Identification of the defects in the hemagglutinin gene of two temperature-sensitive mutants of A/WSN/33 influenza virus

Two temperature-sensitive mutants of WSN influenza virus, ts-61S and ts-134, possess defects in the hemagglutinin (HA) gene. These defects are characterized as a defective intracellular transport of the HA at the nonpermissive temperature and a marked thermolability. The nucleic acid sequences of the HA gene of these two viruses, as well as a series of revertant viruses, were determined. The deduced amino acid sequences demonstrate that the HA of ts-61S varied from the wild type protein by three amino acids while that of ts-134 differed by two residues. For both mutants, analysis of revertant viruses indicated that the phenotype of transport inhibition at the nonpermissive temperature and heat lability were associated with a single amino acid change in the globular portion of the molecule. In the case of ts-61S, the critical change in the HA was the replacement of a serine residue at position 110 with that of a proline. The mutational defect in the HA of ts-134 was due to the substitution of a tyrosine residue at position 159 with that of a histidine residue. Four of five revertants of ts-134 were suppressor revertants, of which some of the compensatory changes did not restore thermostability to the HA.     

Inability of Japanese rubella vaccines to induce antibody response in rabbits is due to growth restriction at 39° C

Summary We have compared the kinetic growth patterns of To-336, MEQ₁₁, KRT, and SK₂ rubella vaccine strains licensed in Japan at 37° and 39° C with those of progenitor wild strains of rubella virus. The growth of vaccine strains was depressed at 39° C to a level about 3 log₁₀ lower than that at 37° C. The difference in virus titer attained by wild strains at 37° and 39° C was less than tenfold. The growth potential at 39° C paralleled the immunogenic marker of rubella virus,i.e. the capability of virus to induce antibody response upon subcutaneous injection in rabbits for all wild and vaccine strains examined, including one strain at an intermediate level of attenuation. Several clones were isolated from the progeny produced by a vaccine strain during the growth at 39° C. Among them were partial revertants in immunogenic marker as well as in the growth potential at 39° C. It was concluded that the immunogenic marker of rubella virus in rabbits represented its capability to replicate at the body temperature of the animal.With 6 Figures     

Morphological study of disordered myelination and the degeneration of nerve fibers in the spinal cord of mice lacking complex gangliosides

Gangliosides, a family of glycosphingolipids that contain sialic acid, are abundant on the neuronal cell membranes, but their precise functions in the central nervous system remain largely undefined. In a previous study of GalNAc-T(-/-) mice engineered to lack beta1,4-N-acetylgalactos-aminyltransferase (GM2/GD2 synthase) to abolish any, complex gangliosides, we observed the reduction of nerve conduction velocity but did not find any obvious morphological change in the brain. In the present study, we observed morphological changes in the nerve fiber tracts of the spinal cord in these mice. In GalNAc-T(-/-) mice, the number of degenerated axons was markedly increased in the dorsal funiculus, tract of Lissauer, and dorsolateral funiculus of the cervical segment of the spinal cord as well as the dorsal funiculus and tract of Lissauer of the lumbar segment of the spinal cord. There were also increased numbers of unmyelinated fibers in GalNAc-T(-/-) mice. Loosened myelin sheaths and myelin sheaths separated from axons by wide spaces were also observed in GalNAc-T(-/-) mice. These results provide a morphological basis for the previously observed reduction in the nerve conduction velocity and suggest that complex gangliosides are essential for the maintenance of myelin and the integrity of nerve fibers of the spinal cord.     

The cytokines NAP-1 (IL-8), MCP-1, IL-1 beta,and GRO in rabbit inflammatory skin lesions produced by the chemical irritant sulfur mustard

