Chimeric Mos1 and piggyBac transposases result in site-directed integration.

Genetic transformation systems based on Mos1 and piggyBac transposable elements are used to achieve stable chromosomal integration. However, integration sites are randomly distributed in the genome and transgene expression can be influenced by position effects. We developed a novel technology that utilizes chimeric transposases to direct integration into specific sites on a target DNA molecule. The Gal4 DNA binding domain was fused to the NH(2) terminus of the Mos1 and piggyBac transposases and a target plasmid was created that contained upstream activating sequences (UAS), to which the Gal4 DBD binds with high affinity. The transpositional activity of the Gal4-Mos1 transposase was 12.7-fold higher compared to controls where the Gal4-UAS interaction was absent and 96% of the recovered transposition products were identical, with integration occurring at the same TA site. In a parallel experiment, a Gal4-piggyBac transposase resulted in an 11.6-fold increase in transpositional activity compared to controls, with 67% of the integrations occurring at a single TTAA site. This technology has the potential to minimize nonspecific integration events that may result in insertional mutagenesis and reduced fitness. Site-directed integration will be advantageous to the manipulation of genomes, study of gene function, and for the development of gene therapy techniques.

Prostate cancer gene therapy and the role of radiation.

Even though prostate cancer is detected earlier than in the pre-PSA era, prostate cancer is the second leading cause of cancer mortality in the American male. Prostate cancer therapy is not ideal, especially for high-risk localized and metastatic cancer; therefore, investigators have sought new therapeutic modalities such as angiogenesis inhibitors, inhibitors of the cell signaling pathway, vaccines, and gene therapy. Gene therapy has emerged as potential therapy for both localized and systemic prostate cancer. Gene therapy has been shown to work supra-additively with radiation in controlling prostate cancer in vivo. With further technological advances in radiation therapy, gene therapy, and the understanding of prostate cancer biology, gene therapy will potentially have an important role in prostate cancer therapy.

Defining a future role for radiogenic therapy.

The goal of cancer therapy is to eliminate the cancer and/or to arrest further growth while decreasing normal tissue toxicity, i.e. to increase the therapeutic ratio. This review focuses on a group of therapeutics that are either (1) directly stimulated by radiation to produce either directly or indirectly cytotoxic agents (i.e. genes under the control of a radiation inducible promoter that produce a cytotoxic protein or an enzyme that converts a prodrug to an active form, respectively); (2) auger-electron emitting radiolabelled oligonucleotides, antibodies, nucleotide analogues, or other small molecules that are internalized; (3) radiation inducible genes that produce a ligand or transporter (or the like) which then can be targeted by cytotoxic agents (e.g. radiolabelled substance). We have termed this group of therapeutics radiogenic therapy.

Synthesis and inhibition of human acrosin and trypsin and acute toxicity of aryl 4-guanidinobenzoates.

The aryl 4-guanidinobenzoate, 4'-nitrophenyl 4-guanidinobenzoate (NPGB), is a potent inhibitor of sperm acrosin, an enzyme with an essential function in the fertilization process. NPGB prevents fertilization in a number of animal species and is a good lead compound for the development of contraceptive agents. In order to assess the efficacy of other aryl 4-guanidinobenzoates as acrosin inhibitors, 24 of these compounds were synthesized. Their inhibitory activity toward human acrosin was determined and compared with their activity toward human pancreatic trypsin in order to assess whether inhibitor sensitivity differed between these similar enzymes. Nine of the inhibitors were synthesized from phenols approved by the FDA for therapeutic use. The acute toxicity of these inhibitors in mice was determined and compared to that of nonoxynol-9, the most commonly used active ingredient in today's vaginal contraceptive preparations. All of the compounds proved to be potent inhibitors of human acrosin although 3 orders of magnitude difference were observed between the most and least effective inhibitors. Little specificity was present in regard to their inhibition of acrosin and trypsin. All the aryl 4-guanidinobenzoates synthesized from FDA-approved phenols were less toxic than nonoxynol-9, and it is concluded that these 4-guanidinobenzoates are of interest for further development and testing as nonhormonal contraceptive agents.

Human sperm acrosin. Further studies with the clinical assay and activity in a group of presumably fertile men.

The goals of this study were to determine the effect of the nonionic detergent, Triton X-100, on the recovery of acrosin in the clinical assay (Kennedy et al, 1989), since previous investigations have used higher concentrations for acrosin extraction from spermatozoa; and to establish the minimal acrosin activity in fertile men. The recovered acrosin activity was dependent on the concentration of Triton. A peak in acrosin activity was obtained at 0.01% to 0.02% Triton, the approximate critical micelle concentration (CMC; 0.015%). The bimodal effect of Triton was not due to substrate/buffer alterations, to the degree of acrosomal disruption as assessed by light and transmission electron microscopic examination, or to its effect on the kinetic properties of acrosin, as determined by spectrophotometric analysis of acid-extracted enzyme. However, Triton affected the conversion of proacrosin to acrosin, with peak activation occurring at 0.01% to 0.02% detergent. The acrosin activity of a group of presumably fertile men (as established by the production of offspring under natural conditions) varied from 18 to 42 microIU/10(6) spermatozoa, as assessed by the clinical assay containing 0.01% Triton. Furthermore, men who had initial acrosin values in the low normal range (18 to 25 microIU/10(6) sperm) were observed for 11 months. The acrosin activity of their ejaculates never fell below 17 microIU/10(6) sperm. Thus, it can be tentatively assumed that the minimal levels of acrosin for naturally fertile men are 17 to 18 microIU acrosin/10(6) sperm in this assay.

A simple, clinical assay to evaluate the acrosin activity of human spermatozoa.

Acrosin, a sperm-specific acrosomal proteinase, has an essential role in the fertilization process. Low levels of acrosin appear to be associated with subfertility and infertility, and the acrosin activity of spermatozoa may potentially be a useful indicator of semen quality. The standard acrosin tests employed by research laboratories are too complicated and/or time consuming for clinical use; therefore, a simple assay has been developed to assess total acrosin activity (acrosin and activatable proacrosin). To perform the test, liquefied semen is centrifuged over Ficoll, the washed sperm pellet is suspended in a detergent (Triton X-100)-substrate (N-alpha-benzoyl-DL-arginine p-nitroanilide) buffer, pH. 8.0, and the amidase activity is determined spectrophotometrically after a 3-hour incubation period. Amidase activity can be inhibited with benzamidine, indicating that the activity is primarily or entirely due to acrosin. The absence of detergent in the incubation medium results in greatly reduced activity. The assay is repeatable, linear with increasing sperm concentration, sensitive to a lower limit of 2 x 10(6) spermatozoa, and the results correspond to those obtained with a standard acrosin extraction and assay technique. Storage of ejaculates at 3 to 6 C or at 22 to 24 C for 24 hours does not affect the acrosin activity significantly but much higher temperatures can cause a loss of activity. Freezing ejaculates results in a large decrease in sperm acrosin activity. Leukocytes show minimal activity in the assay. Sperm populations prepared by a swim-up procedure average approximately a 2-fold higher acrosin activity than the original ejaculates. Preliminary experiments indicate that the average sperm acrosin activity of ejaculates whose spermatozoa successfully fertilize human eggs in vitro is significantly higher than those that do not fertilize eggs.

Vaginal contraceptive activity of aryl 4-guanidinobenzoates (acrosin inhibitors) in rabbits.

Nine aryl 4-guanidinobenzoates were synthesized as inhibitors of the sperm enzyme acrosin. These esters were prepared from 4-guanidinobenzoic acid and a number of phenols which had been approved by the FDA for clinical use. The vaginal contraceptive activity of the inhibitors was evaluated in the rabbit at nonspermicidal concentrations (0.1 mg/ml). All the inhibitors except the 2'-carboxamidophenyl and the 2'-isopropyl-5'-methylphenyl 4-guanidinobenzoates caused significant reductions in fertilization compared to the controls. Several of the aryl 4-guanidinobenzoates appeared to be particularly effective. Nonoxynol-9, under the same conditions but at 10- and 100-fold higher concentrations, also showed an antifertility effect. However, even at these increased dose levels, the contraceptive efficacy of nonoxynol-9 was no higher than that of most of the inhibitors and was less consistent than that of the most active aryl 4-guanidinobenzoates. The relatively high in vivo antifertility activity exhibited by several of the aryl 4-guanidinobenzoates encourages their further evaluation as vaginal contraceptive agents.

Development of the polyurethane sponge as a delivery system for aryl 4-guanidinobenzoates.

The Today Contraceptive Sponge was evaluated as a vehicle for the delivery of aryl 4-guanidinobenzoates (AGs) which are highly active sperm acrosin inhibitors. Studies in animals have shown that several AGs are more potent vaginal contraceptives and less irritating to the vagina than nonoxynol-9 (N-9), the most frequently used active ingredient in commercial vaginal contraceptive formulations. Neither nonoxynol-9 nor the material that could be solubilized from the sponge matrix altered the enzyme-inhibitory activity of 4'-acetamidophenyl 4-guanidinobenzoate HCl (AGB), 4'-carboxyphenyl 4-guanidinobenzoate HCl (EGB) or 4'-carbomethoxyphenyl 4-guanidinobenzoate HCl (MSGB). Besides being acrosin inhibitors, all three AGs exhibited antimotility activity towards human spermatozoa, EGB being as potent as N-9. The antimotility effects of the AGs and N-9 were additive. For subsequent studies, AGB was used as the model compound. Manufacture of the AGB-containing sponges did not affect the chemical structure of AGB. Good release rates of AGB were obtained from the sponges over a 7-day period. The release rates were 20-50% higher when the sponges also contained N-9. These results indicate that certain AGs exert a dual contraceptive action on spermatozoa by inhibiting both the sperm enzyme acrosin and sperm motility. Furthermore, the polyurethane sponge appears to be a convenient and satisfactory long-term delivery system for the AGs. A mixture of N-9 and AG can be used clinically because these compounds have no adverse effects on each other.

Metastatic meningioma to the lung with multiple pleural metastases.

Meningiomas with both malignant clinical behavior and cytology are rare. Meningiomas comprise approximately 15% of the primary brain tumors. The majority are benign; less than 1% metastasize, most commonly to the lung (61%) followed by liver, lymph node, and bone. Approximately 130 cases of extracranial metastatic meningiomas have been described. Only 13% had more than three metastases, with few cases reported with extensive pleural involvement. We report an interesting case of a malignant meningioma that invaded through the skull in the frontal sinus that later metastasized to the left lung with multiple pulmonary and pleural nodules.

Urinary bladder calculus formation on sutures in rabbits, cats and dogs.

Cystotomies were performed upon 152 animals with various suture materials. The bladders were examined for the presence of concretions at intervals ranging from three to 120 days. Calculi were found with regularity in rabbits but not in cats or dogs, irrespective of the suture material used. Urinary calculi persisting later than seven days postoperatively were seen only in those rabbit bladders repaired with nonabsorbable sutures. Despite major differences in the geometry and chemical composition of the suture, the two absorbable sutures polyglycolic acid and catgut showed an equivalent incidence of early, reversible calculus formation in the rabbit urinary bladder. The property of suture materials that contributes most to the formation of persistent urinary calculi is nonabsorbability. Surface characteristics and cross sectional geometry appear to play little, or no, role in the calculus formation in the bladders of animals.

Intracranial fibrosarcoma arising 5 years after chemotherapy alone for glioblastoma multiforme in a child.

We present a child diagnosed with glioblastoma multiforme during infancy and successfully treated with the 'eight-in-one' chemotherapy regimen, who developed an intracranial fibrosarcoma 5 years later. After resection of the fibrosarcoma, she received cranial radiation therapy and high dose chemotherapy with bone marrow transplant. She remains alive and recurrence-free 7 years following the diagnosis of her second intracranial malignancy.

Effect of aryl 4-guanidinobenzoates on the acrosin activity of human spermatozoa.

Certain aryl 4-guanidinobenzoates (AGs; inhibitors of proteinases, including the sperm enzyme acrosin) have been shown to be more potent vaginal contraceptives in rabbits and less toxic than nonoxynol-9, the active ingredient of most marketed vaginal contraceptive formulations. To determine if these AGs can contact sperm and inhibit acrosin when mixed with the entire human ejaculate for a short period of time (roughly imitating clinical conditions), the inhibitors were added to semen at various concentrations for 2 min, after which the seminal plasma and unbound inhibitor were removed from the sperm by Ficoll centrifugation. Subsequently, the total arginine amidolytic activity of the spermatozoa was determined spectrophotometrically after a combined treatment that resulted in extraction, proacrosin activation, and reaction with substrate. Dose-response curves were prepared. All AGs studied were effective inhibitors of the amidolytic activity under these conditions, with ED50 values (the dose levels at which half of the acrosin associated with 10(6) sperm is inhibited) ranging from 10(-5) to 10(-7) M. To determine the effect on the proteolytic activity of individual spermatozoa, the experiment was repeated with 4'-acetamidophenyl 4-guanidinobenzoate (AGB), and the protease released from the sperm was measured by the gelatin-plate assay. The inhibition results were similar to those obtained by extraction of the spermatozoa and measurement of amidolytic activity. Thus, when mixed with the human ejaculate, AGs interact rapidly with spermatozoa to inhibit both their arginine amidolytic and proteolytic activity (probably due primarily or only to inhibition of acrosin) and remain bound even after removal of the seminal plasma. These data encourage further study of the compounds for contraceptive purposes.