Retinoic Acid Deficiency Underlies the Etiology of Midfacial Defects.

Embryonic craniofacial development depends on the coordinated outgrowth and fusion of multiple facial primordia, which are populated with cranial neural crest cells and covered by the facial ectoderm. Any disturbance in these developmental events, their progenitor tissues, or signaling pathways can result in craniofacial deformities such as orofacial clefts, which are among the most common birth defects in humans. In the present study, we show that Rdh10 loss of function leads to a substantial reduction in retinoic acid (RA) signaling in the developing frontonasal process during early embryogenesis, which results in a variety of craniofacial anomalies, including midfacial cleft and ectopic chondrogenic nodules. Elevated apoptosis and perturbed cell proliferation in postmigratory cranial neural crest cells and a substantial reduction in Alx1 and Alx3 transcription in the developing frontonasal process were associated with midfacial cleft in Rdh10-deficient mice. More important, expanded Shh signaling in the ventral forebrain, as well as partial abrogation of midfacial defects in Rdh10 mutants via inhibition of Hh signaling, indicates that misregulation of Shh signaling underlies the pathogenesis of reduced RA signaling-associated midfacial defects. Taken together, these data illustrate the precise spatiotemporal function of Rdh10 and RA signaling during early embryogenesis and their importance in orchestrating molecular and cellular events essential for normal midfacial development.

Superoxide dismutase and thioredoxin restore defective p34cdc2 kinase activation in mouse two-cell block.

We recently showed that superoxide dismutase (SOD), a free radical scavenger, and thioredoxin, a potent protein disulfide reductase, release mouse two-cell stage developmental block in vitro. To elucidate the mechanism underlying the two-cell block and the effects of these enzymes, we studied the chronological changes in the kinase activity and the immunoblotting pattern of p34cdc2, a key regulator of the cell cycle, during the first and second cell cycles of the mouse embryonic development. In vivo embryos were freshly collected at fixed times, and in vitro embryos cultured from the pronuclear stage were also sampled with the same time-course. A marked elevation of p34cdc2 kinase activity was observed in vivo at 27-32 and 51 h after an injection of human chorionic gonadotropin. These times coincide with the M-phases of embryo cleavage. In vitro embryos showed high kinase activity during the M-phase of the first cleavage, but this activity was not elevated during the second cell cycle. The addition of recombinant human SOD (200 micrograms/ml) or thioredoxin from Escherichia coli (500 micrograms/ml) to the medium enabled kinase activation with a time course similar to that of in vivo embryos. The immunoblotting patterns suggested the dephosphorylation of p34cdc2 at the M-phase of the first and the second cleavages in vivo. Although p34cdc2 was dephosphorylated at the M-phase of the first cleavage and then rephosphorylated for embryos cultured in vitro, the second dephosphorylation was not observed during the second cell cycle. The addition of SOD or thioredoxin permitted the dephosphorylation at the M-phases of both the first and the second cleavage. These results suggest that one of the chief causes of two-cell block in vitro is the impairment in p34cdc2 dephosphorylation, recently shown to be catalyzed by the cdc25 homologue. This impairment is thought to be due to oxidative stress, because both SOD and thioredoxin are known to play a defensive role against it.

Role of protein supplements in the culture of mouse embryos.

The effect of various types of proteins used in single protein supplements for Bigger-Whitten-Whittingham (BWW) medium on the in vitro development of mouse preimplantation embryos was evaluated. Thioredoxin, superoxide dismutase (SOD), and apotransferrin showed prominent growth-promoting activity, whereas bovine serum albumin (BSA), fatty acid-free BSA, and catalase showed moderate promoting effects. beta-lipoprotein, ovalbumin and hemoglobin were ineffective, and holo-type transferrin and ceruloplasmin were actually toxic to the embryos. These results suggest that the growth-promotive effect of proteins on mouse pronuclear stage embryos is due to their antioxidative action, or to the removal of some free metal ion(s) such as Fe(3+). The mild growth promoting effect of both BSA and fatty acid free BSA suggest that the effect mediated by BSA is not dependent on bound fatty acids, but more likely is due to their antioxidative effect or chelating effect.

[3 Cases of blunt chest trauma].

3 Cases of blunt chest trauma were reported, including aortic isthmus rupture, diaphragmatic injury and right main bronchial rupture. All cases were injured by traffic accidents. The first case was a 21-year-old man complicated with aortic injury and femoral bone fracture and died of intrapleural rupture before thoracotomy. The second case was a 49-year-old man who had right 11th rib fracture and he received emergent thoracotomy due to hemorrhagic shock. We found accidentally arterial massive bleeding by the diaphragmatic injury at thoracotomy and ligated directly. The third case was a 19-year-old man and he was unconscious on admission and rupture of the right main bronchus was found by bronchofiberscope and performed pneumectomy. One patient died and other two patients have survived. Blunt chest trauma sometimes lead to fatal damage of vital organs. So it is necessary to make early diagnosis and perform the emergent operations for life salvage.

[Study on management of low potential malignancy ovarian tumors].

Thirty-six patients with low potential malignancy ovarian tumors were treated at our hospital from 1972 to 1986. Of these, 80.6% were classified as stage I, 5.6% as stage II, and 13.9% as stage III. Sixteen patients were treated by simple total hysterectomy and bilateral salpingo-oophorectomy, 15 patients by unilateral salpingo-oophorectomy, 2 patients by enucleation of the tumor, and 3 patients by exploratory laparotomy. In stage I no difference between the survival rates for the conservative therapy group and the radical therapy group was seen. Postoperative radiation therapy was given to 4 patients with dysgerminoma, and chemotherapy was given to 13 other patients. The five-year survival rate for stage I was 91.7%, better than for stage I malignant ovarian tumors, which was 78.9%. But the five-year survival rate for stage II and stage III was 0%. Analysis indicated that: 1. Prognosis of stage I patients is so good that treatments may be done in consideration of the patient's fertility. 2. The importance of adequate postoperative treatment and of strict follow up to guard against recurrence of malignancy is important in patients with stage II or stage III disease.

Analysis of oviduct-derived embryonic growth stimulator activity.

A co-culture study was carried out using primary cultures of hamster oviduct tissues to analyze oviduct-derived embryonic growth stimulating factor. In co-cultures of mouse pronuclear embryos and hamster oviduct tissue, the 4-cell rate after 48 hours (53.2%, 109/205) and blastocyst rate after 96 hours (22.4%, 46/205) were significantly higher than those of the standard culture group (4-cell rate, 41.8%, 76/182; blastocyst rate, 4.4%, 8/182), suggesting an embryonic growth stimulating effect of the co-culture. Similar effects were observed in co-cultures of both 2-cell mouse embryos collected 40 or 48 hours after the hCG administration and morulae collected 64 hours after hCG, but no such effect was observed in co-cultures of hamster embryos. When mouse embryos were cultured in a conditioned medium, which was the supernatant of the hamster oviduct tissue culture in Biggers-Whitten-Whittingham's (BWW) solution, no embryonic growth stimulating effect was observed. Further, no stimulating effect was observed in the conditioned medium obtained from 24-hour co-cultures of embryos and oviduct tissue. From these results, we considered the embryonic growth stimulating factor to be labile, with concentration-dependent effects. The absence of a stimulatory effect of co-culturing on the growth of hamster embryos also suggests the presence of species variations in the in vitro arrest of embryogenesis.

Experimental local administration of CDDP to in vitro models of gynecological malignant tumors transplanted into nude mice (compared with medroxyprogesterone acetate orally administered).

In order to develop a new method of administration for CDDP, in vitro models of malignant tumors in the field of gynecology were prepared using two cell lines maintained by the authors, and fundamental experiments on the topical injection of CDDP were carried out. In experimental topical injection of CDDP in tumor-bearing nude mice, the test drug demonstrated an excellent tumor regression effect and an inhibitory effect on tumor growth. In the histopathologic examinations, specific necrosis of tumor cells was observed. It was confirmed that this is a highly safe method, as tissue separation, ulceration, or hemorrhagic lesions attributable to the local administration of CDDP were not observed. In the present study, treatment with oral medroxyprogesterone acetate was also used. At the doses used in this study, however, no inhibitory effect on tumor growth or synergism between medroxyprogesterone acetate and CDDP was observed. Topical injection is an excellent pharmacodynamic method that permits the injection of free platin into the tumor itself or in the boundary area between the tumor and normal tissues, with no loss of the drug, and it is considered a safe and effective mode of local administration. Intra-arterial injection of this drug alone or in conjunction with OK-432 can also be used, even though further studies will be required to determine the optimum dosage and reduce side effects. At present, data are being collected on terminal cancer patients for whom no other therapy is available. In the near future this method of administration is expected to be utilized in the clinical treatment of malignant tumors, be it early tumor or progressive cancer.

[Local injection of high-dose CDDP to the advanced gynecological cancer].

