Homeobox genes in the Australian lungfish, Neoceratodus forsteri.

The aim of the present study was to determine whether the postulated gnathostome duplication from four to eight Hox clusters occurred before or after the split between the actinopterygian and sarcopterygian fish by characterizing Hox genes from the sarcopterygian lungfish, Neoceratodus forsteri. Since lungfish have extremely large genomes, we took the approach of extracting pure high molecular weight (MW) genomic DNA to act as a template for polymerase chain reaction (PCR) of the conserved homeobox domain of the highly conserved Hox genes. The 21 clones thus obtained were sequenced and translated in a BLASTX protein database search to designate Hox gene identity. Fourteen of the clones were from Hox genes, two were Hox pseudogenes, four were Gbx genes, and one most closely resembled the homeobox gene, insulin upstream factor 1. The Hox genes identified were from all four tetrapod clusters A, B, C, and D, confirming their presence in lungfish, and there is no evidence to suggest more than these four functional Hox clusters, as is the case in teleosts. A comparison of Hox group 13 amino acid sequences of lungfish, zebrafish, and mouse provides firm evidence that the expansion of Hox clusters, as seen in zebrafish, occurred after separation of the actinopterygian and sarcopterygian lineages. J. Exp. Zool. (Mol. Dev. Evol.) 285:140-145, 1999.

The anthracycline resistance-associated (ara) gene, a novel gene associated with multidrug resistance in a human leukaemia cell line.

Multidrug resistance (MDR) in cancer cells is a major contributor to the failure of chemotherapy treatment. This paper describes a novel protein named the anthracycline resistance associated (ARA) protein. The ara gene is amplified in the MDR leukaemia line CCRF-CEM/E1000 and its mRNA is overexpressed. ARA belongs to the ATP binding cassette (ABC) family of proteins. Another ABC protein, the multidrug resistance-associated protein (MRP), has previously been reported to be overexpressed in the CEM/E1000 subline. The primary amino acid sequence of ARA indicates that it is 49.5 kDa without glycosylation, and that it has one potential glycosylation site. ARA has one ATP binding site and associated transmembrane regions. This is in contrast to MRP (190 kDa, 172 kDa deglycosylated) and most other higher eukaryote ABC proteins, which consist of two similar halves, each having one ATP binding site. In addition to ARA being coexpressed with MRP, comparison of amino acid sequences showed that, among known proteins, ARA is most similar to the C-terminal half of MRP.

Antigen binding and cytotoxic properties of a recombinant immunotoxin incorporating the lytic peptide, melittin.

BACKGROUND: The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein. OBJECTIVES: We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin. STUDY DESIGN: The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human kappa myeloma and lymphoma cells, and on human free kappa Bence Jones protein (BJP). Melittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of K121 was constructed by PCR to link VH and VL genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2). RESULTS: Expression of the recombinant scFv-mel gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original K121 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone. CONCLUSION: The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression. Linking of melittin to an antibody fragment overcame the non-specific toxicity of melittin as the recombinant immunotoxin exhibited specific toxicity towards antigen-bearing target cells. The observation that the immunotoxin exhibited enhanced cytotoxic activity over the free toxin indicates the potential of this approach for the development of an effective therapeutic agent.

Conformation of the T-cell antigen receptor-beta chain C-domain contributes to V beta 3 epitope recognition by monoclonal antibody KJ25.

The clonotypic T-cell antigen receptor (TCR)-beta chain contains two extracellular intrachain disulfide bonds. It belongs to the immunoglobulin gene superfamily and is subdivided into variable (V), joining (J), diversity (D) and constant (C) region. Monoclonal antibody (MoAb) KJ25 is believed to recognize an epitope in the V-domain of TCR-beta (V beta 3) chain, but its epitope requirements are unknown. In this study of TCR-alpha beta chain interactions using chimeric recombinant TCR-beta chains, the authors found that partial substitution of the C beta-domain with that of interleukin-2 receptor alpha chain (Tac) sequences led to the loss of TCR-V beta 3 epitope recognition by KJ25. These results suggest that epitope recognition of the TCR-V beta 3 by KJ25 MoAb is dependent not only on the V-domain, but also on the close contact with the extracellular C-domain which influences the conformation and epitope recognition of the V beta 3-region. This may not be unique to V beta 3 and may be a general feature of TCR-beta protein folding.

Quality assessment and patient participation in care by means of a touch-screen computer.

Hospital characteristics vary greatly across a geographic area such as a state. Hospital peer groups internally exhibit similar characteristics and can be used as a basis for the analysis of data, the dissemination of information, and the adoption of continuous quality improvement project results. This paper reflects the efforts made toward the identification of hospital peer groups within the state of Michigan. Hospital characteristics data for fiscal year 1992 were obtained from the American Hospital Association's Annual Survey of Hospitals and the Health Care Financing Administration's MEDPRO database. Thirteen peer group clusters have been identified, reviewed, and commented on by the state's hospital association and have met general approval by hospital administrators across the state. The established peer groups are being used to identify the differences in patterns of care among hospitals in the state. The peer groups also are being used for the feedback of comparable data and the identification of hospitals for participation in continuous quality improvement projects. The next research objective is to experiment with other clustering techniques and other inpatient populations. The consistency of the peer groupings across all clustering techniques and across both Medicare and total inpatient populations will be studied.

Drug resistance mechanisms and MRP expression in response to epirubicin treatment in a human leukaemia cell line.

