Identification and primary structural analysis of peptide II, an end-product of precursor processing in cells R3-R14 of Aplysia

Peptide II, which is encoded on a gene for a precursor protein in abdominal ganglion neurons R₃-R₁₄, was purified from extracts of abdominal ganglia of Aplysia californica. Native peptide II comigrates with synthetic standards on HPLC under isocratic conditions. Amino acid sequence and composition analyses indicate that the sequence of peptide II is Glu-Ala-Glu-Glu-Pro-Ser-Phe-Met-Thr-Arg-Leu, as predicted from the precursor. The molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-amide was also identified in abdominal ganglion extracts by similar means. The large amount of peptide II recovered (100 ng/ganglion), and its location on the precursor between two pairs of basic residues, strongly suggest that the precursor is processed into peptide II and at least two other peptides. Although cells R₃-R₁₄ have been postulated to play a role in cardiovascular control, peptide II was without effect at ≤10⁻⁴ M concentrations on identified abdominal ganglion neurons, the gastroesophageal artery or the heart. The physiological role of peptide II therefore remains to be elucidated.     

Effects of acute upper-body vibration on strength and power variables in climbers

Whole-body vibration training has recently received a lot of attention with reported enhancements of strength and power qualities in athletes. This study investigated whether upper-body vibration would be able to augment muscular attributes for climbing performance. Twelve healthy active climbers volunteered for the study. All participants underwent 3 treatments--arm cranking (AC), upper-body vibration (UBV), and non-UBV (NUBV)--in a balanced random order, conducted on separate days. Upper-body vibration was generated via a commercialized electric-powered dumbbell with a rotating axis that delivered oscillatory movements to the shoulders and arms. The UBV treatment consisted of performing 5 upper-body exercises for a total duration of 5 minutes. The UBV frequency was set at 26 Hz, amplitude 3 mm. For the NUBV treatment, the participants performed the exact exercises and time constraints as UBV; however, the vibration dumbbell was set at 0 Hz and 0 mm amplitude. The third treatment consisted of AC, which was performed at 75 k.min(-1) for 5 minutes. Pre- and postmuscular performance measures of medicine ball throw, hand grip strength, and a specific climbing maneuver were performed after each treatment. There were no significant treatment differences on medicine ball throw, hand grip strength, and the specific climbing maneuver. Acute UBV exposure did not demonstrate the expected potential neuromuscular enhancements on the climbing performance tests selected for this study.     

Identification and validation of SRC and phospho-SRC family proteins in circulating mononuclear cells as novel biomarkers for pancreatic cancer

There is an urgent need to develop novel markers of pancreatic cancer to facilitate early diagnosis. Pancreatic carcinoma is characterized by marked stroma formation with a high number of infiltrating tumor-associated macrophages (TAMs) that originate from circulating mononuclear cells (MNCs). We hypothesized that differential analysis of protein expression and phosphorylation in circulating MNCs from healthy nude mice and nude mice bearing orthotopic human pancreatic cancer would identify a surrogate marker of pancreatic cancer. These differences were analyzed by two-dimensional gel electrophoresis followed by Western blot analysis using antibody against phosphorylated tyrosine proteins (pY). Protein and phosphorylated protein spots of interest were identified by mass spectrometry and validated by Western blot analysis as candidate markers for pancreatic cancer. We found that the expression and phosphorylation of Src family proteins were significantly higher in circulating MNCs from mice bearing pancreatic cancer than in circulating MNCs from healthy mice. TAMs in mice with pancreatic tumors also had higher Src family protein expression and phosphorylation than resident macrophages in the pancreas of healthy mice. The expression and phosphorylation of Src family proteins were correlated with tumor weight; however, increased Src expression and phosphorylation also occurred in MNCs from mice with chronic pancreatitis. This is the first report to explore novel pancreatic tumor markers in circulating MNCs. Although the specificity of the marker for pancreatic cancer was low, it could be used to monitor the disease or to select high-risk patients with chronic pancreatitis.     

