Restoration by the rac locus of recombinant forming ability in recB? and recC? merozygotes of Escherichia coli K-12

Summary The recombinant forming ability of recB ^– or recC ^– strains of E. coli K12 is almost totally recovered in merozygotes which are heterozygous for a genetic locus denoted rac which is located five minutes clockwise from trp on the genetic map. This transient recovery phenomenon only occurs when the donor strain is rac ⁺ (wild type) and the recipient strain is rac ^-. The recombinants derived from such crosses all have the normal phenotype characteristic of recB ^– (or recC ^–) strains, and they are almost always rac ^-. The results imply that the rac ⁺ locus (or loci) is zygotically expressed and excised from the chromosome in a manner which is analogous to the zygotic induction of a prophage.     

Isolation and properties of Tn10 insertions in the rac locus of Escherichia coli

Summary Two Tn10 insertions that are in the rac locus of the chromosome of Escherichia coli have been isolated and characterized. These insertions are located at min 29.7 and min 30.0. The insertions are stable when an F123 rac::Tn10 episome is transferred to an F^- rac ⁺ recipient, but they are lost at a high frequency when transferred to an F^- rac ^- recipient. This latter condition has been previously, demonstrated to cause the excision of the rac locus. The Tn10 insertions are also lost at a high frequency when strains containing them are lysogenized with reverse. If the lysogens that have lost the Tn10 insertion are subsequently cured of reverse, the cells no longer contain sequences homologous with rac locus DNA. These strains were rac ^- when tested for recombination activation (Low 1973), and this procedure consequently provides a simple means to make isogenic rac ^- and rac ^- strains.     

Characterization of a hemin-storage locus ofYersinia pestis

Summary The pigmentation phenotype (Pgm⁺) ofYersinia pestis refers to temperature-dependent storage of hemin as well as expression of a number of other physiological characteristics. Spontaneous mutation to a Pgm^– phenotype occurs via a large chromosomal deletion event and results in the inability to express the Pgm⁺ characteristics. In this study, we have used transposon insertion mutants to define two regions of a hemin-storage (hms) locus. A clone (pHMSI) encompassing this locus reinstates expression of hemin storage (Hms⁺) inY. pestis spontaneous Pgm^– strains KIM and Kuma but not inEscherichia coli. Complementation analysis using subclones of pHMS1 inY. pestis transposon mutants indicates that both regions (hmsA andhmsB), which are separated by about 4 kb of intervening DNA, are essential for expression of the Hms⁺ phenotype. The 9.1-kb insert of pHMS1 contains structural genes encoding 90-kDa, 72-kDa, and 37-kDa polypeptides. Two-dimensional gel electrophoresis analysis of cells from Pgm⁺, spontaneous Pgm^–, and Hms^– transposon strains, as well as a spontaneous Pgm^– strain transformed with pHMS1, indicated that two families of surface-exposed polypeptides (of about 87 and 69-73 kDa) are associated with the Hms⁺ phenotype.     

Genetic evidence for plasmid-encoded lactococcin production inLactococcus lactis subsp.lactis 484

Lactococcus lactis subsp.lactis 484 produced a proteinaceous antibacterial substance designated as lactococcin capable of inhibiting members of theLactococcus group,Bacillus cereus, Staphylococcus aureus, andSalmonella typhi. Growth of this culture in the presence of 2–30 g/ml of ethidium bromide or acriflavin or novobiocin, and at elevated temperature (39° and 41°C), could not produce any lactococcin-negative (Lap^–) variants. However, protoplast-induced curing with lysozyme was successful in developing Lap^– derivatives. Two types of cured derivatives, namely Lac^– Lap⁺ and Lac^– Lap^–, were obtained. Lap^– variants were also lacking sucrose-fermenting ability (Suc⁺) and lactococcin resistance (Lap^r). The lactose-negative (Lac^–) variants and Lap⁺ were clearly lacking the largest (65 Md) plasmid. However, Lap^– Suc^– Lap^s variants lost a 2 Md plasmid.L. lactis subsp.lactis 484 transferred lactose-fermenting ability as well as Lap⁺ Suc⁺ Lap^r phenotypes simultaneously toL. lactis subsp.lactis LM 2306 and LM 0230 by surface mating at a frequency of 10^–4 and 10^–1 per donor respectively. However, cured Lac^– Lap^– transconjugants could not transfer Lac⁺ Lap⁺ Suc⁺ Lap^r phenotypes to any of these recipient strains. Our results indicate that Lac⁺ and Lap⁺ Suc⁺ Lap^r phenotypes are associated with 65 Md and 2 Md plasmids respectively. Conjugal transfer of 2 Md plasmid is possible only in the presence of a conjugative 65 Md plasmid.     

