Copurification of osteolytic and transforming growth factor beta activities produced by human lung tumor cells associated with humoral hypercalcemia of malignancy.

The SK-Luci-6 cell line, established from a large-cell anaplastic lung tumor of a patient with humoral hypercalcemia of malignancy (HHM), was investigated to identify osteolytic factors produced that might mediate HHM. Most HHM-associated tumors are thought to produce parathyroid hormone-related proteins or transforming growth factor (TGF) alpha. SK-Luci-6 cells formed s.c. tumors and induced hypercalcemia in athymic nude mice. Serum-free conditioned medium from SK-Luci-6 cultures induced bone resorption in neonatal mouse calvariae in vitro, and also contained TGF-beta activity and mitogenic activity. SK-Luci-6 cell conditioned medium did not displace [125I]epidermal growth factor binding to cell receptors or stimulate cyclic AMP formation in rat osteosarcoma cells, suggesting that the conditioned medium did not contain TGF-alpha or parathyroid hormone-related proteins. The osteolytic, TGF-beta, and mitogenic activities copurified in several chromatographic separations: gel filtration in acid and then in guanidine HCl; ion exchange; and reverse phase. The results suggest that in the HHM-associated SK-Luci-6 tumor, the causative osteolytic factor produced by the tumor cells is not a parathyroid hormone-related protein or TGF-alpha but, rather, may be a TGF-beta.

Primary structure of human skeletal growth factor: homology with human insulin-like growth factor-II.

Human skeletal growth factor (human SGF) extracted from human bone has been purified to homogeneity by hydroxyapatite chromatography and gel filtration under dissociative conditions followed by FPLC heparin-Sepharose affinity chromatography and reverse phase HPLC. Human SGF was homogeneous except that in each preparation about 30% of SGF molecules lacked the N-terminal alanine. 75% of the human SGF sequence has been determined. The amino acid sequences of the N-terminal 20 amino acids and of several tryptic fragments were identical to the corresponding sequences of human insulin-like growth factor-II (IGF-II) purified from serum. However, since the C-peptide (variable region) of human SGF has not yet been sequenced, we cannot conclude that SGF is identical to IGF-II. Comparison of the amino acid sequence of human SGF with that of IGF-II variants that have been described in the literature revealed that human SGF is not one of the known IGF-II variants. IGF-I was also found in human bone extract but was several-fold less abundant than SGF/IGF-II. The relative abundance of SGF/IGF-II and IGF-I in bone corresponded to the relative rates of production of these two mitogens by human bone cells in vitro. Regarding the physiological significance of IGF-II in bone, previous studies on the biological actions of SGF in vitro suggest that this growth factor can have both paracrine and autocrine functions on cells of the osteoblast line. In addition, we have proposed the concept that SGF is a mediator of the coupling of bone formation to bone resorption, an important bone volume regulatory mechanism. In as much as SGF is very similar (if not identical) to IGF-II, it seems likely that these proposed regulatory functions of SGF in bone are attributable to IGF-II.

Isolation and purification of a low-molecular-weight skeletal growth factor from human bones.

A low-molecular-weight potent bone cell mitogen termed human skeletal growth factor (human SGF) was purified to homogeneity from human bone matrix. Extraction and initial purification steps were done under dissociative conditions to separate human SGF from high-molecular-weight complexes of bone matrix proteins. SGF activity was extracted from human femoral heads by demineralization with 10% EDTA in the presence of 4 M guanidine-HCl and proteinase inhibitors and was purified by hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was homogeneous by HPLC reverse-phase chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of human SGF purified under dissociative conditions was 11,000. Human SGF stimulated bone cell proliferation ([3H]thymidine incorporation and cell number) at picomolar concentrations, with half maximum activity at 2-3 ng/ml (180-270 pM). Human SGF constitutes 0.00024% of organic bone matrix by weight.

Characterization and Solubilization of Kaurenoic Acid Hydroxylase from Gibberella fujikuroi.

