Thyroglobulin biosynthesis in a larval (ammocoete) and adult freshwater lamprey (Lampetra planeri Bl.).

1. The biosynthesis of 18-19S thyroglobulin has been studied in a larval and adult freshwater lamprey (Lampetra planeri Bl.). 2. In vivo and in vitro experiments have been performed by injecting into the coelomic cavity or by incubating branchial region labeled constituents of Tg of higher vertebrates (125I, [3H]leucine and various [3H]carbohydrates). 3. Larvae (ammocoetes) and adults incorporate all labels into thyroglobulin (18-19S Tg), containing a small proportion of labeled T3 and T4, as identified by paper chromatography, and very minute amounts of stable iodine. 4. In adults, the biosynthesis of 18-19S Tg proceeds much more rapidly and the labels are incorporated in higher percentage than in larvae. 5. The demonstration of the biosynthesis of the specific thyroid protein, 18-19S Tg, in larvae indicates that the biochemical mechanism of hormonogenesis is present in larval endostyle before the morphological differentiation of thyroid cells and follicles occurring during metamorphosis. 6. Some 18-19S Tg is apparently stored in the endostyle.     

The picoplankton in Antarctic lakes of northern Victoria Land during summer 1989–1990

Summary Photoautotrophic picoplankton is reported from some lakes located near the Italian Antarctic station of Terra Nova. Observations, carried out by both flow cytometry on water samples and electron microscopy on micro-organisms in cultures from each lake, have confirmed the occurrence in all the environments studied of this fraction accounting, in several cases, for more than the 50% of the phytoplankton, measured as chlorophyll. Cultures of the picoplankton fraction from these waters contained known prokaryotic (Synechococcus) and eukaryotic (Chlorella) genera as well as two unidentified entities, possibly prochlorophytes.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl− absorption

Summary Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,V _e , which attends net Cl^– absorption,J _Cl ^net , and to assess how ADH might augment bothJ _Cl ^met andV _e in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba⁺⁺ for 5mm K⁺ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,R _c /R _bl , and increasing luminal K⁺ from 5 to 50mm in the presence of luminal 10^–4 m furosemide resulted in a 53-mV depolarization of apical membrane voltage,V _a . Thus K⁺ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10^–4 m luminal furosemide reducedV _e andJ _Cl ^net to near zero values and hyperpolarized bothV _a andV _bl , the voltage across basolateral membranes; however, the depolarization ofV _bl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofV _a , Thus ADH-dependent increases inV _b were referable to greater depolarizations ofV _bl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofV _a was referable to a decrease in the K⁺ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofV _bl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl^– current. Thus an increase in intracellular Cl^– activity may have contributed to the depolarization ofV _bl during net Cl^– absorption, and the intracellular Cl^– activity was likely greater with ADH than without hormone. Since ADH increases apical K⁺ conductance and since the chemical driving force for electroneutral Na⁺,K⁺,2Cl^– cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba⁺⁺-sensitive conductance K⁺ channels and electroneutral Na⁺,K⁺,2Cl^– cotransport units in apical plasma membranes.     

Strict co-linearity of genetic and protein folding domains in an intragenically duplicated rat lens γ-crystallin gene

Two complementary DNA clones pRLγ-2 and pRLγ-3 of different rat lens γ-crystallin messenger RNAs have been used to identify γ-crystallin gene sequences in rat genomic DNA. Subsequently, the DNA present in the 18,000 to 20,000 bases region of the EcoRI digest, giving rise to a strong doublet hybridization signal, was cloned in λ phage Charon-4A. One of the clones, λRCHγ-3, carrying an insert of 17,500 bases has been characterized in detail. From analysis at the restriction enzyme level with 5′-, “middle” and 3′-specific subprobes of pRLγ-3 it could be deduced that λRCHγ-3 contains only one γ-crystallin gene. The coding sequences of this gene are interrupted by intronic DNA. The primary structure of this gene and its flanking regions have been established by sequencing the relevant regions of a subclone of λRCHγ-3, designated pRCHγ-3.1. The sequence data show that the γ-crystallin gene extends over 2700 bases of rat genomic DNA. The gene is split by two introns, one of 87 base-pairs after the third translation codon and a large one of 1880 base-pairs after codon 84. The mosaic structure of the gene is strictly co-linear with the structure of the γ-crystallin polypeptide in that the large intron is positioned in a region which specifies the so-called “connecting peptide” and which links the two highly symmetrical and homologous protein domains. Although expected from the cDNA and protein sequence no introns were observed between the coding regions in the DNA specifying the two homologous folding motifs present in each protein domain. The relevance of this phenomenon in terms of the evolution of the mature γ-crystallin gene is discussed.     

