Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

We have previously partially purified the basolateral Na⁺/HCO⁻ ₃ cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na⁺/HCO⁻ ₃ cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na⁺/HCO⁻ ₃ cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na⁺/HCO⁻ ₃ cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na⁺/HCO⁻ ₃ cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO₃ or Na₂CO₃) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na⁺/HCO⁻ ₃ cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na⁺/HCO⁻ ₃ cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na⁺/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues. Received: 27 January 1996/Revised: 23 July 1996     

Polymerization of τ into Filaments in the Presence of Heparin: The Minimal Sequence Required for τ - τ Interaction

Abstract: Paired helical filaments isolated from the brains of patients with Alzheimer's disease are composed of a major protein component, the microtubule-associated protein termed τ, together with other nonprotein components, including heparan, a glycosaminoglycan, the more extensively sulfated form of which is heparin. As some of these nonprotein components may modulate the assembly of τ into filamentous structures, we have analyzed the ability of the whole τ protein or some of its fragments to self-assemble in the presence of heparin. Different τ fragments, all of them containing some sequences of the tubulin-binding motif, can assemble in vitro into filaments. We have also found formation of polymers with the 18-residue-long peptide corresponding to the third tubulin-binding motif of τ. This suggests that the ability of τ for self-assembly could be localized in a short sequence of amino acids present in the tubulin-binding repeats of the τ molecule.     

Physiological studies and determination of chromosome number in Maca,Lepidium Meyenii (Brassicaceae)

Experiments based on four accessions of maca (Lepdium meyenii) disclosed higher developmental rates in plants grown in neutral pH (6.6) soil when compared with those grown in acidic soil (5.3). Photoperiod response studies revealed similar growth rate for plants grown under either long day or short day condition. Plants in the field and growth chambers completed their life cycle in 11 months or less, therefore maca can be considered an annual crop. These results suggest that the range of adaptation of maca is not as narrow as previously believed, and therefore it can be successfully produced outside its natural habitat. Chromosome counts and predominance of bivalents in diakinesis and metaphase I disclosed that maca is a disomic octoploid of 2n=8x=64 chromosomes. Field and growth chamber observations and morphological uniformity of the plants within accessions indicate that maca relies mainly on self-fertilization for its reproduction.     

Trinuclear iridium and rhodium complexes: the solution to a puzzle involving the multiple coordination possibilities of 1,8-naphthyridine-2-one ligands

