Age differences in responsiveness of brainstem chemosensitive neurons to extracellular pH changes

The sensitivity of neurons in the caudal chemosensitive area on the ventrolateral surface of the medulla oblongata (VMS) to extracellular pH changes was examined in newborn and young developing kittens and compared to that of adult cats. The pH was varied by superfusion of the VMS with mock cerebrospinal fluid (CSF) of pH 7.4 (control), 7.0 (acid) and 7.8 (alkaline). A total of 97 neuronal units in the three age groups changed their firing rates inversely in response to extracellular fluid (ECF) pH changes. The greatest sensitivity was found in the adult group where acid superfusion caused an increase in neuronal activity. The least sensitivity was observed in the newborn group (1-6 days old), whereas the young kitten group (4-6 weeks old) exhibited an intermediate sensitivity. Neurons of kittens older than 7 weeks of age demonstrated a response pattern characteristic of the adult group. Neurons of neonates older than seven days, exhibited a response pattern characteristic of the young kitten group.     

Confirmation of the Cholinergic Specificity of the Chol-1 Gangliosides in Mammalian Brain Using Affinity-Purified Antisera and Lesions Affecting the Cholinergic Input to the Hippocampus

An antiserum raised to Torpedo electromotor synaptosomal membranes (anti-TSM antiserum) induces a cholinergic-specific immune lysis of mammalian brain synaptosomes and recognizes a group of minor gangliosides appeared, therefore, to be specific to the cholinergic neuron and were designated Chol-1. To confirm the cholinergic specificity of the Chol-1 gangliosidic antigens, we have shown that not only does a mammalian ganglioside fraction that is enriched with respect to the Chol-1 gangliosides inhibit the cholinergic-specific immune lysis induced by the anti-TSM antiserum, but also it can be used to affinity-purify a subpopulation of immunoglobulins from the anti-TSM antiserum that also induce a cholinergic-specific lysis. Furthermore, we have demonstrated that fimbrial lesions, which cause a massive degeneration of cholinergic terminals in the ipsilateral hippocampus, lead to a loss of the Chol-1 gangliosides concomitant with that shown by choline acetyltransferase activity and that lesions to the entorhinal cortex, which cause a loss of mainly glutamergic synapses in the ipsilateral dentate gyrus leading to cholinergic sprouting from adjacent hippocampal areas and an increase in cholinergic markers in the dentate gyrus, produce concomitant increases in choline acetyltransferase activity and Chol-1 content. These results provide strong evidence in favour of the cholinergic specificity of the Chol-1 gangliosides.     

Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy

The family of a male with Duchenne muscular dystrophy (DMD) and a deletion within the dystrophin gene has been studied. Polymerase chain reaction analysis of ectopic mRNA from peripheral blood T+B lymphocytes and the use of (CA) _n repeat polymorphisms in and around the deleted region showed the proband's mother to be both a germline mosaic and a somatic mosaic for the deletion seen in her son. The mutation therefore occurred as a mitotic event early in embryogenesis.     

Two-Dimensional Crystallization of Brush Border Myosin I

Brush border myosin-I (BBMI) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells, where it links the core bundle of actin filaments to the plasma membrane. An association of BBMI with anionic phospholipids has been shown to be mediated by a carboxy-terminal domain which is rich in basic amino acids. We have exploited this natural affinity of BBMI for negatively charged lipids to form two-dimensional (2D) crystals of this protein which are suitable for structural analysis by electron crystallographic techniques. The 2D crystals which we have obtained belong to one of two space groups, p22₁2₁or p2. We present here projection maps calculated from images of negatively stained crystals for each of these crystal types to a resolution of 20 Å and show that the asymmetric unit is the same in both crystal types.     

Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus.

The matrix (M1) protein of influenza virus is a major structural component, involved in regulation of viral ribonucleoprotein transport into and out of the nucleus. Early in infection, M1 is distributed in the nucleus, whereas later, it is localized predominantly in the cytoplasm. Using immunofluorescence microscopy and the influenza virus mutant ts51, we found that at the nonpermissive temperature M1 was retained in the nucleus, even at late times after infection. In contrast, the viral nucleoprotein (NP), after a temporary retention in the nucleus, was distributed in the cytoplasm. Therefore, mutant M1 supported the release of the viral ribonucleoproteins from the nucleus, but not the formation of infectious virions. The point mutation in the ts51 M1 gene was predicted to encode an additional phosphorylation site. We observed a substantial increase in the incorporation of 32Pi into M1 at the nonpermissive temperature. The critical role of this phosphorylation site was demonstrated by using H89, a protein kinase inhibitor; it inhibited the expression of the mutant phenotype, as judged by M1 distribution in the cell. Immunofluorescence analysis of ts51-infected cells after treatment with H89 showed a wild-type phenotype. In summary, the data indicated that the ts51 M1 protein was hyperphosphorylated at the nonpermissive temperature and that this phosphorylation was responsible for its aberrant nuclear retention.     

A ligand field model for MCD spectra of biological cupric complexes.

A ligand field calculation of magnetic circular dichroism (MCD) spectra is described that provides new insights into the information contained in electronic spectra of copper sites in metalloenzymes and synthetic analogs. The ligand field model uses metal-centered p- and f-orbitals to model sigma, pi LMCT mixing mechanism for intensity, allowing the basic features of optical absorption, MCD, and electron paramagnetic resonance spectra to be simultaneously computed from a single set of parameters and the crystallographically determined ligand coordinates. We have used the model to predict changes in spectra resulting from the transformation of electronic wavefunctions under systematic variation in geometry in pentacoordinate ML5 complexes. The effectiveness of the calculation is demonstrated for two synthetic copper model compounds and a galactose oxidase enzyme complex representing limiting coordination geometries. This analysis permits immediate recognition of characteristic patterns of MCD intensity and correlation with geometry. A complementarity principle between MCD and CD spectra of transition metal complexes is discussed.     

Evaluation of cleaning strategies for removal of biofilms from reverse-osmosis membranes

An evaluation was made of the efficiency of five classes of chemical cleaning agents for removing biofilm from spirally wound cellulose acetate reverse-osmosis membranes receiving influent with high or low levels of combined chlorine. Each cleaning regimen utilized one or more of the following types of chemical: (i) surfactants and detergents, (ii) chaotropic agents, (iii) bactericidal agents, (iv) enzymes, and (v) antiprecipitants. Cleaning efficiency was tested in the laboratory on membrane material removed from operations at various intervals (2 to 74 days). Cleaning effectiveness was evaluated against nontreated control membranes and was scored by scanning electron microscopy and enumeration of surviving bacteria after treatment of the membranes. The combinations of classes which were most effective in biofilm removal were the anionic and chaotropic agent combination and combinations involving enzyme-containing preparations. Membranes receiving influent with high levels of combined chlorine were easier to clean but more susceptible to structural damage from prolonged exposure to combined chlorine. No treatment or combination of treatments was completely effective or effective at all stages of biofilm development.     

Autonomy of acetylcholinesterase differentiation in muscle lineage cells of ascidian embryos

The two muscle lineage blastomeres were removed surgically from Ciona intestinalis embryos at the eight-cell stage and allowed to develop in isolation. Acetylcholinesterase, an enzyme that occurs only in muscle cells of the developing larva, was detected histochemically in progeny cells of these isolated blastomers. Acetylcholinesterase differentiation in muscle lineage cells is not, therefore, dependent on inductive interactions with embryonic tissues derived from other eight-cell stage blastomeres.