Development of an autoimmune syndrome affecting the skin and internal organs in P-selectin glycoprotein ligand 1 leukocyte receptor-deficient mice.

OBJECTIVE: To define and characterize the progression of the spontaneous autoimmune disease that develops in mice in the absence of the leukocyte adhesion receptor P-selectin glycoprotein ligand 1 (PSGL-1). METHODS: Skin-resident immune cells from PSGL-1-deficient mice and C57BL/6 control mice of different ages were isolated and analyzed by flow cytometry. Biochemical parameters were analyzed in mouse serum and urine, and the presence of serum autoantibodies was investigated. Skin and internal organs were extracted, and their structure was analyzed histologically. RESULTS: Skin-resident innate and adaptive immune cells from PSGL-1(-/-) mice had a proinflammatory phenotype with an imbalanced T effector cell:Treg cell ratio. Sera from PSGL-1(-/-) mice had circulating autoantibodies commonly detected in connective tissue-related human autoimmune diseases. Biochemical and histologic analysis of skin and internal organs revealed skin fibrosis and structural and functional abnormalities in the lungs and kidneys. Furthermore, PSGL-1(-/-) mice exhibited vascular alterations, showing loss of dermal vessels, small vessel medial layer remodeling in the lungs and kidneys, and ischemic processes in the kidney that promote renal infarcts. CONCLUSION: Our study demonstrates that immune system overactivation due to PSGL-1 deficiency triggers an autoimmune syndrome with characteristics similar to systemic sclerosis, including skin fibrosis, vascular alterations, and systemic organ involvement. These results suggest that PSGL-1 expression contributes to the maintenance of the homeostasis of the immune system and could act as a barrier for autoimmunity in mice.

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana.

Transcription factors containing a conserved DNA-binding domain similar to that of the proto-oncogene c-myb have been identified in nearly all eukaryotes. MYB-related proteins from plants generally contain two related helix-turn-helix motifs, the R2 and R3 repeats. It was estimated that Arabidopsis thaliana contains more than 100 R2R3-MYB genes. The few cases where functional data are available suggest an important role of these genes in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone responses. To determine the full regulatory potential of this large family of regulatory genes, a systematic search for the function of all genes of this family was initiated. Sequence data for more than 90 different A. thaliana R2R3-MYB genes have been obtained. Sequence comparison revealed conserved amino acid motifs shared by subgroups of R2R3-MYB genes in addition to the characteristic DNA-binding domain. No significant clustering of the genes was detected, although they are not uniformly distributed throughout the A. thaliana genome.

Rhizobium meliloti mutants unable to synthesize anthranilate display a novel symbiotic phenotype.

Analyses of Rhizobium meliloti trp auxotrophs suggest that anthranilate biosynthesis by the R. meliloti trpE(G) gene product is necessary during nodule development for establishment of an effective symbiosis. trpE(G) mutants, as well as mutants blocked earlier along this pathway in aromatic amino acid biosynthesis, form nodules on alfalfa that have novel defects. In contrast, R. meliloti trp mutants blocked later in the tryptophan-biosynthetic pathway form normal, pink, nitrogen-fixing nodules. trpE(G) mutants form two types of elongated, defective nodules containing unusually extended invasion zones on alfalfa. One type contains bacteroids in its base and is capable of nitrogen fixation, while the other lacks bacteroids and cannot fix nitrogen. The trpE(G) gene is expressed in normal nodules. Models are discussed to account for these observations, including one in which anthranilate is postulated to act as an in planta siderophore.

Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants.

The acidic exopolysaccharide (EPS I) produced by Rhizobium meliloti during symbiosis with Medicago sativa has been shown to be required for the proper development of nitrogen-fixing nodules. Cloned DNA from the exo region of R. meliloti is shown to stimulate production of the low-molecular-weight form of this exopolysaccharide, and in this report we show that the symbiotic deficiencies of two exo mutants of R. meliloti, the exoA and exoH mutants, can be rescued by the addition of this low-molecular-weight material at the time of inoculation. For exoA and exoH mutants, rescue with a preparation containing low-molecular-weight exopolysaccharide induces the formation of nitrogen-fixing nodules which appear somewhat later and at a reduced efficiency compared with wild-type-induced nodules; however, microscopic analysis of these nodules reveals similar nodule morphology and the presence of large numbers of bacteroids in each.

Morphogenesis of African swine fever virus in monkey kidney cells after reversible inhibition of replication by cycloheximide.

The late cytoplasmic phases of African swine fever virus (ASFV) morphogenesis in monkey kidney cells have been studied by transmission electron microscopy, focusing attention on the synthesis of viral envelopes. Morphogenesis was studied after reversible cycloheximide blockage of monkey kidney cells infected with ASFV. ASFV appears to synthesize its external and internal envelopes within the cellular cytoplasm, at the same time as the capsid is formed, with intracellular and extracellular virions showing similar structure and polypeptide composition.

Degradation of cellular proteins during poliovirus infection: studies by two-dimensional gel electrophoresis.