We investigated the efficacy of local injection of high-dose CDDP. The subjects were 16 patients with advanced gynecological cancer or tumor recurrence, in whom systemic administration of CDDP was inadvisable because of advanced age or associated complications (12 cases of cervical carcinoma, 2 cases of endometrial carcinoma, 1 case of ovarian carcinoma, and 1 case of vulvar carcinoma). In 14 cases, CDDP was injected locally to the tumor mass, using a single dose of 50-300 mg. In 2 cases, a single dose of 10-20 mg of CDDP was infused into the uterine cavity. The effects of the therapy were evaluated by cytodiagnosis, tumor markers, CT, and performance status. In all cases, an antitumor effect was noted, and seven subjects survived for at least 24 months following these therapy with CDDP. One patient developed vesicovaginal and rectovaginal fistulae after local injection of CDDP following high-dose radiotherapy. We investigated the plasma concentrations of free and total platinum after CDDP application with doses from 60-200 mg/body. Plasma concentrations showed a biphasic pattern (phase alpha and phase beta), and the peak plasma concentration of CDDP was lower than that following intravenous administration of the same dose. From these results, it was suggested that a large dose of CDDP can be injected into the tumor tissue itself and the surrounding tissue with comparatively few side effects. It will be possible to administer large dose of CDDP in this way to the terminal patients to whom there is currently no other appropriate method of treatment. The performance status of our subjects was improved, and we expect that wider use of this method will improve the quality of life for end-stage patients.

Release of two-cell block by reduction of protein disulfide with thioredoxin from Escherichia coli in mice.

The development of mouse pronuclear-stage embryos in media containing various concentrations of thioredoxin was monitored and the influence of antithioredoxin immunoglobulin G (IgG) and heat-treated thioredoxin on the thioredoxin-induced effects was evaluated. A significant increase in the number of four-cell embryos (76.3%) and blastocysts (37.3%) was observed when embryos were cultured in the medium containing 50 micrograms thioredoxin ml-1 compared with the rates (55.8 and 3.8%, respectively) in the basic medium. The number of blastocysts increased significantly to a maximum of 70.2% at 500 micrograms ml-1. The biological activity of thioredoxin was evident after dialysis, but was markedly impaired by the addition of anti-thioredoxin IgG to the culture medium. Treatment at 60 degrees C for 5 min did not affect the enzymatic and biological activity of thioredoxin. More severe heat treatment (121 degrees C for 30 min) attenuated the enzymatic activity to 40% of its initial value and reduced the biological activity (number of blastocysts, from 77.8 to 51.6%). These results indicate that the effect of thioredoxin on the two-cell block is due to the thioredoxin molecule itself, and suggest that disulfide formation within or between proteins resulting from oxidative stress is one of the major causes of the two-cell block.

Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development.

We developed a non-radioisotopic quantitative RT-PCR method with high sensitivity and reproducibility. The results of this RT-PCR were in agreement with those of the Northern blot analysis. We measured the mRNA levels of beta-actin, transferrin receptor, and two cell cycle-related genes, cyclin B and cdc25, in early mouse embryos by the RT-PCR. In late two-cell stage embryos, beta-actin, transferrin receptor and cyclin B mRNA levels were 10-20% of those in MII stage oocytes. In contrast, the cdc25 mRNA levels were not different between these stages. When we cultured mouse embryos, the presence of an RNA polymerase inhibitor, alpha-amanitin, in the medium did not affect the mRNA levels at the two-cell stage, indicating that most of the detected mRNAs in two-cell embryos were maternally derived. These results suggest that the rate of mRNA degradation is different between cyclin B and cdc25 during early embryogenesis.

High incidence of uterine inversion in mast cell-deficient osteopetrotic mutant mice of mi/mi genotype.

Mutations at either W or mi (microphthalmia) loci in the mouse can lead to a deficiency in melanocytes and mast cells. In addition, W mutants can be anemic and sterile, whereas mi mice are osteopetrotic because of a monocyte/macrophage/osteoclast defect. Since c-kit receptor tyrosine kinase is the gene product of the W locus and mi mutation has been suggested to affect the transduction of signals from the c-kit and c-fms receptors, we here examined the effect of mi mutation on fertility. Testes and ovaries from mi/mi mice were histologically normal, and the pattern of c-kit protein expression was not different from that of +/+ mice. Homozygous mutant crosses (mi/mi x mi/mi) were fertile, but inversion of the uterus occurred in 86% of the deliveries. In some cases, the placenta was found still attached to the inverted uterus after delivery. Decidual cells were present and expressed c-kit protein normally in the placenta of mi/mi mice. The inversion was also observed in mi/mi females mated to +/+ males. No uterine inversion was noted when +/mi females were crossed with mi/mi or +/mi males, suggesting that the genotype of the mother but not of the father or fetus is important for the pathogenesis. The numbers and body weights of mi/mi newborns were less than those of +/mi littermates. Mast cells were absent, but c-kit-positive cells were present, in the uterine muscle layers of pregnant mi/mi mice.(ABSTRACT TRUNCATED AT 250 WORDS)