The multiple coordination possibilities of 1,8-naphthyridine-2-one (HOnapy) and 5,7-dimethyl-1,8-napthyridine-2-one (HOMe₂napy) ligands allow the synthesis of a variety of tri- di- and mononuclear complexes, showing fluxional behaviour and frequent exchange of the coordinated ML₂ fragments. Thus, reactions of [M₂(μ-OMe)₂(cod)₂] (cod = 1,5-cyclooctadiene) with HOnapy and HOMe₂napy yield the compounds of the general formula [M(μ-OR₂napy) (cod)]_n (M = Ir, R = Me (1a, 1b, H (2); M = Rh, R = Me (3a, 3b). They crystallise as inconvertible yellow (a) and purple/orange (b) forms and also show a puzzling behaviour in solution. X-ray diffraction studies on both forms (3a, 3b) and spectroscopic data reveal that the yellow forms are mononuclear complexes whilst the dark-coloured crystals contain dinuclear complexes. In solution, the nuclearity of the complexes depends on the solvent. In addition both types of complexes are fluxional. The mixed-ligand complexes [M₂(μ-OMe₂napy)₂(CO)₂(cod)] M = Ir (5), Rh (6) have been isolated and characterised; they are found to be intermediates in the synthesis of the trinuclear complexes [M₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]⁺ M = Rh (8), Ir (9). Reactions of [IrCl(CO)₂(NH₂-p-tolyl] with the complexes [Rh(μ-OR₂napy)(diolefin)]_n followed by addition of a poor donor anion is a general one-pot synthesis for the hetertrinuclear complexes [Rh₂Ir(μ₃-OR₂napy)₂(CO)₂(diolefin)₂]⁺ (R=Me, DIOLEFIN = cod (10), tetrafluorobenzo-barrelene (tfbb) (11), 2,5-norbornadiene (nbd) (12); R=H, DIOLEFIN=cod (13)). This synthesis follows a stepwise mechanism from the mononuclear to the trinuclear complexes in which mixed-ligand heterodinuclear complexes are involved as intermediates of the type [(diolefin)Rh(μ-OMe₂napy)₂Ir(CO)₂]. Heteronuclear complexes which possess the core [RhIr₂]³⁺, such as [RhIr₂(μ₃-OR₂napy)₂(CO)₂(cod)₂]BF₄ (R=Me (14), H (15)), result from the reaction of 1 or 2 with [Rh(CO)₂S_x]⁺ (S = solvent). The trinuclear complexes undergo two chemically reversible one-electron oxidation processes. The chemical oxidation of 10, 14 and 9 with silver salts gives the mixed-valence trinuclear radicals [Rh₂Ir(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (16), [RhIr₂(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (17) and [Ir₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (18), which have been isolated as the perchlorate and tetrafluoroborate salts. The EPR spectrum of 16 indicates that the unpaired electron is essentially in an orbital delocalised on the metals. The molecular structures of the complexes 3a, 3b, 6, 10b and 16a are described. Crystals of 3a are triclinic, P-1, with a = 9.7393(2), b = 14.0148(4), c = 16.0607(4) Å, α = 88.122(3), β = 83.924(3), γ = 87.038(3)°, Z = 4; 3b crystallises in the Pna2_i orthorhhombic space group, with a = 16.7541(3), B = 11.7500(8), c = 17.7508(7) Å, Z = 4; complex 6 is packed in the monoclinic space group P2_i/c, a = 9.6371(1), b = 11.8054(4), c = 27.2010(9) Å, β = 90.556(4)°, Z = 4; crystals of 10b are monoclinic, P2₁/n, with a = 17.546(7), b = 13.232(6), c = 17.437(8) Å, β = 106.18(1)°, Z = 4; crystals of 16a are triclinic, P-1, with a = 10.318(4), b = 12.562(6), C = 19.308(8) Å, α = 92.12(8), β = 97.65(9), γ = 90.68(5)°, Z = 2. The five different structures show the coordination versatility of the OMe₂napy molecule as ligand, which behaves as a N,N′-chelating (3a), bidentate N,O-donor (3b, 6), or as a tridentate N,N′,O-donor bridging ligand (10b, 16a).     

Effect of CRF injected into the median eminence on GH secretion in female rats under different steroid status

To evaluate whether the median eminence (ME) is a site of action of CRF (corticotropin releasing factor) on GH secretion and to determine the possible role of estradiol and progesterone in modifying theses secretion, we injected CRF (0.25, 0.75, 1, and 1.5 nmol of peptide dissolved in 1 μl of water) directly into the ME in three experimental groups of rats: Long-term ovariectomized (OVX); OVX primed by estradiol (OVX±E) and OVX primed by estradiol plus progesterone (OVX±EP). Blood was collected to determine GH (30, 60, 90, and 120 min postinjection) Serum T₃, T₄, and glucose levels were measured in OVX±E rats 30 min postinjection. CRF at all doses studied significantly decreased serum GH levels in the three experimental groups. Serum T₃, T₄, and glucose levels were unchanged after CRF administration. The results suggest that: CRF inhibits “per se” GH secretion, at least in part, by a central action in the ME. The inhibitory effect of CRF on GH is independent of the estrogen/progesterone status of the animal. CRF at ME levels may participate in a variety of stress-related responses, including growth inhibition, through GH suppression.     

