Single channel studies of the phosphorylation of K+ channels in the squid giant axon. II. Nonstationary conditions

The effects of phosphorylation on the properties of the 20-pS channel of the squid giant axon were studied using the cut-open axon technique. Phosphorylation of the channel was achieved by photoreleasing caged ATP (inside the patch pipette) in the presence of the catalytic subunit of the protein kinase A. An inverted K+ gradient (500 K+ external parallel 5 K+ internal) was used to study the activation process. Phosphorylation decreased the frequency of openings of the channel at most potentials by shifting the probability vs. voltage curve toward more positive potentials. The mean open times showed no voltage dependence and were not affected by phosphorylation. The distribution of first latencies, on the other hand, displayed a sharp voltage dependence. Phosphorylation increased the latency to the first opening at all potentials, shifting the median first latency vs. voltage curve toward more positive potentials. The slow inactivation process was studied in the presence of a physiological K+ gradient (10 K+ external parallel 310 K+ internal). Pulses to 40 mV from different holding potentials were analyzed. Phosphorylation increases the overall ensemble probability by decreasing the number of blank traces. A single channel inactivation curve was constructed by computing the relative appearance of blank traces at different holding potentials before and after photoreleasing caged ATP. As determined in dialyzed axons, the effect of phosphorylation consisted in a shift of the inactivation curve toward more positive potentials. The 20-pS channel has the same characteristics as the delayed rectifier current in activation kinetics, steady-state inactivation, and phosphorylation effects.     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Oculocerebral syndrome with hypopigmentation (Cross syndrome): the mixed pattern of hair pigmentation as an important diagnostic sign.

The present report describes two female siblings of mixed ancestry (Cape Coloured) with consanguineous parents as further examples of Cross oculocerebral hypopigmentation syndrome. The mixed pattern of hair pigmentation as an important diagnostic sign is emphasized.     

Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55.

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp. BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named 'Manganese-Inhibited Peroxidase'.     

Stabilization of the nuclear matrix by disulfide bridges: identification of matrix polypeptides that form disulfides.

The molecular structure of the nuclear matrix is still poorly understood. We have tried to assess which proteins are important structural elements by examining the process of stabilization of the nuclear matrix by sodium tetrathionate. Sodium tetrathionate stabilizes the nuclear matrix by oxidizing sulfhydryl groups to disulfides. We show that tetrathionate-stabilized matrices are disassembled in buffers containing SDS, indicating that the stabilized nuclear matrix is not a continuous network of cross-linked proteins. Using monobromobimane, a thiol-specific fluorescent reagent, we show that many protein thiols in the stabilized matrix are oxidized. By chromatography on activated thiol-Sepharose we estimated that about 50% of the matrix proteins had oxidized sulfhydryl groups. The protein composition of the material bound to activated thiol-Sepharose was similar to that of the not-bound material. A few proteins are highly enriched in the fraction that was bound to the column. This indicates that many matrix protein species are partially oxidized and that some proteins are completely oxidized. The oxidized protein thiols are found in relatively large complexes as determined by SDS gel-electrophoresis under nonreducing conditions. These results are interpreted in terms of protein-protein interactions in the matrix. The possible role of thiols and disulfides in the in vivo organization of the nucleus is discussed.     

Evolution of the V3 envelope domain in proviral sequences and isolates of human immunodeficiency virus type 1 during transition of the viral biological phenotype.

The third variable domain (V3) of the envelope gene of human immunodeficiency virus type 1 contains a major neutralization epitope and determinants of syncytium-inducing (SI) capacity and replication rate (reviewed by J. P. Moore and P. L. Nara, AIDS Suppl. 2:S21-S33, 1991). Sequences were generated from DNA of samples taken 3 months apart over a period of 24 and 30 months from peripheral blood mononuclear cells (PBMC) of two individuals, both before and after cocultivation with uninfected donor PBMC. The isolated virus shifted from the non-syncytium-inducing (NSI) phenotype to the SI phenotype during the study period. This shift was associated with distinct changes in the V3 domain in both patients. The association of the phenotype shift with the V3 sequence changes was confirmed by construction of viruses with chimeric V3 loops. The shift from NSI- to SI-associated V3 variants was also seen in the uncultured PBMC of both patients, but not until 3 and 9 months after the detection of SI virus in culture. In the samples of uncultured PBMC DNA, several subgroups of sequences were found, indicating that the process of evolution may not be gradual and that several distinct populations can coexist. The paucity of intermediate sequences indicated that strong selection pressure was exerted on this part of the envelope. The early emergence of disease-associated SI variants in cultured material indicates that virus culture may have relevance for the in vivo situation.     

The carboxy-terminal lysine of alpha B-crystallin is an amine-donor substrate for tissue transglutaminase.

