Right atrial and right ventricular transmural pressures in dogs and humans. Effects of the pericardium

BACKGROUND: To determine the transmural pressure-dimension relations of the right atrium (RA) and right ventricle (RV) before and after pericardiectomy, six open-chest dogs were instrumented with pericardial balloons placed over the RA and RV free walls. METHODS AND RESULTS: PA appendage dimensions and RV free-wall segment lengths were measured using sonomicrometry. Intact-pericardium RA and RV transmural pressures were calculated by subtracting the pericardial pressures (measured using balloons) from the cavitary pressures. Pooled data from six animals with pericardium intact indicate that at RA and RV cavitary pressures of 5, 10, and 15 mm Hg, RV pericardial pressure was 4.3 +/- 0.3, 8.6 +/- 1.0, and 13.3 +/- 1.5 mm Hg, respectively, and RA pericardial pressure was 4.8 +/- 0.3, 9.6 +/- 0.6, and 14.6 +/- 0.6 mm Hg, respectively (mean +/- SD). With calculated unstressed dimensions, the cavity dimension data were normalized to strain (in percent). We determined that in the dog, RV strain would increase by 14% and RA by 68% to maintain cavitary pressure at 10 mm Hg on pericardiectomy. To compare these results with clinical data, RV (n = 7) and RA (n = 6) transmural pressures were measured using balloons in patients (age, 19 to 76 years) undergoing cardiac surgery. RA transmural pressure of six patients was 1.0 +/- 1.5 mm Hg when central venous pressures (CVPs) ranged from 3 to 16 mm Hg. RV transmural pressure equaled 1.2 +/- 1.9, 2.3 +/- 1.9, and 3.4 +/- 2.0 mm Hg when CVP was 5, 10, and 15 mm Hg, respectively. CONCLUSIONS: Pericardial constraint (as evaluated by the ratio of pericardial to intracavitary pressures when CVP is 10 mm Hg) accounted for 96% of RA cavitary pressure in the dog and 89% in humans and at least 86% of RV cavitary pressure in the dog and 77% in humans.     

Profiling signaling peptides in single mammalian cells using mass spectrometry

The peptide content of individual mammalian cells is profiled using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Both enzymatic and nonenzymatic procedures, including a glycerol cell stabilization method, are reported for the isolation of individual mammalian cells in a manner compatible with MALDI MS measurements. Guided microdeposition of MALDI matrix allows samples to be created with suitable analyte-to-matrix ratios. More than 15 peptides are observed in individual rat intermediate pituitary cells. The combination of accurate mass data, expected cleavages by proteolytic enzymes, and postsource decay sequencing allows identification of 14 of these peptides as pro-opiomelanocortin prohormone-derived molecules. These protocols permit the classification of individual mammalian cells by peptide profile, the elucidation of cell-specific prohormone processing, and the discovery of new signaling peptides on a cell-to-cell basis in a wide variety of mammalian cell types.     

Discovering new invertebrate neuropeptides using mass spectrometry

Neuropeptides are a complex set of messenger molecules controlling a wide array of regulatory functions and behaviors within an organism. These neuromodulators are cleaved from longer protein molecules and often experience numerous post-translational modifications to achieve their bioactive form. As a result of this complexity, sensitive and versatile analysis schemes are needed to characterize neuropeptides. Mass spectrometry (MS) through a variety of approaches has fueled the discovery of hundreds of neuropeptides in invertebrate species in the last decade. Particularly successful are direct tissue and single neuron analyses by matrix-assisted laser desorption/ionization (MALDI) MS, which has been used to elucidate approximately 440 neuropeptides, and examination of neuronal homogenates by electrospray ionization techniques (ESI), also leading to the characterization of over 450 peptides. Additional MS methods with great promise for the discovery of neuropeptides are MS imaging and large-scale peptidomics studies in combination with a sequenced genome.     