Sedimentation loss of phytoplankton cells from the mixed layer: effects of turbulence levels

In this paper, we describe a study of the role of turbulencein the loss by sedimentation of phytoplankton cells from themixed layer. The approach presented allows the quantificationof the sedimentation rate of phytoplankton in the whole rangeof turbulence levels of this layer. Two types of phytoplanktercan be distinguished according to the effect that turbulencecan exert on their sedimentation rate. The rate of those cellswhose settling velocity is lower than –1 m day^–1will not be modified by turbulence. The sedimentation rate ofcells with higher settling velocities can, however, be modifiedby the level of turbulence. A set of dimensionless numbers isgiven to delimit several processes that are important in thedynamics of phytoplankton sedimentation in a turbulent regime.The use of these dimensionless numbers suggests that an increasein the turbulence level in the mixed layer does not always implya decrease in the sedimentation rate of phytoplankton cells.     

Leishmania infantum Nicolle, 1908 from southern Spain: Characterisation of the strains from human visceral and cutaneous leishmaniasis and from sandflies; with a numerical analysis of the isoenzymatic data

This study presents the results of the characterisation of 17 strains ofLeishmania by isoenzyme electrophoresis from a focus of leishmaniasis in southern Spain: two from human visceral leishmaniasis, four from human cutaneous leishmaniasis and 11 from sandflies. The 17 strains are grouped in 6 zymodemes characterised by their variability as regards to the electrophoretic mobility of the enzymes MDH, G6PD, NP and ME. Thus, we confirm the high intraspecific variability ofLeishmania (L.) infantum in a focus of southern Spain, as already suggested by previous studies. Zymodemes GR-15 and GR-17 are also described for the first time in Spain, and they characteristically possess the same relative electrophoretic mobility in the enzyme ME (93). Sixteen zymodemes of theL. infantum complex found in southern Spain were numerically analysed on the basis of the enzymatic profiles of 122Leishmania strains characterised from this area.     

The Jurassic succession in west-central Argentina: stratal patterns, sequences and paleogeographic evolution

Jurassic rocks in west-central Argentina are predominantly marine and marginal-marine siliciclastics, associated with prominent but volumetrically subordinate carbonates and evaporites. Facies developments were ruled by paleogeographic persistence within the southern-infratropical latitude belt (within 40–50°S) and by siliciclastic derivation from the Patagonian hinterland to the southwest. Minor volcanic and volcaniclastic supply arrived from the west, out of a magmatic belt related to Circum-Pacific convergence. Timing of the main marine-flooding events and general correspondence with the high-rank stratal packaging recorded in western North America, suggest that global eustasy was also a factor in controlling the local stratigraphic record. Early Jurassic sedimentation occurred within a series of semi-isolated depocentres linked to fault-bounded Triassic troughs. The Sinemurian-Toarcian deposits record depocentre expansion and coalescence. These trends were coeval with progressively more widespread marine invasions from the northwest and west, leading to an elongate marine seaway which connected central Patagonia with the Pacific domain. During the Aalenian-Bajocian the region was subject to a more subdued tectonism and the foreland side of the basin became fringed by an extensive clastic embankment. Bathonian and Early Callovian were times when coarse clastics prograded into the basin, while the marine embayment shrank as a result of stepwise forced regressions. During Late Callovian to Oxfordian globally rising sea-level, the depocentre witnessed the appearance of cosmopolitan invertebrates and a stratal pattern of basin widening and depositional underbalance, that promoted cratonward onlap and inception of widespread carbonate deposition. Ooidal-coralline carbonate development was terminated after a relatively sudden (Messinian-style) event that desiccated a large tract of the Andean basin, and favored massive precipitation of anhydrite. In the course of the Kimmeridgian the evaporite basin was largely flooded by siliciclastics and turned into a broad and featureless mudflat-salina complex, linked to a widespread erg and to an ephemeral drainage system. By the Tithonian, at a period of peak oceanic stand, marine connection was reinstated and recorded as the most widespread Jurassic transgression across the Neuquén-Aconcagua embayment. Shelfal deposition consisted of molluscs and ooid-dominated carbonate terraces that grew in pulses tuned to eustatic fluctuations. Like in other prolific petroleum provinces around the world the Tithonian basinal strata involve widespread euxinic deposits featuring unusually high organic content.