Developing and healing dermal inflammatory lesions were produced in rabbits by the topical application of dilute sulfur mustard (SM),⁹ the military vesicant. In tissue sections of such lesions, cells containing the mRNA of important cytokines were identified with in situ hydridization techniques. These cytokines were neutrophil attractant/activation protein-1 (NAP-1 (also called IL-8)), monocyte chemoattractant (activating) protein 1 (MCP-1), interleukin 1 (beta) (IL-1 (beta)), and GRO (a growth factor and chemokine). Mononuclear cells (mainly macrophages and activated fibroblasts) contained the mRNA of all four of these cytokines. A higher percentage of cytokine-producing mononuclear cells (macrophages and activated fibroblasts) was present in lesions at 2 days (their peak size) than at 6 days, when they were almost healed. Granulocytes emigrated from the bloodstream, passed through the lesions, and were the major constituent of the protective crust. This sequence correlated with the distribution of cells able to produce NAP-1: At 2 days and 6 days, the mononuclears that contained messenger RNA for this granulocyte chemoattractant were found mainly in the upper part of the dermis. At 2 days and 6 days, cells containing the mRNA of IL-1, a primary cytokine, were also found predominantly in the upper dermis, i.e., nearest the site of injury. In contrast, mononuclears containing the mRNA of MCP-1 (a monocyte chemoattractant), and the mRNA of GRO (a granulocyte chemoattractant) were more equally distributed throughout the dermis. SM stimulated hair follicle epithelial cells to up-regulate GRO mRNA and, to a lesser degree, NAP-1 mRNA. Apparently, the irritation produced by SM directly or indirectly induces such epithelial cells to manufacture these growth factors. In the rabbit, hair follicles are known to be the main source of new epithelial cells after the covering epithelium has been destroyed. Therefore, GRO is probably a major autocrine-paracrine stimulus for such repair. A brief review of the role of cytokines in dermal inflammation is presented.Abbreviations SM Sulfur mustard: bis(2-chloroethyl)sulfide - GM-CSF Granulocyte-Macrophage Colony Stimulating Factor - GRO A member of the CXC subfamily of chemokines that promotes the multiplication of cells, formerly called melanoma growth stimulating activity (MGSA) - IFN (gamma)-Interferon-gamma - IL-1 Interleukin 1 - IL-8 Interleukin 8 (same as NAP-1)-a CXC chemokine - MCP-1 Monocy te Chemoattractant (Activating) Protein-1-a C-C chemokine - NAP-1 Neutrophil Attractant/Activating Protein-1 (same as IL-8)-a C-X-C chemokine - TGF (beta) Transforming Growth Factor (beta) - TNF (alpha) Tumor Necrosis Factor (alpha) - EDTA Ethylenediamine tetraacetate - DEPC Diethylpyrocarbonate - PBS Phosphate-buffered saline solution - PGE₂ Prostaglandin E₂ - PGI₂ Prostaglandin I₂ (prostacyclin) - SSC Sodium chloride-sodium citrate solution On leave of absence from the Institute for Medical Immunology, Kumamoto University School of Medicine, Kumamoto, Japan.On leave of absence from the Department of Internal Medicine, Oita Medical University, Oita, Japan.     

Retrograde dopaminergic neuron degeneration following intrastriatal proteasome inhibition

Recent studies have suggested that defects in the ubiquitin-proteasome system (UPS) contribute to the etiopathogenetic mechanisms underlying dopaminergic neuronal degeneration in Parkinson's disease. The present study aims to study the effects of proteasome inhibition in the nerve terminals of nigrostriatal dopaminergic neurons in the substantia nigra pars compacta (SNpc). Following a unilaterally intrastriatal injection of lactacystin, a selective proteasome inhibitor, dopaminergic neurons in the ipsilateral SNpc progressively degenerated with alpha-synuclein-immunopositive intracytoplasmic inclusions. When lactacystin was administered at a high concentration, the striatum was simultaneously involved, and alpha-synuclein-immunopositive extracytoplasmic granules appeared extensively within the SN pars reticulata (SNpr). In addition, during the retrograde neuron degeneration in SN, the level of heme oxygenase-1 immunopositivity, an oxidative stress marker, was markedly increased in SNpc neurons. These results reveal that intrastriatal proteasome inhibition sufficiently induces retrograde dopaminergic neuronal degeneration with abundant accumulation of alpha-synuclein in the SN.     

Arrangement of D-periodic collagen fibrils and association of proteoglycans with fibrils in the synovium of the mouse temporomandibular joint

The present study was performed to examine changes in the arrangement of D-periodic collagen fibrils in the synovium of the growing temporomandibular joint in mice. At 1 week of age, the mandibular condyle was undeveloped, and only a few collagen fibrils were recognizable in the subintimal layer of the synovium. At 8 weeks, the mandibular condyle was structurally developed with an increase of collagen fibrils in the synovium; a fully mature condyle was observed at 6 months of age. The close association of proteoglycans with collagen fibrils in the synovium of the growing joint was examined by both conventional and energy-filtering transmission electron microscopy of cupromeronic blue-stained specimens. Proteoglycans were associated with D-periodic collagen fibrils in the short filamentous form in random fashion at 1 week of age, but in a regular pattern with D-periodicity at 6 months. These associations in the synovium could be correlated with the mechanical character of the temporomandibular joint.     

Cardiomyopathy characterized by abnormal accumulation of desmin-type intermediate filaments in cardiac muscle fibers. A case report and review of the literature

A 42-year-old Japanese male, who had been suffering from congestive heart failure and electrocardiographic abnormalities (A-V block, intraventricular conduction disturbance, ventricular tachycardia), died after a clinical course of 2 years and 1 month. Macroscopic investigation revealed dilation of the left ventricle and thickening of the right ventricular wall. The unique finding in this case was a circumferential fibrous scar in the median circular layer and outer oblique layer of the left ventricular wall. Biopsy and autopsy materials revealed diffuse loss of myofibrils in the central zone of cardiac muscle fibers, and replacement with aniline blue-positive homogeneous material (17-35% of the area of one muscle fiber). Electron microscopy revealed abnormal accumulation of fine filamentous material (7.5-25 nm in diameter), which was immunohistochemically proved to be desmin-type intermediate filament. Moreover, sarcoplasmic reticulum-like material was detected in the degenerated area. At autopsy, degeneration was detected all over the heart. The ventricular muscle fibers were more severely affected than the atrial muscle fibers. The conduction system was also affected, in some parts more severely than the surrounding ordinary muscle fibers. The pathogenesis of this disorder remains to be clarified.