Delivery of recombinant product from subcutaneous implants of encapsulated recombinant cells in canines.

Delivering recombinant therapeutic proteins from a universal microencapsulated cell line is an alternate method for gene therapy. It has proved effective in the treatment of several murine models of human genetic diseases. However, in scaling up to large animal models, intraperitoneal Implantations of these microcapsules in canines were associated with excessive Inflammatory response and rapid degradation. We now show that subcutaneous implantation of microencapsulated cells in canines is effective in delivering recombinant product systemically for extended periods, provides a surgically benign site, leads to less inflammatory response, and permits longer-term survival of microcapsules. Allogeneic MDCK cells engineered to secrete human growth hormone (hGH) were microencapsulated in alginate-poly-L-lysine-alginate and implanted subcutaneously. Systemic delivery of hGH was evident within 4 hours and peaked by day 1 after implantation in all dogs. The gradual decline of hGH in the circulation in the first 2 weeks coincided with the development of anti-hGH antibodies by day 11. The high titer persisted for more than 1 month, demonstrating indirectly the persistent delivery of hGH. Microcapsules retrieved from the subcutaneous implant maintained their structure throughout the experiment and were free of host cellular adhesions. The mechanical integrity of the subcutaneously implanted microcapsules also appeared superior to that of the intraperitoneal implant. Hence the subcutaneously implanted microcapsules required minimal surgical intervention and led to a low level of inflammatory response, and the implant survived for at least 1 month, thus demonstrating the feasibility of systemic delivery of recombinant products via subcutaneous implantation in large animals.

Transforming growth factor-alpha-mediated growth pathways in human gastro-intestinal cell lines in relation to the gastrin autocrine pathway.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) increase transcription of the gastrin gene, and the gastrin peptide may be phosphorylated by EGF-stimulated tyrosine kinase. Our aims were to compare EGF/TGF-alpha interactions in 2 human gastro-intestinal cell lines: MGLVA1, with a strong gastrin autocrine pathway, and C170HM2, with a weak pathway. Both cell lines expressed the TGF-alpha gene. MGLVA1 expressed TGF-alpha protein as determined by immuno-cytochemistry, which was absent in C170HM2. Both cell lines expressed the same level of EGF receptors, as assessed by flow cytometry; however, MGLVA1 did not have enhanced in vitro proliferation in response to EGF or TGF-alpha, unlike C170HM2. The basal growth of MGLVA1 was inhibited by anti-sera against TGF-alpha, the EGF receptor and G17. C170HM2 was not inhibited by any of the anti-sera. Neutralisation of TGF-alpha resulted in undetectable cell-associated progastrin levels in MGLVA1 (untreated had 391.7 fmol/5 x 10(6) cells). The progastrin level of C170HM2 remained unaffected. Tyrosine kinase activity, as assessed by phosphopeptide concentration, of unstimulated MGLVA1 was 2.6 times higher than that of C170HM2 in the cell membrane fraction (0.097 compared to 0.037 microg/mg protein, p < 0.001) and 4.8 times higher in the cytosolic fraction (0.269 compared to 0.056 microg/mg protein, p < 0.05). Following treatment with EGF, the phosphopeptide concentration increased in both the membrane and cytosolic fractions of both cell lines. Tyrphostin B42, which inhibits autophosphorylation of the EGF receptor, inhibited the basal growth of MGLVA1 (IC(50) 1.3 microM) and C170HM2 (9.5 microM, p < 0.05 from MGLVA1). Herbimycin, which inhibits pp60(c-src) kinase, reduced the basal growth of MGLVA1 (0.67 microM) but not C170HM2. Immunofluorescence studies confirmed the presence of tyrosine-phosphorylated proteins and pp60(c-src) within the cytoplasm of unstimulated MGLVA1 cells. There was no specific immunofluorescence for either parameter in C170HM2 cells until after treatment with EGF.

5,10,15,20-Tetrakis(4-N-methylpyridyl)porphyrinato-palladium(II ) as a differentiation probe for sensing binding modes with B-DNA duplexes: electronic MCD and CD spectra.

