Role of Shp2 in forebrain neurons in regulating metabolic and cardiovascular functions and responses to leptin.

OBJECTIVE: We examined whether deficiency of Src homology 2 containing phosphatase (Shp2) signaling in forebrain neurons alters metabolic and cardiovascular regulation under various conditions and if it attenuates the anorexic and cardiovascular effects of leptin. We also tested whether forebrain Shp2 deficiency alters blood pressure (BP) and heart rate (HR) responses to acute stress. DESIGN: Forebrain Shp2(-/-) mice were generated by crossing Shp2(flox/flox) mice with CamKIIα-cre mice. At 22-24 weeks of age, the mice were instrumented for telemetry for measurement of BP, HR and body temperature (BT). Oxygen consumption (VO2), energy expenditure and motor activity were monitored by indirect calorimetry. RESULTS: Shp2/CamKIIα-cre mice were heavier (46±3 vs 32±1 g), hyperglycemic, hyperleptinemic, hyperinsulinemic and hyperphagic compared to Shp2(flox/flox) control mice. Shp2/CamKIIα-cre mice exhibited reduced food intake responses to fasting/refeeding and impaired regulation of BT when exposed to 15 and 30 °C ambient temperatures. Despite being obese and having many features of metabolic syndrome, Shp2/CamKIIα-cre mice had similar daily average BP and HR compared to Shp2(flox/flox) mice (112±2 vs 113±1 mm Hg and 595±34 vs 650±40 b.p.m.), but exhibited increased BP and HR responses to cold exposure and acute air-jet stress test. Leptin's ability to reduce food intake and to raise BP were markedly attenuated in Shp2/CamKIIα-cre mice. CONCLUSION: These results suggest that forebrain Shp2 signaling regulates food intake, appetite responses to caloric deprivation and thermogenic control of body temperature during variations in ambient temperature. Deficiency of Shp2 signaling in the forebrain is associated with augmented cardiovascular responses to cold and acute stress but attenuated BP responses to leptin.

A conjugate of alpha-amanitin and monoclonal immunoglobulin G to Thy 1.2 antigen is selectively toxic to T lymphoma cells.

A covalent conjugate of an alpha-amanitin azo derivative and a monoclonal immunoglobulin G to the Thy 1.2 antigen on murine T lymphocytes was synthesized. The conjugate was 375- to 750-fold more inhibitory to murine T lymphoma S49.1 cells than the unconjugated derivative. At 0.7 X 10(-7) to 1.5 X 10(-7) M and at 4 X 10(-7) M amanitin equivalents, the conjugate inhibited protein synthesis in S49.1 cells by 50 percent and 80 to 96 percent, respectively. At these concentrations, mutant Thy l-deficient S49 cells and other murine lymphoma lacking Thy l altogether or carrying Thy 1.1 antigens were unaffected. This result demonstrated the potential for targeting amanitin to specific cell types.

Does the serum peptidome reveal hemostatic dysregulation?

There is a significant need for markers that are diagnostic of disease, particularly cancer. For these biomarkers to be useful they would need to be able to detect disease early in its progression with high sensitivity and specificity. Many approaches are being undertaken to attempt to find such biomarkers using the tools of systems biology, i.e., parallel measurement techniques including proteomics (parallel protein measurements). Often the premise behind such an approach was to cast a wide net and then design an assay for specific elements that were found to be diagnostic. One such approach has utilized matrix-assisted laser desorption/ionization-mass spectrometry to interrogate the low-molecular-weight component of serum (the fluid component of blood following clotting), the serum peptidome. This approach has the appealing characteristic of speed of analysis but has a number of shortcomings mostly due to signal:noise and mass resolution in some instruments, making peak analysis difficult. Of course, experimental design and statistical analysis have to be conducted with the system limitations in mind. These points have been addressed by others, but few have focused on a potentially larger issue with serum peptidome analysis - are the signals being measured informing us about the disease state directly or indirectly through measurement of another physiological process such as hemostatic dysregulation? This article will present evidence that points to careful measures of the serum peptidome revealing differences in clotting time in disease states and not direct measures of tumor proteolytic activity on blood proteins.

Analysis of peptide mixtures by capillary high performance liquid chromatography: a practical guide to small-scale separations.

Capillary HPLC is a very effective means of separating small amounts of peptides and proteins. Capillary columns ranging from 0.01 mm to 0.5 mm in diameter can be constructed using recycled supports and inexpensive fused silica capillary tubing. Commercial pumping systems and UV detectors can be readily converted for operation in the flow rate range of 0.5-50 microL/min. Detailed procedures are given for the construction of columns and UV detector flow cells. A mixture of peptides derived from the endo Lys C digest of horse heart cytochrome c was used to illustrate various aspects of capillary chromatography of peptides and compares the performance of various-sized capillary columns and UV detector flow cell types.

Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer.

A methodology has been developed for the rapid identification of gel separated proteins. Following in gel protein digestion with trypsin, the resulting peptide mixture is analyzed by on-line liquid chromatography, electrospray mass spectrometry (LC/MS). The mass spectral data containing either accurate mass values or sequence specific fragment ion information is then matched to a database of known protein sequences. Key features of the LC/MS system are the use of a novel integrated, microscale LC column-electrospray interface and variable flow solvent delivery to optimize the efficiency of sample loading and gradient elution. With these enhancements, only 10 min is required to analyze each sample. The method is routine for sample amounts ranging from 50 to 500 fmol. The analysis parameters for the ion trap mass spectrometer have to be carefully adjusted in order to keep pace with the rapidly eluting LC peaks. Although designed for rapid LC separations, the integrated column-electrospray interface is also able to provide extended analyses of selected components using a technique known as "peak parking".

Low flow high-performance liquid chromatography solvent delivery system designed for tandem capillary liquid chromatography-mass spectrometry.

A solvent delivery system is described that is designed to increase the efficiency of liquid chromatography-mass spectrometry (LC/MS) analyses. Gradients formed by using two low pressure syringe pumps are stored in a length of narrow bore tubing (gradient loop) mounted on a standard high pressure switching valve. The preformed gradient is pushed through the column by using a high pressure syringe pump. The system is fully automated and can be controlled with either a personal computer or the mass spectrometer data system. Advantages include gradient operation without the use of split flows, pressure programed flow control for rapid sample loading and recycling to initial conditions, and a flow rate range of 0.1-20 µL/min, which is suitable for packed capillary columns 50-500 µm in diameter. The system has been used extensively for rapid molecular weight determinations of intact protein samples, as well as LC/MS and liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures.

Data-controlled automation of liquid chromatography/tandem mass spectrometry analysis of peptide mixtures.

The structural characterization of proteins and peptides isolated in minute quantities requires the most efficient use of available sample. A mass spectrometer data system was programmed to continuously evaluate incoming liquid chromatography/mass spectrometry data against a user-defined array of information. The resulting conclusions were used to automatically set and modify acquisition parameters in real time to collect collision-induced dissociation spectra for selected ions (tandem mass spectrometry). This approach has provided a mechanism to target specific subsets of masses in a complex mixture and/or to discriminate selectively against masses that are known or not of interest. Masses of contaminants or peptide masses derived from known proteins can be automatically recorded and removed from further consideration for collision-induced dissociation analysis. Once recorded, these "libraries" of masses can be used across multiple analyses. This technique directs the mass spectrometer data system to focus on the analysis of masses significant to the user, even if their signal intensities are well below the intensities of contaminating masses. When combined with a database search program to correlate tandem mass spectra to known protein sequences, the identity of the protein can be established unequivocally by using less than 100 fmol of sample.

Degradation of vasoactive intestinal polypeptide by rabbit gastric smooth muscle membranes.

