Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes.

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.

Site-specific charge interactions of alpha-conotoxin MI with the nicotinic acetylcholine receptor.

We have tested the importance of charge interactions for alpha-conotoxin MI binding to the nicotinic acetylcholine receptor (AChR). Ionic residues on alpha-conotoxin MI were altered by site-directed mutagenesis or by chemical modification. In physiological buffer, removal of charges at the N terminus, His-5, and Lys-10 had small (2-4-fold) effects on binding affinity to the mouse muscle AChR and the Torpedo AChR. It was also demonstrated that conotoxin had no effect on the conformational equilibrium of either receptor, as assessed by the effects of the noncompetitive antagonist proadifen on conotoxin binding and, conversely, the effect of conotoxin on the affinity of phencyclidine, proadifen, and ethidium. Conotoxin displayed higher binding affinity in low ionic strength buffer; neutralization of Lys-10 and the N terminus by acetylation blocked this affinity shift at the alphadelta site but not at the alphagamma site. It is concluded that Ctx residues Lys-10 and the N terminal interact with oppositely charged receptor residues only at the alphadelta site, and the two sites have distinct arrangements of charged residues. Ethidium fluorescence experiments demonstrated that conotoxin is formally competitive with a small cholinergic ligand, tetramethylammonium. Thus, alpha-conotoxin MI appears to interact with the portion of the binding site responsible for stabilizing agonist cations but does not do so with a cationic residue and is, consequently, incapable of inducing a conformational change.

A tail of protein folding.

This review describes the use of a simple genetic system that has provided important insight into the process of folding and, of its flipside, that of protein aggregation. These studies make use of the tail protein of the bacterial virus P22 which infects Salmonella typhimurium. This folding system serves as a model for a number protein structural elements and may also provide important insights into disease-related protein folding defects at a time when an increasing number of diseases are being shown to be due to protein folding alterations.

Nonextractive spectrofluorimetric determination of aluminium in real, environmental and biological samples using chromotropic acid.

An ultra-sensitive and highly selective nonextractive fluorimetric method is presented for the rapid determination of aluminium at nano-trace levels using chromotropic acid as a fluorimetric reagent [lambda(ex) = 360 nm and lambda(em) = 390 nm] in the pH range of 4.1-4.7. The fluorescence intensity of the metal chelate (2:3 complex) reaches a constant value within 1/2 hr and remains unchanged for over 48 hr. The fluorescence intensity aluminium concentration calibration curve is collinear between 1 and 300 ng/ml of Al. A constant fluorescence intensity is obtained over a wide range (1:50-1:1500) of Al:reagent molar concentrations. Large excesses of over 60 cations, anions and complexing agents (like tartrate, oxalate, phosphate, thio-urea, SCN(-), etc.) do not interfere in the Al determination. The developed method was successfully used in assaying aluminium in several standard reference materials (Al-bronze, brass, stainless steel) as well as in some environmental and biological samples. The method is very precise and accurate (S.D = +/-0.001 on 10 ng/ml; 11 determinations).

Monoclonal antibody to amphomycin. A tool to study the topography of dolichol monophosphate in the membrane.

Understanding the topographical orientation of dolichol monophosphate (Dol-P) in the membrane of the endoplasmic reticulum (ER) is of utmost importance for studying the regulation of asparagine-linked protein glycosylation in eukaryotic cells. This was practically impossible due to the nonavailability of a suitable probe. Recent studies on the specific interaction between a lipopeptide, amphomycin, and Dol-P, provided an insight to develop a monospecific antibody to amphomycin which could recognize the amphomycin-Dol-P complex in order to detect Dol-P immunocytochemically in the ER membrane. We report herein the successful production of a monoclonal antibody to amphomycin. The antibody belongs to the IgG+IgM subclasses and is specific for amphomycin when analyzed by the enzyme-linked immunoassay and immunoblot procedures. The antibody recognizes with equal potency both the native amphomycin and also mild acid-hydrolyzed amphomycin from which N-terminal fatty acylated aspartic acid has been removed. Preincubation of amphomycin with the antibody partially reduced the inhibitory action of amphomycin on dolichol phosphate mannosyltransferase (EC 2.4.1.83). Furthermore, exposure of capillary endothelial cells to amphomycin, followed by the monoclonal antibody to amphomycin, followed sequentially by staining with FITC-conjugated goat anti-mouse IgG and examination under a fluorescent microscope gives intense fluorescence at the perinuclear region of the cell with a structure reminiscent of the ER.

