml x kg(-1) x min(-1) in boys and men, respectively (p>0.05). In the men, VO(2) max (53.3+/-5.4 ml x kg(-1) x min(-1) ) was higher (p<0.05) in the faster protocol. The percentage of men achieving the criterion was nearly double the percentage of boys (23.8 vs 12.5%), although the difference was not significant. Age-specific criteria heart rate (HR) and respiratory exchange ratio (RER) were achieved in a similar manner; however, more men (100%) than boys (86.7%) achieved an age-specific blood lactate (BLa) criterion (p<0.05). Plateau achievement increased to 33.0% in the 11.26 km x hr(-1) protocol, but was not significantly different from 8.04 km x hr(-1). HR, RER and BLa criteria achievement were comparable. CONCLUSIONS: Maturation may influence the achievement of a plateau and BLa criteria, but not age-specific criteria for RER or HR.

Running performance in middle-school runners.

AIM: This study examined the relationship of 3-km run time to indices of aerobic and anaerobic ability in 9 male runners (13.4+/-0.6 years, mean+/-SD). METHODS: Anthropometric measurements were made, and an exercise test to determine running economy at 187 m x min(-1) and (.-)VO(2max) were assessed on a treadmill. On a separate day, 2 55-m sprints followed by a 3-km run were performed on a 200-m indoor track. Capillary blood samples were obtained from a finger tip immediately after the run to determine blood lactate level. Fractional utilization (%(.-)VO(2max) used during the 3-km run) was calculated. Correlations were used to examine the relationship between run time and the physiological measurements. RESULTS: Mean values for (.-)VO(2), HR and RER at maximal exercise were 61.7+/-4.4 ml x kg(-1)xmin(-1), 198.9+/-6.7 b x min(-1), and 1.16+/-0.04, respectively. The average time to run 3 km was 13.27+/-0.97 min (90.1+/-7.2% of (.-)VO(2max)). Post-run blood lactate level was 8.3+/-3.2 mmol x L(-1) and was significantly related (r=-0.73, p=0.02) to 3-km time. Fractional utilization tended to be related (r=-0.56, p=0.12) to time. CONCLUSIONS: In this age group the ability to run at a high percentage of (.-)VO(2max) and tolerate a high blood lactate appear to be important determinants of running performance in young male runners.

Framing treatment options: a method to enhance informed consent.

Advanced practice nurses are challenged with counseling and providing information to clients about treatment options during the informed consent process. In the clinical area of oncology, clients are faced with choosing treatment options that are undesirable or affect quality and quantity of life directly. Framing as a nursing intervention to assist advanced practice nurses during the informed consent process is explored in this article. Framing provides a method to present information from different vantage points and perspectives with a focus on the client's values and perceptions of quality of life. Parse's man-living-health theory is used to guide nursing practice. Quality of life is addressed with an assessment tool by Ferrans. The use of framing to present treatment options from the client's perspective of health and quality of life, as well as from the biomedical and nursing perspectives, can result in a consent that is truly informed.

Marketer's forgotten challenge: physician recruitment.

As marketing department missions continue to expand, more and more people are finding themselves not just recruiting customers, but doctors. The good news: May of the tips and tricks of direct marketing can be used to reach out to in-demand physicians.

Senior market tough to crack. MacNeal Health Network, Berwyn, IL.

Seniors, often thought of as a stodgy, conservative market, actually constitute one of the most vibrant, diverse age groups of all. Here's how people who have discovered the secrets of the mature market use that diversity to their advantage.

Identification of the reactive sulfhydryl and sequences of cysteinyl-tryptic peptides from beef heart aconitase.

