Protein S-nitrosylation: a physiological signal for neuronal nitric oxide.

Nitric oxide (NO) has been linked to numerous physiological and pathophysiological events that are not readily explained by the well established effects of NO on soluble guanylyl cyclase. Exogenous NO S-nitrosylates cysteine residues in proteins, but whether this is an important function of endogenous NO is unclear. Here, using a new proteomic approach, we identify a population of proteins that are endogenously S-nitrosylated, and demonstrate the loss of this modification in mice harbouring a genomic deletion of neuronal NO synthase (nNOS). Targets of NO include metabolic, structural and signalling proteins that may be effectors for neuronally generated NO. These findings establish protein S-nitrosylation as a physiological signalling mechanism for nNOS.

Haem oxygenase-1 prevents cell death by regulating cellular iron.

Haem oxygenase-1 (HO1) is a heat-shock protein that is induced by stressful stimuli. Here we demonstrate a cytoprotective role for HO1: cell death produced by serum deprivation, staurosporine or etoposide is markedly accentuated in cells from mice with a targeted deletion of the HO1 gene, and greatly reduced in cells that overexpress HO1. Iron efflux from cells is augmented by HO1 transfection and reduced in HO1-deficient fibroblasts. Iron accumulation in HO1-deficient cells explains their death: iron chelators protect HO1-deficient fibroblasts from cell death. Thus, cytoprotection by HO1 is attributable to its augmentation of iron efflux, reflecting a role for HO1 in modulating intracellular iron levels and regulating cell viability.

Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer's disease.

Amyloid precursor protein (APP) generates the beta-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer's disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimer's disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimer's disease.

Insulin restores neuronal nitric oxide synthase expression and function that is lost in diabetic gastropathy.

Gastrointestinal dysfunction is common in diabetic patients. In genetic (nonobese diabetic) and toxin-elicited (streptozotocin) models of diabetes in mice, we demonstrate defects in gastric emptying and nonadrenergic, noncholinergic relaxation of pyloric muscle, which resemble defects in mice harboring a deletion of the neuronal nitric oxide synthase gene (nNOS). The diabetic mice manifest pronounced reduction in pyloric nNOS protein and mRNA. The decline of nNOS in diabetic mice does not result from loss of myenteric neurons. nNOS expression and pyloric function are restored to normal levels by insulin treatment. Thus diabetic gastropathy in mice reflects an insulin-sensitive reversible loss of nNOS. In diabetic animals, delayed gastric emptying can be reversed with a phosphodiesterase inhibitor, sildenafil. These findings have implications for novel therapeutic approaches and may clarify the etiology of diabetic gastropathy.

Atypical neural messengers.

Notions of what constitutes a neurotransmitter have changed markedly with the advent in the past decade of synaptic molecules, which satisfy key neurotransmitter criteria but differ radically from classical transmitters. Thus, NO and carbon monoxide are neither stored in synaptic vesicles nor released by exocytosis. These gases do not act via traditional receptors on postsynaptic membranes. In addition, zinc, stored together with glutamate in synaptic vesicles, appears to act as an 'antagonist' co-transmitter at the NMDA receptor, and although localized exclusively to glia, D-serine fulfills most neurotransmitter criteria as an endogenous ligand for the 'glycine' site of NMDA receptors.

Novel neurotransmitters and their neuropsychiatric relevance.

OBJECTIVE: The purpose of this review is to integrate insights regarding novel neurotransmitters or neuromodulators of neuropsychiatric significance. METHOD: Evolving concepts of neurotransmitter criteria are reviewed in light of the unexpected properties displayed by recently identified transmitters. RESULTS: Classic criteria for transmitters were based on the properties of acetylcholine but were markedly revised with the recognition of the catecholamines, serotonin, gamma-aminobutyric acid (GABA), and other amino acid transmitters and neuropeptides. Nitric oxide and carbon monoxide are notably atypical, as they are not stored in synaptic vesicles, are not released by exocytosis, and do not act at postsynaptic membrane receptor proteins. D-Serine, recently appreciated as the endogenous ligand for the glycine site of the glutamate N-methyl-D-aspartate (NMDA) receptor, overturns fundamental axioms of biology as well as those of neuroscience. It is a D-amino acid, and it is synthesized and stored in glia rather than neurons. Released glutamate acts on receptors on the protoplasmic astrocytes closely apposed to the synapse to release D-serine, which coactivates postsynaptic NMDA receptors together with glutamate. D-Serine is formed by serine racemase, which directly converts L-serine to D-serine. Inhibitors of this enzyme should reduce NMDA neurotransmission and might be therapeutic in stroke and other conditions associated with glutamate excitotoxicity. CONCLUSIONS: The diversity of novel neurotransmitters and venues of their activity afford multiple opportunities for therapeutic intervention.

Amplifier design considerations for blood cell counter sampling probes.

Blood cell counters that operate on the Coulter principle of an electrical resistance change when a cell passes through a small sampling orifice are especially sensitive to electrical noise. The sampling probe is immersed in an electrolyte (isotonic diluent for blood cells), which in itself presents an electrochemically noisy environment. The probe, owing to its large size, acts as an antenna for environmental electrical noise up to 60 Hz. Additionally, the basic Coulter method for cell counting requires a dc potential across the sampling orifice electrodes contained within the probe. This potential produces electrolysis of the diluent and generation of gas bubbles at the electrode surfaces. Sampling (counting) time must be short (less than 30 seconds) to avoid sample heating and an intolerably high noise level as a consequence of ionic motion and gas bubble generation. The resistance change that takes place when a cell passes through the sampling orifice is only a small fraction of a percent, thus noise is a serious problem. Electric noise produces false counts and general degradation of counting function. This paper presents a discussion of a currently used method for signal acquisition and some of the problems encountered in the clinical laboratory. A novel alternative design has been implemented using integrated circuit components, which eliminates many of the problems associated with the use of small bench-type counters. Design philosophy is discussed in detail including presentation of the final circuitry developed. Performance characteristics of the signal acquisition circuitry are presented.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrofit designs for small bench-type blood cell counters.

This paper describes several retrofit designs to correct operational problems associated with small bench-type blood cell counters. Replacement electronic circuits as well as modifications to the vacuum systems are discussed.

Hand-held instrument for evaluation of breast engorgement.

A second-generation instrumentation system is described for obtaining qualitative (and inferred quantitative) measurements of breast engorgement in postpartum nursing mothers. It also has application in evaluating the extent of edema in tissue. The system uses an LVDT and associated electronics.

Instrumentation system for breast engorgement evaluation.

An instrumentation system is described for obtaining qualitative and quantitative measurements of breast engorgement in postpartum nursing mothers. The system uses an LVDT and associated electronics.

True constant-current stimulators for biomedical applications.

Several designs for true constant-current stimulators are described in this paper. These designs, which include both analog signal and pulse operation, center about the LM334 integrated circuit adjustable current source. Depending upon configuration, these circuits will drive resistive-component loads varying from 500 omega to 10 k omega at current levels up to 10 mA.

Compact anisotropic bone: elastic constants, in vitro aging effects and numerical results of a mathematical model.

This paper briefly examines the experimentally observed effects in, and symmetry arguments for, the constituents of bone which lead to a transversely isotropic configuration for the elastic behavior of bone. Based on this, a regime for computing the anisotropic elastic constants is considered in terms of ultrasonic transit time measurements. To extend the usefulness of ultrasonic techniques to an in vivo situation, a mathematical model for bone is developed in outline form and numerical results for the characteristic frequency equation discussed.

The mobility analog for modeling the intra-arterial pressure wave parameters.

To assist in the identification of physical/physiological parameters obtained from in vivo rat aortic artery dynamic pressure data, the natural (mobility) mechanical circuit model was constructed. The direct electrical analog of the model thus obtained was then analyzed using SPICE. The experimental data were obtained using a Multifunction Pressure Generator (MPG), appropriate pressure probes, and a high-speed video camera. Two 486 computers were used for system control and data recording and computation. Transfer functions in rational form of the ratio of the MPG input pressure (Pi) to the intra-arterial pressure (Po) were then generated in the s-domain. The mechanical circuit described by these rational functions was then constructed and transformed into its equivalent electrical model for analysis. On this basis, physiological pressures are represented by electrical currents, and volume flow rates by electrical voltages. The results obtained through steady-state (Bode plot) and transient analysis of the model developed suggest a compartmental model that explains the experimental observations. The mobility model is an improvement over previous models in that the mass element is referred to a single frame of reference, which agrees with the physical property that mass is a one-terminal device.

Orientation of Euglena gracilis by electromagnetic fields: theory and experiment.

Computer data derived from theoretical treatments of the orientation of ellipsoidal particles in alternating fields is compared with experimental data from microscopic studies on Euglena gracilis, and elongated free-living flagellate. Computed data based upon a theoretical treatment by SAITO, SCHWAN and SCHWARZ showed good agreement with experimental results, predicting the relationship between cellular orientation, the frequency of the impressed field, and the conductivity of the suspending medium.

A sampling and storage system for arbitrary biomedical waveforms.

This paper describes a microcontroller based system which can sample, monitor and store, and generate biomedical waveforms. The frequency of the output waveform is controllable. The inherent advantage of this system is the programming flexibility offered by the microcontroller. Applications include patient monitoring, test signal generator for repair and maintenance of EEG or ECG monitoring equipment, and a laboratory simulation device for student educational use.

Prosthetic communication device.

A code-generating and display system is described which permits an individual who has lost vocal voluntary motor ability to communicate. The subject sustained severe brain damage in an automobile accident which left him without the power of speech or any significant motor ability, although he is not by definition quadriplegic. He lacks reliable control of eye blink, and the oral movements associated with sucking and blowing are severely impaired; thus, the usual foundations upon which prosthetic devices for quadriplegic patients are designed were not applicable in this case. The subject is confined to a bed, wheelchair, or an exercise table. The system described here has provided the subject with a simple and reliable means of communicating with his family and it permits him to communicate with persons who do not know the Morse code.

Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites.

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.

Cephalosporin antibiotics accelerate gastric emptying in mice.

Gastroparesis is a common debilitating complication in many diabetic patients. While several drugs are available for gastroparesis, many patients are not adequately treated. Many patients do not respond to available drugs or appear to develop tachyphylaxis after an initial response. New agents are needed. Erythromycin is a macrolide antibiotic that accelerates gastric emptying through interaction with motilin receptors. Many antibiotics, like erythromycin itself, have significant gastrointestinal side effects. We investigated the ability of cephalosporin antibiotics to alter gastric emptying in mice by employing phenol red spectrophotometry to monitor gastric emptying. Our results indicate that several cephalosporin antibiotics, particularly cefazolin, accelerate gastric emptying. In some cases these drugs appear more efficacious than either erythromycin or metoclopramide. At very high doses, many drugs, including erythromycin, appear to delay gastric emptying. We hypothesize that the gastrointestinal side effects of nausea and vomiting may result from delayed gastric emptying occurring at high doses while lower doses are prokinetic in the stomach.