A key step in gibberellin biosynthesis is the conversion of ent-kaurenoic acid to ent-7[alpha]-hydroxykaurenoic acid, mediated by the enzyme kaurenoic acid hydroxylase. A cell-free system obtained from Gibberella fujikuroi (Saw.) Wr. was used to characterize kaurenoic acid hydroxylase activity. Microsomal preparations from disrupted fungal cells, in the presence of O2 and NADPH, converted [17-14C]ent-kaurenoic acid to oxidation products that were separated by high-performance liquid chromatography and identified as ent-7[alpha]-hydroxykaurenoic acid and gibberellin A14 by combined gas chromatography-mass spectrometry. Flavin adenine dinucleotide and the chloride salts of several monovalent cations stimulated the conversion of ent-kaurenoic acid to these products, whereas CO and a number of known inhibitors of cytochrome P-450-dependent reactions, including paclobutrazol, tetcyclacis, BAS 111.W, flurprimidol, triarimol, metyrapone, and 1-phenylimida-zole, significantly reduced kaurenoic acid hydroxylase activity. Kaurenoic acid hydroxylase was solubilized from fungal microsomes by treatment with 1 M KCl. The properties of the enzyme noted above suggest that kaurenoic acid hydroxylase from G. fujikuroi is a cytochrome P-450-dependent monooxygenase.

Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin.

Human bone matrix is known to contain a battery of polypeptide growth factors. Since dentin is a mineralized tissue similar to bone in composition and perhaps in formation, human dentin was assayed for the presence of similar growth factors. Root dentin proteins were extracted by demineralization in 4 M guanidine hydrochloride (Gu) and 30 mM Tris (pH 7.4) containing 20% EDTA and proteinase inhibitors. Gu-EDTA extracts were desalted and used for the following assays: (1) bone cell proliferation in chick calvarial cell mitogenic assay using the incorporation of [3H]thymidine into TCA-insoluble material; (2) osteocalcin by radioimmunoassay (RIA); (3) insulin-like growth factor I (IGF-I) by RIA; (4) skeletal growth factor/insulinlike growth factor II (SGF/IGF-II) by radioreceptor assay; and (5) transforming growth factor beta (TGF-beta) by bioassay. Gu-EDTA extracts stimulated bone cell proliferation. At 10 micrograms/ml, dentin proteins increased the incorporation of [3H]thymidine by calvarial cells to 320% of that by BSA-treated control cells. Consistent with the presence of mitogenic activity, growth factors were found in dentin in the following concentrations (ng/micrograms Gu-EDTA protein): (1) IGF-I, 0.06; (2) SGF/IGF-II, 0.52; and (3) TGF-beta, 0.017. All three growth factors were present in concentrations lower than that found in human bone. Osteocalcin was detected at a concentration of 3.0 mg/g Gu-EDTA protein, also much lower than that in bone.

Heterogeneity of latent transforming growth factor-beta isolated from bone matrix proteins.

Transforming growth factor-beta (TGF beta) is a family of 25-kDa peptides that appear to be important regulators of cell proliferation, differentiation, and differentiated function in many tissues. TGF beta is present in platelets and serum and is released by cultured cells as several distinct large mol wt complexes of TGF beta with other proteins. These complexes are biologically inactive and are generically called latent TGF beta (L-TGF beta). Large quantities of TGF beta are present in bone matrix. This study was undertaken to determine whether the TGF beta in bone matrix was present as a free 25-kDa peptide or a large mol wt L-TGF beta complex. TGF beta activity was determined by inhibition of [3H]methylthymidine incorporation in mink lung epithelial cells. The specificity of inhibition was determined by treating fractions with a polyclonal rabbit antiporcine TGF beta-blocking antibody before assay. Latency was examined by assaying untreated and acid-treated fractions for TGF beta activity. Acid treatment of EDTA extracts of the bovine bone matrix proteins increased TGF beta activity from a mean of 0.8 pg/microgram protein to 56 pg/micrograms. Under native conditions L-TGF beta eluted from S400 between the 600-400 kDa mol wt standards. No activity eluted in the fractions with authentic 25-kDa TGF beta. Eighty-five percent of the L-TGF beta bound to lentil lectin, and this separated into four discrete L-TGF beta peaks (I-IV) at 0.22, 0.25, 0.35, and 0.42 M NaCl with Mono-Q anion exchange chromatography. Mono-Q pools II and III were reseparated by molecular size on Superose-12 under native and dissociative conditions. Under native conditions TGF beta activity was latent and eluted in the large mol wt fractions. No 25-kDa TGF beta was present. With dissociating conditions (4 M GuHCl) all TGF beta activity eluted in the small mol wt fractions identical to the elution position of authentic 25-kDa TGF beta. The active fractions from the dissociative separation of Mono-Q pool III were separated by C4 reverse phase HPLC. There were three discrete peaks of TGF beta activity corresponding to TGF beta 1, TGF beta 2, and an unidentified form of TGF. Maximum activation of L-TGF beta in each Mono-Q peak occurred at pH 3-3.5. There was partial activation at pH 4.5, but no additional activation at pH 1.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Beta 2-microglobulin is not a bone cell mitogen.

During the purification of skeletal growth factor/insulin-like growth factor-II and transforming growth factor-beta (TGF beta) from EDTA extracts of bovine bone matrix significant mitogenic activity for cultured osteoblast (Ob)-like cells eluted in fractions that contained a nearly homogeneous peptide with a mol wt of about 14,000. This peptide has been purified to apparent homogeneity and identified as bovine beta 2-microglobulin (beta 2M) by amino-terminal amino acid sequencing. During the final purification of beta 2M by CN reverse phase HPLC the mitogenic activity for bone cells separated from the beta 2M protein peak. In spite of this the apparently homogeneous beta 2M preparation retained some mitogenic activity. The ED50 of the bone-derived beta 2M (4,890 +/- 462 ng/ml) was several orders of magnitude (2 x 10(3) to 1.4 x 10(5) times) greater than the ED50 of simultaneously assayed purified growth factors and was no different from the ED50 of the crude EDTA matrix extract (3,350 +/- 890 ng/ml). The beta 2M accounted for less than 0.002% of the total mitogenic activity for Ob-like cells present in the extracted matrix proteins. The following lines of evidence suggest that the mitogenic activity of bone matrix-derived beta 2M (BMD-beta 2M) is due to contamination of the BMD-beta 2M with TGF beta rather than an intrinsic property of beta 2M: 1) the coelution of TGF beta through four successive purification procedures and purification of TGF beta from adjoining fractions from C4 reverse phase HPLC; 2) the abolition of biological activity of BMD-beta 2M and TGF beta with reducing agents; 3) the lack of additive stimulation of [3H] methylthymidine incorporation into bone cells when beta 2M was added to maximally active concentrations of purified TGF beta; 4) the reduction of mitogenic activity when the BMD-beta 2M was incubated with anti-TGF beta; and 5) the inhibition of mink lung epithelial proliferation by the beta 2M preparation. Based on these findings we conclude that although beta 2M is present in bovine bone matrix extracts, it is not a mitogen for Ob-like cells.

Reduced cell-cell communication in experimentally induced autoimmune thyroid disease.

We have recently described a spontaneous murine model of autoimmune thyroid disease. The disorder was in part characterized by reduced thyroid epithelial cell-cell communication that was associated with abnormalities in three major connexins. To compare whether this finding was a common secondary occurrence in autoimmune thyroid disease, or unique to the spontaneous development in the MRL mice, we induced thyroiditis in Lewis rats. Immunization with thyroid extract and thyroglobulin resulted in extensive lymphocytic infiltration and increased expression of major histocompatibility gene complex (MHC) class II surface antigen in the diseased thyroid. Both experimental and control rat thyroid tissues produced gap junction proteins connexin 43, connexin 32, and connexin 26. The connexins in nondiseased tissue was located in the plasma membrane at points of cell-cell contact and labeled as discrete arrays of punctate fluorescence. The quantity of all three connexins were reduced in the diseased thyroid tissue. More importantly, the connexin proteins were not distributed as gap junctions at contacting cell interfaces. Both nondiseased and diseased thyroid tissue expressed messenger RNA (mRNA) for the three connexins, but the diseased tissue had reduced levels of mRNA for connexin 43 (45%), and to a lesser extent, connexin 26 (25%) and connexin 32 (20%). The reduced connexin mRNA, protein, and lack of assembled gap junctions measured in the diseased tissue were obtained under conditions where the infiltrating cells and their potent cytokine products were continuously present. To determine if this difference persisted when these inflammatory components were absent, primary cultures of thyroid cells from control and experimental rats were established and connexin localization experiments repeated. The diseased thyroid cells, like the diseased tissue, lacked plasma membrane associated connexin protein. The lack of gap junction assembly in the thyrocytes cultured from the diseased tissue was accompanied by a loss of functional coupling. Collectively, the data document that autoimmune diseased thyroid tissue from both the spontaneous mouse and induced rat models have reduced plasma membrane assembled gap junctions and deficient intercellular communication as determined by the inability to transfer lucifer yellow dye to contiguous cells. Nondiseased cultured thyrocyte monolayers and follicles transferred dye to second and third order neighboring cells in 80 and 95% of trials, respectively. In contrast, only 5-10% of the diseased thyrocytes transferred microinjected dye, and in these cases the transit was limited to primary contacting cells. Culturing removed inequities introduced by the infiltrating cells and their products. However, the established cultures of diseased thyroid cells retained their communication deficiency. This suggests that the loss of communication may be a common abnormality in autoimmune disease, and furthermore, this uncoupling could contribute to the loss of coordinated hormonal regulation (hypothyroidism) in the diseased thyroid gland in the absence of thyroid cell destruction.