载货汽车悬架系统频率响应仿真

悬架系统是汽车底盘的重要部件,它的设计参数直接关系到载货汽车行驶的平顺性和操纵稳定性.为了对载货汽车行驶平顺性进行科学评价,建立了载货汽车1/2车辆五自由度仿真模型,采用频域分析法、以1/3倍频带加速度均方根值作为评价指标、用Matlab编制了随机路面输入下的悬架系统性能仿真程序,通过仿真得到空载和满载情况下加速度功率谱及加速度均方根值仿真曲线田,较好地反映了载货汽车行驶平顺性的实际状况.所开发的仿真程序可用于对汽车悬架系统进行平顺性评估以及后续的参数优化设计.     

One-step sampling, extraction, and storage protocol for peptidomics using dihydroxybenzoic Acid

The isolation and extraction of natively occurring signaling peptides (SPs) from tissue is a critical first step in characterizing these peptides. Recent studies have outlined several approaches designed to preserve and extract SPs from tissue. Here, we demonstrate a surprisingly simple method to extract SPs from tissue samples, ranging from cell clusters to brain punches to intact brain regions, using a matrix often employed in matrix-assisted laser desorption/ionization mass spectrometry-2,5-dihydroxybenzoic acid (DHB). DHB allows for the effective extraction of endogenous peptides from tissue as well as long-term preservation of tissue samples and extracts. Using the mouse pituitary gland as a model, the extraction protocol effectively recovers 24 known and many additional putative peptides from individual samples. Peptide extracts stored in the DHB extraction media are stable for years without freezing. The approach is also effective for other neuronal tissues; the complement of neuropeptides in bag cell neuron clusters from the Aplysia central nervous system, the rat cerebellum, and rat dorsal striatum also have been examined. Advantages of this new extraction procedure are its technical simplicity, reproducibility, ease of remote preparation of samples, and long-term sample preservation without freezing.     

Single-neuron analysis using CE combined with MALDI MS and radionuclide detection

Capillary electrophoresis (CE) has been combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and radionuclide detection to assay mass-limited biological samples. Nanovial sampling techniques enable injections into the CE capillary from 50 to 150-nL volume samples; after the separation, nanoliter fraction collection combines the CE effluent with a MALDI matrix and minimizes sample spreading, thus allowing both MALDI MS and radionuclide detection on the CE fractions. MALDI MS complements the elution time information of CE by providing accurate molecular mass data, and radionuclide detection provides zeptomole limits of detection with quantitative information. While MALDI MS detects all fully processed peptides at sufficient concentration, culturing the neuron in media containing 35S-Met provides selective radionuclide detection of newly synthesized methionine-containing peptides. The analysis and detection of the expected neuropeptides and hormones in a single 40-microm bag cell neuron from Aplysia californica with CE/MALDI MS/radionuclide detection demonstrates the ability of this hyphenated approach to work with chemically complex mass-limited samples.     

Collection of peptides released from single neurons with particle-embedded monolithic capillaries followed by detection with matrix-assisted laser desorption/ionization mass spectrometry

Characterization of the stimulated release of neuropeptides from brain slices and individual cultured neurons requires efficient collection of the releasate from relatively large volumes of physiological saline. Here, several collection approaches are optimized using particle-embedded monolithic capillaries (PEMCs) with poly(stearyl methacrylate-co-ethylene glycol dimethacrylate) monolith acting as a "glue". Two distinct extraction particles, with either pyrrolidone (PY) or ethylenediamine (EDA) as the functional group on polystyrene backbone, have been embedded into capillaries having an inner diameter of 250 μm. The capillaries act as collection devices for sampling neuropeptide release; the collection protocols are described, and the extraction efficiency of the probes are characterized. Specifically, the binding of angiotensin II from a peptide mixture onto the PY and EDA columns was 16 and 28 pmol, respectively, in a volume of 20 μL of saline. The peptides released from these columns have been characterized via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with low femtomole detection limits. When the PEMC columns were positioned in close proximity to individual neurons and 50 mM KCl was used as the secretagogue, peptides released from individual identified cultured neurons isolated from Aplysia californica were collected and characterized.     

trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP

Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.