Neither high-pass filtering nor mathematical differentiation of the EMG signals can considerably reduce cross-talk.

Using mathematical simulation of motor unit potentials (MUPs), detected by a point and rectangular plate electrode, we have shown that the muscle tissue does not act like a low-pass frequency filter on MUPs. Depending on the electrode type and its longitudinal position, the relative weight of the terminal phases (reflecting the excitation extinction) in MUPs and thus of high frequencies in the MUP power spectrum, increase with the MU depth. Therefore, high-pass filtering or differentiating signals detected neither monopolarly nor bipolarly could eliminate the cross-talk produced by high frequency components of MUPs from deep MUs. Such methods could be effective against the main components but not against the MUP leading edge and terminal phases. To reduce the cross-talk, position of the detecting electrodes should correspond to anatomy of muscles producing the cross-talk. Monopolar electrode should be located above the ends of the muscles. Cross-talk of the muscles located beyond the muscle of interest could be higher than that produced above the end-plate of deep muscles. On the contrary, under detection by a longitudinal bipolar electrode, the cross-talk is much smaller above the end-plate region or beyond deep muscles. The cross-talk is the greatest above the ends of the deep muscles.     

Induced androgenesis in tomato (Lycopersicon esculentum Mill.) I. Influence of genotype on androgenetic ability

The androgenetic ability of 85 Lycopersicon esculentum Mill. genotypes was tested. Callus was induced from anthers of 53 lines and hybrids. Regeneration of plants was obtained only from calli of 15 genotypes. The data obtained clearly showed that the genotype affects induced androgenesis in tomato. The in vitro response of anthers from the cultivars Roma, Pearson, San Marzano, Por, Sar, Vigapol, Day, David and Start, containing the ms 10³⁵ gene, which is responsible for male sterility in tomato, confirm the strongly expressed dependence of both callus induction and organogenetic potential on the homozygous or heterozygous state of that gene. A protocol of callus induction, organogenesis and plant regeneration has been developed. More than 6000 regenerants have been obtained. Most of them showed different morphological alterations and variations in chromosome number (n, 2n, 4n). Some are interesting as source material for tomato breeding. Received: 17 April 1997 / Revision received: 4 August 1997 / Accepted: 15 October 1997     

The NMR solution structure and characterization of pH dependent chemical shifts of the β-elicitin, cryptogein

The NMR structure of the 98 residue -elicitin, cryptogein, which induces a defence response in tobacco, was determined using 15N and 13C/15N labelled protein samples. In aqueous solution conditions in the millimolar range, the protein forms a discrete homodimer where the N-terminal helices of each monomer form an interface. The structure was calculated with 1047 intrasubunit and 40 intersubunit NOE derived distance constraints and 236 dihedral angle constraints for each subunit using the molecular dynamics program DYANA. The twenty best conformers were energy-minimized in OPAL to give a root-mean-square deviation to the mean structure of 0.82 Å for the backbone atoms and 1.03 Å for all heavy atoms. The monomeric structure is nearly identical to the recently derived X-ray crystal structure (backbone rmsd 0.86 Å for residues 2 to 97) and shows five helices, a two stranded antiparallel -sheet and an -loop. Using 1H,15N HSQC spectroscopy the pKa of the N- and C-termini, Tyr12, Asp21, Asp30, Asp72, and Tyr85 were determined and support the proposal of several stabilizing ionic interactions including a salt bridge between Asp21 and Lys62. The hydroxyl hydrogens of Tyr33 and Ser78 are clearly observed indicating that these residues are buried and hydrogen bonded. Two other tyrosines, Tyr47 and Tyr87, show pKa's >12, however, there is no indication that their hydroxyls are hydrogen bonded. Calculations of theoretical pKa's show general agreement with the experimentally determined values and are similar for both the crystal and solution structures.     

Photoinactivation and kinetics of membrane fusion mediated by the human immunodeficiency virus type 1 envelope glycoprotein.

The fusion kinetics of cells expressing the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein with CD4 target cells was continuously monitored by image-enhanced Nomarski differential interference contrast optics. The analysis of the videotape recordings showed that (i) cells made contact relatively rapidly (within minutes), in many cases by using microspikes to "touch" and adhere to adjoining cells; (ii) the adhered cells fused after a relatively long waiting period, which varied from 15 min to hours; (iii) the morphological changes after membrane fusion, which led to disappearance of the interface separating the two cells, were rapid (less than 1 min); and (iv) the process of syncytium formation involved subsequent fusion with other cells and not simultaneous fusion of many cells. To measure the kinetics of early stages of cell fusion, we used the recently developed very stable membrane-soluble dye, PKH26, which redistributes between labeled and unlabeled membranes after fusion but does not exchange spontaneously between membranes for prolonged periods. We found that photoactivation of this dye by illumination with green light inhibits fusion of cell membranes as indicated by the lack of dye transfer from the labeled HIV-1 envelope-expressing cells to unlabeled CD4 cells. The inhibitory effect was localized in space and time, which allowed us to develop a new assay for measuring the kinetics of membrane fusion by illuminating the cell mixture at different times after coculture. This assay has also been used to monitor the fusion kinetics of HIV-1 and recombinant vaccinia virus. The photoactivation of nonexchangeable membrane-soluble fluorescent dyes may be useful for development of new assays for measuring the kinetics of membrane fusion and could also be important in designing new antiviral approaches.     