Crude membrane fractions prepared from rabbit gastric fundic muscle degraded vasoactive intestinal polypeptide (VIP) with an average specific activity of 0.96 nmol/min/mg protein at 37 degrees C, pH 7.5, and at [S]o = 0.05 mM. The relative activities towards [Leu5]enkephalin, substance P, VIP, and neurotensin were approximately 7.7, 2.0, 1.0, and 0.54, respectively. The VIP degradation was inhibited by metal chelators EDTA and o-phenanthroline. CaCl2 at 0.3-1.0 mM enhanced VIP degradation up to twofold. Phosphoramidon, captopril, and bestatin, the specific inhibitors for endopeptidase-24.11, angiotensin-converting enzyme, and aminopeptidase M, respectively, did not affect VIP degradation significantly. However, the complex mixtures of VIP fragments generated implicates action of multiple peptidases including the aforementioned three peptidases and other unidentified peptidase(s).

Structural characterization of calcitonin gene-related peptide purified from rabbit intestine.

Calcitonin gene-related peptide (CGRP) immunoreactive material has been found in extracts of the intestine, however, the structure of intestinal CGRP is not known. Analytical reverse phase HPLC and ion-exchange FPLC revealed one predominant immunoreactive CGRP peak in rabbit intestinal extracts. This material was purified from rabbit intestine by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence and mass spectral analysis of the purified peptide and its chymotryptic fragments were consistent with the structure: GCNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSEAF-amide. Rabbit intestinal CGRP is identical to human CGRP-II in 35 of 37 amino acid residues. Two amino acid differences were detected at position 1, with Gly in rabbit CGRP instead of Ala in human CGRP-II, and at position 35, with Glu instead of Lys, respectively. Rabbit CGRP differed from human CGRP-I by three additional amino acids at positions 3, 22, and 25. This report shows that a CGRP form which closely resembles human CGRP-II, by means of chemical characterization, is the predominant form in rabbit intestine. Rabbit CGRP is the only CGRP form which has Gly as the amino terminal amino acid. Since the amino terminus of CGRP seems to be important for expression of bioactivity, the biological activity of rabbit CGRP may differ from human, rat and porcine CGRP.

Neuropeptide Y 3-36 is an endogenous ligand selective for Y2 receptors.

Neuropeptide Y (NPY 1-36) binds to Y1 and Y2 receptors with similar affinity. No endogenous molecular form of NPY with selectivity for Y1 or Y2 receptors has been described so far. We report the presence of an endogenous fragment of NPY in porcine brain, NPY 3-36, which lacks the amino-terminal dipeptide Tyr-Pro of NPY 1-36. NPY 3-36 accounts for 35% of NPY-like immunoreactivity in porcine brain. We have compared binding of NPY 3-36 and NPY 1-36 in model systems of Y1-like (SK-N-MC cells) and Y2-like receptors (CHP234 cells). NPY 3-36 and NPY 1-36 had similarly high affinity for Y2-like receptors on CHP234 cells, but NPY 3-36 had a 1000-fold lower affinity than NPY 1-36 for Y1-like receptors on SK-N-MC cells. Thus amino-terminal cleavage of NPY 1-36 generating NPY 3-36 converts an unselective Y1/Y2 receptor ligand into a highly Y2 selective ligand. This may be a means of fine tuning NPY biological actions.

Incorporation of deoxycytidine into deoxyribonucleic acid deoxycytidylate in Lactobacillus acidophilus R-26.

Lactobacillus acidophilus R-26 a strain deficient in ribonucleotide reductase, was grown with [G-14C]deoxycytidine as the only source of deoxyribose in the medium. Of the radioactivity incorporated into deoxyribonucleic acid, a fifth moved directly into deoxyribonucleic acid deoxycytidylate, without deamination. Furthermore, deoxycytidine and thymidine nucleotides had similar sugar/base ratios, suggesting a direct conversion of deoxycytidine nucleotides to thymidine nucleotides through deamination, without further dilution by glycosyl transfer. Although radioactivity was incorporated into both the sugar and base moieties of deoxyribonucleic acid pyrimidine deoxyribonucleotides, only the sugar moiety of purine deoxyribonucleotides was labeled. Purine deoxyribonucleotides probably were synthesized by glycosyl transfer from [G-14C]deoxycytidine to unlabeled purines, followed by phosphorylation of the deoxynucleotides.

