Production of offspring after sperm chromosome screening: an experiment using the mouse model.

STUDY QUESTION: Is it possible to produce offspring after sperm chromosome screening? SUMMARY ANSWER: It is possible to produce zygotes after examining the genome of individual spermatozoa prior to embryo production. WHAT IS KNOWN ALREADY: Chromosomal aberrations in gametes are a major cause of pregnancy loss in women treated with assisted reproductive technology. However, to our knowledge, there are no reports on the successful genomic screening of spermatozoa, although some attempts have been made using the mouse as a model. STUDY DESIGN: To prevent the transmission of chromosomal aberrations from fathers to offspring, we performed sperm chromosome screening (SCS) prior to fertilization using the mouse as a model. The production of offspring after SCS consists of (i) replication of the sperm chromosomes, (ii) analysis of one copy of the replicated sperm chromosomes, (iii) construction of a zygote using another set of chromosomes and (iv) production of a transferable embryo. MATERIALS, SETTING, METHODS: A single spermatozoon of a male mouse, with or without a Robertsonian translocation, was injected into an enucleated oocyte to allow the replication of sperm chromosomes. One of the sister blastomeres of a haploid androgenic 2-cell embryo was used for chromosome analysis. The other blastomere was fused with an unfertilized oocyte, activated and allowed to develop to a blastocyst before transfer to a surrogate mother. MAIN RESULTS AND ROLE OF CHANCE: With high efficiency, we were able to analyze sperm chromosomes in a blastomere from the androgenic 2-cell embryos and culture zygotes, with and without aberrant chromosomes, to the blastocyst stage before embryo transfer. The karyotypes of the offspring faithfully reflected those of the blastomeres used for SCS. LIMITATIONS, REASONS FOR CAUTION: This study was conducted using a mouse model; whether or not the method is applicable to humans is not known. WIDER IMPLICATIONS OF THE FINDINGS: This study has shown that it is possible to produce zygotes without any paternally inherited aberrations by examining the genome of individual spermatozoa prior to embryo production.

Osteomyelitis due to trimethoprim/sulfamethoxazole-resistant Edwardsiella tarda infection in a patient with X-linked chronic granulomatous disease.

Edwardsiella tarda, a catalase-positive bacillus widely distributed throughout nature, is generally susceptible to trimethoprim/sulfamethoxazole. We describe osteomyelitis due to trimethoprim/sulfamethoxazole-resistant E. tarda in a patient with chronic granulomatous disease (CGD). Once E. tarda acquires antibiotic resistance, infected CGD patients may develop severe infections with unforeseeable consequences.

Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes.

BACKGROUND: Although mouse spermatozoa can be freeze-dried without losing their reproductive capacity, the technique needs further improvements to reduce the incidence of chromosomal damage to spermatozoa. Effects of freeze-drying on human spermatozoa are unknown. METHODS: Mouse spermatozoa were suspended in a Tris-buffered EGTA solution briefly (10 min at 37 degrees C) or for 1-7 days at 4 degrees C before freeze-drying. Freeze-dried spermatozoa were maintained for up to 1 year at 4 degrees C before injection. Sperm chromosomes were examined during the first mitosis (cleavage) of zygotes. The ability of sperm to support embryo development was assessed by examining mid-gestation fetuses (Day 14) after transfer of 2-cell embryos to surrogate mothers. Chromosome integrity of freeze-dried human spermatozoa was examined by injecting individual spermatozoa into mouse oocytes which were previously enucleated. RESULTS: When mouse spermatozoa were freeze-dried immediately after suspension in Tris-buffered EGTA solution, only c.40% had normal chromosomes. When the mouse spermatozoa were kept in the same solution for 3-7 days before freeze-drying, 85-95% had normal chromosomes and they were able to support embryo development better than those which were in the solution briefly (P < 0.05). Freeze-dried human spermatozoa well maintained their chromosomes regardless of the duration of pre-freeze-drying incubation of spermatozoa in the Tris-buffered EGTA solution. CONCLUSIONS: Prior incubation of mouse spermatozoa in Tris-buffered EGTA solution for several days makes sperm chromosomes more resistant to freeze-drying. As the consequence, spermatozoa freeze-dried this way support embryo development better than those exposed to Tris-buffered EGTA solution only briefly. Freeze-dried human spermatozoa well maintained their chromosomes without pre-freeze-drying incubation in Tris-buffered EGTA solution.

Extracellular glutamate: on-line monitoring using microdialysis coupled to enzyme-amperometric analysis.

An enzyme-amperometric detector cell is described for flow analysis of glutamate in dialysate emerging from an implanted microdialysis probe. Its small size allows it to be placed within a few centimetres of the animal preparation, reducing the delay for data acquisition to around 2 min. The selectivity is provided by glutamate oxidase, immobilised with glutaraldehyde on surfaces adjacent to the 3-electrode system. A film of 1,2-diaminobenzene, electropolymerized on the platinum working electrode, eliminates interference from ascorbic acid and other endogenous electroactive compounds. The high sensitivity (< 0.5 mumol/l) and fast response time of the cell (90% of maximum response in 30 s) make it particularly suitable for investigating conditions that produce rapid changes in brain extracellular glutamate. This is illustrated by monitoring changes in extracellular glutamate subsequent to cardiac arrest, and K(+)-induced local depolarization.

A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates.

We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.

The determination of the extracellular concentration of brain glutamate using quantitative microdialysis.

Quantitative microdialysis with two enzyme-based assays was used to determine the extracellular concentration of glutamate in the striatum of freely moving rats. From the difference between infused and dialysate glutamate a value of 3.0 +/- 0.6 microM for the extracellular glutamate concentration was computed by regression analysis. The in vivo recovery, derived from the slope of the regression line, was 50%.

Expression of lung resistance protein in epithelioid sarcoma in vitro and in vivo.

The incidence of epithelioid sarcoma among patients with malignant soft tissue tumors is small, but the rates of recurrence and metastasis of this type of sarcoma are high. To date, effective chemotherapy for advanced epithelioid sarcoma has not been established and, furthermore, epithelioid sarcoma is known to exhibit multidrug resistance (MDR). The chemosensitivities to anticancer agents of two cell lines established from epithelioid sarcoma were examined in this study. The results showed that the ES-OMC-MN and SFT-8606 cell lines were resistant to vincristine (IC50 1190 nM and 872 nM, respectively) and Adriamycin (IC50 921 nM and 650 nM, respectively), but sensitive to actinomycin D (IC50 < 10 nM). P-glycoprotein (p-Gp) and MDR-associated protein (MRP) were not expressed in these cell lines, but a high expression level of lung resistance protein (LRP) was observed. The original tumor tissues from which the two cell lines were established were also found to be LRP-positive but not to express p-Gp or MRP. Their chemosensitivities to Adriamycin were not significantly altered in the presence of 2.5 microg/ml anti-LRP antibody (LRP-56), but the IC50 of vincristine was much less (IC50 128 nM and 27 nM, respectively) than that for an untreated cell line. It is thus suggested that the vincristine resistance in the two cell lines is LRP-mediated. Since cyclosporin A, known to be a modifier of p-Gp, also induced reversal of vincristine resistance in the ES-OMC-MN and SFT-8606 cell lines (IC50 6.2 nM and 17 nM, respectively), it is suggested that cyclosporin A acts as a modifier of MDR mediated by LRP.

Establishment and characterization of an epithelioid sarcoma cell line with an autocrine response to interleukin-6.

A novel epithelioid sarcoma (ES) cell line, designated as ES-OMC-MN, was established from a 44-year-old woman with recurrence and metastasis of ES. The cells were spindle-shaped or polygonal and were positive for cytokeratin and vimentin on immunohistochemical staining. Electron microscopy revealed desmosome-like structures between the cells. These characteristics were also noted in the original tumor. Southern blot analysis of HindIII digests showed an additional 8.0 kb band and an 8.8 kb band in DNA from the cultured cells and the original tumor as well as the peripheral blood cells of the patient, while only an 8.8 kb band was observed in control human placental DNA. There were no point mutations of N-ras codons 12, 13, and 61, suggesting that the abnormality of N-ras was not due to mutation but to polymorphism. Interleukin-6 (IL-6) was secreted into the culture medium at high levels. Recombinant IL-6 augmented the proliferation of these cells, while cell growth was inhibited by incubation with an anti-IL-6 antibody. The cells also expressed surface IL-6 receptors, indicating that IL-6 acted on this cell line in an autocrine manner. IL-6 and IL-6 receptors were also found in the original tumor. These results demonstrate that ES-OMC-MN cells retained all the morphological and biochemical characteristics of the original tumor and suggest that an autocrine effect of IL-6 may be involved in the development of ES.

Comparative studies of MCL-5 cells and human lymphocytes for detecting indirect-acting clastogens.

The MCL-5 cell line was established from human lymphoblastoid TK+/- cells transfected with cDNAs of human cytochrome P450s (CYP1A2, CYP2A6, CYP2E1, and CYP3A4) and microsomal epoxide hydrolase. The TK+/- cells constitutively express a relatively high level of endogenous CYP1A1. To study metabolic activities to indirect-acting clastogens, MCL-5 cells were treated with four clastogens, i.e. aflatoxin B1 (AFB1), diethylnitrosamine (DEN), cyclophosphamide (CPA), and 7,12-dimethylbenz[a]anthracene (DMBA). Human lymphocytes from peripheral blood were used as control cells under the assay conditions with or without induced rat liver metabolic activation (S9). All chemicals tested without S9 induced chromosomal aberrations (CA) in MCL-5 cells but not in human lymphocytes. All chemicals induced CA in both cell types in the presence of S9.

Detection of neocarzinostatin-induced translocations in human sperm chromosomes using fluorescence in situ hybridization of chromosome 2.

Mature sperm and late spermatid are known to be sensitive stages to clastogens in mammalian spermatogenesis. Certain types of chromosomal damage induced in these stages will pass to successive generations as heritable translocations. In the present study, we employed whole chromosome 2 painting with the fluorescence in situ hybridization (FISH) technique to detect the chemically induced translocations in human sperm. Mature human sperm were treated in vitro with an antitumor drug, neocarzinostatin (NCS), and fertilized in vitro with golden hamster oocytes. Sperm pronuclear chromosome slides were prepared at the first cleavage metaphase. To compare the characteristics of translocations between somatic and germ cells, human lymphocytes in peripheral blood treated with NCS in vitro were analyzed at first round metaphase after PHA-stimulation. From the analysis of translocations by whole chromosome 2 painting, frequencies of the haploid genomic translocations (FhG) were predicted for both sperm and lymphocytes. At 1.0 micrograms/ml, the actual percentages of sperm and lymphocytes with chromosome 2 translocations were almost identical (11.9% and 12.0%). At the same dose, however, the FhG of the sperm (1.15) was considerably higher than that of the lymphocytes (0.58), indicating that complex translocations having two or more rearranged sites were induced by NCS more frequently in sperm than in lymphocytes.

Mutagenic reactivities of 3,4-dinitrobiphenyl derivatives.

3,4-Dinitrobiphenyl derivatives were mutagenic in Salmonella typhimurium TA98, TA98/1,8-DNP6 and in TA98NR. We describe here the specific reactivity of 3,4-dinitrobiphenyl derivatives with diluted sodium hydroxide solution and the determination of the amounts of released nitrous ion. 3,4-Dinitrobiphenyl derivatives begin to release nitrous ions when treated with NaOH solution at a concentration of 10(-3) N. The behavior of 4NQO and o-dinitrobenzene was the same as that of 3,4-dinitrobiphenyl derivatives. The residues of 3,4-dinitrobiphenyl derivatives, after releasing nitrous ions, were estimated to be hydroxy-nitrobiphenyls, as by GC/MS, we found the formation of o-nitrophenol in the reaction mixture of o-dinitrobenzene with aqueous NaOH solution. 3,4,4'-Trinitrobiphenyl, 3,4,3',4'-tetranitrobiphenyl and 4NQO had reduced mutagenic potency in Salmonella typhimurium TA98 following treatment with diluted NaOH. In order to elucidate the ultimate forms of 3,4-dinitrobiphenyl derivatives, we investigated the reaction of o-dinitrobenzene as a basic model substance of 3,4-dinitrobiphenyl, with nucleic bases in the presence of NaOH in nonaqueous solvent. o-Nitrophenyl guanine and adenine adducts were obtained.

Relationship between mutagenic potency in Salmonella typhimurium strains and the chemical structure of nitro biphenyls.

Most of the positional isomers of mono-, di-, tri- and tetranitrobiphenyls were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8DNP6 in the absence of S9 mix. In mono- and dinitrobiphenyls, the structure requirements favoring mutagenic activity are the presence of a nitro group at the 4-position and its absence at the 2-position. TA98 and TA98/1,8DNP6 were reverted by 2-position-free 4-nitro analogues, but TA98NR was not reverted. The results suggest that direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes. Some of the tri- and tetranitrobiphenyls e.g. 3,4,3'-, 3,4,4'-, 3,4,3',4'- and 3,4,2',4'-derivatives reverted not only TA98 and TA98/1,8DNP6 but also TA98NR. Those derivatives commonly have 2 nitro groups at an adjoining position (3,4-dinitro group), whereas 2,4,2',4'-tetranitrobiphenyl, which has strong potency not only in TA98 and TA98/1,8DNP6 but also in TA98NR, possesses 2 nitro groups at the 2-position of each benzene ring.

Re-evaluation of chromosomal aberration induction on nine mouse lymphoma assay "unique positive' NTP carcinogens.

In a collaborative study organized under the JEMS MMS, nine mouse lymphoma assay (MLA) "unique positive' NTP rodent carcinogens were re-evaluated by an in vitro chromosomal aberration assay using Chinese hamster lung fibroblast cells (CHL/IU). Six of nine chemicals induced chromosomal aberrations; bromodichloromethane, chlorendic acid and isophorone induced structural aberrations, and chlorodibromomethane, pentachloroethane and 1,1,1,2-tetrachloroethane induced numerical aberrations (polyploidy). These six chemicals, therefore, are not uniquely positive in the MLA. The difference between the NTP results and ours might be due to the use of different cell lines and protocols, and in some cases, to different interpretations of polyploidy. The remaining three chemicals, benzyl acetate, cinnamyl anthranilate and trichloroethylene, were negative in this study.

Taxonomic studies on Kitasatosporia cystarginea sp. nov., which produces a new antifungal antibiotic cystargin.

Taxonomic studies on a new species, Kitasatosporia cystarginea are presented. Among the several species already described in this genus, this strain is characteristic in forming distinct spirals of spore chains. A significant properties of the species is the production of a new antifungal antibiotic, cystargin.

Epiderstatin, a new inhibitor of the mitogenic activity induced by epidermal growth factor. I. Taxonomy, fermentation, isolation and characterization.

Inhibitors of mitogenic activity induced by epidermal growth factor (EGF) were screened from culture broths of soil microorganisms. A strain of actinomycetes has been found to produce a new glutarimide antibiotic named epiderstatin which inhibits the incorporation of [3H]thymidine into quiescent animal cells stimulated by EGF. Taxonomic studies have revealed that the producing strain belongs to a subspecies of Streptomyces pulveraceus, thus the name, Streptomyces pulveraceus subsp. epiderstagenes was given to this strain. The molecular formula (C15H20N2O4) and UV profile (lambda max 295 nm) of the antibiotic are distinct from other known antibiotics. It inhibited the incorporation of [3H]thymidine into quiescent cells stronger than into growing cells.

A new inhibitor of protein kinase C, RK-286C (4'-demethylamino-4'-hydroxystaurosporine). I. Screening, taxonomy, fermentation and biological activity.

In the course of our screening program using a bleb-forming assay, a new inhibitor of protein kinase C (PKC) was found in the fermentation of a streptomycete. The inhibitor, RK-286C (4'-demethylamino-4'-hydroxystaurosporine), inhibited the morphological change of K562 cells, a human chronic erythroleukemia cell, induced by phorbol 12,13-dibutylate at the concentration of 3 microM. The same concentration of the compound inhibited the activity of PKC in vitro and the aggregation of rabbit platelets induced by collagen and arachidonic acid.

A new antifungal antibiotic, cystargin: fermentation, isolation, and characterization.

A new sulfur-containing peptide antifungal antibiotic, cystargin, was isolated from the fermentation broth of a new species of genus Kitasatosporia, designated as Kitasatosporia cystarginea. On acid hydrolysis, cystargin (C60H77N19O17S6) gave equimolar glycine, proline, aspartic acid and arginine. By performic acid oxidation, cysteic acid was detected after hydrolysis. It showed a growth inhibitory activity against various phytopathogenic fungi and inhibition of beta-1,3-glucan synthetase from Saccharomyces cerevisiae.

New types of liposidomycins that inhibit bacterial peptidoglycan synthesis and are produced by Streptomyces. I. Producing organism and medium components.

Liposidomycins are atypical lipid-bearing nucleoside antibiotics that inhibit bacterial peptidoglycan synthesis. A producing strain was identified as a Streptomyces sp. from its cultural characteristics and physiological properties. It produced new types of liposidomycins that lacked sulfate and/or 3-methylglutaric acid moieties present in known liposidomycins by changing medium components. Sucrose and malt extract were particularly suitable sources for specific production of the new types of liposidomycins.

Isolation and characterization of phosmidosine. A new antifungal nucleotide antibiotic.

A new nucleotide antibiotic, phosmidosine was isolated from a culture filtrate of a newly isolated streptomycete identified as Streptomyces sp. RK-16. HRFAB-MS and elemental analysis established the molecular formula of C16H24N7O8P. 1H, 13C and 31P NMR indicated the presence of a methyl phosphate group and UV spectra were similar to those of 8-hydroxyadenosine. The antibiotic inhibited spore formation of Botrytis cinerea at the concentration of 0.25 micrograms/ml.

[Administration of vindesine sulfate for the treatment of malignant hematological tumors].

Forty-six cases with hematological malignancies were treated with vindesine sulfate (VDS); a new semisynthetic vinca alkaloid. Six cases with ALL, 5 cases CML in blastic crisis, 3 Hodgkin's disease (HD) and 4 non-Hodgkin's lymphoma (NHL) were treated with VDS alone. Five out of 6 cases ALL, 2 out of 5 CML in blastic crisis were induced into partial remission with VDS alone. All of 3 HD, and 4 NHL were induced in complete remission (CR) or partial remission (PR). Out of 5 cases AML in CR who received VDS as the maintenance therapy in combination with cyclophosphamide, 6-MP and prednisone, one case relapsed during the treatment, but other four cases maintained CR for 4 to 24 months. One case of APL in relapse, which was treated with VDS and 6-MP, reinduced into CR after one month. Out of 16 cases with malignant lymphoma treated by combination chemotherapy including VDS, eleven cases entered in CR or PR. Out of four cases in which the disease became refractory to vincristine (VCR) or vinblastine (VLB) clinically, two achieved PR. VDS was administered intravenously with 3 mg/body/week. When undesirable effect such as leucocytopenia was observed, the dose was reduced to 2-2.5 mg/body/week or 3 mg/body/2 weeks or month. Neurotoxicity (i.e. Paresthesia 21.7%), alopecia (21.7%), leucocytopenia (19.6%), constipation (10.9%) and fever (6.5%) were main side effects of VDS. The neurotoxicity of VDS, however, seemed far less intensive than VCR.