Characterization of mitogenic activities extracted from bovine bone matrix.

The mitogenic activity in the unfractionated mixture of proteins released from adult bovine bone matrix during demineralization with ethylenediaminetetraacetate (EDTA) has been examined. Bovine bone extract (BBE) from 1 to 25 micrograms protein per ml stimulated proliferation of chick embryo calvaria bone cells, newborn mouse bone cells, and osteoblastlike cell lines MMB-1 and ROS 17/2.8. BBE also stimulated DNA synthesis in cells from chick embryo cartilage, skin and skeletal muscle tissues and fibroblastlike BALB/c 3T3 and NRK cells. BBE contained beta transforming growth factor (TGF) activity (NRK cell colony formation in soft agar in the presence of epidermal growth factor EGF). The cell specificity results suggest that BBE contains more than one growth factor, including a beta TGF and a factor that is not specific for bone cells, and all of the bone derived growth factor activities that have been described previously, including SGF, are apparently present in BBE. Maximal stimulation of chick embryo calvarial cell DNA synthesis by BBE was equal to or exceeded maximal stimulation by nonosseous growth factors that have been reported to stimulate DNA synthesis in bone organ cultures (EGF, fibroblast growth factor, platelet-derived growth factor, insulinlike growth factor I, and multiplication stimulating activity). Combinations of BBE with maximally stimulatory concentrations of each growth factor stimulated DNA synthesis to a greater magnitude than did each growth factor alone. These results suggest that combinations of bone derived and systemic factors can coordinately stimulate bone cell proliferation.

In vitro fertilisation. A review of drug therapy and clinical management.

Since the first in vitro fertilisation (IVF) pregnancy was delivered in 1978, this procedure has resulted in thousands of pregnancies and opened a vast new frontier of research and treatment for the infertile couple. Pregnancy rates with IVF improve as the number of high quality embryos available for transfer increases; therefore, ovarian stimulation agents to produce multiple oocysts for IVF are advantageous. Clomifene (clomiphene citrate), human menopausal gonadotrophin (hMG; menotropins), and subsequent generations of products are commonly used as stimulation agents. In conjunction with the stimulation agents, gonadotrophin-releasing hormone (GnRH) agonists and human chorionic gonadotrophin (hCG) serve as adjuvants for successful control of all events in the induction process. Clomifene, an estrogen agonists/antagonist, occupies the estrogen receptor for a longer period of time than estrogen (weeks versus hours). Because this signal is interpreted as low estrogen, GnRH is released, which produces a rise in circulating levels of follicle-stimulating hormone (FSH) and luteinising hormone (LH) and subsequent ovarian follicular development. Menotropins is collected by passing urine from menopausal donors over a Sepharose column, followed by removal of high molecular weight impurities by chromatography. The mixture of FSH and LH is biologically standardised. This product stimulates multiple ovarian follicular development. Urofollitrophin is produced using antibodies to hCG anchored to a separation column. LH then can be excluded from the eluate by binding to the hCG antibodies (LH immunoaffinity column). Highly purified FSH is obtained by passing menopausal urine over a column with monoclonal antibodies to FSH. The isolated FSH is then eluted from the column by a highly basic solution and crystallised. This product delivers FSH at a 90% purity and can be administered subcutaneously rather than intramuscularly. Dosage is standardised on a mg/kg basis. Recombinant human FSH is completely free of LH and offers the advantages of better batch consistency, greater purity, and absence of any human contaminants. It may be given both subcutaneously and intravenously. Genetically engineered FSH combines portions of the native protein with another protein (hCG) which enhances its potency and extends the half-life compared with wild-type FSH. Short, medium and ultra-long activity analogues of genetically engineered FSH may be used to tailor stimulation protocols in various clinical situations. Growth hormone is an adjuvant to ovarian stimulation which results in a decreased number of ampoules of menotropins being required to achieve ovulation in poor responders. Ovulation triggers include both hCG and GnRH agonists. Progesterone supplementation is generally used in the luteal phase of the IVF cycle and is administered by intramuscular injection or vaginal suppository. It appears that conscious sedation with midazolam, pethidine (meperidine) and fentanyl is nontoxic for oocyte recovery. If full anaesthesia is required for gamete intrafallopian tube transfer (GIFT) or zygote intrafallopian tube transfer (ZIFT), balanced anaesthesia with nitrous oxide and an opioid appears to be the most appealing option. Appropriate information on the clinical use of the drugs used in IVF greatly reduces patient stress associated with the complex multidrug regimens associated with the procedure.

