Iodocyclization reactions for the desymmetrization of cyclohexa-1,4-dienes.

Fifteen examples are presented showing that various modes of cyclization (5-endo, 5-exo, 6-endo, 6-exo, and 7-endo) can be used for the desymmetrization of cyclohexa-1,4-dienes. All take place with complete diastereocontrol and good yield.

Breast carcinoma and the role of iron metabolism. A cytochemical, tissue culture, and ultrastructural study.

Transferrin receptors on proliferating and malignant cells are well documented. Iron is an essential micronutrient for cell growth that plays an important role in energy metabolism and DNA synthesis. Malignant cells requiring more iron modulate a transferrin receptor. Iron-bound transferrin interacts with this receptor, facilitating the transport of iron across the cell membrane. Transferrin is a glycoprotein and is the chief iron transport protein in mammalian blood. The more aggressive the tumor, the higher the transferrin receptor levels and the greater the proliferative index. We have found by cytochemical and ultrastructural studies that ferritin, an iron storage protein, is increased in breast cancer tissue. Anaplastic tumors have higher tissue ferritin levels. Tissue ferritin concentration may be an indirect method of measuring transferrin receptors and thus might be an index of proliferation and a prognostic indicator. Transferrin may be used as a carrier to target toxic therapy selectively to tumor tissue. A platinum transferrin complex (MPTC-63) has been developed and shown to be cytostatic in tissue culture, animal, and human studies. It also sensitizes tissue to agents that produce free radicals, such as adriamycin, and thus is synergistic with other drugs and radiation. Other transferrin complexes and conjugates of gallium, indium, and daunorubicin have also shown growth inhibition in tissue culture and animals. Human studies are in progress. By studying iron metabolism in breast cancer, we may be able to selectively inhibit tumor growth without toxic effects, and with other tumor biologic data be better able to select the stage I patient for adjuvant therapy.

pBECKS. A flexible series of binary vectors for Agrobacterium-mediated plant transformation.

A series of binary T-DNA vectors (pBECKS) has been created for use in the Agrobacterium-mediated genetic transformation of plants. The pBECKS series has corrected the undesirable features of the popular pBIN19 vector; the deleterious mutation within the coding sequence of nptII has been amended and the cloning sites are now adjacent to the right border repeat in order to reduce the possibility of producing truncated sequences of novel genes within transformants. One set of vectors incorporates various combinations of the marker genes gusA, C1/Lc, nptII, hph, and bar, for pursuit of early and stable transformation events. A set of constructs which contain deleted T-DNA borders in various combinations and display predictably altered efficacies for gene transfer has also been created. A modular set of vectors has been designed to facilitate the insertion and transfer of novel gene sequences by providing a nptII-linked plant expression cassette or lacZ-multiple cloning site. A range of antibiotic resistance genes has been incorporated into the non-T-DNA part of the vectors in order to facilitate their selection across the range of Agrobacterium virulence strains.

Agrobacterium-mediated transformation of élite indica and japonica rice cultivars.

A rapid, efficient, routine system has been established for Agrobacterium tumefaciens-mediated production of hundreds of fertile transgenic plants from commercially important rice cultivars, including an indica cultivar, Pusa Basmati 1. Calli induced from embryos of mature rice seeds were cocultivated with A. tumefaciens strain LBA4404 carrying the plasmid pTOK233, then exposed to hygromycin selection followed by an efficient regeneration system. Based on the total number of calli co-cultivated, the transformation frequencies of independent transgenic rice plants including cultivars Pusa Basmati 1, E-yi 105, E-wan 5 and Zhong-shu-wan-geng, were 13.5, 13.0, 9.1, and 9.3%, respectively. T1 seeds were harvested within 7-8 mo of initiation of mature embryo cultures. Data from Southern hybridization analysis proved that foreign genes on T-DNA were stably integrated into the rice genome at low copy/site numbers. Mendelian inheritance of the transgenes was confirmed in T1 progeny.

A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation.

The introduction of binary plasmids into Agrobacterium hosts for Agrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations of Agrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformed Agrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of three Argobacterium tumefaciens (EHA101, LBA4404, C58) and two Agrobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin(res), kanamycin(res), and gentamycin(res)).

The plant cell cycle in context.

Biological scientists are eagerly confronting the challenge of understanding the regulatory mechanisms that control the cell division cycle in eukaryotes. New information will have major implications for the treatment of growth-related diseases and cancer in animals. In plants, cell division has a key role in root and shoot growth as well as in the development of vegetative storage organs and reproductive tissues such as flowers and seeds. Many of the strategies for crop improvement, especially those aimed at increasing yield, involve the manipulation of cell division. This review describes, in some detail, the current status of our understanding of the regulation of cell division in eukaryotes and especially in plants. It also features an outline of some preliminary attempts to exploit transgenesis for manipulation of plant cell division.

Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.).

Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate "escapes" and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3-11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2-5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.

Stereotaxic localization and core needle biopsy of nonpalpable breast lesions: two-year follow-up of a prospective study.

In August 1989, we began a prospective study of patients with abnormal mammograms to determine the clinical efficacy of stereotaxic localization and needle biopsy of nonpalpable breast lesions. By August 1990, 115 patients had undergone stereotaxic localization and needle biopsy of nonpalpable breast lesions using the Mammotest II (Fischer Imaging, Denver, CO) and an 18-gauge needle in a Bard Biopty gun (Bard Urological, Covington, GA). In 19 per cent (22 of 115) of the cases, the pathologist suggested open surgical biopsies of the lesions due to clinical judgment, or atypical or cancer cells in the core biopsy specimen. Of these 22 cases, 12 (54%) were found to be cancer on open surgical biopsy--10 per cent of all the patients. Of the remaining 93 patients with benign pathology, 9 were lost to follow-up by the end of the second year after closure of the study. The remaining 84 patients were followed by mammography and physical exam at 3 months, at 12 months, and yearly thereafter to determine whether cancer had been missed or developed at the biopsy site. The median follow-up was 24 months (mean, 23.3 months), and all 84 patients were found to have no evidence of malignant disease at follow-up. The small group (10 cases) of patients who were determined to have benign disease by open surgical biopsy were also found on follow-up to have no evidence of malignant disease (median follow-up, 20.5 months; and mean, 18.3 months). The accuracy of this stereotaxic procedure, combined with an approximately 80 per cent decrease in the more expensive and more invasive open surgical biopsy procedure, provides strong support for the use of this procedure on all nonpalpable breast lesions that would normally proceed to open surgical biopsy.

Accelerated partial breast irradiation: initial experience with the Intrabeam System.

Failure after breast conserving surgery (BCS) and total breast irradiation usually occurs at the site of the original tumor. This has caused an increased interest in accelerated partial breast irradiation (APBI), because if radiation is delivered directly to the tumor bed there should be better local control. Patients greater than age 50 with core biopsy confirmed invasive ductal carcinoma were enrolled. They had preoperative ultrasound defining margins of less than 3.5 cm. Intraoperative ultrasound was also performed in an effort to ensure good surgical margins. After excision of the tumor, intraoperative radiotherapy (IORT) with the Intrabeam System was delivered to the tumor bed. The procedure has been performed on 67 patients. Sixty-one patients had it with the original surgery, while 6 had the procedure after re-exploration of the segmental mastectomy site. Because of the final pathology (surgical margins, tumor biology, and nodal status) 4 patients later had total mastectomy and 11 received total breast irradiation. When total breast irradiation is done the IORT serves as the radiation boost. The cosmetic results have been good to excellent, and there have been no serious surgical or radiation complications. To date there have been no local failures. IORT with the Intrabeam System is feasible, user friendly, versatile, with few complications, good cosmetic results, and great patient acceptance. It is practical and excellent for breast IORT in the community setting.

The logMAR Kay picture test and the logMAR acuity test: a comparative study.

PURPOSE: To compare the level of visual acuity with crowded and uncrowded versions of the logMAR acuity test and the Kay picture test in amblyopia. METHODS: A prospective study was carried out on 51 participants with amblyopia (strabismic n=17; anisometropic n=10; combined n=24), mean age 10 years 8 months. The amblyopia was defined as severe/moderate (< 0.250 logMAR), n=41 or mild (> or = 0.250 logMAR), n=10. Visual acuity was assessed uniocularly using the crowded and uncrowded logMAR acuity tests and the logMAR crowded and uncrowded Kay picture tests in random orders. RESULTS: The mean visual acuity outcome using the logMAR crowded Kay picture test (0.343+/-0.150) was comparable (P=0.084) with the mean outcome using the crowded logMAR acuity test (0.402+/-0.188). However, the mean acuity difference between these two tests in the subgroup with severe/moderate amblyopia (0.074+/-0.036) was statistically significant (P=0.0382). The uncrowded logMAR acuity test significantly overestimated visual acuity when compared with the logMAR crowded Kay picture test (P<0.005) by a mean of 0.088+/-0.008. CONCLUSION: The logMAR crowded Kay picture test is a useful tool in clinical practice. The test design takes the crowding phenomenon into account. It provides visual acuity measures more comparable with the gold standard crowded logMAR acuity test than the uncrowded logMAR acuity test. However, the outcomes in poorer acuities should still be viewed with caution.

Indole Compounds Related to Auxins and Goitrogens of Woad (Isatis tinctoria L.).

Five conspicuous indole derivatives are present in leaves and other tissues of woad (Isatis tinctoria L.). They were identified as tryptophan, isatan B, glucobrassicin, neoglucobrassicin, and glucobrassicin-1-sulfonate. The latter three indole glucosinolates are present at levels of at least 260, 69, and 200 milligrams per kilogram fresh weight and were isolated as crystalline salts. Comparison of physical and chemical properties, particularly NMR spectral analysis, confirms that the 1-methoxyglucobrassicin structure suggested for neoglucobrassicin is correct, whereas further evidence for the even more unusual sulfonation of the ring nitrogen in glucobrassicin-1-sulfonate was obtained. Glucobrassicin-1-sulfonate has an enzymic degradation pattern identical to that of glucobrassicin. As it too releases thiocyanate, it must be added to the list of known plant goitrogens. These studies and the techniques described establish woad as exceptionally suitable higher plant material for metabolic studies of indoles related to goitrogens and auxins.

Distribution and Variation of Indole Glucosinolates in Woad (Isatis tinctoria L.).

The exceptionally high levels in woad (Isatis tinctoria L.) of three indolic goitrogens, namely glucobrassicin, neoglucobrassicin, and glucobrassicin-1-sulfonate, permit the facile study of their distribution in the plant and their changes during its development. Woad seeds contain as much as 0.23% fresh weight of glucobrassicin but no other indole glucosinolate, while 1-week-old seedlings also contain substantial amounts of neoglucobrassicin and glucobrassicin-1-sulfonate in their shoots whether grown in the light or dark. The sulfonate is not found in roots, and light depresses neoglucobrassicin levels in shoots. Sterile root cultures synthesize glucobrassicin and neoglucobrassicin, and significant quantities of these were even found to be excreted by the roots of intact sterile seedlings in culture. This may explain the long known deleterious effect of woad and other cruciferous crops on subsequent plantings and the observation could be of ecological importance. Long term changes in levels of all three substances in the plant are similar and are compatible with earlier suggestions that the compounds could be auxin precursors at the time of flower stem elongation. Since sterile seedlings readily incorporate (35)SO(4) (2-) into indole glucosinolates and relative specific radioactivities suggest that glucobrassicin is the precursor of the other two compounds, pathways of goitrogen biosynthesis should be relatively easily determined in this material.

Tryptophan and indole-3-acetic acid accumulation in Acer cell cultures and its relationship with cell autolysis.

The accumulation and decline of 'free indole-3-acetic acid (IAA)' and tryptophan has been monitored in cells of Acer pseudoplatanus L. grown in batch suspension cultures. The period of maximal IAA accumulation per cell or per unit dry weight of tissue was found to precede the peak of tryptophan accumulation by several days. A study of cell viability throughout a growth passage indicated the presence of a basal level of non-viable cells of 5-7%, with only minor increases occurring during the first week of the three-week growth passage. The results suggest that IAA biosynthesis is not regulated by substrate availability arising from proteolysis in dead cells.

Ontogenetic Changes in the Transport of Indol-3yl-acetic Acid into Maize Roots from the Shoot and Caryopsis.

The quantities of endogenous indol-3yl-acetic acid (IAA) in endosperms and scutella of 6-day-old maize seedlings (Zea mays L. cv Giant White Horsetooth) were determined by a fluorimetric method. Endosperms were found to contain 33.4 nanograms IAA per plant, and scutella 7.5 nanograms IAA per plant. [5-(3)H]IAA applied to endosperms of 6-day-old seedlings moved into the roots and radioactivity accumulated at the apex of the primary root within 8 hours. Two to 7-day-old seedlings were treated simultaneously with [5-(3)H]IAA in the endosperm and [2-(14)C] IAA on the shoot apex. The patterns of transport into the root were found to change during ontogeny: in successively older plants, transport from the shoot into the roots increased relative to transport from the endosperm into the roots. The auxin required for the growth of maize roots could, therefore, partially be contributed by the shoot and endosperm. Ontogenetic changes in the relative importance of these two supplies could be of significance for the integration of growth and development between shoot and root.

Acid growth effects in maize roots: Evidence for a link between auxin-economy and proton extrusion in the control of root growth.

The role of proton excretion in the growth of apical segments of maize roots has been examined. Growth is stimulated by acidic buffers and inhibited by neutral buffers. Organic buffers such as 2[N-morpholino] ethane sulphonic acid (MES) - 2-amino-2-(hydroxymethyl)propane-1,3 diol (Tris) are more effective than phosphate buffers in inhibiting growth. Fusicoccin(FC)-induced growth is also inhibited by neutral buffers. The antiauxins 4-chlorophenoxyisobutyric acid (PCIB) and 2-(naphthylmethylthio) propionic acid (NMSP) promote growth and H(+)-excretion over short time periods; this growth is also inhibited by neutral buffers. We conclude that growth of maize roots requires proton extrusion and that regulation of root growth by indol-3yl-acetic acid (IAA) may be mediated by control of this proton extrusion.

Endogenous and exogenous auxin in the control of root growth.

The endogenous indol-3yl-acetic acid (IAA) of detipped apical segments from roots of maize (cv ORLA) was greatly reduced by an exodiffusion technique which depended upon the preferential acropetal transport of the phytohormone into buffered agar. When IAA was applied to the basal cut ends of freshly prepared root segments only growth inhibitions were demonstrable but after the endogenous auxin concentration had been reduced by the exodiffusion technique it became possible to stimulate growth by IAA application. The implications of the interaction between exogenous and endogenous IAA in the control of root segment growth are discussed with special reference to the role of endogenous IAA in the regulation of root growth and geotropism.

pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates "clean-gene" facilities.

A new plasmid series has been created for Agrobacterium-mediated plant transformation. The pBECKS2000 series of binary vectors exploits the Cre/ loxP site-specific recombinase system to facilitate the construction of complex T-DNA vectors. The new plasmids enable the rapid generation of T-DNA vectors in which multiple genes are linked, without relying on the availability of purpose-built cassette systems or demanding complex, and therefore inefficient, ligation reactions. The vectors incorporate facilities for the removal of transformation markers from transgenic plants, while still permitting simple in vitro manipulations of the T-DNA vectors. A 'shuttle' or intermediate plasmid approach has been employed. This permits independent ligation strategies to be used for two gene sets. The intermediate plasmid sequence is incorporated into the binary vector through a plasmid co-integration reaction which is mediated by the Cre/loxP site-specific recombinase system. This reaction is carried out within Agrobacterium cells. Recombinant clones, carrying the co-integrative binary plasmid form, are selected directly using the antibiotic resistance marker carried on the intermediate plasmid. This strategy facilitates production of co-integrative T-DNA binary vector forms which are appropriate for either (1) transfer to and integration within the plant genome of target and marker genes as a single T-DNA unit; (2) transfer and integration of target and marker genes as a single T-DNA unit but with a Cre/loxP facility for site-specific excision of marker genes from the plant genome; or (3) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes.

Preliminary evaluation of platinum transferrin (MPTC-63) as a potential nontoxic treatment for breast cancer.

Transferrin receptors on proliferating and malignant cells are well documented. Faulk et al. demonstrated transferrin receptors in breast carcinoma by immunofluorescence. Malignant cells requiring more iron modulate a transferrin receptor and the iron transporting protein transferrin delivers iron to the cell. We have developed a physiologically active platinum transferrin complex that has been tested on several cell lines in culture, a tumor model in the Fischer rat, and five human patients with advanced breast carcinoma. The complex slowed the rate of growth of feline lymphoma cells to one-half that of controls and killed human HeLa cell cultures in 7 days. Growth of the rat tumor was slightly impaired, but treated rats never got systemic disease and controls died. Two patients had dramatic responses to treatment. One had systemic disease and the other advanced locoregional disease. Both patients were on Tamoxifen, as receptors were positive for estrogen. Disease was progressing in the former with little improvement in the latter. After treatment both had a marked response. We postulate that MPTC-63 may work synergistically with Tamoxifen and be an effective nontoxic antitumor agent. More studies are indicated.

Auxin gradient along the root of the maize seedling.

[5-(3)H]Indol-3yl-acetic acid (IAA) applied to the shoot apices of intact 6-day-old maize (Zea mays L.) plants moved into the primary root and accumulated at the root apex. IAA from the shoot could partially satisfy the requirement of the primary root for IAA for growth.

PCR and non-isotopic labeling techniques for plant virus detection.

PCR technology permits the detection of viruses at levels several orders of magnitude lower than is possible by other methods. This high sensitivity facilitates detection of virus sequences during the early stages of infection of plants and in soil and vector samples. Early detection of beet necrotic yellow vein virus (BNYVV) in Beta vulgaris is an important part of the strategy for prevention of the spread of rhizomania, a commercially significant disease of sugar beet. A diagnostic test for BNYVV has been developed. This test involves amplification of the viral genome by PCR coupled with non-isotopic labeling and detection of specific sequences. The PCR amplification of BNYVV sequences has been optimized with respect to primer design, sample preparation and reaction conditions. Several non-isotopic labeling strategies for signal amplification have been compared. Hybridization with digoxigenin-labelled cDNA permits the most sensitive detection of PCR products and is the most appropriate method for routine diagnosis. These observations are discussed in the context of the application of PCR for detecting a wide range of viruses.