Transformation of Saccharopolyspora erythraea by electroporation of germinating spores: construction of propionyl Co-A carboxylase mutants

The introduction of plasmid DNA into germinating spores of an industrially improved strain of Saccharopolyspora erythraea was accomplished by electroporation. Various parameters affecting the efficiency of electroporation were examined. The most critical factor was the extent of spore germination. Electrocompetence was limited to a 4-h period following the initial emergence of the germ tube. Electroporation efficiencies as high as 2 × 10⁵ CFU μg⁻¹ of plasmid DNA were obtained using electrocompetent germlings. The optimal field strength was 12–14 kV cm⁻¹ with a pulse duration of 15–20 ms. Electrocompetent germlings were stored at −80°C without a significant decrease in transformation efficiency. The utility of this protocol was demonstrated by isolating a propionyl-CoA carboxylase mutant through targeted gene disruption and replacement. Received 3 April 1998/ Accepted in revised form 28 September 1998     

Effect of anion transport blockers on CFTR in the human sweat duct

Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A (PKA) and ATP regulated Cl- channel. Studies using mostly ex vivo systems suggested diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and glybenclamide inhibit CFTR Cl- conductance (CFTR GCl). However, the properties of inhibition in a native epithelial membrane have not been well defined. The objective of this study was to determine and compare the inhibitory properties of the aforementioned inhibitors as well as the structurally related anion-exchange blockers (stilbenes) including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) in the microperfused intact and basilaterally permeabilized native sweat duct epithelium. All of these inhibitors blocked CFTR in a dose-dependent manner from the cytoplasmic side of the basilaterally permeabilized ducts, but none of these inhibitors blocked CFTR GCl from the luminal surface. We excluded inhibitor interference with a protein kinase phosphorylation activation process by "irreversibly" thiophosphorylating CFTR prior to inhibitor application. We then activated CFTR GCl by adding 5 mM ATP. At a concentration of 10(-4) M, NPPB, DPC, glybenclamide, and DIDS were equipotent and blocked approximately 50% of irreversibly phosphorylated and ATP-activated CFTR GCl (DIDS = 49 +/- 10% > NPPB = 46 +/- 10% > DPC = 38 +/- 7% > glybenclamide = 34 +/- 5%; values are mean +/- SE expressed as % inhibition from the control). The degree of inhibition may be limited by inhibitor solubility limits, since DIDS, which is soluble to 1 mM concentration, inhibited 85% of CFTR GCl at this concentration. All the inhibitors studied primarily blocked CFTR from the cytoplasmic side and all inhibition appeared to be independent of metabolic and phosphorylation processes.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

Normal CFTR Activity and Reversed Skin Potentials in Pseudohypoaldosteronism

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl⁻ channel function is required for activating amiloride-sensitive epithelial Na⁺ channels (ENaC) in salt-absorbing human sweat duct. It is unclear whether ENaC channel function is also required for CFTR activation. The dysfunctional ENaC mutations in type-1 pseudohypoaldosteronism (PHA-1) provided a good opportunity to study this phenomenon of ion channel interaction between CFTR and ENaC. The PHA-1 ducts completely lacked spontaneous ENaC conductance (gENaC). In contrast, the normal ducts showed large spontaneous gENaC (46 ± 10 ms, mean ± SE). After permeabilization of the basolateral membrane with α-toxin, cAMP + ATP activation of CFTR Cl⁻ conductance (gCFTR) or alkalinization of cytosolic pH (6.8 to 8.5) stimulated gENaC of normal but not PHA-1 ducts. In contrast, both spontaneous gCFTR in intact ducts and (cAMP + ATP)-activated gCFTR of permeabilized ducts appeared to be similar in normal and PHA-1 subjects. Lack of gENaC completely blocked salt absorption and caused dramatic reversal of skin potentials associated with pilocarpine-induced sweat secretion from significantly negative in normal subjects (−13 ± 7.0 mV) to significantly positive (+22 ± 11.0 mV) in PHA-1 patients. We conclude that virtual lack of ENaC in PHA-1 ducts had little effect on CFTR activity and that the positive skin potentials could potentially serve as a diagnostic tool to identify type-1 pseudohypoaldosteronism. An erratum to this article is available at .     

cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct

It is generally believed thatcAMP-dependent phosphorylation is the principle mechanism foractivating cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. However, we showed that activating Gproteins in the sweat duct stimulated CFTR Cl conductance(G_Cl) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G_Cl is independent ofprotein kinase A. We activated G proteins and monitored CFTRG_Cl in basolaterally permeabilized sweat duct.Activating G proteins with guanosine5'-O-(3-thiotriphosphate) (10-100 µM) stimulated CFTRG_Cl in the presence of 5 mM ATP alone withoutcAMP. G protein activation of CFTR G_Cl requiredMg²⁺ and ATP hydrolysis (5'-adenylylimidodiphosphate couldnot substitute for ATP). G protein activation of CFTRG_Cl was 1) sensitive to inhibition bythe kinase inhibitor staurosporine (1 µM), indicating that theactivation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM)and SQ-22536 (100 µM); and 3) independent ofCa²⁺, suggesting that Ca²⁺-dependent proteinkinase C and Ca²⁺/calmodulin-dependent kinase(s) are notinvolved in the activation process. Activating AC with10⁶ M forskolin plus 10⁶ M IBMX (in thepresence of 5 mM ATP) did not activate CFTR, indicating that cAMPcannot accumulate sufficiently to activate CFTR in permeabilized cells.We concluded that heterotrimeric G proteins activate CFTR G_Cl endogenously via a cAMP-independent pathwayin this native absorptive epithelium.

     

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.     

Disruption of epidermal specific gene expression and delayed skin development in AP-2 gamma mutant mice

Summary Sentence: Conditional ablation of AP-2γ results in a delay in skin development and abnormal expression of p63, K14, K1, filaggrin, repetin and secreted Ly6/Plaur domain containing 1, key genes required for epidermal development and differentiation.The development of the epidermis, a stratified squamous epithelium, is dependent on the regulated differentiation of keratinocytes. Differentiation begins with the initiation of stratification, a process tightly controlled through proper gene expression. AP-2γ is expressed in skin and previous research suggested a pathway where p63 gene induction results in increased expression of AP-2γ, which in turn is responsible for induction of K14. This study uses a conditional gene ablation model to further explore the role of AP-2γ in skin development. Mice deficient for AP-2γ exhibited delayed expression of p63, K14, and K1, key genes required for development and differentiation of the epidermis. In addition, microarray analysis of E16.5 skin revealed delayed expression of additional late epidermal differentiation genes: filaggrin, repetin and secreted Ly6/Plaur domain containing 1, in mutant mice. The genetic delay in skin development was further confirmed by a functional delay in the formation of an epidermal barrier. These results document an important role for AP-2γ in skin development, and reveal the existence of regulatory factors that can compensate for AP-2γ in its absence.     

Terminal uridyltransferase enzyme Zcchc11 promotes cell proliferation independent of its uridyltransferase activity

Zcchc11 is a uridyltransferase protein with enzymatic activity directed against diverse RNA species. On the basis of its known uridylation targets, we hypothesized that Zcchc11 might regulate cell proliferation. Confirming this, loss-of-function and complementary gain-of-function experiments consistently revealed that Zcchc11 promotes the transition from G(1) to S phase of the cell cycle. This activity takes place through both Rb-dependent and Rb-independent mechanisms by promoting the expression of multiple G(1)-associated proteins, including cyclins D(1) and A and CDK4. Surprisingly, a Zcchc11 construct with point mutations inactivating the uridyltransferase domain enhanced cell proliferation as effectively as wild-type Zcchc11. Furthermore, truncated mutant constructs revealed that the cell cycle effects of Zcchc11 were driven by the N-terminal region of the protein that lacks the RNA-binding domains and uridyltransferase activity of the full protein. Therefore, the N-terminal portion of Zcchc11, which lacks nucleotidyltransferase capabilities, is biologically active and mediates a previously unrecognized role for Zcchc11 in facilitating cell proliferation.