A hexapeptide, corresponding to the sequence around the glutamine in beta A3-crystallin that functions as amine-acceptor for transglutaminase, was synthesized. This peptide was biotinylated and used as a probe to identify amine-donor substrates for transglutaminase among lens proteins. It was found that Ca(2+)-activated transglutaminase linked this peptide not only to several beta-crystallins but, unexpectedly, also to alpha B-crystallin. The C-terminal lysine residue of alpha B-crystalline could be identified as the site of linkage. This strengthens the notion that, at least in crystallins, all transglutaminase substrate residues are located in terminal extensions of the polypeptides. It was shown that in lens homogenate, alpha B-crystallin can be covalently crosslinked to beta-crystallins by transglutaminase. The transglutaminase-mediated crosslinking of alpha B-crystallin may have implications for its involvement in normal and pathological processes in lens and other tissues.     

Maturation- and differentiation-dependent responsiveness of human CD4+ T helper subsets.

The human CD45R0+ (memory) CD4+ T cell population can be subdivided into a large (82%) CD27+ and a small (18%) CD27- subset. Within the CD45R0+CD27- subset, cells accumulate that have been persistently stimulated by Ag in vivo. As an apparent consequence, TLC with a differentiated functional phenotype, producing either high levels of IFN-gamma (Th1-like), high levels of IL-4 (Th2-like) or high amounts of both these cytokines (here referred to as Thx) can primarily be generated from the CD27- memory CD4+ T cell subset. In this study we examined the requirements for induction of proliferation of distinct CD4+CD45R0+ Th subsets. Immobilized CD3 mAb induced proliferation with comparable magnitude and kinetics in all types of TLC. However, interference with intracellular signaling pathways in this activation system, resulted in a strong inhibition of proliferation in TLC derived from CD27+ cells whereas, in contrast, TLC from CD27- cells were relatively resistant to elevation of [cAMP]i, inhibition of protein kinase C activation and the immunosuppressive effects of cyclosporin A. Stimulation with CD3 mAb in soluble form resulted in Il-4 secretion by Th2-like and Thx-type TLC but did not induce IFN-gamma or Il-2 secretion in any Th subset. Interestingly, Th2-like cells but not Thx-type cells were able to use endogenously produced Il-4 for proliferation. These data demonstrate a differential sensitivity of CD45R0+CD4+ Th subsets for immune activation and suppression, which correlated with their maturation stage, as reflected by CD27 membrane expression, as well as with their effector phenotype.     

Minimal requirements for the human immunodeficiency virus type 1 V3 domain to support the syncytium-inducing phenotype: analysis by single amino acid substitution.

The third variable domain (V3) of the human immunodeficiency virus type 1 external envelope contains determinants of cell tropism, cytopathicity, and infectivity and elicits antibodies able to block infectivity in vitro and in vivo. Our study encompassed point-mutational analysis of HXB-2 viruses containing patient-derived V3 regions and expressing a non-syncytium-inducing, low-replicating phenotype in T-cell line SupT1. The mutation within V3 of a serine at position 306 into an also naturally occurring arginine (S to R) required an additional, naturally occurring mutation at position 320 (aspartate to glutamine, D to Q) or 324 (aspartate to asparagine, D to N) for full expression of the syncytium-inducing, high-replicating (SI) phenotype. The naturally occurring mutation of an aspartate into an arginine at position 320 (D to R) was sufficient for production of the SI phenotype. This study proves that introduction of a positively charged amino acid at position 306 or 320, previously shown to be strongly associated with the SI phenotype in field isolates (R.A.M. Fouchier, M. Groenink, N.A. Kootstra, M. Tersmette, H.G. Huisman, F. Miedema, and H. Schuitemaker, J. Virol. 66:3183-3187, 1992), is minimally required for production of SI viruses. In addition, naturally occurring mutations at residue 324 also modulate the virus phenotype.     

Plant size,geitonogamy and seed set in Ipomopsis aggregata

Summary We used powdered fluorescent dyes to estimate receipt of self vs. outcross pollen in the self-incompatible species Ipomopsis aggregata (Polemoniaceae). Flowers on small and large plants received equal amounts of outcross pollen, whereas flowers on large plants received more self pollen, so the proportion of self pollen delivered through geitonogamy increased with plant size. In natural populations emasculation of all flowers on a plant raised average seed set per flower from 5.19 to 6.99 and also raised fruit set, though not significantly. From these results one expects a negative correlation between plant size and seeds per flower. The opposite trend was observed in a sample of plants in the field, suggesting that deleterious effects of geitonogamy on female fecundity in large plants can be overruled by other factors such as size-related fruit or seed abortion. Results are discussed in relation to the evolution of gynodioecy.