Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.     

Effect of phosphorus supply to the surface roots of wheat on root extension and rhizosphere chemistry in an acidic subsoil

Seedlings of two cultivars of wheat (Triticum aestivum L.) differing in tolerance to aluminium (Al) were grown using a split-root sand/soil culture technique. Each culture tube was divided horizontally into a surface (0–150 mm) compartment and a subsurface (150–250 mm) compartment separated by a root-permeable paraffin wax barrier. Thus phosphorus (P) supplied to surface roots could not percolate or diffuse into the soil in the subsurface compartment. The soil in the subsurface compartment was divided into ‘rhizosphere’ and ‘non-rhizosphere’ zones using a porous (5 μm) membrane. Root growth of both cultivars into the subsurface zone was enhanced by increased P supply to surface roots, but did not conform to known relationships between root growth and soil pH, extractable-Al, or pH, Al or P concentrations in soil solution. Concentrations of Al in soil solution in the rhizosphere were greater than those in solution in the bulk soil. Concentrations of Al reactive with pyrocatechol violet (30s-RRAI) in the rhizosphere soil solution were generally greater than those in non-rhizosphere soil. With the Al-sensitive cultivar, root dry weight and length increased as concentrations of RRAl in the rhizosphere soil solution increased. Increased concentrations of Al in rhizosphere soil solutions were not related to the presence of organic ligands in solution. The effect of P in promoting root penetration into the acidic subsurface stratum was not related to differential attainment of maturity by the plant shoots, but appeared to be related to the effect of P in enhancing the rate of root growth. Thus, suboptimal supply of P to the surface roots of a plant, even at levels sufficient to preclude development of nutritional (P) stress symptoms, may seriously reduce tolerance to Al, and hence diminish the ability of roots to penetrate into acidic subsoils.     

Acquisition of phosphorus and copper by VA-mycorrhizal hyphae and root-to-shoot transport in white clover

White clover (Trifolium repens L.) plants were grown in a calcareous soil in pots with three compartments, a central one for root growth and two outer ones for growth of vesicular-arbuscular (VA) mycorrhizal (Glomus mosseae [Nicol. & Gerd.] Gerdemann & Trappe) hyphae (hyphal compartments). Phosphorus (P) was applied at three levels (0, 20 and 50 mg kg⁻¹ soil) in the outer compartments in mycorrhizal treatments. Root and shoot dry weight were increased in mycorrhizal plants with hyphal access to outer compartments. Growth of the mycorrhizal hyphae in the outer compartments was not significantly affected by variation in P level in these compartments. However, both concentration and amount of P in roots and shoots sharply increased with increasing P supply in the outer (hyphal) compartments. With increasing P levels the calculated delivery of P by the hyphae from the outer compartments increased from 34% to 90% of total P uptake. Hyphal access to the outer compartments also significantly increased both concentration and quantity of Cu in the plants. The calculated delivery of Cu by the hyphae from the outer compartments ranged from 53% to 62% of total Cu uptake, irrespective of the P levels and the amounts of P taken up and transported by the hyphae. However, the distribution of Cu over roots and shoots was largely dependent on P levels. With increase in P level in the outer compartments the calculated hyphal contribution to the total amount of Cu in the shoots increased from 12% to 58%, but decreased in the roots from 75% to 46%. In conclusion, uptake and transport by VA-mycorrhizal hyphae may contribute substantially not only to P nutrition, but also to Cu nutrition of the host.     

Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Lithium treatment of affective disorders: effects of lithium on the inositol phospholipid and cyclic AMP signalling pathways

The effects of lithium (Li⁺) on the adenylyl cyclase and inositol phospholipid receptor signalling pathways were compared directly in noradrenergic and carbachol stimulated rat brain cortical tissue slices. Li⁺ was a comparatively weak inhibitor of noradrenaline-stimulated cyclic AMP accumulation with an IC₅₀ of approx. 20 mM. By contrast, half-maximal effects of Li⁺ on inositol monophosphate (InsP) accumulation in [³H]inositol labelled tissue slices occurred at about 1 mM. A similar IC₅₀ for Li⁺ of about 1 mM was also obtained for noradrenaline-stimulated accumulation of CMP-phosphatidate (CMPPA), a sensitive indicator of intracellular inositol depletion, in tissue slices that had been prelabelled with [³H]cytidine. The effect of myo-inositol (inositol) depletion on the prolonged activity of phosphoinositidase C (PIC) was examined in carbachol-stimulated corticol slices using a novel mass assay fro InsP. Exposure to a maximal dose of carbachol for 30 min in the presence of 5 mM Li⁺ caused a 10-fold increase in the level of radioactivity associated with the InsP fraction, but only a 2-fold increase in InsP mass. During prolonged incubations in the presence of both carbachol and Li⁺ the accumulation of InsP mass was enhanced if 30 mM inositol was included in the medium. The results are comptable with the inositol depletion hypothesis of Li⁺ action but do not support the concept that adenylyl cyclase or guanine nucleotide dependent proteins represent therapeutically relevant targets of this drug.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

Experimental effects of elevated salinity on three benthic invertebrates in Pyramid Lake,Nevada

Salinity of Pyramid Lake increased from 3.7 to 5.5 between 1933 and 1980. Concern over future reductions in overall species richness prompted experiments to assess responses of dominant lake organisms to elevated salinity. Salinity tolerances of three important benthic invertebrates, Hyalella aztecta, Chironomus utahensis, and Heterocypris sp., were tested in controlled laboratory bioassays and also in a semi-natural environment consisting of large (47 m³) mesocosms.Densities of H. azteca in mesocosms were significantly lower at salinities of 8.0 and 11.0 compared with 5.6 controls in year one, but not in 8.5 salinity mesocosms in year two. The 96-h LC₅₀ for H. azteca was high at 19.5. Short-term mortalities of C. utahensis were 100% at salinities of 13.3 and greater. Fifty-seven percent fewer larvae matured from third to fourth instar at 8.9 than at 5.5 salinity in 17 day subacute bioassays. Furthermore, larval chironomid densities and emergence of adults from mesocosms were significantly reduced at salinities of 8.0 and higher compared with controls. Mortality of Heterocypris sp. was 50% at a salinity of 18.6 in laboratory bioassays and populations in mesocosms ranged between 40 and 100% lower at salinities of 8.0 and 11.0 than in controls.Multiple generation mesocosm experiments indicated all three invertebrates were more sensitive to elevated salinity than results of short-term bioassays. Our studies suggest populations of these invertebrates may be reduced from present levels if Pyramid Lake's salinity were to double, although none are expected to be extirpated. Food habit shifts and reduced production of lake fishes are likely consequences of salinity-induced disruption in the benthic invertebrate forage base.     

Phosphatases; origin,characteristics and function in lakes

Phosphatases catalyze the liberation of orthophosphate from organic phosphorus compounds. The total phosphatase activity in lake water results from a mixture of phosphatases localized on the cell surfaces of algae and bacteria and from dissolved enzymes supplied by autolysis or excretion from algae, bacteria and zooplankton. External lake water phosphatases usually have pH optima in the alkaline region. Acid phosphatases generally seem to be active in the internal cell metabolism. The synthesis of external alkaline phosphatases is often repressed at high phosphate concentrations and derepressed at low phosphate concentrations. Phosphatase activity has therefore been used as a phosphorus deficiency indicator in algae and in natural plankton populations. The possibilities for this interpretation of phosphatase activity in lake water are limited, however, and this is discussed. The in situ hydrolysis capacity, i.e. the rate by which orthophosphate is released from natural substrates, is unknown. However, we advocate that this process is important and that the rate of substrate supply, rather than phosphatase activity, limits the enzymatic phosphate regeneration.     

Trace element distributions in some saline lakes of the Vestfold Hills,Antarctica

The distributions of trace elements in Shield, Ace and Burton Lakes of the Vestfold Hills were investigated. Three aspects are discussed as follows: (1) the vertical distribution of 18 trace elements in the three lakes, (2) the behaviour of trace elements in the lakes, especially that of manganese in Shield Lake, and (3) the origin of trace elements in antarctic saline lakes.High concentrations of trace elements were found in these coastal saline lakes, when compared to open ocean water.We suggest that the peak of total extractable manganese, found at 20 m in Shield Lake, was related to the oxic/anoxic water interface brought about by microbiological activity. Solid phase manganese at the upper oxic layer may have precipitated and then reached the anoxic boundary to be there reduced to manganese ion. This dissolved manganese may then have diffused upwards to be reoxidized to a solid form. This cycle, repeated many times, may have produced the Mn profile.The alkali, alkaline earth elements and Cl were probably derived from relict seawater. Other elements were present in similar concentration ratios to those of South Polar aerosols. Residence time calculations indicate that fallout of aerosol particles, themselves derived from various sources, is capable of accounting for the measured concentrations of some trace elements in Shield Lake. This source of trace elements may be significant for other antarctic saline lakes.