We report our detailed electronic MCD, CD, and optical spectroscopic measurements and analysis of the porphyrin Soret (B(o)) region of four-coordinate 5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrinatopalladium( II), PdP(4), and its bound states with B-DNA duplexes poly(A-T)2, poly(G-C)2, and calf thymus DNA (CT DNA). For system PdP(4)/poly(A-T)2 it was possible to conclude that the porphyrin is bound edge-on in the major groove, specifically at the 5'AT3' site. For this orientation the porphyrin's electric dipole transition moments (edtm), mu(x) (most perturbed direction) and mu(y) (least perturbed direction), have tilt angles alpha approximately 90 degrees and approximately 45 degrees , respectively, relative to the helix axis. It was further concluded from the small shifts of B(o) optical and MCD band intensities and wavelengths and from the net MCD (+) A-term sign retention upon binding that the porphyrin's frontier ppi MOs (1a(1u) 3a(2u) 4eg) are only weakly perturbed by the heterocyclic bases of poly(A-T)2, and therefore that the LUMO (4eg) splitting is less than the |1a(1u)-3a(2u| energy separation, deltaHOMO, that is, deltaLUMO < deltaHOMO for the bound state in PdP(4)/poly(A-T)2. For intercalation systems PdP(4)/ poly(G-C)2 and /CT DNA, with PdP(4) centered in the intercalation "pocket" and having two of its 4-N-methylpyridyls extending into each of the major and minor grooves, the edtms mu(x) and mu(y) were determined to be oriented perpendicular (gamma approximately 0 degrees) and parallel (gamma approximately 90 degrees) to the hydrogen bonds of the base pairs, respectively. Intercalation is characterized by a much stronger binding interaction, viz., the B(o) optical band and net MCD extrema wavelength shifts are relatively large, and the net MCD (+) A-term of PdP(4) is substantially quenched as it becomes the (-) pseudo-A-term of intercalated PdP(4)/poly(G-C)2. This A-term sign reversal informs that the porphyrin MOs are so strongly perturbed by the GC base pairs that deltaLUMO > deltaHOMO, which gives rise to a (-) pseudo-A-term. Also, the findings demonstrate (1) the potential of PdP(4) as a sensitive, discriminating analytical probe of DNA sequences and (2) the diagnostic capability of the composite of five spectra [net MCD, CD, and optical of free and bound PdP(4)] in differentiating the site and sequence selectivity and preferred binding mode of this porphyrin.

Modification of flower color in florist's chrysanthemum: production of a white-flowering variety through molecular genetics.

Chimeric chalcone synthase (CHS) constructs were prepared in both anti-sense and sense orientations, and introduced into the chrysanthemum cultivar Moneymaker, along with a T-DNA vector lacking a CHS construct. For both the anti-sense and sense constructs, the majority of the plants produced pink flowers typical of Moneymaker itself. Of 133 sense and 83 anti-sense transgenic individuals 3 of each set produced fully white or very pale pink flowers. No white-flowering transgenic plants were obtained in control transformations. The white flowers were found to accumulate higher levels of chalcone synthase precursors and to have reduced levels of chalcone synthase message. A small-scale field trial was performed to evaluate the stability of the phenotype throughout a series of vegetative propagation steps and during plant growth. The white-flowering trait was maintained well through vegetative propagation; however, during growth of individual white-flowering plants, some pink color was found in some flowers. At one site 2% of the white-flowering plants produced a few pink flowers; at two other sites, as many as 10-12% of the plants produced pale pink flowers.

Differential physiological and morphological responses of inbred lines to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid by cultured Helianthus annuus (sunflower) shoot tips.

The four Helianthus annuus (sunflower) inbred lines examined showed different abilities to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene and different morphological responses to exogenous ACC, however, ACC had no effect on precocious flowering. The greatest effect of ACC was seen with inbred SS405B where it suppressed shoot growth and induced hypocotyl enlargement and callus induction. The greatest response did not correlate with the highest ethylene production. Although each inbred responded differently, callus induction and hypocotyl enlargement observed in hypocotyl segments treated with naphthalene acetic acid and benzyladenine could be partially explained as ethylene-mediated effects of the two hormones. It is suggested that inbred differences could be due to different endogenous hormone levels and/or different sensitivities to them.

Is there still a role for knee arthrography?

To assess the continued value of arthrograms in the wake of arthroscopic diagnosis, an analysis of arthrographic data on 240 knees was carried out. The accuracy, sensitivity, and specificity of the arthograms were compared with the arthroscopic findings for each knee. For meniscal lesions, the arthrogram is reasonably accurate. An algorithm is presented that defines the relative roles of arthrography and arthroscopy.

Correlations of arthrography with arthroscopy.

Arthrography and arthroscopy were compared on a prospective basis in 100 consecutive and unselected knees, all in patients who underwent arthrography, arthroscopy, and surgery. Arthroscopy was found to be more accurate than arthrography in the diagnosis of medial and lateral meniscus and anterior cruciate ligament lesions. Because of its easy availability and relatively low cost, arthrography continues to be a useful extension of the physical examination of the knee, serving as a further diagnostic screening technique. However, arthroscopy is recommended in all patients prior to surgery because it provides more complete and more accurate delineation of intra-articular lesions.

Ossicle of the meniscus.

Two patients with an ossicle of the meniscus are described. Radiologic differentiation from osteochondral loose body or chondrocalcinosis can be made by its ossified appearance and its location within the meniscus. Correct diagnosis is important so that unnecessary surgery is avoided and a protracted search for a free fragment is not carried out.