Content of continuing education courses in obstetrics and gynecology.

The objective of this study was to determine the emphases of continuing medical education courses in obstetrics and gynecology. Eighty programs for obstetricians and gynecologists were evaluated for location, accreditation, sponsorship, cost, faculty composition, teaching methods, and curriculum content. The programs' curricula were compared through classification of courses and individual topic presentations as emphasizing primary care, maternal-fetal medicine, gynecologic oncology, reproductive endocrinology and infertility, introduction of new technology, or general review. All 80 programs were in acceptable locations and had valid accreditation. Universities, hospitals, industrial corporations, and professional organizations were among the programs' sponsors. Tuition costs per credit hour averaged $46.48 and ranged from $4.64-238.09; programs emphasizing new technology cost the most. Ninety percent of the programs' 880 faculty had medical school affiliations. Of the teaching methods, lectures accounted for 77.9%, laboratory instruction 9.1%, panel discussions 7.0%, workshops 4.9%, and other methods 1.0%. The total 1592 credit hours consisted of 563 in primary care, 181 in maternal-fetal medicine, 99.5 in gynecologic oncology, 122.5 in reproductive endocrinology and infertility, and 626 in introduction of new technology. Although these programs were diversified, many emphasized the introduction of new technology, which does not enhance the designation of the specialty as "primary care."

Abnormal uterine bleeding.

Physicians who care for female patients cannot avoid the frequent complaint of abnormal uterine bleeding. Knowledge of the disorders that cause this problem can prevent serious consequences in many patients and improve the quality of life for many others. The availability of noninvasive and minimally invasive diagnostic studies and minimally invasive surgical treatment has revolutionized management of abnormal uterine bleeding. Similar to any other disorder, the extent to which a physician manages abnormal uterine bleeding depends on his or her own level of comfort. When limitations of either diagnostic or therapeutic capability are encountered, consultation and referral should be used to the best interest of patients.

Quantitation of growth factors in ossein-mineral-compound.

Study was undertaken to identify polypeptide factors in the commercially available ossein-mineral-compound and to see if they are present in a biologically relevant quantity. Using the guanidine-EDTA extraction, 35.7 +/- 0.1 mg proteins were obtained from 1 g of the ossein-mineral-compound. At concentration 1 micrograms/ml, guanidine-EDTA-extractable proteins stimulated the incorporation of thymidine into DNA by human bone cells to 581 +/- 122% (p less than 0.001) of that by bovine serum albumin-treated control cells, decreasing thereafter. Similarly, it stimulated the activity of alkaline phosphatase in the human bone cells. Growth factors IGF-I, IGF-II, and TGF-beta were identified in the ossein-mineral-compound. This leads to speculation regarding possible role of growth factors in explaining the beneficial effects of the compound in retarding bone loss in patients with osteoporosis.

Osteoporosis in children and adolescent girls: case report of idiopathic juvenile osteoporosis and review of the literature.