Formation of Polar Bundles of Pili and the Behavior of Azospirillum brasilenseCells in a Semiliquid Agar

This paper describes the formation of single polar bundles of pili on Azospirillum brasilensecells, the twitching motility of cell aggregates, and a new type of social behavior—the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri⁺phenotype)—which is an alternative to swarming (the Swa phenotype). The wild-type A. brasilensecells occurring in a semiliquid agar may show either the Swa⁺Gri^–, or Swa^–Gri^–, or Swa^–Gri⁺phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilensecells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.     

Chloramphenicol resistance in Streptomyces coelicolor A3(2): Possible involvement of a transposable element

Summary The transfer of a Chl element, causing resistance to chloramphenicol in Streptomyces coelicolor A3(2), was studied in NF x SCP1^– superfertile crosses. When the Chl element is on the donor side (NF) its transfer to the recombinant cells was virtually total as if the element acted as a second concomitant transfer origin. When the Chl element was on the recipient side (SCP1^–) it was never displaced by the immigrant chromosome even when the region facing chl ⁺ was selected for. A fraction of the original Chl^– mutants presented a requirement for arginine (ArgB^–). A Chl^– mutant gave rise spontaneously to ArgB^– derivatives at high frequency. The same ArgB^– requirement come out at high frequency among Chl^– derivatives from a cross NFChl^– x SCP1^–Chl⁺ in which neither parent required arginine or produced spontaneously arginineless derivatives. It is suggested that the Chl element is a transposable element (Tn) presumably associated with insertion sequences (IS). The insertional inactivation of the Chl element may be accompanied or followed by a deletion in the adjacent ArgB gene.     

Natural killer (NK) cell immunodeficiency in patients with chronic myelogenous leukemia

Summary Defective natural killer (NK) cell populations from patients with chronic myelogenous leukemia (CML), that reacted with both HNK-1⁺ and B73.1⁺ antibodies, were obtained by a flluorescence-activated cell sorter (FACS). These fractions, along with NK fractions from normal donors which reacted with both antibodies, were expanded as bulk cultures or clones by limiting dilution, for 4 weeks in the presence of 10% interleukin 2 (IL 2), human type AB plasma, and irradiated human allogeneic mononuclear cells. Successfully established clones from patients with CML, with lytic activity against autologous and more differentiated neoplastic granulocytes, were generated more efficiently from B73.1⁺ than from HNK-1⁺ subsets. However, there were no significant differences among the generations of B73.1⁺ and HNK-1⁺ clones for both patients and normal donors with lytic activity against NK susceptible K-562 targets. Fresh myeloblast preparations from a blast crisis were found to be more susceptible to lysis by IL 2-proliferative B73.1⁺ and HNK-1⁺ clones than were fresh myelocyte preparations from a chronic phase CML patient, which were lytically susceptible to only B73.1⁺ clones. B73.1⁺ and HNK-1⁺ subsets from CML patients demonstrated major histocompatibility complex nonrestricted killing, and showed the following predominant phenotypes: B73.1⁺T3⁺T8⁺ or B73.1⁺T3⁺T8^– from B73.1⁺ subsets; and HNK-1^–T3⁺T8⁺ (initially HNK-1⁺) from HNK-1⁺ subsets. In contrast, B73.1⁺ and HNK-1⁺ clones from normal donors showed the following predominant phenotypes: B73.1⁺T3^–T8^–; and HNK-1^–T3^–T8^– or HNK-1^–T3^–T8⁺ (initially all HNK-1⁺). Short-term in vitro IL 2 or interferon treatment of fresh NK defective subsets from CML patients resulted in minimal cytotoxic augmentation. In contrast, defective NK cells from CML patients, whether HNK-1⁺ or B73.1⁺ subsets, proliferated with complete regeneration of cytolytic activity after a 3–4 week exposure to IL 2, but differed in phenotypic profiles as compared to those of normal donors. These observations imply that not only fresh defective NK cells but also the cytotoxically restored clones from CML patients are derived from different NK subsets and may represent undifferentiated forms of NK cells that may be arrested at an early stage of development by yet unknown mechanism(s). In vitro substantiation of autologous leukemia cell killing by IL 2-proliferative NK cell clones is encouraging and may allow for new in vivo immunotherapeutic modalities in CML patients.     