The effect of valinomycin on the electrical properties of solutions of red cell lipids in n-decane

This paper reports the electrical properties of thick lipid membranes in the absence and presence of valinomycin. The thick lipid membranes were formed by placing a solution of sheep red cell lipids in decane between two cellophane partitions which formed the interfaces between the membrane and the two aqueous bathing solutions. The DC electrical resistance of these structures was found to be directly proportional to the reciprocal of the concentration of lipids in the decane (C_L). The limiting resistance, as (C_L ^-1) approached zero, was 3 x 10⁸ ohm-cm². Resistance was also found to be linearly related to membrane thickness. The limiting resistance at zero thickness was again 1–3 x 10⁸ ohm-cm². These data are interpreted to indicate that the DC resistance of thick lipid membranes comprises two surface resistances (R_S) at each interface with the aqueous bathing solutions, and a bulk resistance (R_B) of the lipid-decane solution, arranged in series. Measurements of the effect of variations of area on resistance were consistent with this interpretation. Valinomycin reduced R_S but had no effect on R_B. Under certain conditions, thick lipid membranes containing valinomycin behaved like highly selective K⁺ electrodes.     

The formation and properties of thin lipid membranes from HK and LK sheep red cell lipids

Lipids were obtained from high potassium (HK) and low potassium (LK) sheep red cells by sequential extraction of the erythrocytes with isopropanol-chloroform, chloroform-methanol-0.1 M KCl, and chloroform. The extract contained cholesterol and phospholipid in a molar ratio of 0.8:1.0, and less than 1% protein contaminant. Stable thin lipid membranes separating two aqueous compartments were formed from an erythrocyte lipid-hydrocarbon solution, and had an electrical resistance of ∼10⁸ ohm-cm² and a capacitance of 0.38–0.4 µf/cm². From the capacitance values, membrane thickness was estimated to be 46–132 A, depending on the assumed value for the dielectric constant (2.0–4.5). Membrane voltage was recorded in the presence of ionic (NaCl and/or KCl) concentration gradients in the solutions bathing the membrane. The permeability of the membrane to Na⁺, K⁺, and Cl^- (expressed as the transference number, T _ion) was computed from the steady-state membrane voltage and the activity ratio of the ions in the compartments bathing the membrane. T _Na and T _K were approximately equal (∼0.8) and considerably greater than T _Cl (∼0.2). The ionic transference numbers were independent of temperature, the hydrocarbon solvent, the osmolarity of the solutions bathing the membranes, and the cholesterol content of the membranes, over the range 21–38°C. The high degree of membrane cation selectivity was tentatively attributed to the negatively charged phospholipids (phosphatidylethanolamine and phosphatidylserine) present in the lipid extract.     

Cl− channels in basolateral renal medullary memnbranes: III. Determinants of single-channel activity

Summary We evaluated the effects of vawrying aqueous Cl^– concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl^– channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for, channel activity. and the efects of varying Cl^– concentrations and/or PGO on the relation between holding voltageV _H -mV) and open-time probability (P _o).Reducingcis Cl^– concentrations, in the range 50–320mm, produced a linear reduction in fractional open time (P _v) with a half-maximal reduction inP _o atcis Cl^–170mM. Channel activity was sustained by equimolar replacement ofcis Cl^– with F^–, but not with impermeant isethionate. Fortrans solutions, the relation between Cl^– concentration andP ₀ at 10mm Cl^–. Reducingcis Cl^– had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent, energy (G) for channel opening.Phenylglyoxal (PGO) reducedZ and altered G for Cl^– channel activity when added tocis, but nottrans solutions, Furthermore, in the presence ofcis PGO, reducing thecis Cl^– concentration had no effect onZ but altered G. Thus we propose thatcis PGO and,cis Cl^– concentrations affect separate sites determining channel activity at the extracellular faces of, these Cl^– channels.     

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.