An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences.

Nonoverlapping deletions that eliminated the 5' (HIV-1US/603del), middle (HIV-1U5/206del), and 3' (HIV-1U5/604del) thirds of the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (transient transfection of HeLa cells) and infectivity (T-cell lines and peripheral blood mononuclear cells). All three mutants exhibited a wild-type phenotype in directing the production and release of virus particles from transfected HeLa cells. In infectivity assays, HIV-1U5/206del was usually indistinguishable from wild-type virus whereas HIV-1U%/603del was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1U5/603del particles revealed a packaging defect resulting in a 10-fold reduction of encapsidated genomic RNA. The HIV-1U5/604del mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type and multiplicity of infection. Quantitative competitive PCR indicated that HIV-1U5/604del synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1U5/604del mutant that displayed rapid infection kinetics emerged. Nucleotide sequence analysis indicated that the original 26-nucleotide deletion present in HIV-1U5/604del had been extended an additional 19 nucleotides in the revertant virus. Characterization of the HIV-1U5/604del mutant LTR in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutant virus.     

Quantitation of human immunodeficiency virus type 1 infection kinetics.

Tissue culture infections of CD4-positive human T cells by human immunodeficiency virus type 1 (HIV-1) proceed in three stages: (i) a period following the initiation of an infection during which no detectable virus is produced; (ii) a phase in which a sharp increase followed by a peak of released progeny virions can be measured; and (iii) a final period when virus production declines. In this study, we have derived equations describing the kinetics of HIV-1 accumulation in cell culture supernatants during multiple rounds of infection. Our analyses indicated that the critical parameter affecting the kinetics of HIV-1 infection is the infection rate constant k = Inn/ti, where n is the number of infectious virions produced by one cell (about 10(2)) and ti is the time required for one complete cycle of virus infection (typically 3 to 4 days). Of particular note was our finding that the infectivity of HIV-1 during cell-to-cell transmission is 10(2) to 10(3) times greater than the infectivity of cell-free virus stocks, the inocula commonly used to initiate tissue culture infections. We also demonstrated that the slow infection kinetics of an HIV-1 tat mutant is not due to a longer replication time but reflects the small number of infectious particles produced per cycle.     

Venom exonuclease. IV. Intrinsic fluorescence of the exonuclease from Crotalus adamanteus venom

The intrinsic fluorescence of the exonuclease isolated from Crotalus adamanteus venom, was studied. The position of its maximum at 335 nm and half-width of the emission band 55 nm (lambda exc. 295 nm) suggested the existence of at least two types of tryptophan residues in the enzyme molecule. Differential analysis of the fluorescence spectra obtained by excitation at 280 and 295 nm revealed about 12.5% contribution of the tyrosine fluorescence in the overall emission excited at 280 nm. The environment of the tryptophan residues in the exonuclease was studied by quenching of their fluorescence with various ionic (NO3-, NO2-, I-, Br- and Cs+) and non-ionic agents (acrylamide, chloroform-methanol). On this basis, fractions of inner (non-polar) and surface tryptophan residues located in charged and neutral regions of the enzyme molecule were evaluated. More than half of the residues (60%) was found in the inner part of the exonuclease while most of its surface tryptophans--in a neutral region(s).     

Kinetics of soluble CD4 binding to cells expressing human immunodeficiency virus type 1 envelope glycoprotein.

The high-affinity interaction between the envelope glycoprotein (gp120-gp41) of the human immunodeficiency virus type 1 and its receptor, CD4, is important for viral entry into cells and therapeutical approaches based on the soluble form of CD4 (sCD4). Using flow cytometry, we studied the kinetics of binding of sCD4 to gp120-gp41 expressed on the cell surface. sCD4 binding was dependent on sCD4 concentration and temperature and exhibited bimolecular reaction kinetics. Binding was very slow at low sCD4 concentrations (below 0.2 micrograms/ml) and low temperatures (below 13 degrees C) but increased sharply with increasing temperature. The rate constant for association at 37 degrees C (1.5 x 10(5) M-1 s-1) was 14-fold higher than at 4 degrees C, but the affinity of sCD4 to membrane-bound gp120-gp41 was not significantly affected. The activation energy at higher temperatures (28 to 37 degrees C) was less than at lower temperatures (4 to 13 degrees C). After long periods of incubation, we observed a decrease of surface-bound sCD4 and gp120, even at low temperatures, which was attributed to sCD4-induced shedding of gp120. The rate of gp120 shedding was much lower than the rate of sCD4 binding and was dependent on sCD4 concentration and temperature. The finding that sCD4 binding is slow, especially at low sCD4 concentrations, can be of critical importance for efficient blocking of viral infection by sCD4 and should be considered when designing new protocols in the therapy of AIDS patients.