A drug resistant series of sublines were developed by treating the human leukaemia CCRF-CEM cell line with 16-1000 ng/ml of the anthracycline, epirubicin. The sublines developed resistance in two stages, neither involving detectable levels of P-glycoprotein. Treatment with up to 50 ng/ml epirubicin produced sublines with cross resistance limited to the anthracyclines and etoposide. Treatment with 100-1000 ng/ml epirubicin produced sublines with increased expression of the mrp gene, increased resistance to the anthracyclines and etoposide, additional cross resistance to vincristine and colchicine, decreased drug accumulation and reversal of resistance by verapamil and by buthionine sulphoximine (BSO; an inhibitor of glutathione synthesis). Our results indicate an interaction between MRP and glutathione metabolism as a mechanism for multidrug resistance.

Full-length cDNA sequence of X-linked G6PD of an Australian marsupial, the wallaroo.

In order to study the mechanism of X Chromosome (Chr) inactivation in marsupials, the cDNA for glucose-6-phosphate dehydrogenase (G6PD) from an Australian marsupial, the wallaroo (Macropus robustus), was cloned. A partial clone containing the 3' half of the cDNA was obtained by screening a liver cDNA library. The majority of the coding region was obtained by polymerase chain reaction of cDNA with primers designed from regions of conservation between human and opossum G6PD. The 5' end was obtained by rapid amplification of cDNA ends. High homology was observed between mammalian species in the coding region. The 5' untranslated region is highly G+C rich, and appears to be part of a CpG island, as is the case in the human and mouse genes. This is the first report of the full sequence of the cDNA for any marsupial X-linked gene.

Structure of the tomato Adh2 gene and Adh2 pseudogenes, and a study of Adh2 gene expression in fruit.

A cDNA library was constructed from RNA from the pericarp of ripe tomato fruit and four cDNAs encoding ADH2 were isolated and characterized. The cDNAs encode a peptide 379 amino acids in length. They hybridized strongly with a 1.8 kb RNA species well represented in RNA from ripe, but not from mature, unripe fruit, and strongly to a similar RNA species present in hypoxic, but not in aerobic roots. Northern analysis showed that the mRNA for ADH2 in fruit increased in abundance through ripening, particularly during late ripening. In pericarp tissue of fruit, the Adh2 mRNA level increased to a maximum within 8-16 h of exposure to atmospheres with 3% (v/v) oxygen, and returned to the basal level within 16 h of a return to air. The mRNA level was sensitive to the oxygen level in the atmosphere, increasing 20-fold in 12% (v/v) oxygen and 100-fold in 3% oxygen. The homologous tomato Adh2 gene was isolated from a genomic library. The gene has an overall length of 2334 bp from transcription start site to poly(A) addition site and includes eight introns. Southern blot analysis of tomato genomic DNA identified multiple Adh2-related sequences. Two of these, PSA1 and PSA2, were cloned and found to have 94% similarity with each other and 77% similarity with the tomato Adh2 gene over a 1000 bp region. The homologous regions include introns and exons but the equivalent exons contain frame shifts, deletions and stop codons. The two regions are therefore presumptive pseudogenes.

Self-assessment and impairment in adult/elderly hearing screening--recent data and new perspectives.

Because self-assessment measures are helpful in identifying handicap/communication difficulties, they have an important place in hearing screening protocols for adult/elderly persons. When impairment is used as the criterion, questionnaire results can be used to calculate sensitivity, specificity, predictive values, and efficiency for a variety of fences. Findings are summarized for 2825 persons on three different questionnaires that reveal adequate, reasonably similar, and predictable relationships between impairment and self-assessment findings. However, evidence on "deny-ers" (persons with impairment who deny their handicap) and "complainers" (persons who complain about handicap but have no impairment) are also presented. These latter findings show a strong rationale for using self-assessment measures in their own right, and not simply as an alternate, less effective method for measuring pure-tone impairment.

Self-assessment of hearing in rehabilitative audiology: developments in the USA.

Self-assessment of hearing is being used for screening, diagnostic, and rehabilitative audiology. Hearing handicap/disability measures were derived on a large number of patients who were classified based on pure tone findings into Pure Tone Groups (PTGs). A systematic relationship was found between handicap/disability as measured with Self-Assessment of Communication (SAC) and PTG. Handicap/disability ratings were also found to be systematically related to use of hearing aids.

Communication in retarded adolescents: response to listener feedback.

Retarded adolescents' response to listener feedback was studied using an interpersonal description task. Thirty-six adolescent retarded speakers were asked to describe nonsense line figures for a confederate adult listener so that the listerner could select the same stimuli from an array. The listener responded to the descriptions with a predetermined random order of success and failure. Communication failure was reported to the speaker by three methods: gestural, implicit, and explicit feedback. Redescriptions given by the speaker following each type of listener feedback were counted and analyzed as to type of redescription. Significant differences were found among intelligence level groups for number of redescriptions after communication failure and also for type of redescription.

The applied communication game: a comment on Muma's "communication game: dump and play".

We now have technology available to teach speech and language skills to children. We cannot always expect children to carry over or generalize this training and spontaneously use this "trained language" in communication situations, so we now must develop technology for teaching interpersonal communication skills. This report commented on a model presented by Muma (1975). Illustrations were presented that should help clinicians train more effective interpersonal communication. The applied communication game provides an objective procedure that utilizes the natural consequences in communication situations to improve communication skill in handicapped children.