Expression of human T cell receptor-gamma delta structural forms

The human TCR-gamma delta occurs in three biochemically distinct forms (forms 1, 2bc, and 2abc). A 40-kDa TCR gamma-chain is disulfide-linked to the TCR delta-chain in form 1, whereas 40-kDa or 55-kDa TCR-gamma polypeptides are noncovalently associated with the TCR delta-chain in forms 2bc and 2abc, respectively. Sequence analysis of TCR-gamma cDNA clones indicates that form 1 utilizes the C gamma 1 gene segment, whereas forms 2bc and 2abc appear to use allelic C gamma 2 gene segments containing either two copies (b and c) or three copies (a, b, and c) of the CII exon, respectively. We transfected TCR-gamma cDNA encoding form 1 or form 2abc into the MOLT-13 cell line that expresses form 2bc. The transfected TCR gamma-chains associate with the resident MOLT-13 TCR-delta, normally part of form 2bc, to yield CD3-associated TCR-gamma delta heterodimers identical to those seen on the donor cell lines (form 1 or 2abc). These transfection experiments show directly that, 1) when a single TCR-delta subunit is available, the presence or absence of disulfide linkage between TCR gamma- and TCR delta-chains is controlled by the TCR gamma-chain, and 2) the difference in the amount of N-linked carbohydrate attached to the transfected TCR-gamma proteins of form 2bc vs form 2abc is influenced by the presence or absence of CII exon copy "a" which appears to alter the secondary and/or tertiary structure of these TCR gamma-chain constant regions, thereby affecting the attachment of N-linked glycans. In contrast to the similar structure and usage of C beta 1 and C beta 2, TCR-gamma delta forms show striking differences in structure and are not equally represented in peripheral blood. Although the role of each form is unknown, it is possible that variable or joining-gene segment selection events or functional differences account for their unequal usage.     

Cytohistologic correlation of urothelial lesions secondary to photodynamic therapy

Qualitative cytologic evaluations of urinary bladder washings were performed on a selected population following photodynamic therapy for recurrent transitional cell carcinoma of the bladder. The seven patients were monitored trimonthly by cystoscopy, multiple biopsies and cytopreparations. Cancers reappeared in two of the five patients who initially responded to therapy. In the remaining two patients, the recurrent neoplasms were therapeutically refractory. Cytology detected recurrent cancer prior to biopsy confirmation and/or cytoscopic identification. Exfoliative cytology was correlated with the histopathology of the concurrent biopsies; a possible source for a false-positive cytodiagnosis was the cellular atypia of reepithelialized bladder mucosa. Dysplasia was not identified cytologically or histologically.     

Apparent phytotoxicity of mononuclear hydroxy-aluminum to four dicotyledonous species

It has long been assumed that Al³⁺ is an important rhizotoxic ion in acid soils around the world, but the toxicity of Al³⁺ relative to mononuclear hydroxy-Al [AlOH²⁺ and Al(OH)⁺₂] has been examined in detail only for an Al-sensitive wheat variety ( Triticum aestivum L. cv. Tyler). That plant appears to be sensitive to Al³⁺ but not to AlOH²⁺ and Al(OH)⁺₂. New experiments, and reanalyses of previously published experiments, provide evidence that dicotyledonous species may be sensitive to mononuclear hydroxy-Al and that Al³⁺ may be nontoxic, or less toxic, to those plants. Despite these consistently measured differences between wheat and the dicotyledons, the determination of relative toxicities (Al³⁺ vs mononuclear hydroxy-Al) may be an intractable problem. Because of hydrolysis equilibria, (AlOH²⁺) and (Al(OH)⁺₂) are equivalent to (Al³⁺)k₁(H⁺)⁻¹ and (l³⁺)k₂(H⁺)⁻², respectively, in which k₁ and k₂ are the first and second hydrolysis constants (braces denote activities). Thus, any expression of root elongation as a function of mononuclear hydroxy-Al can be alternatively expressed as a function of (Al³⁺) and (H⁺). Toxicity attributed to mononuclear hydroxy-Al may actually be Al³⁺ toxicity that increases as pH rises (i.e. Al³⁺ toxicity ameliorated by H⁺).     

