1. methyl-(14)C-labelled 1,3-dihydroxy-4,5-dimethylbenzene, 5-methylorcylaldehyde and 5-methylorsellinic acid were synthesized from orcinol and sodium [(14)C]cyanide and tested for activity as precursors of gliorosein. ring-(14)C-labelled orcylaldehyde was also prepared. 5[(14)C]-Methylorcylaldehyde was incorporated into gliorosein (36% conversion); all the radioactivity was located in the C-methyl groups. 5-Methylorsellinic acid was decarboxylated by Gliocladium roseum and the resulting phenol was secreted into the medium. 2. The formation of an enzyme-bound derivative of 5-methylorsellinic acid as the first aromatic compound in the biosynthesis of gliorosein is suggested to explain these results. 3. ring-(14)C-labelled 3,4-dihydroxy-6-methyltoluquinone was also effectively incorporated into gliorosein and related products (20% conversion). 4. Sodium [(14)C]formate and [Me-(14)C]-methionine were incorporated into gliorosein and related products (15.4 and 22.2% conversion respectively). Isolation and estimation of the radioactivity in the O-methyl and C-methyl groups in the (14)C-labelled gliorosein thus formed showed an appreciable difference in the specific activities of the two types of methyl group (14 and 15% respectively). The results in the doubly-labelled methionine experiment indicate that the C-methyl group arises in the same manner as that in ergosterol; one of the original hydrogen atoms of the methyl group is lost. This confirms that C-methylation occurs at an ethylenic group at the aliphatic level. 5. The sequence of reactions at the aromatic level leading to the formation of gliorosein is proposed as 5-methylorsellinyl-enzyme-->3-hydroxy-5-methylorsellinyl-enzyme-->3,4-dihydroxy-6-methyltoluquinol-->3,4-dimethoxy-6-methyltoluquinol-->gliorosein.

Aldehydes↗

PubMed1967

Studies on the biosynthesis of phenols in fungi. Biosynthesis of 3,4-dimethoxy-6-methyltoluquinol and gliorosein in Gliocladium roseum I.M.I. 93 065.

1. Gliorosein was obtained in excellent yield (150mg./200ml. of Raulin-Thom medium) from surface cultures of Gliocladium roseum. Its nuclear-magnetic-resonance spectrum showed conclusively that it is 1,6-dihydro-3,4-dimethoxy-6-methyltoluquinone. 2. Sodium [2-(14)C]acetate was incorporated into gliorosein and the related products (3.3% conversion). The specific activities of these substances increased in the order gliorosein, 3,4-dimethoxy-6-methyltoluquinol, the related quinhydrone and quinone, indicating that gliorosein was the actual metabolite that was secreted and that the other compounds were derived from it in the medium. 3. 6-Methylsalicylic acid was not taken up by the mycelium and could be recovered unchanged. Orsellinic acid was decarboxylated by G. roseum and an equivalent amount of orcinol was secreted into the medium. The methyl esters of 6-methylsalicylic acid and orsellinic acid were both hydrolysed by an esterase present in the mycelium. Some of the 6-methylsalicylic acid thus produced was secreted into the medium and the orsellinic acid was decarboxylated. 4. Washed mycelium of G. roseum converted aurantiogliocladin and 3,4-dimethoxy-6-methyltoluquinol quantitatively into gliorosein within 18hr. More critical experiments with (14)C-labelled substrates demonstrated that 3-hydroxy-4-methoxy-6-methyltoluquinol and 3,4-dimethoxy-6-methyltoluquinol, and their respective quinones, were effectively incorporated into gliorosein and related products (49, 68, 30 and 57% respectively). 5. The following sequence of reactions is proposed for the biosynthesis of gliorosein: acetyl-CoA+3 malonyl-CoA+S-adenosyl-methionine --> 5-methylorsellinic acid --> 3-hydroxy-4-methoxy-6-methyltoluquinol --> 3,4-dimethoxy-6-methyltoluquinol --> gliorosein. 6. Since gliorosein is optically active (dextrorotatory), the final tautomerization reaction leading to its formation must be enzyme-catalysed.

Acetates↗

PubMedDate not supplied

The development of a mimotope-based synthetic peptide vaccine against respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide and the development of a synthetic peptide epitope-based vaccine to induce virus-neutralising antibodies against RSV would seem to be a valid approach to the production of an effective vaccine against infection.A combinatorial solid-phase peptide library has been screened with a virus-neutralising, protective monoclonal antibody (MAb19) directed towards a conserved and conformationally-determined epitope of the Fusion (F) protein of the virus. Two of the sequences identified from the peptide library using MAb19 reacted specifically with the antibody and amino acid substitution experiments identified four sequences from one of the mimotopes which showed increased reactivity with MAb19. Immunisation of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb19 to the virus and neutralised viral infection in vitro, with titres equivalent to those in sera from RSV-infected animals. Following RSV challenge of mimotope-immunised mice, a significant reduction in the titre of virus and a greatly reduced cell infiltration into the lungs of immunised mice compared to that in controls was observed. The induction of virus-specific cytotoxic T-lymphocyte responses as well as virus-specific antibodies are likely to be necessary in an effective vaccine. The incorporation of a peptide representing a CTL epitope from the M2 protein of the virus together with peptides inducing T-helper and anti-mimotope responses in a peptide cocktail vaccine resulted in a more effective clearance of the virus from immunised, challenged mice than peptide-induced humoral or cellular immunity alone.

Amino Acid Sequence↗

PubMedDate not supplied

Protection against measles virus-induced encephalitis by antibodies from mice immunized intranasally with a synthetic peptide immunogen.

Balb/c mice were immunized intranasally (i.n.) with a chimeric synthetic peptide containing two copies of a T- and one copy of a B-cell epitope (TTB) from measles virus (MV) fusion protein, plus cholera toxin B (CTB) adjuvant. The antibodies induced cross-reacted with, and neutralized MV and on passive transfer, protected mice against encephalitis induced by neuroadapated MV. Immunization with TTB alone induced antibodies which increased survival but not significantly compared to controls. Furthermore, i.n. immunization with TTB plus CTB induced TTB-specific IgA antibodies in saliva and nasal washes. Co-administration of CTB increased the affinity of antibodies to the B-cell epitope of TTB and caused a relative increase in the level of anti-peptide antibodies of the IgG2a subclass and the overall titre of IgG antibodies. These results indicate the potential of the i.n. route for immunization with synthetic peptide immunogens for induction of both local and systemic anti-peptide antibody responses.

Administration, Intranasal↗

PubMedDate not supplied

Variations in host responses and the pathogenesis of human onchocerciasis.

Different immune responses to Onchocerca volvulus cause considerable variation in clinical manifestations of human onchocerciasis. Onchocercal lesions result from inflammatory reactions involving immunologic mechanisms; the role of the immune system in pathogenesis is emphasized by the phenomena accompanying accelerated worm destruction during microfilaricidal chemotherapy (e.g., eosinophilia, changes in total immunoglobulin level, and anaphylactic symptoms). Although most pathologic changes are associated with the microfilarial stage, the extent to which circulating antibodies are directed against antigens in the adult worm or its uterine constituents is unknown. Microfilarial destruction can be mediated by antibody to the surface-associated antigens of the worms and enhanced by complement; a correlation exists between the presence of these antibodies and punctate keratitis. Heterogeneous immunologic components are associated with the surface of dermal and nodular microfilariae in vivo. Preliminary findings indicate that the level of O. volvulus-specific immune complexes is inversely proportional to the microfilarial load. To monitor a patient's clinical status and immunologic response, a quantitative system dividing symptoms into those associated with active responses to the microfilariae and those representing long-term consequences of these reactions is suggested.

Antibody Formation↗