Multiphoton fluorescence lifetime imaging shows spatial segregation of secondary metabolites in Eucalyptus secretory cavities.

Multiphoton fluorescence lifetime imaging provides an excellent tool for imaging deep within plant tissues while providing a means to distinguish between fluorophores with high spatial and temporal resolution. Ideal candidates for the application of multiphoton fluorescence lifetime imaging to plants are the embedded secretory cavities found in numerous species because they house complex mixtures of secondary metabolites within extracellular lumina. Previous investigations of this type of structure have been restricted by the use of sectioned material resulting in the loss of lumen contents and often disorganization of the delicate secretory cells; thus it is not known if there is spatial segregation of secondary metabolites within these structures. In this paper, we apply multiphoton fluorescence lifetime imaging to investigate the spatial arrangement of metabolites within intact secretory cavities isolated from Eucalyptus polybractea R.T. Baker leaves. The secretory cavities of this species are abundant (up to 10 000 per leaf), large (up to 6 nL) and importantly house volatile essential oil rich in the monoterpene 1,8-cineole, together with an immiscible, non-volatile component comprised largely of autofluorescent oleuropeic acid glucose esters. We have been able to optically section into the lumina of secretory cavities to a depth of ∼80 µm, revealing a unique spatial organization of cavity metabolites whereby the non-volatile component forms a layer between the secretory cells lining the lumen and the essential oil. This finding could be indicative of a functional role of the non-volatile component in providing a protective region of low diffusivity between the secretory cells and potentially autotoxic essential oil.

Myelodysplasia in adult life: the value of an outreach nursing service.

In the state of South Australia, infants with myelodysplasia have been treated by early intervention since 1963, and the majority have survived. Many are now adults, leading active lives despite having severe disabilities. A pilot study confirmed that this population has a high incidence of preventable ill health because of recurrent urinary infections, renal damage, and pressure ulcers. Many of these complications can be prevented by an outreach nursing service aimed at providing education in self-care, counseling, advocacy, and other appropriate nursing interventions, thus allowing clients to avoid hospitalization and maintain their independence. The study also showed that many clients were unaware of important social benefits, the knowledge of which should promote their financial independence. The outreach nursing service is an essential part of an integrated long-term support program for adults with myelodysplasia and other forms of congenital spinal paralysis.

Serodiagnosis of Mycobacterium bovis infection in badgers: development of an indirect ELISA using a 25 kDa antigen.

A 25 kDa antigen of Mycobacterium bovis has previously been identified as immunodominant during badger infections. This 25 kDa antigen was partially purified from sonicated M bovis bacilli by using water precipitation and ion exchange chromatography, and its purification was monitored with a mouse monoclonal antibody, MBS43, which was specific for the antigen. The partly purified antigen was used to develop an ELISA for the assay of badger sera for the presence of specific antibodies. A presumed negative badger population was used to calculate the assay's threshold of seropositivity and using this value, its sensitivity (37 percent) and specificity (98 percent) were determined in a second population of known culture status. The results indicate that it may be possible to develop a specific and cost effective serological field assay for the diagnosis of M bovis infection in living badgers.

Production and characterization of a monoclonal badger anti-immunoglobulin G and its use in defining the specificity of Mycobacterium bovis infection in badgers by western blot.

A mouse monoclonal anti-badger IgG antibody was produced to investigate the specificity of the antibody response of badgers infected with Mycobacterium bovis. The monoclonal antibody generated was directed against badger IgG heavy chain and appeared to be species restricted, reacting only with badger and dog IgGs but not cat, rabbit, mouse, guinea pig, bovine or ferret IgGs. This monoclonal antibody detection system functioned well in both ELISA and Western blot analyses and was successfully used to investigate the humoral response of the badger to M. bovis infection. Sera from infected badgers detected a 25 kDa antigen which was not detected by sera from M. bovis culture-negative animals. This antigen was conserved in all field strains of M. bovis tested and seroconversion to it was detected during experimental infection. The immunodominance of this antigen in the badger during infection with M. bovis suggests that this 25 kDa polypeptide is a suitable candidate on which to base an antibody detection test for M. bovis infection.

Cloning and characterization of the gene for the '19 kDa' antigen of Mycobacterium bovis.

Monoclonal antibody CMA134.1 reacted with a protein antigen of apparent molecular mass 22 kDa from Mycobacterium bovis and Mycobacterium tuberculosis, and with an apparently 24 kDa antigen of Mycobacterium kansasii, but not with other mycobacteria or related species. This antibody was used to screen a gene library of M. bovis in lambda gt11 and identified a recombinant clone that expressed a protein with an apparent molecular mass of 19-20 kDa. Gene expression occurred from the lac promoter in lambda gt11, but used an unidentified vector promoter, possibly that of the replication primer RNA, in the final plasmid construct. The sequence of an 840 bp fragment was determined and shown to code for a product of 15 kDa. This sequence is identical to that, independently determined, of a gene from M. tuberculosis, usually referred to as the 19 kDa antigen. The reasons for the apparent size discrepancies are discussed.