Dynamics of Endogenous Cytokinins during the Growth Cycle of a Hormone-Autotrophic Genetic Tumor Line of Tobacco

The profile of endogenous cytokinins in a genetic tumor line of tobacco, namely, Nicotiana glauca (Grah.) × Nicotiana langsdorffii (Weinm.), following 1 to 10 weeks of growth on solid medium was determined by radioimmunoassay. ³H-labeled cytokinins of high specific activity were added during tissue extraction to correct for the purification losses. Following subculture (of 4-week-old tissues when their cytokinin content is high) onto fresh medium the total cytokinin content continued to be high during the first week (1470 picomoles per gram fresh weight) when the tissue fresh weight remained essentially unchanged (lag phase). The cytokinin levels then declined by about half in 2- and 3-week-old tissues (626 and 675 picomoles per gram fresh weight, respectively), a period when rapid increase in tissue fresh weight was recorded. Increments of 840% and 2780% over initial fresh weight were obtained in 2- and 3-week-old cultures, respectively. The cytokinin content then increased to initial high levels in 4-week-old tissues (1384 picomoles per gram fresh weight) after which it gradually declined with tissue age. The lowest cytokinin levels (432 picomoles per gram fresh weight) were observed in 10-week-old tissues. Maximal tissue fresh weight (4030% increase over initial fresh weight) was recorded in 5-week-old cultures after which it decreased slowly to 77.5% of the highest tissue fresh weight in 10-week-old cultures. Zeatin appeared to be the dominant endogenous cytokinin in tissues of all ages. Other cytokinins quantified were dihydrozeatin, zeatin riboside, and dihydrozeatin riboside; the values may include contributions from aglucones derived from the hydrolysis of corresponding O-glucosides, since the entire basic fraction was treated with β-glucosidase before analysis. In addition the levels of isopentenyladenine, isopentenyladenosine, and the nucleotides of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine were also determined.     

Selection and characterization of sethoxydim- tolerant maize tissue cultures

`Black Mexican Sweet' (BMS) maize (Zea mays L.) tissue cultures were selected for tolerance to sethoxydim. Sethoxydim, a cyclohexanedione, and haloxyfop, an aryloxyphenoxypropionate, exert herbicidal activity on most monocots including maize by inhibiting acetyl-coenzyme A carboxylase (ACCase). Selected line B10S grew on medium containing 10 micromolar sethoxydim. Lines B50S and B100S were subsequent selections from B10S that grew on medium containing 50 and 100 micromolar sethoxydim, respectively. Growth rates of BMS, B10S, B50S, and B100S were similar in the absence of herbicide. Herbicide concentrations reducing growth by 50% were 0.6, 4.5, 35, and 26 micromolar sethoxydim and 0.06, 0.5, 5.4, and 1.8 micromolar haloxyfop for BMS, B10S, B50S, and B100S, respectively. Sethoxydim and haloxyfop concentrations that inhibited ACCase by 50% were similar for BMS, B10S, B50S, and B100S. However, ACCase activities were 6.01, 10.7, 16.1, and 11.4 nmol HCO₃⁻ incorporated per milligram of protein per minute in extracts of BMS, B10S, B50S, and B100S, respectively, suggesting that increased wild-type ACCase activity conferred herbicide tolerance. Incorporation of [¹⁴C]acetate into the nonpolar lipid fraction was higher for B50S than for BMS in the absence of sethoxydim providing further evidence for an increase in ACCase activity in the selected line. In the presence of 5 micromolar sethoxydim, [¹⁴C]acetate incorporation by B50S was similar to that for untreated BMS. The levels of a biotin-containing polypeptide (about 220,000 molecular weight), presumably the ACCase subunit, were increased in the tissue cultures that exhibited elevated ACCase activity indicating overproduction of the ACCase enzyme.     