Picornaviruses encode for their own proteinases, which are responsible for the proteolytic processing of the polyprotein encoded in the viral genome to produce the mature viral polypeptides. The two poliovirus proteinases, known as proteins 2A and 3C, use the poliovirus-encoded polyprotein as a substrate. The possibility that these poliovirus proteinases also degrade cellular proteins remains largely unexplored. High-resolution two-dimensional gel electrophoresis indicates that a few cellular proteins disappear after poliovirus infection. Thus, at least nine acidic and five basic cellular proteins, ranging in Mr from 120 to 30 kilodaltons, are clearly degraded during poliovirus infection of HeLa cells. The degradation of these cellular polypeptides is very specific because it does not occur upon infection of HeLa cells with encephalomyocarditis virus or Semliki Forest virus. Moreover, inhibitors of poliovirus replication, such as cycloheximide or 3-methylquercetin, block the disappearance of these polypeptides. These results suggest that the input virions are not responsible for this degradation and that active poliovirus replication is required for the proteolysis to occur. Analysis of the time course of the disappearance of these polypeptides indicates that it does not occur during the first 2 h of infection, clearly suggesting that this phenomenon is not linked to the poliovirus-induced shutoff of host protein synthesis. This conclusion is strengthened by the finding that 3-methylquercetin blocks proteolysis without preventing shutoff of host translation.

Post-translational modifications of poliovirus proteins.

The post-translational modifications of poliovirus proteins have been investigated by analysis of glycosylation, sulphation, phosphorylation and acylation of the proteins made in the infected HeLa cells. No glycosylation or sulphation of proteins specific for virus-infected cells was apparent. A number of changes in the pattern of phosphorylated proteins took place. The specific myristylation of the structural protein VP4 and its precursors was clearly apparent. Acylation of viral proteins with oleic or palmitic acid was not detected. Myristylation took place in the presence of the protease inhibitor ZnCl2, but not in the presence of inhibitors of translation, such as cycloneximide and anysomycin.

The P2 and P3 regions of the poliovirus genome are preferentially translated at alkaline pH in infected HeLa cells.

HeLa cells infected with poliovirus exclusively synthesized proteins coded by the P2 and P3 regions of the viral genome when they were placed in an alkaline medium lacking sodium ions. The amount of viral protein synthesized was augmented by increasing the concentration of KCl. Also in the presence of KCl, the cells continued synthesizing this altered pattern of proteins for longer times. This effect occurred in the presence of guanidine, it was reversible, and the normal pattern of poliovirus proteins reappeared when control medium was added, even when guanidine was present. These findings suggest that truncated viral RNA is not made under these conditions. Pactamycin and sodium fluoride, two known inhibitors of the initiation of translation, blocked protein synthesis in cells placed in alkaline medium, indicating the possibility that initiation at internal sites of the viral mRNA may take place under these conditions. Finally, the proteins synthesized were analysed by two-dimensional gel electrophoresis. The proteins migrated as authentic viral proteins indicating that if internal initiation takes place, at least some of these initiation events occur in phase with the initiation codon present in the poliovirus genome.

The heat-shock response in Trypanosoma cruzi.

When Trypanosoma cruzi epimastigotes are exposed to temperatures of 37-41 degrees C there is a drastic decline in total protein synthesis. Analysis of the proteins synthesized at 41 degrees C by one-dimensional gel electrophoresis showed three major bands of Mr 83,000, 70,000 and 60,000. A similar pattern of heat-shock proteins was found in two different strains of T. cruzi (Tulahuen and GM strains) and in exponentially growing or in stationary epimastigotes. Actinomycin D prevented the appearance of these polypeptide bands, suggesting that the heat-shock proteins in T. cruzi epimastigotes are induced at the level of transcription. Analysis of the proteins synthesized by metacyclic forms at different temperatures suggests that heat-shock proteins in these cells are already synthesized at 27 degrees C. Elevation of temperature above 37 degrees C blocks the synthesis of most proteins in metacyclic forms except for major bands of Mr 83,000, 70,000, 60,000 and 55,000. More detailed analyses by high-resolution two-dimensional gel electrophoresis of the proteins synthesized at 27 degrees C or 37 degrees C by epimastigotes indicates that the heat-shock protein pattern is more complex than that demonstrated by one dimension, and at least ten new polypeptides are identified in two-dimensional gels. A similar analysis of metacyclic forms shows that most if not all the proteins present at 39 degrees C are also present at 27 degrees C. This result led us to the suggestion that the differentiation of T. cruzi to metacyclic forms involves the induction of heat-shock proteins, which prepares the parasite to infect the mammalian host.

Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis.

At least 74 acidic and 37 basic proteins are synthesized in African swine fever virus (ASFV)-infected monkey cells not detected in uninfected cells analyzed by two-dimensional gel electrophoresis. Essentially all the proteins synthesized early during infection are also observed at late times. The use of inhibitors such as cycloheximide and phosphonoacetate has led to the identification of 34 immediate early and 13 delayed early polypeptides. Therefore 64 proteins were classified as late polypeptides. Several ASFV-induced proteins are phosphorylated as proteins a1, a4, a20, a41, a48, a49, a51, a52, a55, a58, a67, b2, b12, b28, and b32.