Sample-hold technique for analysis of respiratory gas composition at high breathing frequencies

A gas sampling device is described for continuous monitoring of respiratory gas composition that is applicable to experimental conditions when the breathing frequency is very high (greater than 2 Hz) and the response time of conventional gas analyzers becomes a critical limiting factor. The system utilizes the principle of discontinuous gas collection at any selected point of the respiratory cycle facilitated by ultraspeed piezoelectric valves and includes provision for sample-hold characteristics. Two distinct modes of operation are supported. In phase-locked mode gas sampling is synchronous with breathing frequency. In scanning mode gas collection is asynchronous with breathing frequency. Phase-locked mode may be used for continuous monitoring of end-tidal gas concentrations, whereas scanning mode is intended for assessing the gas concentration profile throughout the respiratory cycle. The system may be applied to steady breathing encountered in mechanical ventilation at high frequency or during quasi-steady breathing observed in panting animals. Combined with a respiratory mass spectrometer, the system has been used for measurement of gas concentrations in alveolar gas mixtures at breathing frequencies ranging from 3 to 30 Hz that were otherwise not amenable to rapid measuring techniques.     

Human debrisoquine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22

The enzyme P450db1 (db1) is responsible for the common human defect in drug oxidation known as the "debrisoquine/sparteine polymorphism." Polyclonal antibody against the rat db1 protein was used to screen a human liver lambda gt11 library for the db1 cDNA clone. A cDNA containing the full protein coding sequence was isolated; the deduced NH2-terminal sequence of this cDNA was identical to that derived from direct sequencing of the purified human db1 protein. Comparison of the human db1 with rat db1 revealed 71 and 73% similarities of nucleotides and amino acids, respectively. By use of human-rodent somatic cell hybrids the db1 gene was localized to human chromosome 22 (CYP2D locus).     

The amino acid substrate of bovine tyrosine hydroxylase.

Tyrosine hydroxylase catalyzes the tetrahydropterin-dependent hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine. Several nonphysiological aromatic amino acids have been examined as inhibitors and substrates for bovine adrenal tyrosine hydroxylase. The Ki values for para-substituted phenylalanines increase as the size of the substituent increases. For each A2 increase in surface area of the substituent, the free energy of binding becomes 50 cal more positive. Replacement of the phenyl ring with a pyridyl ring decreases the affinity about one order of magnitude. A number of these aromatic amino acids are also substrates for the enzyme. The KM values again increase in size with increasing size of the substituent, but the Vmax value is independent of the reactivity of the amino acid. The effect of size on binding is consistent with a tight interaction between the para position region of the substrate and the enzyme. The lack of a change in the Vmax value is consistent with the rate-limiting step in catalysis by bovine tyrosine hydroxylase being formation of the hydroxylating intermediate rather than hydroxylation of the amino acid. These results will be useful in designing mechanism-based inhibitors of catecholamine biosynthesis and establish that the mechanisms of rat and bovine tyrosine hydroxylase do not differ significantly.     

Amphiphysin, a novel protein associated with synaptic vesicles.

To obtain access to novel proteins of the neuronal synapse, we have raised antisera against proteins of synaptic plasma membranes and used them for immunoscreening brain cDNA expression libraries. One of the newly isolated cDNAs encodes an acidic protein of 75 kDa with a distinct architecture of structural domains and multiple potential phosphorylation sites. Light and electron microscopy employing monospecific antisera raised against the expression product indicate a synapse-specific, presynaptic localization of this protein in many synapses of the chicken and rat nervous system. Its overall distribution in brain is very similar to that of synaptophysin, a ubiquitous protein of synaptic vesicles. In addition to brain, the protein or its mRNA is expressed in adrenal gland and anterior and posterior pituitary, but was not detected in a variety of other tissues. In controlled pore glass chromatography the native protein copurifies with synaptic vesicles and largely remains associated with them under various washing conditions. However, its amino acid sequence is very hydrophilic and it segregates into the aqueous phase in detergent phase partition. An earlier step of synaptic vesicle purification, sucrose cushion centrifugation, separates a vesicle-bound fraction of this protein from an unbound fraction. This seems to be a new, perhaps peripheral, protein of synaptic vesicles for which we propose the name, amphiphysin.     

Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.

The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.     

Detection of a cytotoxic substance produced by the cyanobacteriumMicrocystis aeruginosa strain PCC 7806: Isolation and differentiation from the peptide toxin microcystin-LR by cytotoxicity assays

Exposure of four permanent cell lines to crude aqueous extracts of the cyanobacteriumMicrocystis aeruginosa (strain PCC 7806) resulted in rounding and lysis of the cells within a few minutes. Cell damage was quantified by determination of lactate dehydrogenase (LDH) activity in the cell culture supernatants. By gel filtration, cation exchange chromatography and SDS-PAGE, the cytotoxic effects could be related to a defined substance with an apparent molecular weight of about 35 kDa, which was not heat resistant. The physicochemical properties very clearly mark off this compound from the peptide toxin microcystin-LR, which did not show any detectable cytotoxic effects.     

A non-alphoid repetitive DNA sequence from human chromosome 21

Summary A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40–50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specifity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.     

Progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus.

Within 6 months of infection with the Petaluma isolate of feline immunodeficiency virus, specific-pathogen-free domestic cats exhibited a decrease in the percentage and number of circulating CD4+ lymphocytes and in the CD4+/CD8+ T-cell ratio, along with a marginally significant depression of pokeweed mitogen-induced lymphocyte proliferation in vitro. There was no loss of responsiveness to concanavalin A during this stage, and the cats were capable of mounting a satisfactory antibody response to a T-dependent, synthetic polypeptide immunogen. The pokeweed mitogen response deficit became clearly demonstrable by 11 to 12 months postinfection. A decline in the lymphocyte proliferative response to concanavalin A and a diminished ability to mount an in vivo antibody response to the T-dependent immunogen evolved by 25 to 44 months postinfection. Virus infection did not affect the ability of cats to mount an antibody response to a T-independent synthetic polypeptide immunogen. These data indicate that feline immunodeficiency virus produces a slowly progressive deterioration of T-cell function but does not affect the ability of B cells to recognize and respond to a T-independent antigenic stimulus.