Chloride movement across the basolateral membrane of proximal tubule cells

Summary Electrophysiologic and tracer experiments have shown that Cl^– entersNecturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na⁺, and that Cl^– entry is electrically silent. The mechanism of Cl^– exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl^– ions across the basolateral membrane, the relative conductance of Cl^– to K⁺ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl^– and K⁺ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl^– conductance was about 10% of K⁺ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl^– activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl^– activity was virtually unaffected by the PD changes. We conclude that Cl^– leavesNecturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na⁺ or K⁺.     

Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase

Summary Antibodies which were raised against highly purified membrane-bound (Na⁺–K⁺)-ATPase from the outer medulla of rat kidneys inhibit the (Na⁺–K⁺)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na⁺–K⁺)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na⁺–K⁺)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity (n=0.77). By measuring the Na⁺ dependence of the (Na⁺–K⁺-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t _0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na⁺–K⁺)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na⁺–K⁺)-ATPase reaction and on the ATP binding of the enzyme.V _max of the (Na⁺–K⁺)-ATPase reaction and the number of ATP binding sites were reduced whileK _{0.5 ATP} for the (Na⁺–K⁺)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK _0.5 value for the Na⁺ stimulation of the (Na⁺–K⁺)-ATPase activity, but they did not alter the homotropic interactions between the Na⁺-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na⁺–K⁺)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na⁺–K⁺)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na⁺ dependence of the (Na⁺–K⁺)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.     

The Lyt phenotype of the T cells responsible for in vivo tumor rejection in syngeneic mice

Summary Spleen cells of BALB/c mice hyperimmunized with a transplantable methylcholanthrene-induced sarcoma Meth A (Meth A-Im-SPL) inhibited the growth of Meth A tumor in vivo in a tumor neutralizing test. Meth A-Im-SPL did not neutralize another antienically distinct sarcoma, Meth 1, indicating that the antitumor activity is tumor specific. Lyt-1⁺2^– cells of Meth A-Im-SPL (Im-Lyt-1⁺2^–) were the effectors since in vitro treatment of Meth A-Im-SPL with anti-Thy 1.2 or anti-Lyt 1.2 antibody plus complement completely abrogated their neutralizing activity, whereas treatment with anti-Lyt 2.2 plus complement did not. To further confirm the effector activity of Im-Lyt-1⁺2^– cells, T cell subpopulations were separated from Meth A-Im-SPL by the panning method. The purified Im-Lyt-1⁺2^–, but not Im-Lyt-1⁺2⁺ cells neutralized the tumor in athymic nu/nu mice as efficiently as in +/+ mice, suggesting that the donor Im-Lyt-1⁺2^– cells but not recipient T cells were primarily responsible for neutralizing the tumor. The present study, however, did not exclude the possible contribution of recipient T cells to the tumor neutralization and this is open to further investigation.Abbreviations Meth A-Im-SPL Meth A-immune mouse spleen cells - Meth 1-Im-SPL Meth 1-immune mouse spleen cells - sIg⁺ cells surface immunoglobulin positive cells - moAb monoclonal antibody     

Efficient transformation of beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis>) and production of plants with improved salt-tolerance

