Structural alteration of glycosaminoglycan side chains and spatial disorganization of collagen networks in the skin of patients with mcEDS-CHST14

Musculocontractural Ehlers-Danlos syndrome (mcEDS) due to CHST14/D4ST1 deficiency (mcEDS-CHST14) is a recently delineated type of EDS caused by biallelic loss-of-function mutations in CHST14, which results in the depletion of dermatan sulfate (DS). Clinical characteristics of mcEDS-CHST14 consist of multiple malformations and progressive fragility-related manifestations, including skin hyperextensibility and fragility. Skin fragility is suspected to result from the impaired assembly of collagen fibrils caused by alteration of the glycosaminoglycan (GAG) chain of decorin-proteoglycan (PG) from DS to chondroitin sulfate (CS). This systematic investigation of the skin pathology of patients with mcEDS-CHST14 comprised both immunostaining of decorin and transmission electron microscopy-based cupromeronic blue staining to visualize GAG chains. Collagen fibrils were dispersed in the affected papillary to reticular dermis; in contrast, they were regularly and tightly assembled in controls. Moreover, the fibrils exhibited a perpendicular arrangement to the affected epidermis, whereas fibrils were parallel to control epidermis. Affected GAG chains were linear, stretching from the outer surface of collagen fibrils to adjacent fibrils; in contrast, those of controls were curved, maintaining close contact with attached collagen fibrils. This is the first observation of compositional alteration, from DS to CS, of GAG side chains, which caused structural alteration of GAG side chains and resulted in spatial disorganization of collagen networks; this presumably disrupted the ring-mesh structure of GAG side chains surrounding collagen fibrils. McEDS-CHST14 provides a critical example of the importance of DS in GAG side chains of decorin-PG during assembly of collagen fibrils in maintenance of connective tissues.     

Influence of adjuvants on the antibody specificity to the Plasmodium falciparum major merozoite surface protein, gp195

The effect of adjuvants on the specificity of immune responses to the Plasmodium falciparum gp195 protein was investigated using adjuvant formulations based on synthetic muramyl dipeptide and monophosphoryl lipid A derivatives, in parallel with CFA and alum. Although these immunomodulators were as effective as CFA in inducing an antibody response to gp195, there were distinct differences in the recognition of B cell epitopes by these antibody populations. We have also demonstrated that MHC control of antibody specificity can be related to the adjuvant used for immunization. In general, the potency of adjuvants, their ability to induce antibodies of a particular specificity, or their ability to overcome MHC control of immune responsiveness varied independently. These findings suggest a critical role of adjuvants in the determination of the specificity of the immune response to protein Ag. Thus, the influence of adjuvants should be a major consideration in studies on immunologic recognition, as well as in the design of modern subunit vaccines.     

Turnover of enzymes of peroxisomal β-oxidation in rat liver

Male Wistar rats were given a diet containing 2% (w/w) di-(2-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal β-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal β-oxidation was 5–7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the β-oxidation system and of catalase decreased to the control levels with a half-life of 2–3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.     

Biosynthesis of enzymes of peroxisomal beta-oxidation

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for 2 weeks. The increases in the individual enzymes of the hepatic peroxisomal beta-oxidation system after administration of DEHP were 31- to 33-fold. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rates of synthesis of the enzymes were 16- to 20-fold higher and those of degradation were 1.7- to 1.9-fold lower in the DEHP group. The translation rates of these enzymes in vitro with liver RNA in the reticulocyte-lysate system were 12- to 14-fold higher in the DEHP group. Short-term kinetic labeling experiments on acyl-CoA oxidase consisting of three subunits were conducted in vivo to explore the biogenesis of peroxisomes. The label was found in the biggest subunit of the enzyme in the supernatant fraction shortly after the label injection, but was distributed to the smaller subunits later. The labeling in the smaller subunits in the peroxisomal fraction was greater than that of the supernatant. The distribution of the label among the subunits in these subcellular fractions was the same as that of the protein amounts 1 day after the label injection. This paper reports that the increase in the quantities of peroxisomal enzymes upon administration of DEHP is mainly due to the increase in their synthesis rates caused by the increase in amounts of mRNA coding for these enzymes.     

Gene recombination and segregation of resistance factor R in Escherichia coli

Hashimoto, Hajime (Osaka University, Osaka, Japan), and Yukinori Hirota. Gene recombination and segregation of resistance factor R in Escherichia coli. J. Bacteriol. 91:51-62. 1966.-Independent chloramphenicol-sensitive (CM(s)) mutants of the drug-resistance factor R were isolated. Introduction of two different R factor CM(s) mutants into a single bacterium, by conjugation or transduction, gave chloramphenicol-resistant (CM(r)) colonies when such strains were plated on a medium containing chloramphenicol (Cm). These CM(r) colonies resulted from recombination between two R factors contained within the same cell. Most of the CM(r) colonies were heterogeneous, and segregation of drug-resistance markers was observed among the progeny. Segregated bacteria which still carried the recombinant R factor were stable for resistance to Cm as well as for other markers of R. All the markers of recombinant R factors were cotransducible with high coincidence and at the same frequency as wild-type R. Sensitive mutants of R which had lost all the resistance markers of the R factor were found also. A mutation of R, referred to as SMA, which was sensitive to streptomycin and sulfanilamide, was capable of reverting to resistance to both of these drugs simultaneously. The sensitive alleles for SMA, CM, and TC were shown to be recessive to the resistance alleles. Mutants of R having multisite mutations or deletions in the CM gene were isolated and used to analyze the pattern of linked segregation of unselected markers of the recombinant R factor. The drug resistance factor R was shown to have two linkage groups, CM-SMA and TC-m.     

Activation of c-Ha-ras proto-oncogene by in vitro chemical modification with 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 4-nitroquinoline N-oxide (4NQO)

Chemical modification of a plasmid containing the human c-Ha-ras proto-oncogene (pSVMBras-gpt) in vitro with the ultimate carcinogens N-acetoxy-2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (N-OAc-Glu-P-1) and N-acetoxy-4-aminoquinoline N-oxide (N-OAc-4AQO) generated an activated oncogene that transformed NIH3T3 cells. As DNA is only cellular macromolecule present in the reactions, the results clearly show that the chemical modification of DNA with carcinogens alone can cause the induction of transformation of mammalian cells.     

Drug Resistance of Enteric Bacteria

Drug resistance of 3,000 Shigella strains isolated in 1965 were investigated. These strains originated from 10 City Hospitals and 4 Prefectural Health Centers, which are located in different parts of Japan. One hundred and seventy strains which were resistant to 4 drugs, chloramphenicol (CM), tetracycline (TC), dihydrostreptomycin (SM), and sulfanilamide (SA), were selected at random from these stock cultures in this laboratory and the distribution of R factors in these isolates was examined. It was found that the strains all harbored R factors which were capable of transferring drug resistance by usual conjugal process. Among the strains carrying R factors, 85 per cent harbored a single type of R factor and 15 per cent carried two types of R factor in a cell. The latter is called the hetero-R state. Among the strains in the hetero-R state, isolation of strains harboring both R (SM.SA) and R (TC.CM.SM.SA) factors was most frequent. It was found that 25 R (SM.SA) factors isolated from strains in hetero-R had the genetic determinant iR^?, while most of the R (TC.CM.SM.SA) factors isolated from natural sources were iR⁺. When two types of R factor, R (SM.SA) and R (TC.CM.SM.SA) derived from the same host cells, were brought together in a host cell by superinfection with both factors, they were found to exist stably in a host bacterium. These results confirmed the stable existence of both factors in Shigella strains isolated from dysenteric patients.     

Effect of passive administration of alloantiserum containing antibody against putative acceptor(s) for T cell-replacing factor (TRF) in the neonatal stage on development of B cell activity responsive to TRF

Previously, we showed that the antiserum raised in male (DBA/2Ha X BALB/c)F1(DCF1) mice (T cell-replacing factor [TRF]-low response animals) by immunizing them with activated B cells from BALB/c mice (TRF-high-responders) contained antibodies against putative TRF-acceptor site(s). We have now evaluated the hypothesis that neonatal treatment of mice with the above antiserum suppresses the development of B cells responsive to TRF. Male DCF1 mouse anti-BALB/c B-cell antiserum or normal DCF1 mouse serum as a control was injected into BALB/c mice within 24 hr after birth. In the antiserum-treated mice, no augmented primary immunoglobulin M (IgM) antibody responses to sheep red blood cells (SRBC) were observed under the conditions in which markedly augmented IgM anti-SRBC responses were induced in control BALB/c mice, suggesting that development of B cells reacting with male DCF1 mouse anti-BALB/c B-cell antiserum is suppressed by the neonatal treatment with the antiserum. Furthermore, the development of B cell activity responsible for helper factors derived from T cells, such as TRF, was markedly suppressed in the neonatally antiserum-treated mice, whereas activity of B cells capable of interacting directly with helper T cells through antigen-bridges was not significantly affected by the same treatment. Such suppression of the B cell activity could be induced only when the antiserum was administered within 48 hr after birth. Moreover, neonatal treatment of mice with the antiserum induced suppressed responsiveness of B cells to a T-independent type 2 antigen, TNP-Ficoll. Neither serum-borne suppressive serum components nor suppressor cells were detected by the system employed. These results support the hypothesis that TRF responsive B cells constitute a subpopulation distinct from the other B cells capable of cooperating with helper T cells via cognate interaction.