Distribution and Subcellular Localization of High-Molecular-Weight Microtubule-Associated Protein-2 Expressing Exon 8 in Brain and Spinal Cord

Abstract: The expression of high-molecular-weight (HMW) microtubule-associated protein-2 (MAP-2) expressing exon 8 (MAP-2+8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP-2+8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP-2+8 comigrated with MAP-2a. In human adult brain, HMW MAP-2+8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium-sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultra-structural level, HMW MAP-2+8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP-2+8. The presence of HMW MAP-2+8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.     

rG-CSF reduces endotoxemia and improves survival during E.coli pneumonia

Freeman, Bradley D., Zenaide Quezado, Fabrice Zeni, CharlesNatanson, Robert L. Danner, Steven Banks, Marcello Quezado, YvonneFitz, John Bacher, and Peter Q. Eichacker. rG-CSF reduces endotoxemia and improves survival during E. coli pneumonia. J. Appl.Physiol. 83(5): 1467-1475, 1997.We investigatedthe effects of recombinant granulocyte colony-stimulating factor(rG-CSF) during canine bacterial pneumonia. Beagles with chronictracheostomies received daily subcutaneous rG-CSF (5 µg/kg body wt)or placebo for 14 days, beginning 9 days before intrabronchialinoculation with E. coli. Animalsreceived antibiotics and fluid support; a subset received humidifiedoxygen (fractional inspired O₂0.40). Compared with controls, rG-CSF increased circulating neutrophil counts (57.4 vs. 11.0 × 10³/mm³,day 1 after infection;P = 0.0001), decreased plasmaendotoxin (7.5 vs. 1.1 EU/ml at 8 h; P < 0.01) and serum tumor necrosis factor- (3,402 vs.729 pg/ml at 2 h; P = 0.01) levels,and prolonged survival (relative risk of death = 0.45, 95% confidenceinterval 0.21-0.97; P = 0.038).Also, rG-CSF attenuated sepsis-associated myocardial dysfunction(P < 0.001). rG-CSF had no effect onpulmonary function or on blood and lung bacteria counts (allP = not significant). Other animalschallenged with endotoxin (4 mg/kg iv) after similar treatment withrG-CSF had lower serum endotoxin levels (7.62 vs. 5.81 log EU/ml at 6 h; P < 0.01) and less cardiovasculardysfunction (P < 0.05 to < 0.002)but similar tumor necrosis factor- levels (P = not significant) compared withcontrols. Thus prophylactic rG-CSF sufficient to increase circulatingneutrophils during bacterial pneumonia may improve cardiovascularfunction and survival by mechanisms that in part enhance the clearanceof bacterial toxins but do not improve lung function.

     

Dissociation of photoreceptors from whole heads of the fruit fly,Drosophila melanogaster

Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple smash technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.     

Treating tumor-bearing mice with vitamin D3 diminishes tumor-induced myelopoiesis and associated immunosuppression,and reduces tumor metastasis and recurrence

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colonystimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1,25-dihydroxyvitamin D₃ reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D₃ reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D₃ treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D₃ after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D₃ treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.This study was supported in part by grants from the Medical Research Service of the Department of Veterans Affairs and by grants CA-45080 and CA-48080 from the National Institutes of Health     

Identification and chromosomal mapping of a third mouse runt-like locus

The Drosophila runt gene, which controls early events in embryogenesis, has been shown to have homologues in human and mouse. The human gene on 21q22 is involved in the t(8;21) associated with acute myeloid leukemia. Two mouse runt-like loci encoding DNA-binding proteins have been identified. We report here the isolation and partial sequence of a molecular clone of a third mouse runt-like locus. By using a panel of somatic cell hybrids and interspecific backcross mice, we map the novel locus to the telomeric region of mouse chromosome 4.     

Physical mapping of the human carbonic anhydrase gene cluster on chromosome 8

A cluster of genes encoding the three cytoplasmic carbonic anhydrase isozymes CAI, CAII, and CAIII lie on the long arm of chromosome 8 (8q22) in humans. These genes have been mapped using pulsed-field gel electrophoresis. The genes lie in the order CA2, CA3, CA1. CA2 and CA3 are separated by 20 kb and are transcribed in the same direction, away from CA1. CA1 is separated from CA3 by over 80 kb and is transcribed in the direction opposite to CA2 and CA3. The arrangement of the genes is consistent with proposals that the duplication event which gave rise to CA1 predated the duplication which gave rise to CA2 and CA3. The order of these three genes differs from that suggested for the mouse based on recombination frequency.     

Polarity in higher plant dictyosomes

Summary Dictyosomes in three higher plant cell secretory systems,Zea root cap cells,Tradescantia pollen tubes and digestive glands ofDionaea fly traps, are shown to possess a structural polarity in terms of either the production of secretory vesicles, or the reaction of individual cisternae to the osmium-zinc-iodide impregnation procedure. These observations contradict previous claims that higher plant dictyosomes lack structural polarity. Doubts about the applicability of the endomembrane flow concept to higher plant cell dictyosomes are discussed in relation to the relative balance between carbohydrate and protein in secreted products. The lack of dictyosome-associated endoplasmic reticulum in some of these plant cell systems is confirmed.     

The ability of nonspecific T-cell stimulators to induce helper-cell-dependent increases in either polyclonal or isotype-restricted Ig production in vivo

In order to delineate the various roles of T cells in B-cell activation, mice were exposed to a variety of specific or nonspecific T-cell stimuli including mitogens, e.g., concanavalin A, adjuvants, e.g., complete Freund's adjuvant, and colchicine plus nonimmunogenic doses of antigen, anti-lymphocyte serum, and pathogens and their spleens analyzed for total class-specific immunoglobulin-secreting cells as indicators of helper cell generation. The results demonstrate that, depending on the mode of stimulation, markedly different Ig-secreting cell response patterns were induced, differing with respect to their kinetics and the isotype induced. In contrast to polyclonal T-cell stimuli such as concanavalin A and 17X lethal malaria which induced increases in all classes of Ig-recruiting cells, injection of many T-cell-activating agents resulted in the selective production of IgG clones in particular IgG 1. Such findings are discussed in terms of the different mechanisms of T-cell help and provide further evidence for functional heterogeneity in the T-helper-cell pool.