Characterization of rat pulmonary monoamine oxidase.

The substrate specificities and kinetics of rat lung monoamine oxidase (MAO) have been studied. Utilizing the irreversible MAO inhibitors, clorgyline and deprenyl, rat lung was shown to possess at least two types of MAO, A and B. Tyramine was a substrate for both forms of the enzyme, whereas 5-hydroxytryptamine (5-HT) was a preferred substrate for the A-form. In contrast to most other tissues, 2-phenylethylamine was not solely a B-type substrate for the rat lung MAO and some metabolism by the A-type was apparent $\text{(}\text{B}\text{A}\text{=}\text{80}\text{20}\text{)}$. Using tyramine as substrate the ratio A/B was shown to be $\text{55}\text{45}$. Rat pulmonary MAO-B was inhibited by deprenyl and the kinetics of MAO-A studied. The Km values for the A-form for tyramine, phenylethylamine and 5-HT were relatively similar and were 270, 244 and 170 μM respectively. Whereas, when the A-form was inhibited by clorgyline, the Km values for the B-form were found to differ considerably: 330, 42 and 850 μM for tyramine, phenylethylamine and 5-HT respectively.     

Membrane-derived oligosaccharides (MDO's) promote closing of an E. coli porin channel.

The outer membrane of Escherichia coli is a diffusion barrier for macromolecules, but allows the passage of small hydrophilic solutes through non-specific channels, the porins. Some electrophysiological studies find reconstituted porins in a mostly open state, while those done with the patch-clamp technique performed on live cells suggest that the vast majority of the native channels are closed. We present here current measurements through porins from reconstituted outer membrane, which demonstrate that bacterial metabolites, the MDO's, which bathe the periplasmic side of the outer membrane, induce the channels to close. These findings illustrate that the degree of openness of porins can be regulated by compounds naturally found in bacteria.     

Calcium-dependent inactivation of the calcium current activated upon hyperpolarization of Paramecium tetraurelia

The Ca2+ current activated upon hyperpolarization of Paramecium tetraurelia decays over a period of 150-200 ms during sustained steps under voltage clamp. At membrane potentials between -70 and approximately -100 mV, the time course of this inactivation is described by a single exponential function. Steps negative to approximately -100 mV elicit currents that decay biexponentially, however. Three lines of evidence suggest that this current's inactivation is a function of intracellular Ca2+ concentration rather than membrane potential: (a) Comparing currents with similar amplitudes but elicited at widely differing membrane potentials suggests that their time course of decay is a sole function of inward current magnitude. (b) The extent of current inactivation is correlated with the amount of Ca2+ entering the cell during hyperpolarization. (c) The onset and time course of recovery from inactivation can be hastened significantly by injecting cells with EGTA. We suggest that the decay of this current during hyperpolarization involves a Ca(2+)-dependent pathway.     

Possible repetitive DNA markers for Eusorghum and Parasorghum and their potential use in examining phylogenetic hypotheses on the origin of Sorghum species

Genomic structures of two major species in section Eusorghum (Sorghum), Sorghum bicolor and Sorghum halepense, and their phylogenetic relationships with a species in section Parasorghum, Sorghum versicolor, were studied by using cloned repetitive DNA sequences from the three species. Of the five repetitive DNA clones isolated from S. bicolor and S. halepense, four produced qualitatively similar hybridization patterns with detectable variations in copy numbers of some of the restriction fragments on the Southern blots of the two genomic DNAs. One clone was shown to be diagnostic for S. halepense. Molecular analysis at the DNA level indicates that S. bicolor and S. halepense have similar but not identical genomes, consonant with differences in karyotypes, meiotic chromosome behaviors, morphology, and physiology of the species. In addition to five repetitive clones isolated from S. bicolor and S. halepense, eight more sequences were cloned from S. versicolor. Nine clones were found to be specific for either S. bicolor and S. halepense or S. versicolor. The remaining four had a moderate to strong homology with sequences present in all Sorghum species studied. We speculate that the genome in the common ancestor of Sorghum has differentiated to give rise to genomes of at least three major chromosome sizes; large, medium, and small, as seen at present. Amplifications, eliminations, rearrangements, and new syntheses of repetitive sequences may have been involved in genome differentiation of these species. The results also suggest that the S. versicolor genome has strongly diverged from the genomes of the two species in section Eusorghum.     

Ion channel activities in the Escherichia coli outer membrane.

The electrical properties of Escherichia coli cells were examined by the patch-clamp technique. Giant cells or giant spheroplasts were generated by five different methods. By electron micrographic and other criteria we determined that the patches are most likely from the outer membrane. We regularly observed currents through at least two types of channels in this membrane. The first current is mechanosensitive and voltage-dependent, and can be observed in single gene mutants of the known major porins (ompF, ompC, phoE, lamB); this channel may represent a minor porin or a new class of outer membrane protein. The possible identity of the second, voltage-sensitive channel with one of the known outer membrane proteins is being explored. The high-resistance seals consistently formed on these patches and the presence of gated ion channels suggest that most of the pores of the outer membrane are not statically open, as commonly held, but are closed at rest and may be openable by physiological stimuli.     

Sensitization of lymphocytes against pooled allogeneic cells. II. Characterization of effector cells cytotoxic for autologous lymphoblastoid cell lines

Stimulation of human lymphocytes in mixed leukocyte culture (MLC) with x-irradiated pooled allogeneic normal cells (poolx) was previously shown to result in generation of effector cells cytotoxic for autologous Epstein-Barr virus- (EBV) transformed lymphoblastoid cell lines (LCL). This study was undertaken to determine whether lysis of the autologous EBV- transformed LCL cells by pool-stimulated cells is mediated by cytotoxic Tc lymphocytes (Tc) or natural killer- (NK) like cells, both of which are generated in MLC. In the first series of experiments, proliferating cells were eliminated by treatment of pool-stimulated cells with 5 X 10(-5) M 5-bromodeoxyuridine (BUdR) and light. The remaining cells failed to lyse allogeneic normal lymphocytes and autologous LCL cells, whereas cytotoxicity against NK-sensitive K562 leukemia cells was retained. In the second series of experiments, pool-stimulated effector cells were treated with monoclonal anti-human Tc cell antibodies, OKT3 or OKT8, and complement (C). The cells recovered after antibody and C treatment were diminished in their ability to lyse allogeneic normal lymphocytes as well as autologous LCL cells, whereas their cytotoxicity against K562 leukemia cells was unaffected. These combined results provide strong evidence that lysis of autologous LCL cells by lymphocytes stimulated with pooled allogeneic normal cells is mediated by Tc rather than NK-like cells.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.