In an accompanying paper (Kennedy, M. C., Spoto, G., Emptage, M. H., and Beinert, H. (1988) J. Biol. Chem. 263, 8190-8193), it was shown that one cysteine per mol of aconitase is modified by a variety of sulfhydryl reagents. We have identified the tryptic peptide that contains the iodoacetamide-reactive cysteine. We have also demonstrated that this cysteine is the primary site of modification by phenacyl bromide (2-bromoacetophenone), a spin label analogue of N-ethylmaleimide (HO-461) and iodoacetate in both the 3Fe and 4Fe forms of aconitase. The amino acid sequence of the peptide containing the reactive cysteine from beef heart aconitase shares no homology with the reactive cysteine-containing peptide reported for pig heart aconitase (Hahm, K.-S., Gawron, O., and Piszkiewicz, D. (1981) Biochim. Biophys. Acta 667, 457-461). We also report the amino acid compositions and sequences of seven other cysteine-containing tryptic peptides from beef heart aconitase. However, none of the cysteinyl peptides isolated were found to correspond to the reported pig heart reactive cysteinyl peptide. Evidence is also presented that no previously unreactive cysteine becomes exposed and reactive to sulfhydryl reagents in the conversion from the [4Fe-4S] cluster of the enzyme to the [3Fe-4S] cluster. We conclude from this that any potential cysteine ligand to the Fea site of the cluster must be inaccessible to solvent in the 3Fe form or, alternatively, that active 4Fe aconitase does not contain a cysteine ligand to the Fea site.

Evaluation of speech following prosthetic obturation of surgically acquired maxillary defects.

The speech intelligibility of patients who have undergone partial surgical resection of the maxilla was investigated. The intelligibility characteristics of connected discourse in each patient were compared under three experimental conditions: presurgical, postsurgical with immediate prosthetic obturation, and postsurgical with definitive prosthetic obturation. The ability of untrained listeners to differentiate between presurgical speech and postsurgical speech following definitive prosthetic treatment was also studied. Each subject recorded a standard passage of connected discourse, the Rainbow Passage, under the three conditions stated above. Each of the 24 recordings was then segmented into 23 phrases and presented over earphones to listeners. A group of 10 listeners evaluated each recording, or a total of 240 listeners was used. The recordings were graded by the number of words correctly written. Analysis of variance showed no significant loss in intelligibility across the three conditions. A group of 50 listeners than evaluated sentence pairs containing samples of presurgical speech and speech produced following surgery and definitive prosthetic obturation. Their task was to judge whether there was a difference between the two speech samples. This was done in a classroom over loudspeakers. Sign-test procedures revealed that listeners could differentiate presurgical speech samples from postsurgical speech with definitive prosthetic obturation in four of the eight patients studied.

Cysteine labeling studies of beef heart aconitase containing a 4Fe, a cubane 3Fe, or a linear 3Fe cluster.

The reactivity of cysteines following cluster destruction by iron chelation was investigated for [4Fe-4S]2+ and cubane [3Fe-4S]+ beef heart aconitase. When the chelator orthobathophenanthroline disulfonate was used, the formation of sulfur-sulfur bonds and the retention of inorganic sulfur from the cluster was observed. For both the 4Fe and 3Fe forms of aconitase, the two cysteines in peptide 7, the cysteine in peptide 3, and the cysteine in peptide 2 were found as the primary constituents of sulfur-sulfur bonds (the peptide sequences and nomenclature are from Plank, D. W., and Howard, J. B. (1988) J. Biol. Chem. 263, 8184-8189). Three of these four cysteines (peptides 3 and 7) correlated with those proposed to be cluster ligands recently determined by x-ray crystallography (Robbins, A. H. and Stout, C. D. (1989) Proteins, in press; Robbins, A. H., and Stout, C. D.,, (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3639-3643) for pig heart aconitase. A mechanism is proposed whereby the greater affinity of orthobathophenanthroline disulfonate for Fe2+ relative to Fe3+ shifts the equilibrium toward reduction of ferric iron through sulfur-sulfur bond formation at the cluster site. Aconitase which has been oxidized with ferricyanide and from which the cluster iron has been removed by EDTA has been shown to have two di- or polysulfides (Kennedy, M. C., and Beinert, H. (1988) J. Biol. Chem. 263, 8194-8198). The cysteines found in the sulfur-sulfur bonds generated by this treatment also were predominantly those from peptides 3 and 7. In addition, the putative thiol ligands for the linear [3Fe-4S]+ cluster of aconitase are reported. The four cysteines of peptides 7 and 9 (two in each peptide) were found to be protected by the cluster from alkylation when the protein was denatured. The difference in the ligands between the cubane and linear forms indicates that a specific thiol exchange occurs during the conversion.

Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the phosphorylation and dephosphorylation of isocitrate dehydrogenase (IDH) are catalyzed by a bifunctional protein kinase/phosphatase. We have determined the nucleotide sequence of aceK, the gene encoding IDH kinase/phosphatase. This gene consists of a single open reading frame of 1,734 base pairs preceded by a Shine-Dalgarno ribosome-binding site. Examination of the deduced amino acid sequence of IDH kinase/phosphatase revealed sequences which are similar to the consensus sequence for ATP-binding sites. This protein did not, however, exhibit the extensive sequence homologies which are typical of other protein kinases. Multiple copies of the REP family of repetitive extragenic elements were found within the intergenic region between aceA (encoding isocitrate lyase) and aceK. These elements have the potential for combining to form an exceptionally stable stem-loop structure (delta G = -54 kcal/mol [ca. -226 kJ/mol]) in the mRNA. This structure, which masks the ribosome-binding site and start codon for aceK, may contribute to the downshift in expression observed between aceA and aceK. Another potential stem-loop structure (delta G = -29 kcal/mol [ca. 121 kJ/mol]), unrelated to the REP sequences, was found within aceK.

Histamine-releasing activity. IV. Molecular heterogeneity of the activity from stimulated human thoracic duct lymphocytes.

Previous studies with the lymphokine, histamine-releasing activity (HRA), showed that HRA consisted of a heterogeneous group of molecules. The possibility of using thoracic duct lymphocytes (TDL) as a source of large quantities of HRA has been investigated. Antigen-stimulated TDL synthesize and release HRA in quantities similar to an equivalent number of peripheral blood lymphocytes (PBL). Streptokinase (SK) antigen routinely caused TDL to produce HRA approximately 15,000 Da. In contrast, staphylococcus enterotoxin B (SEB) induced the formation of a heterogeneous mixture of HRAs with apparent molecular weights of 50,000 and 15,000. Two peaks of activity (HRA I and II) were recovered when the supernatant from SK-stimulated TDL was subjected to ion-exchange chromatography. Interestingly, basophil chemotactic activity (BCA) was also eluted in these two peaks. Although interferon (IFN) is also released by antigen-stimulated TDL, the nonidentity of IFN and HRA was established by fundamental differences in chromatographic properties and specific antisera to IFN. In contrast, these studies suggest that HRA and BCA may be present on the same molecular entity.

Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography.

The production of large quantities of the lymphokine(s) histamine-releasing activity (HRA) and its partial purification by Sephadex G-75 and ion-exchange chromatography on carboxymethyl (CM) Sepharose 6B have been detailed (M. A. Lett-Brown, D. O. Thueson, D. E. Plank, M. P. Langford, and J. A. Grant, Cell. Immunol. 87, 434-444, 1984). Two peaks of activity (HRA I and II) were recovered. Preparations of HRA have now been analyzed by high-performance liquid chromatography (HPLC). Thoracic duct lymphocytes stimulated with 200 U/ml streptokinase were used as a source of HRA. Gel-filtration HPLC on a TSK 3000 column separated HRA into two peaks of activity (10,000-20,000 and 1300 Da). Reverse-phase high-performance liquid chromatography using a Nucleosil C-8 column showed that HRA II (the activity eluted at a conductivity of 18-20 mmho on the CM-Sepharose column) eluted as a single sharp peak, the main protein contaminant being cytochrome c, the carrier protein added to enhance the yield of HRA. High-performance liquid chromatography was found to be a useful analytical tool and may be suitable for the large-scale purification of HRA.