UNLABELLED: The diagnosis and treatment of osteoporosis is an important aspect of gynecologic training and practice. Idiopathic juvenile osteoporosis (IJO) is a rare disease of children and adolescents that resolves after the onset of puberty. A case report is presented and current methods of diagnosis and treatment of IJO are discussed as well as the differential diagnosis. A MEDLINE search was performed of the following terms: idiopathic juvenile osteoporosis, pediatric osteoporosis, adolescent osteoporosis, bisphosphonates pediatric adolescent, and pregnancy osteoporosis, and references from bibliographies of selected papers were used as well. All papers in English, French, and German are considered in this review. There were 114 papers selected as relevant to the topic. Data relevant to the diagnosis, pathogenesis, methods of imaging, laboratory evaluation, differential diagnosis, and treatment of IJO are presented. IJO is a diagnosis of exclusion in the pediatric and adolescent patient with osteoporosis. Although bone density gradually improves after the onset of puberty, treatment of currently affected children and adolescents involves activity restriction, calcium, vitamin D, and bisphosphonate therapy. Future reproductive concerns are discussed and areas requiring additional study are reviewed. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians LEARNING OBJECTIVES: After completion of this article, the reader will be able to describe the condition idiopathic juvenile osteoporosis, compare the clinical features of this condition to other similar conditions, outline the diagnostic workup of a child with this condition, and list the potential therapeutic options for a patient with idiopathic juvenile osteoporosis.

Dendryphion penicillatum and Pleospora papaveracea, Destructive Seedborne Pathogens and Potential Mycoherbicides for Papaver somniferum.

ABSTRACT Dendryphion penicillatum and Pleospora papaveracea were isolated from blighted Papaver somniferum and Papaver bracteatum plants grown in growth chambers and the field in Beltsville, MD. The etiology of the diseases was determined, and the fungi are being investigated as potential mycoherbicides to control the narcotic opium poppy plant. P. papaveracea is known to be a highly destructive seedborne pathogen of Papaver somniferum, causing seedling blight, leaf blight, crown rot, and capsule rot. Single conidia and ascospores were isolated and cultures established from naturally infested seed and diseased foliage and pods of opium poppy from Iran, Colombia, Venezuela, Sweden, India, and the United States (Maryland and Washington). Mycelia and conidia of P. papaveracea and D. penicillatum produced on necrotic leaf tissues appear morphologically similar, and the fungi were previously considered to be anamorph and teleomorph. However, no anamorph/teleomorph connection could be established, and the fungi appear to be distinct taxa. P. papaveracea produced conidia, mature pseudothecia, and chlamydospores in vitro and on infected stems. D. penicillatum produced conidia, microsclerotia, and macronematous conidiophores. Although both fungi were pathogenic to three poppy cultivars, conidial inoculum from P. papaveracea cultures was more virulent than conidial inoculum from D. penicillatum. Eight-week-old plants became necrotic and died 8 days after inoculation with a conidial suspension of P. papaveracea at 2 x 10(5) spores per ml. Disease severity was significantly enhanced by inoculum formulations that contained corn oil, by higher conidial inoculum concentrations, and by increased wetness periods. Symptoms on plants inoculated with either pathogen included leaf and stem necrosis, stem girdling, stunting, necrotic leaf spots, and foliar and pod blight. Inoculated seedlings exhibited wire stem, damping-off, and root rot. Conidia, and less frequently pseudothecia, of P. papaveracea and conidia of D. penicillatum were produced abundantly on inoculated, necrotic foliage, pods, and seedlings. Cultures from conidia or ascospores reisolated from these tissues consistently produced fungi whose morphologies were typical of the fungus from which the inoculum was derived.

Evaluation of Infection Processes and Resulting Disease Caused by Dendryphion penicillatum and Pleospora papaveracea on Papaver somniferum.

ABSTRACT Two pathogenic fungi of opium poppy, Pleospora papaveracea and Dendryphion penicillatum, were isolated from field material in Beltsville, MD. The processes of infection by these two fungi were studied to determine the optimal environmental conditions for infection. Both fungi formed appressoria capable of penetrating directly through the plant epidermal layer. Of the two fungi, P. papaveracea was more aggressive, causing more rapid necrosis. Appressorial formation by P. papaveracea occurred as early as 4 h after application of a conidial suspension to poppy leaves. P. papaveracea formed more appressoria than did D. penicillatum, especially at cool temperatures (7 to 13 degrees C). In greenhouse studies, P. papaveracea caused more damage to opium poppy than did D. penicillatum when applied in 10% unrefined corn oil. In the field, P. papaveracea was more consistent in its effects on opium poppy from a local seed source designated Indian Grocery. P. papaveracea caused higher disease ratings, more stem lesions, and equal or greater yield losses than did D. penicillatum on Indian Grocery. The late-maturing opium poppy variety White Cloud was severely damaged by disease, regardless of formulation or fungal treatment. P. papaveracea was the predominant fungus isolated from poppy seed capsules and the only fungus reisolated from the field the following year. These studies provide a better understanding of the infection process and the differences between these two pathogenic fungi and will be beneficial for the development of the fungi as biological control agents.