Small bud tips of 1–3 mm in length were taken from multiple shoot clumps that derived from immature inflorescence cultures of beet as recipient for the Agrobacterium-mediated transformation and transgenic plants were obtained from eight genotypes. The optimal genetic transformation protocol was established as followed: the buds were immersed in Agrobacterium suspension of OD₆₀₀ =0.3–0.5 for 5–10 min, with vacuum infiltration (0.3–0.5 × 10⁵ Pa) or supplemented with 0.01% Silwet L-77, co-cultured for 2–4 days and followed by 10-day culture on medium containing 100 mg l⁻¹ cefotaxime, then the buds were selected on medium containing 10 mg l⁻¹ hygromycin B for three consecutive generations. The percentage of hygromycin-resistant buds after three selections varied from 13.3 to 30.6% with genotypes. The results of PCR and further Southern blotting of genomic DNA of hygromycin-resistant buds or plants showed that the exogenous hpt and AtNHX1 gene had been integrated into the genomes of some transformed buds or plants. The transgenic buds or plants with AtNHX1 gene encoding Na⁺/H⁺ antiport on the vacuole membrane of Arabidopsis showed improved salt-tolerance than the controls. AtNHX1gene inherited in some transgenic lines as Mendelian segregation. This result revealed that it was feasible to improve salt-tolerance of beets by the introduction of AtNHX1 gene into cultured buds.     

Responsiveness of cardiac Na+ channels to a site-directed antiserum against the cytosolic linker between domains III and IV and their sensitivity to other modifying agents

Elementary Na⁺ currents were recorded in inside-out patches from neonatal rat heart cardiocytes to analyze the influence of a site-directed polyclonal anti-serum against the linker region between the domains III and IV (amino acids 1489–1507 of the cardiac Na⁺ channel protein) on Na⁺ channel gating and to test whether this part of the -subunit may be considered as a target for modifying agents such as the (–)-enantiomer of DPI 201-106.Anti-SLP 1 serum (directed against amino acids 1490–1507) evoked, usually within 10–15 min after cytosolic administration, modified Na⁺ channel activity. Antiserum-modified Na⁺ channels retain a single open state but leave, at –60 mV for example, their conducting configuration consistently with an about threefold lower rate than normal Na⁺ channels. Another outstanding property of noninactivating Na⁺ channels, enhanced burst activity, may be quite individually pronounced, a surprising result which is difficult to interpret in terms of structure function relations. Removal of inactivation led to an increase of reconstructed peak I _Na (indicating a rise in NP _o) and changed I _Na decay to obey second-order kinetics, i.e., open probability declined slowly but progressively during membrane depolarization. The underlying deactivation process is voltage dependent and responds to a positive voltage shift with a deceleration but may operate even at the same membrane potential with different rates. Iodatemodified Na⁺ channels exhibit very similar properties including a conserved conductance. They are likewise controlled by an efficient, voltage-dependent deactivation process. Modification by (–)-DPI 201-106 fundamentally contrasts to the influence of anti-SLP 1 serum and the protein reagent iodate since (–)-DPI-modified Na⁺ channels maintain their open probability for at least 120 msec, i.e., a deactivation process seems lacking. This functional difference suggests that the linker region between the domains III and IV of the -subunit may not be the only target for (–)-DPI 201-106 and related compounds, if at all.This work was supported by a grant of the Deutsche Forschungs-gemeinschaft (Ko 778/2–4), Bonn.     

Properties of strains of Escherichia coli K12 carrying mutant lex and rec alleles

Summary Strains of Escherichia coli K12 have been constructed which carry the lex-3 mutation in combination with recA56, recB21, or recC22. The lex ^– recA ^–strain is equally as sensitive to ultraviolet light (UV) and ionizing radiation and as recombination-deficient as the corresponding lex ⁺ recA ^–strain, whereas the lex ^– recB ^–and lex ^– recC ^–strains are somewhat more sensitive to UV and ionizing radiation than the corresponding lex ^– rec ⁺strain and have approximately the same recombination deficiencies as the respective lex ⁺ recB ^–and Lex ⁺ recC ^–strains. When cultures of the lex ^– recB ^–and lex ^–recC^–strains are UV-irradiated, they degrade their DNA at the low rate characteristic of lex ⁺ recB ^–and lex ⁺ recC ^–single mutants, in contrast to the high rate of degradation seen with lex ^– rec ⁺single mutants.These results imply that the lex ⁺and recA ⁺products act in the same pathway of DNA repair and that both are needed to limit the DNA breakdown due to the recB ⁺/C⁺nuclease.     

