Gas2, a growth arrest-specific protein, is a component of the microfilament network system

In this report we analyze the protein product of a growth arrest-specific gene, gas2, by means of an affinity-purified antibody raised against the protein produced in bacteria. The regulation of Gas2 biosynthesis reflects the pattern of mRNA expression (Schneider, C., R. King, and L. Philipson. 1988. Cell. 54:787-793): its relative level is tightly associated with growth arrest. Gas2 seems to be regulated also at the posttranslational level via a phosphorylation mechanism. Gas2 is well conserved during the evolution with the same apparent molecular mass (36 kD) between mouse and human. We also demonstrate that Gas2 is a component of the microfilament system. It colocalizes with actin fiber, at the cell border and also along the stress fiber, in growth-arrested NIH 3T3 cells. The pattern of distribution, detected in arrested cells, can also be observed in growing cells when they are microinjected with the purified GST-Gas2 protein. In none of the analyzed oncogene-transformed NIH 3T3 cell lines was Gas2 expression induced under serum starvation.     

The Binding Properties of the Particulate and Solubilized Sulfonylurea Receptor from Cerebral Cortex Are Modulated by the Mg2+ Complex of ATP

Glibenclamide closes an ATP-sensitive K+ channel (K-ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonyl-ureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40-50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg-ATP (300 microM) reduced the number of high-affinity sites for [3H]-glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg-ATP was half-maximally effective at 41 microM. Alkaline phosphatase accelerated the reversal of Mg-ATP-induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.     

Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Human τ Detection

Immunoaffinity chromatography with a monoclonal antibody produced against bovine tau protein was used to purify tau proteins from human brain. Fifty grams of brain tissue yielded approximately 2 mg of pure tau proteins. The affinity-purified human tau was used to produce a high-titered rabbit anti-human tau serum. The monoclonal anti-tau antibody and the polyclonal rabbit anti-tau serum were then used to construct a sandwich enzyme-linked immunosorbent assay for detection of human tau proteins, with a sensitivity of 1 ng/ml.     

Online control of evaluation algorithms of the image applied to identification of deglutition function]

Comprehensive software and hardware have been developed for the processing of biosignals. Such automatic signal processing, however not only has advantages, but also drawbacks. The question as to the reliability of the evaluation algorithm arises when the signal is modified, in the presence of interindividual differences, and in particular when noise is superimposed. This is of great interest for long-term recording when the original signal can no longer be inspected visually. The aim of our work was to display the signals on the screen of a monitor simultaneously with lines marking the points (start, end, extreme value, etc.) processed by the specific signal processing algorithm. The program package permits the on-line recording and monitoring of signals, the parallel processing and marking of detected events on the monitor, as well as storage of the parameters extracted. It is a very effective tool for developing, improving and monitoring of algorithms and their efficiency for signal processing.     

Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase.

The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri [Viebrock, A. & Zumft, W. G. (1988) J. Bacteriol. 170, 4658-4668] and the subunit II of cytochrome-c oxidase (COII). Both types of enzymes possess the CuA binding site. The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22-kb PstI fragment (NS220) of nosZ from P. stutzeri. The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A. eutrophus) and 70695 Da (P. aeruginosa). The N-terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport. A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences. Only three out of nine cysteine residues of the NosZ protein (P. stutzeri) are invariant. Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus. Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes. The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences. Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination. Of 23 histidine residues in NosZ (P. stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences. Conserved tryptophan residues are located close to several potential copper ligands. Trp615 may contribute to the observed quenching of fluorescence when the CuA site is occupied.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.     

Auxin-induced H+-pump stimulation does not depend on the presence of epidermal cells in corn coleoptiles

Cell-wall acidification and electrical reactions (depolarization and hyperpolarization) are typical auxin responses in maize (Zea mays L.) coleoptiles. In an attempt to test the role of the outer epidermis in these responses, they have been measured and compared in intact and peeled coleoptile fragments. To exclude interactions between parenchymal and epidermal cells, the coleoptile pieces were completely stripped of their outer epidermis. This preparation was monitored by means of a scanning electron microscope. When externally applied indole-3-acetic acid was tested, we found that neither cell-wall acidification nor the electrical membrane responses depended on the presence of intact epidermal cells.Abbreviations IAA Indole-3-acetic acid - MES 2-[N morpholino-ethane-sulfonic acid - TRIS 2-Amino-2-hydroxymethyl-1,3-propanediol We thank Kuki Kaethner for her excellent technical assistance. This work was supported by the Hessische Graduiertenförderung and the Deutsche Forschungsgemeinschaft.     

Biochemical diversity and genetic distance in two species of the genusSaguinus

A comparative study of 20 blood genetic systems was performed on three populations of genusSaguinus: S. fuscicollis weddelli, S. midas niger, andS. midas midas. Some markers are useful for the characterization of the two species.ADA ⁵, ADA⁶, CA2³, CA2⁴, andES2 ² occur only inS. fuscicollis whileADA ⁴is fixed inS. midas. S. midas midas showed heterozygosity value comparable to those previously obtained for the genusAlouatta. Estimates of genetic distance betweenS. fuscicollis andS. midas species of about 14% are in accordance with those referred to in the literature for interspecific differences. Genetic distances between subspecies ofSaguinus midas were nearly 3%, which is within the range for subspecies.     

Structure of the smooth muscle myosin light-chain kinase calmodulin-binding domain peptide bound to calmodulin

The interaction between the peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light-chain kinase and (Ca2+)4-calmodulin has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. The study was facilitated by the use of 15N-labeled peptide in conjunction with 15N-edited and 15N-correlated 1H spectroscopy. The peptide forms a 1:1 complex with calcium-saturated calmodulin which is in slow exchange with free peptide. The 1H and 15N resonances of the bound have been assigned. An extensive set of structural constraints for the bound peptide has been assembled from the analysis of nuclear Overhauser effects and three-bond coupling constants. The backbone conformation of the bound peptide has been determined using these constraints by use of distance geometry and related computational methods. The backbone conformation of the peptide has been determined to high precision and is generally indicative of helical secondary structure. Nonhelical backbone conformations are seen in the middle and at the C-terminal end of the bound peptide. These studies provide the first direct confirmation of the amphiphilic helix model for the structure of peptides bound to calcium-saturated calmodulin.     

Voltage-gated and calcium-gated calcium release during depolarization of skeletal muscle fibers.

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.