Identification of constitutive and steroid-dependent transactivation domains in the mouse oestrogen receptor

We have identified two transactivation domains in the mouse oestrogen receptor whose activities depend on the target promoter. The major domain is contained within the C-terminal portion of the protein and depends upon oestrogen binding for its activity. The location and oestrogen dependence of this domain has been confirmed using chimaeric receptors containing the Lex A DNA binding domain. Although transactivation by the C-terminal domain is dependent upon ligand binding the analysis of receptor deletion mutants has demonstrated that these two functions are not entirely coincident. The second transactivation domain lies within the N-terminal region and is active in the absence of oestradiol. The differences in oestrogen requirement for the activity of the two transactivation domains may account for the partial agonist activity of certain antihormones.     

Stable transfection of the oestrogen receptor gene into a human osteosarcoma cell line

The oestrogen receptor (ER) gene was introduced into an ER-negative osteoblast-like osteosarcoma cell line HTB 96 by transfection. A number of clones were isolated which expressed ER at levels of up to 70 fmol/mg cytosol protein as determined by immunoassay. Scatchard analysis of the binding of [3H]17 beta-oestradiol in cytosols demonstrated the presence of high affinity binding sites, with a dissociation constant of 0.08-0.13 nM at 4 degrees C. High levels of a 3 kb ER mRNA are produced by the clones, which have gene copy numbers ranging from 2 to greater than 10. Functional receptor activity has been demonstrated by co-transfection of a plasmid containing the chloramphenicol acetyl transferase (CAT) gene linked to an oestrogen response element. Induction of CAT activity is observed in the presence of added oestradiol and is concentration-dependent. The transfected ER is also able to affect endogenous cellular function as several ER-positive clones, but not HTB 96 cells, are growth inhibited by oestradiol in the concentration range 10(-9)-10(-7) M. These effects on growth are not induced by other classes of steroids and are reversible by antioestrogens. No endogenous genes have yet been identified which are oestrogen-regulated in ER-transfected clones.     

Activation of the c-H-ras proto-oncogene by retrovirus insertion and chromosomal rearrangement in a Moloney leukemia virus-induced T-cell leukemia.

A rearrangement of the c-H-ras locus was detected in a T-cell line (DA-2) established from a Moloney leukemia virus-induced tumor. This rearrangement was associated with the high-level expression of H-ras RNA and the H-ras gene product, p21. DNA from DA-2 cells transformed fibroblasts in DNA transfection experiments, and the transformed fibroblasts contained the rearranged H-ras locus. The rearrangement involved one allele and was present in tissue from the primary tumor from which the cell line was isolated. Cloning and sequencing of the rearranged allele and comparison with the normal allele demonstrated that the rearrangement was complex and probably resulted from the integration of a retrovirus in the H-ras locus between a 5' noncoding exon and the first coding exon and a subsequent homologous recombination between this provirus and another newly acquired provirus also located on chromosome 7. These events resulted in the translocation of the coding exons of the H-ras locus away from the 5' noncoding exon region to a new genomic site on chromosome 7. Sequencing of the coding regions of the gene failed to detect mutations in the 12th, 13th, 59th, or 61st codons. The possible reasons for the complexity of the rearrangement and the significance of the activation of the H-ras locus to T-cell transformation are discussed.     

Germanium accumulation byPseudomonas stutzeri AG259

Summary The influence of pH, temperature and catechol concentration on germanium (Ge) accumulation byPseudomonas stutzeri AG259 was investigated. Increasing the incubation temperature or pH of the culture medium markedly enhanced Ge accumulation. High amounts of Ge were accumulated at pH 11 and at 50°C, conditions under whichP. stutzeri cells were non-viable. Ge accumulation was unaffected by treatment with toluene or 2,4-dinitrophenol. These results indicate that Ge was accumulated by an energy-independent process. Ge accumulation increased as the catechol concentration increased. The use of autoclaved catechol solutions consistently increased the amount of Ge accumulated at all concentrations of catechol tested. It is possible that Ge enters the bacterial cells as a Ge-catechol complex and this uptake is enhanced by autoclaved catechol.