Satellite glial cell responses to neuronal firing in the nervous system of Helix pomatia

Patch clamp experiments were conducted on satellite glial cells attached to the cell body of neurons in place within the nervous system of the snail Helix pomatia. The glial cells were studied using cell-attached and whole-cell patch clamp configurations while the underlying neurons were under current or voltage clamp control.The resting potential of the glial cells (–69 mV) was more negative than that of the underlying neurons (–53 mV), due to their high K⁺ selectivity. Densely packed K⁺ channels were present, some of which were active at the cell resting potential. Neuronal firing elicited a cumulative depolarization of the glial cells. Large K⁺ currents flowing from V-clamped neurons depolarized the glial layer by up to 30 mV. The glial depolarization was directly correlated with the size of the neuronal K⁺ current. The glial cells recovered their resting potential within 2–5 sec. The neuronal depolarization induced a delayed (20–30 sec) and persistent (3–4 min) increase in the glial K⁺ channel opening probability. Likewise, pulses of K⁺ (20–50 mM)-rich saline activated the glial channels, unless the underlying neuron was held hyperpolarized. In low Ca²⁺-high Mg²⁺ saline, neuron depolarization and K⁺-rich saline did not activate the glial K⁺ channels.These data indicate that a calcium-dependent signal released from the neuronal cell body was involved in glial channel regulation. Neuron-induced channel opening may help eliminate the K⁺ ions flowing from active neurons.I. Gommerat is recipient of a fellowship from the Ministère de la Recherche et de la Technologie.This work was supported by the CNRS and by a grant from the Fondation pour la Recherche Médicale. We would like to thank Mrs. M. André and Mr. G. Jacquet for technical assistance and Mrs. J. Blanc for improving the English.     

TheChara plasmalemma at high pH. Electrical measurements show rapid specific passive uniport of H+ or OH−

Summary Above a critical external pH (about 10.5), theChara membrane acquires new propertes. In this state the membrane potential is close to the equilibrium potentials for H⁺ and OH^–, hyperpolarizing as external pH increases with a slope of –59 mV/pH unit. The membrane conductance increases by an average factor of 2.4 above the critical pH. These changes are explained by an increase in permeability to OH^– (or H⁺). The establishment of a OH^– (or H⁺ permeable membrane at high pH suggests that the large fluxes of OH^– (or H⁺ which occur in the alkaline band in photosynthesizing cells are passive.     

Cytotoxic functions of blood mononuclear cells in patients with colorectal carcinoma treated with mAb 17-1A and granulocyte/macrophage-colony-stimulating factor

Summary Unconjugated monoclonal antibodies (mAb) may induce tumour regression in patients. The mechanisms of action are complex. Antibody-dependent cellular cytotoxicity (ADCC) is considered one of the effector functions. Augmentation of the killing capacity of cytotoxic cells may thus be a way to increase the therapeutic potential of mAb. Granulocyte/macrophage-colony-stimulating factor (GM-CSF) has been shown to enhance this function in vitro. Eighteen patients with metastatic colorectal carcinoma received GM-CSF (250 µg m^–2 day^–1 s.c.) for 10 days and a single infusion of the anti-(colon carcinoma) mAb 17-1A (mouse IgG2A) (400 mg) on day 3 of the cycle. The cycles were repeated once a month four times. Neutrophils, eosinophils, monocytes and lymphocytes increased significantly in a biphasic way. However, at the fourth cycle the rise in white blood cells was significantly lower compared to the preceding courses. ADCC (SW948, a human CRC cell line, + mAb 17-1A) or peripheral blood mononuclear cells (PBMC) was significantly (P <0.05) augmented by day 6 of a cycle and then declined gradually and, at the end of a cycle, the ADCC activity had returned to the pretreatment level. The spontaneous cytotoxicity of PBMC against the natural-killer-resistant cell line, SW948, varied in a similar way. During GM-CSF treatment there was also a significant increase in FcRI⁺ (CD64), FcRII⁺ (CD32), FcRIII⁺ (CD16) and CD14⁺ cells but not of CD56⁺ cells.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na⁺ + K⁺)-ATPase, leucine aminopeptidase, 5′-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na⁺ + K⁺)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48–108 h and in 5′-nucleotidase activity at 12–24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Thiol-dependent passive K+Cl− transport in sheep red blood cells: VI. Functional heterogeneity and immunologic identity with volume-stimulated K+(Rb+) fluxes

Summary Ouabain-resistant (OR), volume-or N-ethylmaleimide (NEM)-stimulated K⁺(Rb⁺)Cl^– fluxes were measured in low-K⁺ sheep red cells and found to be functionally separate but immunologically similar. In anisosmotic solutions both K⁺ effluxes and Rb⁺ influxes of NEM-treated and control cells were additive. In contrast to the NEM-stimulated K⁺Cl^– flux, metabolic depletion did not reduce K⁺Cl^– flux of normal or swollen cells. The anion preference of OR K⁺ efflux in NEM-treated cells was Br^–>Cl^–>HCO ₃ ^– =F^–I^–=NO ₃ ^– =CNS^–, and hence consistent with a reported Br^–>Cl^–>NO ₃ ^– sequence of the volume-dependent K⁺Cl^– transport. Alloimmune anti-L_l antibodies known to decrease passive K⁺ fluxes in low K⁺ cells reduced by 51% both volume-and NEM-stimulated, furosemidesensitive Rb⁺Cl^– fluxes suggesting their immunologic identity, a conclusion also supported by anti-L₁ absorption studies. Since pretreatment with anti-L₁ prevented the activation of Rb⁺ influx by NEM, and the impermeant glutathionmaleimide-I did not stimulate Rb⁺Cl^– influx, the NEM reactive SH groups must be located apart from the L₁ antigen either within the membrane or on its cytoplasmic face. A model is proposed consisting of a K⁺Cl^– transport path(s) regulated by a protein with two functional subunits or domains; a chemically (C _s) and a volume (V _s)-stimulated domain, both interfacing with the L₁ surface antigen. Attachment of alloanti-L₁ from the outside reduces K⁺Cl^– transport stimulated throughC _s by NEM orV _s by cell swelling.     

Passive sodium movements across the opercular epithelium: The paracellular shunt pathway and ionic conductance

Summary The unidirectional Na⁺, Cl^–, and urea fluxes across isolated opercular epithelia from seawater-adaptedFundulus heteroclitus were measured under different experimental conditions. The mean Na⁺, Cl^–, and urea permeabilities were 9.30×10^–6 cm·sec^–1, 1.24×10^–6 cm·sec^–1, and 5.05×10^–7 cm·sec^–1, respectively. The responses of the unidirectional Na⁺ fluxes and the Cl^– influx (mucosa to serosa) to voltage clamping were characteristic of passively moving ions traversing only one rate-limiting barrier. The Na⁺ conductance varied linearly with, and comprised a mean 54% of, the total tissue ionic conductance. The Cl^– influx and the urea fluxes were independent of the tissue conductance. Triaminopyrimidine (TAP) reduced the Na⁺ fluxes and tissue conductance over 70%, while having no effect on the Cl^– influx or urea fluxes. Mucosal Na⁺ substitution reduced the Na⁺ permeability 60% and the tissue conductance 76%, but had no effect on the Cl^– influx or the urea fluxes. Both the Na⁺ and Cl^– influxes were unaffected by respective serosal substitutions, indicating the lack of any Na⁺/Na⁺ and Cl^–/Cl^– exchange diffusion.The results suggest that the unidirectional Na⁺ fluxes are simple passive fluxes proceeding extracellularly (i.e., movement through a cation-selective paracellular shunt). This pathway is dependent on mucosal (external) Na⁺, independent of serosal (internal) Na⁺, and may be distinct from the transepithelial Cl^– and urea pathways.