Identification by differential display of a protein phosphatase-2A regulatory subunit preferentially expressed in malignant melanoma cells.

We described the occurrence of 4 transcripts differentially displayed between syngeneic murine B16F10 (metastatic melanoma) and Melan-a (immortalised melanocytes) cell lines. We now report that one such transcript, which is B16F10-specific, represents a protein phosphatase-2A B' regulatory subunit. No expression of this transcript was detected in the weakly metastatic B16F1 by Northern blotting. Moreover, the transcript was not expressed by spontaneously immortalised, non-tumorigenic, melanocytes (Melan-Ab and Melan-a2), nor was it expressed by ras-transformed, tumourigenic melanocytes (Melan-Ab-LTR-ras). Cloning of the 5'-end region of this transcript (termed band 8A) from B16F10 cells revealed an intracisternal A-particle insertion, including the long terminal repeat region, which could account for the observed high expression in B16F10 cells. Single cell clones of B16F10 manifested an experimental metastasis capacity, which correlated with band 8A expression with the lowest expressors being least metastatic. The human homologue of the B' regulatory subunit, B56gamma, is expressed preferentially at the mRNA level in human melanoma cell lines compared with normal epidermal melanocytes. In situ hybridisation studies on human clinical samples detected high expression of this gene in a number of malignant melanomas. Our results imply strongly that this protein phosphatase-2A regulatory subunit may have a role in melanoma tumour progression.

Identification by differential display of annexin-VI, a gene differentially expressed during melanoma progression.

To identify genes involved in the melanocyte to malignant melanoma conversion, we have applied differential display to the comparison of syngeneic murine B16F10 (metastatic melanoma) and Melan-a-immortalized melanocyte cell lines. Approximately 7000 bands were analyzed, revealing approximately 80 to be differentially displayed. Reverse Northern blotting and subsequent Northern blotting confirmed the reproducible differential expression of four transcripts. Three B16F10-specific bands encode novel genes or partially sequenced cDNAs of unknown function. One Melan-a-specific band was found to be identical to the 3' end region of the mouse Annexin VI mRNA and shown to have a reduced message expression in B16F10 relative to Melan-a. Differential expression was confirmed at the protein level with Western blotting using a rabbit polyclonal antiserum. Immunohistochemistry of human melanoma specimens with this antiserum revealed a decrease or loss of Annexin VI expression as melanomas progressed from a benign to a more malignant phenotype. Our results provide further evidence for a potential role of Annexin VI in tumor suppression.

Veillonella myositis in an immunocompromised patient.

Infectious myositis is rather uncommon. When caused by anaerobic organisms, myositis is usually polymicrobial. Trauma, ischemia, or a contiguous focus of infection is often an antecedent of myositis. We report a case of monomicrobial veillonella myositis in an immunocompromised patient. The infection responded to debridement and therapy with metronidazole.

Expression and function of the CD44 glycoprotein in melanoma cell lines.

We have analysed a panel of murine and human melanoma cell lines for expression of the glycoprotein CD44. All 12 cell lines examined expressed CD44 at their cell surfaces, as demonstrated by fluorocytometric analysis using monoclonal antibodies (MAbs) IM7 and F10.44.2. Northern analysis revealed three transcript sizes that were 4.5, 2.2, and 1.5 kb in the human cell lines and 4.5, 3.0, and 1.5 kb in the murine cell lines. Levels of mRNA did not correlate with level of surface expression, which was highly variable between the cell lines. RT-PCR analysis of mRNA revealed that the major band identified was the expected 792 bp fragment indicative of the CD44H haemopoietic form, compared to a 1194 bp form found in the human colorectal adenocarcinoma cell line HT29, indicative of the CD44E epithelial form. There was no evidence of variant CD44 mRNA in our panel of melanomas. Functional assays revealed no clear correlation between the level of cell surface CD44 and the ability of the melanoma cell lines to adhere to hyaluronate. Rather adherence appeared to relate to the activation status of CD44 on the different cell lines as a consequence of MAb stimulation (e.g. the 1735P line demonstrated a 46.2 +/- 5.7% adherence in an inactivated state versus 62.4 +/- 5.6% adherence in an activated state to 5 mg/ml hyaluronate) and suggests that the functional capacity of CD44 expressed by melanoma cells may be modified more by activation state than by RNA splicing.

Planned early discharge from New Zealand maternity hospitals.

AIMS: The purpose of the Plunket National Child Health Study was to establish a database on health indicators of New Zealand children and families. This paper examines the issue of early discharge in New Zealand maternity hospitals. METHODS: As part of the study, 4286 parents were interviewed when the child was around six weeks post partum regarding their planned choice for length of post natal hospital stay and their actual length of stay. RESULTS: Most New Zealand mothers choose to stay in hospital for 4-7 days following childbirth. Those mothers choosing early discharge (within 48 hours of delivery), tended to be multiparous, nonEuropean, less educated and of lower socioeconomic status. CONCLUSIONS: The characteristics of those choosing early discharge demonstrate the vulnerability of this group and also have implications for the provision of health care services which are discussed.

An improved metaphase index assay for detecting thyroid growth stimulators using FRTL-5 thyroid cells cultured on a microtitre plate.

The metaphase index assay (MIA) for thyroid growth stimulators, as originally described, used FRTL-5 thyroid cells cultured in Bellco culture chambers and glass microscope slides. The metaphases were observed using the nuclear strain aceto-lacto orcein. However the surface properties of the glass proved to be variable and so polystyrene microscope slides were substituted. The aceto-lacto orcein stain was found to be unsuitable for use with polystyrene because of the solvents and mountant used. Therefore combinations of various other nuclear stains and mounting media were tested. The Giemsa stain, which was found to be the most satisfactory, could be applied to FRTL-5 cells maintained on the large variety of plastic supports now available for tissue culture, e.g., 96 well microtitre plates. This permitted the design of an MIA which is much more convenient, robust and economical in its use of clinical samples. The results with seven IgG preparations derived from the sera of patients with a variety of thyroid disorders are presented. In its revised form, the metaphase index assay provides a rapid screening assay for thyroid growth stimulators, such as autoantibodies and TSH.

Recovery of plant amines with carboxylic ion-exchange substrates and their subsequent analysis by thin-layer chromatography.

Two grades of carboxymethylcellulose (CMC) and five ion-exchange resins were compared for their effectiveness in recovering endogenous amines from plant extracts. If amine loss was to be limited to 10%, the H+ and Na+ forms of CMC could not be loaded with the solutes from more than 0.25 and 1.5 gram fresh weight (gfw) tissue per ml bed volume respectively. The corresponding ionic forms of a typical resin, Amberlite CG-50, would tolerate loadings ca. 5 X higher than this. However, it was then necessary to use very slow flow-rates (13 ml cm-2 h-1) for both applying and eluting the amines and, even so, they could not be quantitatively displaced from any resin tested, with the possible exception of Duolite C433. If the extract was acidic, maximum permissible loadings were reduced by a factor of 3 to 20, depending on substrate and ionic form. The composition of the amine fraction was essentially the same whatever substrate was used for its recovery and whatever percentage of it had been lost.

Enhanced 3-methoxytyramine levels in crown gall tumours and other undifferentiated plant tissues.

An amine, after dansylation, has been isolated from Nicotiana tabacum crown gall tumours for the first time and characterized as 4-hydroxy-3-methoxy-beta-phenylethylamine (3-methoxytyramine). The compound cannot be detected in differentiated N. tabacum tissues but appears in the corresponding callus controls. Its concentration is further increased 5-42-fold when N. tabacum is transformed with all strains of Agrobacterium tumefaciens so far tested.