Effects of Oxygen on Nodule Physiology and Expression of Nodulins in Alfalfa1

Early nodulin 2 (ENOD2) transcripts and protein are specifically found in the inner cortex of legume nodules, a location that coincides with the site of a barrier to O2 diffusion. The extracellular glycoprotein that binds the monoclonal antibody MAC236 has also been localized to this site. Thus, it has been proposed that these proteins function in the regulation of nodule permeability to O2 diffusion. It would then be expected that the levels of ENOD2 mRNA/protein and MAC236 antigen would differ in nodules with different permeabilities to O2. We examined the expression of ENOD2 and other nodule-expressed genes in Rhizobium meliloti-induced alfalfa nodules grown under 8, 20, or 50% O2. Although there was a change in the amount of MAC236 glycoprotein, the levels of ENOD2 mRNA and protein did not differ significantly among nodules grown at the different [O2], suggesting that neither ENOD2 transcription nor synthesis is involved in the long-term regulation of nodule permeability. Moreover, although nodules from all treatments reduced their permeability to O2 as the partial pressure of O2 (pO2) was increased to 100%, the levels of extractable ENOD2 and MAC236 proteins did not differ from those measured at the growth pO2, further suggesting that if these proteins are involved in a short-term regulation of the diffusion barrier, they must be involved in a way that does not require increased transcription or protein synthesis.

Mapping the mouse genome: current status and future prospects.

The mouse is the best model system for the study of mammalian genetics and physiology. Because of the feasibility and importance of studying genetic crosses, the mouse genetic map has received tremendous attention in recent years. It currently contains over 14,000 genetically mapped markers, including 700 mutant loci, 3500 genes, and 6500 simple sequence length polymorphisms (SSLPs). The mutant loci and genes allow insights and correlations concerning physiology and development. The SSLPs provide highly polymorphic anchor points that allow inheritance to be traced in any cross and provide a scaffold for assembling physical maps. Adequate physical mapping resources--notably large-insert yeast artificial chromosome (YAC) libraries--are available to support positional cloning projects based on the genetic map, but a comprehensive physical map is still a few years away. Large-scale sequencing efforts have not yet begun in mouse, but comparative sequence analysis between mouse and human is likely to provide tremendous information about gene structure and regulation.

Cannabinoid CB1 receptors and ligands in vertebrate retina: Localization and function of an endogenous signaling system

CB1, a cannabinoid receptor enriched in neuronal tissue, was found in high concentration in retinas of rhesus monkey, mouse, rat, chick, goldfish, and tiger salamander by using a subtype-specific polyclonal antibody. Immunolabeling was detected in the two synaptic layers of the retina, the inner and outer plexiform layers, of all six species examined. In the outer plexiform layer, CB1 was located in and/or on cone pedicles and rod spherules. Labeling was detected in some amacrine cells of all species and in the ganglion cells and ganglion cell axons of all species except fish. In addition, sparse labeling was found in the inner and/or outer segments of the photoreceptors of monkey, mouse, rat, and chick. Using GC/MS to detect possible endogenous cannabinoids, we found 3 nmol of 2-arachidonylglycerol per g of tissue, but no anandamide was detectable. Cannabinoid receptor agonists induced a dramatic reduction in the amplitude of voltage-gated L-type calcium channel currents in identified retinal bipolar cells. The presence and distribution of the CB1 receptor, the large amounts of 2-arachidonylglycerol found, and the effects of cannabinoids on calcium channel activity in bipolar cells suggest a substantive role for an endogenous cannabinoid signaling system in retinal physiology, and perhaps vision in general.

Effective intercellular communication distances are determined by the relative time constants for cyto/chemokine secretion and diffusion

A cell’s ability to effectively communicate with a neighboring cell is essential for tissue function and ultimately for the organism to which it belongs. One important mode of intercellular communication is the release of soluble cyto- and chemokines. Once secreted, these signaling molecules diffuse through the surrounding medium and eventually bind to neighboring cell’s receptors whereby the signal is received. This mode of communication is governed both by physicochemical transport processes and cellular secretion rates, which in turn are determined by genetic and biochemical processes. The characteristics of transport processes have been known for some time, and information on the genetic and biochemical determinants of cellular function is rapidly growing. Simultaneous quantitative analysis of the two is required to systematically evaluate the nature and limitations of intercellular signaling. The present study uses a solitary cell model to estimate effective communication distances over which a single cell can meaningfully propagate a soluble signal. The analysis reveals that: (i) this process is governed by a single, key, dimensionless group that is a ratio of biological parameters and physicochemical determinants; (ii) this ratio has a maximal value; (iii) for realistic values of the parameters contained in this dimensionless group, it is estimated that the domain that a single cell can effectively communicate in is ≈250 μm in size; and (iv) the communication within this domain takes place in 10–30 minutes. These results have fundamental implications for interpretation of organ physiology and for engineering tissue function ex vivo.