In vivo characterization of the poly(ADP-ribosylation) of SV40 chromatin and large T antigen by immunofractionation.

We have confirmed the poly(ADP-ribosylation) of large T antigen of SV40 by using antibodies to both large T antigen and poly(ADP-ribose) and consequently have begun to characterize how this post-translational nuclear modification of the viral protein modulates its biological functions. SV40 minichromosomal subpopulation containing replicative intermediate DNA was shown to have a significantly higher affinity for anti-poly(ADP-Rib)-Sepharose than viral chromatin fractions containing mature minichromosomal DNA. An anti-large T-Sepharose column was used to isolate T antigen from crude extracts by two different approaches: (1) large T antigen was labeled with [35S]methionine in vivo and the infected cell extract was immunofractionated to isolate large T antigen and (2) large T antigen from infected cell extracts was immunofractionated followed by immunostaining. Using these techniques, 1-10% of the total T antigen from infected cells was found to be poly(ADP-ribosylated). Minichromosome preparations per se were also subjected to immunofractionation on anti-large T-Sepharose. The high level of retention of poly(ADP-ribosylated) species of minichromosomes on this matrix suggested that this post-translational modification of viral chromatin may be related to those steps in viral replication and transcription under regulation by large T antigen.

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.

Genetic evidence that action of cAMP-dependent protein kinase is not an obligatory step for antiviral and antiproliferative effects of human interferon in Chinese hamster ovary cells.

Several Chinese hamster ovary (CHO) cell mutants with altered cAMP-dependent protein kinases were utilized to study the possible involvement of this enzyme in mediating interferon effects in cultured cells. Human fibroblast interferon had a pronounced protective effect for CHO cells against encephalomyocarditis virus (EMCV). This protective effect was also observed in cAMP-resistant CHO mutants with protein kinase defects, although to a slightly lesser extent. EMCV, however, which grows well in wild-type CHO cells, grew poorly in the cAMP-resistant CHO mutants even in the absence of interferon, suggesting that cAMP-dependent protein kinase is involved in the normal growth of this virus. The growth inhibitory and morphological effects of human fibroblast interferon on CHO cells as well as the induction of 2',5'-oligoA synthetase and the level of RNase F activity (which is constitutive in CHO cells) were very similar in wild-type and mutant CHO cells. It is concluded from these studies that cAMP-dependent protein kinase may have a facilitating role in antiviral activity of interferon, but does not have an obligatory role in mediating either the antiviral or antiproliferative effects of interferon in CHO cells.

Interferon-mediated inhibition of adenylate cyclase in mouse cells.

Adenylate cyclase activity was measured in mouse cell plasma membrane after 30 units/ml of interferon (IFN) treatment. The basal and Gpp(NH)p stimulated enzyme activity in LB cell was inhibited by 20%-40% throughout the time course of treatment (30 min to 18 h). The reduced enzyme activity was not associated with enhanced breakdown of cAMP, but was related to alteration in the catalytic component of the enzyme. Moreover, there was no change in the affinity for guanine-nucleotide regulatory unit and 1 X 10(-7) M Gpp(NH)p was required to obtain half-maximal activation with either type of membrane. Interferon action on adenylate cyclase was biphasic, at 10 u/ml it was stimulatory and between 30 and 1000 u/ml it inhibited the enzyme activity to a great extent; but IFN did not exert its effect directly on the enzyme. The reduction in enzyme activity was comparable to a reduction in the intracellular level of cAMP in most of the interferon-sensitive cell lines studied. We propose, therefore, that the inhibitory effect on adenylate cyclase is related to a step in the development of antiviral activity.

Rapid, single-step purification of restriction endonucleases on cibacron blue F3GA-agarose.

After sonication and high-speed centrifugation, crude extracts of B. amyloliquefaciens, P. alcalifaciens, X. holicola, and B. globiggi were adsorbed on the dye Cibacron blue F3GA convalently cross-linked to agarose. The restriction endonucleases BamHI, PalI, XhoI, and BglI together with BglII were isolated by elution of the dye column with linear gradients to 0.5 M NaCl. The enzymes so purified were free of contaminating nucleic acids and other nucleases and were sufficiently concentrated for direct, specific DNA hydrolysis.