An Immunoblot Study of Neurofilament Degradation In Situ and During Calcium-Activated Proteolysis

The degradation of neurofilament (NF) proteins was examined by immunoblot methods to identify, characterize, and monitor the appearance of immunoreactive breakdown products during the loss of NF triplet proteins. Individual NF proteins and their breakdown products were identified using polyclonal and monoclonal antibodies to NF proteins. NF degradation was compared during calcium-activated proteolysis of isolated rat NF, during an experimental influx of calcium into excised rat spinal nerve roots, and during NF breakdown in transected rat peripheral nerve. These different experimental conditions produced similar patterns of NF fragmentation, including the transient appearance of NF immunobands between Mr 150,000-200,000 and 110,000-120,000 as well as the appearance and accumulation of NF immunobands between Mr 45,000 and 65,000. Most immunoreactive NF fragments remained Triton-insoluble. Low levels of the same immunoreactive fragments were present in control neural tissues, suggesting that calcium-activated proteolysis may be operative in the turnover and/or processing of NF proteins in vivo. Very similar patterns of NF degradation during experimental calcium influxes into different CNS and PNS tissues are indicative of the widespread distribution of calcium-activated NF protease in neural tissues.     

Identification of Ia on a subpopulation of human T lymphocytes that stimulate in a mixed lymphocyte reaction

The delineation of discrete subpopulations of human T lymphocytes has permitted preliminary analyses of the complex cellular network regulating the immune response in man. We previously showed that a subset of T lymphocytes, designated as theophylline-sensitive because of their inability to bind sheep red blood cells in the presence of the drug, are responsible for antigen-specific suppression or regulation in an in vitro plaque-forming cell assay. We now show that 25 to 45% of these theophylline-sensitive T cells were Ia-positive by immunofluorescence with a rabbit antiserum raised against purified B lymphoblast surface antigenic material. These data suggested that 4 to 7% of peripheral blood T cells carry Ia determinants. The presence of Ia determinants on this T cell subset was confirmed by gel analysis of radioiodinated surface material. Furthermore, in mixed lymphocyte culture, the theophylline-sensitive cells demonstrated HLA-D determinants and were 10-fold more potent stimulators than equal numbers of B lymphocytes. The presence of Ia determinants on these T cells indicates the expression of major histocompatibility complex-related regulatory gene products on a specific human T lymphocyte subpopulation.     

The precipitation of poly-L-glutamic acid. II. beta-Precipitation.

Infrared and X-ray diffraction studies have established that in the β-precipitation region of poly-L -glutamic acid the chains are in the β-conformation. Therefore, a major molecular conformational change has taken place upon precipitation. It is shown that the size of the α-helical aggregates remains constant with time in the β-region. Strong evidence can be offered to indicate that the transformation involves a transitory random-coil intermediate. Reasons are advanced, in view of the stability of the β-form, as to why two distinct precipitation regions exist.     

Temporal correlation of measurements of airway hyperresponsiveness in ovalbumin-sensitized mice

Airway hyperresponsiveness, airway inflammation, and reversible airway obstruction are physiological hallmarks of asthma. These responses are increasingly being studied in murine models of antigen exposure and challenge, using whole body plethysmography to noninvasively assess airway hyperresponsiveness. This approach infrequently has been correlated with indexes of airway hyperresponsiveness measured by invasive means. Furthermore, correlation with quantitative histological data for tissue infiltration by inflammatory and immune cells, particularly in the wall of airways, during daily airway challenge is lacking. To address these uncertainties, we used C57BL/6 mice that were immunized with ovalbumin or vehicle (saline) and sensitized to aerosolized ovalbumin or vehicle 8 days later. The mice were subsequently exposed to aerosolized ovalbumin or vehicle, respectively, on days 14-22. We assessed airway hyperresponsiveness to methacholine noninvasively on days 14, 15, 18, or 22; we studied the same mice 24 h later while they were anesthetized for invasive analyses of airway hyperresponsiveness. Plasma total IgE concentration was significantly higher in the ovalbumin-treated mice compared with the vehicle-treated mice, but this did not correlate with eosinophil number. Peak airway hyperresponsiveness measured by either approach correlated early during daily antigen challenge (days 14 and 15), but this correlation was lost later during subsequent daily antigen challenges (days 18 and 22). On days 14 and 15, peak airway hyperresponsiveness correlated with transmigration of neutrophils and macrophages, but not lymphocytes, in the peribronchovascular connective tissue sheaths. This extravascular accumulation was found to be focal by three-dimensional microscopy. We conclude that, although ovalbumin treatment changed lung function in mice, correlation between noninvasive and invasive measures of peak airway hyperresponsiveness was inconsistent.     

Species density and associations in Caribbean reef corals

The density of scleractinian corals on portions of two reefs in the Grenadine Islands, W. I. has been investigated by direct observation by SCUBA divers. Grid, transect, and random quadrat methods were compared with total samples to determine the precision and accuracy of these techniques. Transect methods were generally satisfactory provided at least 15% of a 400 m² total grid area was included in the sample. The data strongly indicated clumped distributions of all species, although numerical analysis does not indicate the distinct zones reported by other workers. Species associations based on Jaccard's coefficient and cluster analysis showed possible similarities in physical requirements, although few strong associations were found. Data based on 4 m² quadrats generally provided a more reliable estimate of species associations than did data based on 1 m² quadrats. It is suggested that surveys of these reef types may be better based on a number of parallel transects rather than a single transect, and that well-defined zones are more likely to be the exception than the rule.     

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs^∗7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853^∗), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.     

Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox

Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurring smallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaks of human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear that naturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious health risk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizing antibodies developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible.