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.

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.

Assessment of polysomaty, embryo formation and regeneration in liquid media for various species of diploid annual Medicago.

To avoid polyploidy in regenerants the source of explant material should be monosomatic. Therefore, the leaf and petiole tissue of five diploid Medicago species (Medicago ciliaris, Medicago murex, Medicago orbicularis, Medicago polymorpha and Medicago truncatula cv. Jemalong, and the ecotype R108-1) was assessed for polysomaty by flow cytometry. For the species studied the frequency of 2C nuclei was about 90% in leaves compared with that in petioles. Embryos were readily formed from tissue of leaves in liquid media containing 1 mg l(-1) or 4 mg l(-1) dichlorophenoxyacetic acid (2,4-D). For embryo development two procedures were tested - prolonged use of induction medium and treatment with polyethylene glycol Mw 6000 (PEG). The highly regenerable genotypes M. truncatula cv. Jemalong and R108-1 showed efficient conversion of embryos after maturation in liquid medium. The regenerated plants were diploid and with normal phenotype.

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.

Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation.

The co-transformation of a single plant genome with two independent T-DNA regions provides opportunities for genetic separation in subsequent generations. In an effective strategy, co-delivery events must form a high proportion of the total transformed population. In this study, using the model plant species tobacco (Nicotiana tabacum), it was shown that the frequency of co-transformation within a given To population could be as high as 100% and this was found to be dependent, at least in part, on designing the plasmid vectors so that the kbp size of the first selected T-DNA region was >2-fold that of the designated T-DNA region for co-transfer. Overall, 40-50% of To lines demonstrated the capacity for segregational separation of co-transformed T-DNA regions. Hence, the estimate of the required number of total transformants for such an independent strategy may seem to be as little as 2-fold that for a conventional, single T-DNA strategy, but we strongly temper such estimates with indications that high co-transformation frequencies may be associated with a higher incidence of linkage. In this co-transformation study we used a single (Agrobacterium) strain system in which a single binary plasmid contained either two or three T-DNA regions, each with a selectable marker. This arrangement could reveal that 'read-through' events within the Agrobacterium cells, resulting in the co-transfer of adjacent T-DNA regions as a single linked unit, accounted for up to 20% of co-transformed plant lines. Such read-through co-delivery appeared to be more frequent from the 'supervirulent' EHA101 A. tumefaciens strain, compared to the 'ordinary' LBA4404 strain. By using the binary plasmid with three selectable T-DNA regions, we have been able to consider the frequency of co-integration of a third independent T-DNA within a T0 subpopulation of co-transformants. This was found to be higher than expected. These observations were applied to the co-transfer of (unwanted) plasmid backbone sequences and showed that screening against such sequences may add a significant factor in achieving the desired, final genotype.

Characterization of a Mak subgroup Cdc2-like protein kinase from sugar beet (Beta vulgaris L.).

The Mak-type Cdc2-like protein kinases are, a relatively uncharacterized group of proteins. Bvcrk2 encodes a plant Mak-type kinase. Its highest levels of expression occur in the secondary meristems of developing sugar beet storage organs, suggesting a role, in planta, in the regulation of cell division or early cell differentiation.

RS2: a sugar beet gene related to the latex allergen Hev b 5 family.

A novel gene (RS2) has been isolated from a Beta vulgaris (cv. Regina) cDNA library. The expression of this gene was enhanced in the mature storage organ as compared to leaf tissue. The protein encoded by this gene was found to be alanine- and glutamic acid-rich and it resembles members of the latex allergen Hev b 5 family.

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.

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.

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)).

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.

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.

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.

Cloning novel alfalfa cyclin sequences--a RACE-PCR approach.

Cyclins are a complex group of proteins involved in regulation of the eukaryotic cell division cycle via their interaction with cyclin dependent kinases (Cdks). Cyclin gene sequences have been cloned from a number of plant species, including alfalfa, but the diversity of these genes suggests that there are many plant cyclins which have yet to be characterized. A RACE-PCR strategy has been adopted for cloning cyclin gene sequences expressed during direct somatic embryogenesis in alfalfa. RT-PCR with nested degenerate primers was used to amplify the highly conserved "cyclin box" region of a novel A-like cyclin mRNA sequence expressed after induction of somatic embryogenesis. The sequence of this PCR product was used to design primers for 5'- and 3'-RACE protocols. 5'-RACE using a modified SLIC (single strand ligation to single stranded cDNA) procedure revealed considerable sequence heterogeneity in the N-terminal region of the coding sequence with several closely related sequences apparent. Conventional 3'-RACE generated a single cyclin sequence. The complete coding sequence of one member of this A-like cyclin subgroup has been obtained by this RACE strategy and confirmed by PCR amplification and sequencing of alfalfa genomic DNA.

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.

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.

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.