Depolarization increases the single-channel conductance and the open probability of crayfish glutamate channels.

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from -90 mV to approximately 20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around -75 mV) and depolarization (membrane potential approximately 105 mV). We found that the glutamate dissociation rate constant (k_) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing (k(c)) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening (k(o)) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.     

Tight DNA binding and oligomerization are dispensable for the ability of p53 to transactivate target genes and suppress transformation.

The p53 tumor suppressor protein can bind tightly to specific sequence elements in the DNA and induce the transactivation of genes harboring such p53 binding sites. Various lines of evidence suggest that p53 binds to its target site as an oligomer. To test whether oligomerization is essential for the biological and biochemical activities of p53, we deleted a major part of the dimerization domain of mouse wild-type p53. The resultant protein, termed p53wt delta SS, was shown to be incapable of forming detectable homo-oligomers in vitro and is, therefore, likely to be predominantly if not exclusively monomeric. In agreement with the accepted model, p53wt delta SS indeed failed to exhibit measurable DNA binding in vitro. Surprisingly, though, it was still capable of suppressing oncogene-mediated transformation and of transactivating in vivo a target gene containing p53 binding sites. These findings indicate that dimerization-defective p53 is biologically active and may engage in productive sequence-specific DNA interactions in vivo. Furthermore, p53 dimerization probably leads to cooperative binding to specific DNA sequences.     

Conduction block in a branching axon innervating two muscles under physiological conditions

The escape reflex of the lobster consists of a series of tail flips resulting from alternating activity of the abdominal flexor and extensor muscles. Electromyographic (EMG) activity was recorded from the medial (DEAM) and the lateral (DEAL₁) deep abdominal extensor muscles during free swimming. During the escape response, the muscles were active either synchronously or separately, at frequencies of 100–120 Hz. This activity pattern could be generated either by central programming, or by a peripheral mechanism such as frequency-dependent differential conduction block into one of the two branches of the common excitor axon (C.Ex) innervating these muscles. In order to explore the latter possibility in a living animal, we left the DEAM and DEAL₁ muscles innervated only by the C.Ex from the tested segment. This was accomplished by manually cutting all other axons in the nerve under visual control. During escape responses in six successfully dissected animals, we found 27 sudden failures of the DEAM responses and only three in DEAL₁. The failures were usually preceded by an increase in the delay of the response. These findings strongly suggest that conduction block occurs in the M branch innervating the DEAM under physiological conditions.     

Differential effects of GABA on three muscles innervated by a common inhibitory axon ofOcypode

Summary The effect of GABA (-aminobutyric acid) on three muscles innervated by the common inhibitory axon in the walking leg of the crabOcypode cursor, was studied. The muscles differ in the percentage of fibres responding to GABA by membrane resistance decrease, and in the magnitude of the response (Table 1). In addition to the postsynaptic effect (on muscle fibre membrane) of GABA, a presynaptic effect (on excitatory terminals) was observed in one muscle, resulting in more effective inhibition of excitatory potentials. The presynaptic effect sustained as long as GABA was present, while the postsynaptic effect underwent desensitization (Fig. 2). The data demonstrate differential inhibition of distinct functional units innervated by a common axon. The channeling of inhibitory information results from spatial organization of innervation, differing in location (pre-or postsynaptic) and density.This investigation was supported by grant AZ11 1955 for Stiftung Volkswagenwerk.     

Coupling factor ATPase complex of Rhodospirillum rubrum. Purification and characterization of an oligomycin and N,N'-dicyclohexylcarbodiimide-sensitive (Ca+ + Mg2+)-ATPase.

An ATPase complex sensitive to the energy transfer inhibitors oligomycin, dicyclohexylcarbodiimide and venturicidin has been solubilized from Rhodospirillum rubrum chromatophores with Triton X-100 and further purified by centrifugation on a glycerol gradient. The partially purified RrFo . F1 contains 13 distinct polypeptide subunits, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, including the subunits of the oligomycin-sensitive, water-soluble RrF1 ATPase. The ATPase activity of RrF0 . F1 as that of the membrane-bound enzyme complex depends on Ca2+ or Mg2+ and from detailed kinetic studies it is concluded that the divalent cation-ATP complex is the substrate for both ATPase complexes. Free ATP and free Mg2+ act as competitive inhibitors, with Ki values of 1 mM and 7 muM, respectively. The subunit composition of the purified RrFo . F1 and its similarity to the membrane-bound ATPase with respect to cation dependence and sensitivity to energy transfer inhibitors suggests that it contains all the subunits of the R. rubrum coupling factor-ATPase complex.     

Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA

In various permissive monkey cell lines infected with simian virus 40 there are two major forms of large T antigen which differ in their rate of sedimentation through sucrose gradients. The lighter (5 to 7S) form sedimented slightly more rapidly than the 4S tRNA marker, whereas the heavier (16S) form sedimented slightly more slowly than the 18S rRNA marker. The small t antigen did not form complexes which sedimented as rapidly as those formed by the large T antigen. The 16S T antigen form was converted to the slowly sedimenting 5 to 7S form in the presence of 1.0 M NaCl. The majority of large T antigen synthesized in cell-free protein-synthesizing systems primed by mRNA isolated from infected cells sedimented as the 5 to 7S form even when premixed with excess quantities of cellular T antigen. The formation of the 16S form in infected cells did not require ongoing viral or cellular DNA replication because considerable quantities of this T antigen class were produced in the presence of DNA synthesis inhibitors, such as cytosine arabinoside. Both 5 to 7S and 16S forms could be isolated separately and, therefore, each could be analyzed as to its individual properties. The 5 to 7S T antigen form bound more efficiently and tightly to DNA and had specific affinity for sequences at the viral origin of replication, whereas the 16S form bound less efficiently to DNA and exhibited very little specificity for origin-containing DNA sequences. It is therefore likely that the active DNA-binding species of T antigen isolated from infected cells is the 5 to 7S form.     

SV40 large tumor antigen can regulate some cellular transcripts in a positive fashion

Eleven cDNA clones identified from a cDNA library prepared from the mRNA fraction of SV40 transformed cells detected, by hybridization, higher levels of cellular mRNA in SV40-transformed cells than in nontransformed cells. Three of these cDNA clones detected levels of cellular mRNA that were more than 100-fold greater in SV40tsA transformed cell lines grown at the permissive temperature than in those grown at the nonpermissive temperature. Northern blot hybridizations confirmed these results and in some cases detected RNA species of multiple sizes that were regulated in a temperature-dependent fashion in SV40tsA transformed cell lines. Infection of 3T3 cells with SV40 stimulated the levels of RNAs complementary to these cDNA clones. The results demonstrate that the SV40 large T antigen can regulate the steady state levels of some cellular RNA species.     

Survey and synthesis of intra- and interspecific variation in stomatal sensitivity to vapour pressure deficit

Responses of stomatal conductance (g_s) to increasing vapour pressure deficit (D) generally follow an exponential decrease described equally well by several empirical functions. However, the magnitude of the decrease – the stomatal sensitivity – varies considerably both within and between species. Here we analysed data from a variety of sources employing both porometric and sap flux estimates of g_s to evaluate the hypothesis that stomatal sensitivity is proportional to the magnitude of g_s at low D ( ≤ 1 kPa). To test this relationship we used the function g_s = g_sref–m· lnD where m is the stomatal sensitivity and g_sref = g_s at D = 1 kPa. Regardless of species or methodology, m was highly correlated with g_sref (average r² = 0·75) with a slope of approximately 0·6. We demonstrate that this empirical slope is consistent with the theoretical slope derived from a simple hydraulic model that assumes stomatal regulation of leaf water potential. The theoretical slope is robust to deviations from underlying assumptions and variation in model parameters. The relationships within and among species are close to theoretical predictions, regardless of whether the analysis is based on porometric measurements of g_s in relation to leaf-surface D (D_s), or on sap flux-based stomatal conductance of whole trees (G_Si), or stand-level stomatal conductance (G_S) in relation to D. Thus, individuals, species, and stands with high stomatal conductance at low D show a greater sensitivity to D, as required by the role of stomata in regulating leaf water potential.