Determining whether transgenic and endogenous plant DNA and transgenic protein are detectable in muscle from swine fed Roundup Ready soybean meal.

Questions regarding the digestive fate of DNA and protein from transgenic feed have been raised in regard to human consumption and commercial trade of animal products (e.g., meat, milk, and eggs) from farm animals fed transgenic crops. Using highly sensitive, well-characterized analytical methods, pork loin samples were analyzed for the presence of fragments of transgenic and endogenous plant DNA and transgenic protein from animals fed meal prepared from conventional or glyphosate-tolerant Roundup Ready (RR) soybeans. Pigs were fed diets containing 24, 19, and 14% RR or conventional soybean meal during grower, early-finisher, and late-finisher phases of growth, respectively, and longissimus muscle samples were collected (12 per treatment) after slaughter. Total DNA was extracted from the samples and analyzed by PCR, followed by Southern blot hybridization for the presence of a 272-bp fragment of the cp4 epsps coding region (encoding the synthetic enzyme 5-enolpyruvylshikimate-3-phosphate synthase derived from Agrobacterium sp. strain CP4) and a 198-bp fragment of the endogenous soybean gene le1 (encoding soy lectin). Using 1 microgram of input DNA per reaction, none of the extracted samples was positive for cp4 epsps or le1 at the limit of detection (LOD) for these PCR/Southern blot assays. The LOD for these assays was shown to be approximately one diploid genome equivalent of RR soybean DNA, even in the presence of 10 micrograms of pork genomic DNA. A 185-bp fragment of the porcine preprolactin (prl) gene, used as a positive control, was amplified from all samples showing that the DNA preparations were amenable to PCR amplification. Using a competitive immunoassay with an LOD of approximately 94 ng of CP4 EPSPS protein/g of pork muscle, neither the CP4 EPSPS protein nor the immunoreactive peptide fragments were detected in loin muscle homogenates from pigs fed RR soybean meal. Taken together, these results show that neither small fragments of transgenic DNA nor immunoreactive fragments of transgenic protein are detectable in loin muscle samples from pigs fed a diet containing RR soybean meal.

Attempts to detect transgenic and endogenous plant DNA and transgenic protein in muscle from broilers fed YieldGard Corn Borer Corn.

Questions regarding the digestive fate of DNA and protein from transgenic grain have been raised in regard to human consumption and trade of animal products (e.g., meat, milk, and eggs) from farm animals fed transgenic crops. Using highly sensitive, fully characterized analytical methods, fragments of transgenic and endogenous plant DNA, as well as transgenic protein, were not detected in chicken breast muscle samples from animals fed YieldGard Corn Borer Corn event MON 810 (YG). Total DNA was extracted from breast muscle samples from chickens fed for 42 d with a diet including either 55 to 60% YG grain or 55 to 60% conventional corn grain. DNA preparations were analyzed by PCR followed by Southern blot hybridization for the presence of a 211-bp fragment of the Bacillus thuringiensis (Bt) cry1Ab gene and a 213-bp fragment of the endogenous corn gene sh2 (encoding ADP glucose pyrophosphorylase). By using 1 microg of input DNA per reaction, none of the extracted samples was positive for cry1Ab or sh2 at the limit of detection for these PCR assays. A 396-bp fragment of the chicken ovalbumin (ov) gene, used as a positive control, was amplified from all samples showing that the DNA preparations were amenable to PCR amplification. By using a competitive immunoassay with a limit of detection of approximately 60 ng of CrylAb protein per gram of chicken muscle, neither the CrylAb protein nor immunoreactive peptide fragments were detectable in the breast muscle homogenates from chickens fed YG grain.