The identification of peptide modifications derived from gel-separated proteins using electrospray triple quadrupole and ion trap analyses.

Microspray tandem mass spectrometry (MS/MS) in combination with database search routines has become a powerful tool for the identification of proteins from femtomole amounts of material following gel electrophoresis and in-gel digestion procedures. However, artifactual modification of susceptible residues can arise during gel electrophoresis, leading to unexpected peptide mass shifts during mass analysis. Consequently, collision-induced dissociation (CID) spectra generated from these derivatized peptides can defy direct interpretation by automated database search routines and remain unidentified. Here, we evaluate the MS/MS spectra of peptides carrying oxidized derivatives of tryptophane and methionine residues, and various modifications of cysteine. We demonstrate that certain of these modifications generate characteristic fragmentation patterns or "fingerprints", during CID analysis, the knowledge of which can facilitate the interpretation of the spectra. We will show that these signature fragment ions are predominantly produced during the CID analysis of singly charged ions although they can be observed in the MS/MS spectra of the doubly charged species as well. In other cases, the CID spectrum lacks a characteristic fingerprint and the modification remains silent. However, CID spectra of related peptides, differing only by their modifications, are similar and all or part of the fragment ion spectra will have shifted by a discreet mass, which facilitates the identification of the modified residue. At the same time, the comparison of related spectra can prevent misinterpretations such as the assignment of a residue mass to the wrong amino acid or a neutral loss fragment ion to a gamma- or b-ion.

Microscale immobilized protease reactor columns for peptide mapping by liquid chromatography/mass spectral analyses.

Methods and procedures for the construction and operation of a microscale immobilized protease (trypsin) reactor are described. Optimization of reactor efficiency with regard to reactor flowrate, length, and temperature as well as sample concentration has been examined, and a novel system for sample preconcentration is presented. The structural analysis of a standard protein (cytochrome c) by a combination of trypsin microreactor digestion followed by off-line electrospray ionization MS analysis is demonstrated at the 10-pmol level. The application of these techniques for the rapid confirmation (1-2 h) of a genetic variant (hemoglobin E) including MS/MS analysis of the variant peptide is included.

Molecular cloning of the pheromone biosynthesis-activating neuropeptide in Helicoverpa zea.

Pheromone biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone biosynthesis in female Helicoverpa (Heliothis) zea. Two oligonucleotide probes representing two overlapping amino acid regions of PBAN were used to screen 2.5 x 10(5) recombinant plaques, and a positive recombinant clone was isolated. Sequence analysis of the isolated clone showed that the PBAN gene is interrupted after the codon encoding amino acid 14 by a 0.63-kilobase (kb) intron. Preceding the PBAN amino acid sequence is a 10-amino acid sequence containing a pentapeptide Phe-Thr-Pro-Arg-Leu, which is followed by a Gly-Arg-Arg processing site. Immediately after the PBAN amino acid sequence is a Gly-Arg processing site and a short stretch of 10 amino acids. This 10-amino acid sequence contains a repeat of the PBAN C-terminal pentapeptide Phe-Ser-Pro-Arg-Leu and is terminated by another Gly-Arg processing site. It is suggested that the PBAN gene in H. zea might carry, besides PBAN, a 7- and an 8-residue amidated peptide, which share with PBAN the core C-terminal pentapeptide Phe-(Ser or Thr)-Pro-Arg-Leu-NH2. The C-terminal pentapeptide sequence of PBAN represents the minimum sequence required for pheromonotropic activity in H. zea and also bears a high degree of homology to the pyrokinin family of insect peptides with myotropic activity. It is possible that the putative heptapeptide and octapeptide might be new members of the pyrokinin family, with pheromonotropic and/or myotropic activities. Thus, the PBAN gene products, besides affecting sexual behavior, might have broad influence on many biological processes in H. zea.