Isolation of two distinct type I angiotensin II receptor genes.

A rat genomic Southern blot, probed with a type I angiotensin II receptor probe, demonstrated that two highly homologous type I angiotensin II receptors were present. A rat genomic library was subsequently screened and four clones were isolated. From restriction mapping, differential hybridization, polymerase chain reaction amplification and sequence analyses we have determined that there are two unique type I angiotensin II receptor genes. The first of these genes corresponds to the published rat vascular complementary DNA sequence; the second, corresponds to a novel receptor not previously described.     

Study of water and oil diffusion in rape seeds with sorption-desorption and NMR techniques.

The sorption and desorption of water in rape seeds was measured. From the sorption isotherm it follows that for water content greater than about 6% the water molecules tend to form clusters. The mutual diffusion coefficient of water into and out of the seeds was determined from the time dependence of sorption and desorption. There is a pronounced hysteresis in the sorption-desorption process, desorption proceeds faster than sorption. The self-diffusion of water (at maximum humidity of the seeds) and oil within the seeds was investigated by the pulsed field gradient NMR. The measurement of oil self-diffusion shows restricted diffusion of the oil within droplets and allows the determination of the droplet radii and their distribution width.     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

No evidence for ‘skewed’ inactivation of the X-chromosome as cause of Leber's hereditary optic neuropathy in female carriers

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder of the optic nerves. It has been proposed that the specific mutations in the mitochondrial DNA (mtDNA) that are associated with LHON require and X-chromosomally encoded permissive factor in order to become expressed. This would explain both the preponderance of male patients and the fact that most carriers of specific mtDNA mutations remain unaffected. Although linkage studies have been negative so far, the existence of such a factor has not been ruled out. We investigated the genealogical data of 24 large LHON pedigrees and concluded that the presumed X-linked factor would be recessively inherited and that at least 57% of the affected females would be heterozygous. Therefore, these females must be the victim of nonrandom X-chromosomal inactivation (skewed lyonization). However, analysis of X-chromosomal methylation patterns in 16 LHON-affected females revealed substantial skewing in only 15%–20% of cases, which is not significantly different from the patterns in 49 controls. Moreover, we found the frequency of LHON in daughters of affected heterozygous females to be twice to three times as high as in daughters of unaffected heterozygous females, which cannot be explained by an X-chromosomally inherited factor. We conclude that the results of our investigations do not support the hypothesis that LHON is a digenic disease with an X-linked factor being the main cause of loss of vision in the presence of relevant mtDNA mutations. Received: 1 June 1995 / Revised: 20 September 1995     

Effects of elevated CO2 and increased nitrogen deposition on photosynthesis and growth of understory plants in spruce model ecosystems

We studied the effects of atmospheric CO₂ enrichment (280, 420 and 560 l CO₂ l^–1) and increased N deposition (0,30 and 90 kg ha^–1 year^–1) on the spruce-forest understory species Oxalis acetosella, Homogyne alpina and Rubus hirtus. Clones of these species formed the ground cover in nine 0.7 m² model ecosystems with 5-year-old Picea abies trees (leaf area index of approx 2.2). Communities grew on natural forest soil in a simulated montane climate. Independently of N deposition, the rate of light-saturated net photosynthesis of leaves grown and measured at 420 l CO₂ l^–1 was higher in Oxalis and in Homogyne, but was not significantly different in Rubus compared to leaves grown and measured at the pre-industrial CO₂ concentration of 280 l l^–1. Remarkably, further CO₂ enrichment to 560 l l^–1 caused no additional increase of CO₂ uptake. With increasing CO₂ supply concentrations of non-structural carbohydrates in leaves increased and N concentrations decreased in all species, whereas N deposition had no significant effect on these traits. Above-ground biomass and leaf area production were not significantly affected by elevated CO₂ in the more vigorously growing species O. acetosella and R. hirtus, but the slow growing H. alpina produced almost twice as much biomass and 50% more leaf area per plant under 420 l CO₂ l^–1 compared to 280 l l^–1 (again no further stimulation at 560 l l^–1). In contrast, increased N addition stimulated growth in Oxalis and Rubus but had no effect on Homogyne. In Oxalis (only) biomass per plant was positively correlated with microhabitat quantum flux density at low CO₂, but not at high CO₂ indicating carbon saturation. On the other hand, the less shade-tolerant Homogyne profited from CO₂ enrichment at all understory light levels facilitating its spread into more shady micro-habitats under elevated CO₂. These species-specific responses to CO₂ and N deposition will affect community structure. The non-linear responses to elevated CO₂ of several of the traits studied here suggest that the largest responses to rising atmospheric CO₂ are under way now or have already occurred and possible future responses to further increases in CO₂ concentration are likely to be much smaller in these understory species.