Protein conformation significantly influences immune responses to prion protein

In prion diseases, such as variant Creutzfeldt-Jakob disease normal cellular prion protein (PrPC), a largely alpha-helical structure is converted to an abnormal conformational isoform (PrPSc) that shows an increase in beta-sheet content. Similarly, the recombinant form of PrPC (ralpha-PrP) can be converted to a conformation dominated by beta-sheet (rbeta-PrP) by reduction and mild acidification in vitro, a process that may mimic in vivo conversion following PrPC internalization during recycling. Despite PrPSc accumulation and prion propagation in the lymphoreticular system before detectable neuroinvasion, no Ab response to PrP has been detected, probably due to immune tolerance. To investigate how the immune system may respond to alpha- and beta-PrP, we immunized Prnp(0/0) mice that are not tolerant of PrP with ralpha-PrP and rbeta-PrP. In this study, we show that although T cells stimulated by these differently folded conformers PrP recognize similar immunodominant epitopes (residues 111-130 and 191-210) the cytokine profile in response to ralpha- and rbeta-PrP was different. Challenge with ralpha-PrP elicited a strong response of IL-5 and IL-10, whereas rbeta-PrP led to an early increased production of IFN-gamma. In addition, immunization with ralpha-PrP led to production of predominantly IgG1 isotype Ab in the sera, whereas after immunization with rbeta-PrP, IgG2b was significantly produced. Thus, both humoral and cellular responses to these differently folded isoforms of the same protein are different, indicating a possible involvement of Th1 and Th2 pathway activation. These differences may be exploitable diagnostically and therapeutically for prion diseases, such as variant Creutzfeldt-Jakob disease.     

Immunotherapy as a therapeutic treatment for neurodegenerative disorders

Human neurodegenerative illnesses such as Alzheimer's disease and Creutzfeldt-Jakob disease exact an enormous cost on individuals, families and society. For these and related disorders, current treatment is largely symptomatic without influencing the underlying disease process. Until recently, the development of immunotherapeutic approaches to neurodegenerative disorders had been almost completely ignored despite growing successes against other non-infectious diseases such as cancer. However, since Schenk and colleagues described the antibody-mediated clearance of amyloid plaques in a transgenic mouse model of Alzheimer's disease, a number of studies have confirmed the feasibility of this strategy for several neurodegenerative disorders including Huntington's disease and prion diseases. These reports offer the exciting prospect that either the immune system or its derivative components can be harnessed to fight the misfolded and/or aggregated proteins that accumulate in many neurodegenerative illnesses. If the remarkable power of clonal expansion, specificity and efficiency of the immune system can successfully inactivate these abnormal proteins, real hope exists that effective immunotherapeutic treatments for neurodegenerative illnesses may be available in the near future.     

Primary structure of a glycosylated DNA-binding domain in human plasma fibronectin

The complete amino acid sequence of a DNA-binding domain isolated from human plasma fibronectin after limited trypsin digestion has been obtained. It contains 132 amino acids and one biantennary glycosyl unit at residue 104, for an estimated Mr of 16,931. The fragment can be purified by a two-step procedure consisting of DNA-affinity chromatography and reverse-phase high performance liquid chromatography. It can also be purified by heparin-affinity chromatography. The domain is unusual in its susceptibility to tryptic-like cleavages even by neutral or aromatic residue-specific proteases. It has no cysteine residues and is predicted to favor a beta-sheet structure by Chou and Fasman analysis. Based on this analysis we have proposed a model which exhibits a clustering of aromatic and basic residues, consistent with similar involvement of basic and aromatic residues in other DNA-binding proteins. The net charge of the domain at neutral pH (+1, without sialic acid) argues against a nonspecific charge interaction with polyanionic macromolecules such as DNA and heparin. Internal sequence repeats occur at intervals of 30, 60, and 90 residues, thus suggesting a maximum size for a repetitive building block which gave rise to this domain.