The degree of nonlinearity and anisotropy of blood vessel elasticity

Blood vessel elasticity is important to physiology and clinical problems involving surgery, angioplasty, tissue remodeling, and tissue engineering. Nonlinearity in blood vessel elasticity in vivo is important to the formation of solitons in arterial pulse waves. It is well known that the stress–strain relationship of the blood vessel is nonlinear in general, but a controversy exists on how nonlinear it is in the physiological range. Another controversy is whether the vessel wall is biaxially isotropic. New data on canine aorta were obtained from a biaxial testing machine over a large range of finite strains referred to the zero-stress state. A new pseudo strain energy function is used to examine these questions critically. The stress–strain relationship derived from this function represents the sum of a linear stress–strain relationship and a definitely nonlinear relationship. This relationship fits the experimental data very well. With this strain energy function, we can define a parameter called the degree of nonlinearity, which represents the fraction of the nonlinear strain energy in the total strain energy per unit volume. We found that for the canine aorta, the degree of nonlinearity varies from 5% to 30%, depending on the magnitude of the strains in the physiological range. In the case of canine pulmonary artery in the arch region, Debes and Fung [Debes, J. C. & Fung, Y. C.(1995) Am. J. Physiol. 269, H433–H442] have shown that the linear regime of the stress–strain relationship extends from the zero-stress state to the homeostatic state and beyond. Both vessels, however, are anisotropic in both the linear and nonlinear regimes.

Effect of COMT Val108/158 Met genotype on frontal lobe function and risk for schizophrenia

Abnormalities of prefrontal cortical function are prominent features of schizophrenia and have been associated with genetic risk, suggesting that susceptibility genes for schizophrenia may impact on the molecular mechanisms of prefrontal function. A potential susceptibility mechanism involves regulation of prefrontal dopamine, which modulates the response of prefrontal neurons during working memory. We examined the relationship of a common functional polymorphism (Val108/158 Met) in the catechol-O-methyltransferase (COMT) gene, which accounts for a 4-fold variation in enzyme activity and dopamine catabolism, with both prefrontally mediated cognition and prefrontal cortical physiology. In 175 patients with schizophrenia, 219 unaffected siblings, and 55 controls, COMT genotype was related in allele dosage fashion to performance on the Wisconsin Card Sorting Test of executive cognition and explained 4% of variance (P = 0.001) in frequency of perseverative errors. Consistent with other evidence that dopamine enhances prefrontal neuronal function, the load of the low-activity Met allele predicted enhanced cognitive performance. We then examined the effect of COMT genotype on prefrontal physiology during a working memory task in three separate subgroups (n = 11–16) assayed with functional MRI. Met allele load consistently predicted a more efficient physiological response in prefrontal cortex. Finally, in a family-based association analysis of 104 trios, we found a significant increase in transmission of the Val allele to the schizophrenic offspring. These data suggest that the COMT Val allele, because it increases prefrontal dopamine catabolism, impairs prefrontal cognition and physiology, and by this mechanism slightly increases risk for schizophrenia.

Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na+] and pH but elevated viscosity

Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to be important in normal airway physiology and in the pathophysiology of cystic fibrosis (CF). An in situ fluorescence method was applied to measure the ionic composition and viscosity of freshly secreted fluid from airway glands. Fragments of human large airways obtained at the time of lung transplantation were mounted in a humidified perfusion chamber and the mucosal surface was covered by a thin layer of oil. Individual droplets of secreted fluid were microinjected with fluorescent indicators for measurement of [Na+], [Cl−], and pH by ratio imaging fluorescence microscopy and viscosity by fluorescence recovery after photobleaching. After carbachol stimulation, 0.1–0.5 μl of fluid accumulated in spherical droplets at gland orifices in ≈3–5 min. In gland fluid from normal human airways, [Na+] was 94 ± 8 mM, [Cl−] was 92 ± 12 mM, and pH was 6.97 ± 0.06 (SE, n = 7 humans, more than five glands studied per sample). Apparent fluid viscosity was 2.7 ± 0.3-fold greater than that of saline. Neither [Na+] nor pH differed in gland fluid from CF airways, but viscosity was significantly elevated by ≈2-fold compared to normal airways. These results represent the first direct measurements of ionic composition and viscosity in uncontaminated human gland secretions and indicate similar [Na+], [Cl−], and pH to that in the airway surface liquid. The elevated gland fluid viscosity in CF may be an important factor promoting bacterial colonization and airway disease.

Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease.