Absolute calibration of imaging plate for GeV electrons.

An imaging plate has been used as a useful detector of energetic electrons in laser electron acceleration and laser fusion studies. The absolute sensitivity of an imaging plate was calibrated at 1 GeV electron energy using the injector Linac of SPring-8. The sensitivity curve obtained up to 100 MeV in a previous study was extended successfully to GeV range.

Contribution of glucan-binding protein A to firm and stable biofilm formation by Streptococcus mutans.

Glucan-binding proteins (Gbps) of Streptococcus mutans, a major pathogen of dental caries, mediate the binding of glucans synthesized from sucrose by the action of glucosyltransferases (GTFs) encoded by gtfB, gtfC, and gtfD. Several stress proteins, including DnaK and GroEL encoded by dnaK and groEL, are related to environmental stress tolerance. The contribution of Gbp expression to biofilm formation was analyzed by focusing on the expression levels of genes encoding GTFs and stress proteins. Biofilm-forming assays were performed using GbpA-, GbpB-, and GbpC-deficient mutant strains and the parental strain MT8148. The expression levels of gtfB, gtfC, gtfD, dnaK, and groEL were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, the structure of biofilms formed by these Gbp-deficient mutant strains was observed using confocal laser scanning microscopy (CLSM). Biofilm-forming assay findings demonstrated that the amount formed by the GbpA-deficient mutant strain (AD1) was nearly the same as that by the parental strain, while the GbpB- and GbpC-deficient mutant strains produced lower amounts than MT8148. Furthermore, RT-qPCR assay results showed that the expressions of gtfB, dnaK, and groEL in AD1 were elevated compared with MT8148. CLSM also revealed that the structure of biofilm formed by AD1 was prominently different compared with that formed by the parental strain. These results suggest that a defect in GbpA influences the expression of genes controlling biofilm formation, indicating its importance as a protein for firm and stable biofilm formation.

Dysgerminoma with syncytiotrophoblastic giant cells arising from 46,XX pure gonadal dysgenesis.

BACKGROUND: Dysgerminoma with syncytiotrophblastic giant cells is a rare ovarian tumor. Only ten cases of this tumor have been reported, and all tumors developed in normal ovaries. This report presents a case of dysgerminoma with syncytiotrophoblastic giant cells arising in a patient with 46,XX pure gonadal dysgenesis. CASE: An 18-year-old phenotypic female of normal height without somatic anomalies with nonfunctional ovaries underwent a bilateral gonadectomy for a right ovarian tumor and left streak gonad. The tumor revealed a dysgerminoma containing scattered syncytiotrophoblastic giant cells. Her serum hCG was elevated, and hCG was demonstrated within syncytiotrophoblastic giant cells immunohistochemically. The clinical diagnosis was stage Ia dysgerminoma with syncytiotrophoblastic giant cells. Her karyotype was 46,XX and the sex-determining region Y gene was not detected in tumor DNA by polymerase chain reaction analysis. CONCLUSION: This rare gonadal tumor may arise from dysgenetic gonads in addition to gonadoblastoma and pure dysgerminoma. It is an example of tumorgenesis in pure gonadal dysgenesis with no identifiable Y chromosome component.

Localization of fibronectin on the surface of human spermatozoa and relation to the sperm-egg interaction.

OBJECTIVES: The mechanism of human sperm-oolemma adhesion and penetration as well as localization of fibronectin on the sperm head and its relation to fertilization were investigated. DESIGN: Sperm-oolemma interaction was examined with an in vitro assay of the human sperm-zona-free hamster egg interaction. Localization of fibronectin on the surface of human spermatozoa was observed by the back scattered electron imaging mode of the scanning electron microscopy (SEM). RESULTS: It was confirmed by observations under SEM that the anterior tip of the sperm head is the first to come into contact with the egg plasma membrane but that the equatorial segment of the sperm head is the first to be trapped by microvilli of the plasma membrane and that the postacrosomal region is first incorporated into ooplasm. Localization of fibronectin on the equatorial segment of the human sperm head was detected by SEM. Antifibronectin antibodies inhibited human sperm-oolemma adhesion significantly. CONCLUSIONS: Important involvement of fibronectin in the gamete interaction was made clear by the fact that fibronectin is localized in the region where a spermatozoon is fused first with the egg plasma membrane during fertilization and that the sperm adhering to the egg is inhibited by antifibronectin antibodies.

The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm.

OBJECTIVE: To determine whether the outcome of IVF can be predicted by acridine orange (AO) nuclear fluorescence of sperm. DESIGN: Based on the fact that AO nuclear fluorescence color after acid treatment reflects maturity (green fluorescence) or immaturity (yellow to red fluorescence) of spermatozoa, the relationships between sperm maturity and the outcome of IVF, subzonal insemination, or intracytoplasmic sperm injection (ICSI) were investigated. SETTING: The IVF program at the Obstetrics and Gynecology Hospital, Fukushima Medical College. PATIENTS: Sixty-eight patients undergoing 68 IVF treatment cycles. MAIN OUTCOME MEASURES: Acridine orange fluorescence of sperm nuclei and successful oocyte fertilization. RESULTS: conventional semen parameters (sperm concentration and percentages of motile or morphologically normal spermatozoa in semen) did not correlate with the incidence of spermatozoa with green AO fluorescent (mature) nuclei. When > or = 50% of spermatozoa in semen samples exhibited green AO nuclear fluorescence, IVF was always successful. When green AO nuclear fluorescence was < 50%, only 39% of IVF treatment cycles (13/33) were successful. Only green AO fluorescent spermatozoa were able to bind efficiently human zona pellucida. When the incidence of green AO fluorescent spermatozoa was < 50%, no pregnancy resulted even though an average of 26% of the oocytes could be fertilized by ICSI. CONCLUSIONS: The spermatozoa which fertilized oocytes in vivo and in IVF were limited to those whose nuclei exhibited green AO fluorescence. Intracytoplasmic sperm injection may be the method of choice when the incidence of green AO nuclear fluorescence is low regardless of the results of semen analysis.

Intracytoplasmic sperm injection using immobilized or motile human spermatozoon.

OBJECTIVES: To investigate the efficacy of the treatments for oocyte activation on the results of intracytoplasmic sperm injection using immobilized or motile human spermatozoa. DESIGN: The protocol of intracytoplasmic sperm injection was divided into four groups according to the states of sperm used for microinjection and the treatment for oocyte activation. In group A, immobilized sperm is used. The oocyte is activated merely by aspiration of the cytoplasm into the pipette. In group B, immobilized sperm is used. Microinjected oocyte is treated with A23187. In group C, immobilized sperm is used. Electroporation is performed on the microinjected oocyte. In group D, motile sperm is used. The oocyte is activated merely by aspiration of the cytoplasm into the pipette. SETTING: The Obstetrics and Gynecology Hospital, Fukushima Medical College. PATIENTS: The subjects are the cases that had failed fertilization in standard IVF, cases of severe oligozoospermia, and cases of severe asthenozoospermia. RESULTS: No difference was found between the groups as to the survival rate and fertilizing rates of oocytes after intracytoplasmic sperm injection. The cleavage rate of oocytes was high in order of group D, C, B, A. The cleavage rate for groups D, C, and B was significantly higher than group A. Cases of pregnancy were found in groups D and B. CONCLUSION: Using motile sperm rather than immobilized sperm can be expected to produce better results in human ICSI. Activating oocytes positively is needed when immobilized sperm is used.

Induction of the acrosome reaction in human spermatozoa by human zona pellucida and effect of cervical mucus on zona-induced acrosome reaction.

OBJECTIVES: To investigate the induction of the acrosome reaction in human spermatozoa by the zona pellucida (ZP), cumulus oophorus, and cervical mucus (CM), and to examine the effect of cumulus oophorus and CM on the zona-induced acrosome reaction. DESIGN, PATIENTS, MAIN OUTCOME MEASURES: The acrosome status of spermatozoa from healthy donors that were cultured with salt-stored human ZP, cumulus oophorus, and/or cervical mucus was assessed using fluoresceinated pisum sativum agglutinin. RESULTS: The acrosome reaction rate after sperm attachment to the zona for 6 hours was 35.7% +/- 17.7%, which was higher than controls (2.8% +/- 1.9%). The acrosome reaction was not observed after passage through cumulus oophorus or CM; however, the acrosome reaction rate of spermatozoa passed through CM after the attachment to zona was 51.6% +/- 6.8%, a higher value in comparison with spermatozoa that had not passed through CM (25.6% +/- 9.4%). CONCLUSION: These data indicate that human ZP are capable of inducing the acrosome reaction in human spermatozoa; however, cumulus oophorus and CM are unable to do so. The data further provide evidence that CM promotes the zona-induced acrosome reaction in human spermatozoa.

Introduction of a new scale into reversed-phase high-performance liquid chromatography of pyridylamino sugar chains for structural assignment.

Addition of a monosaccharide residue to a pyridylaminated (PA)-N-linked sugar chain results in an increment or decrement in the elution time on reversed-phase HPLC, the difference being defined as the partial elution time of the residue. Based on this principle, an empirical rule was deduced, which states that the elution time is roughly equal to the sum of the partial elution times of the component sugar residues [Anal. Biochem., 167 (1987) 321-326]. In practice, however, some partial elution times obtained from different pairs of mother PA-sugar chains are found to deviate, and consequently the closeness of the elution times of PA-sugar chains calculated therefrom to the observed times is reduced in such cases. To improve the reliability of the additivity rule and to generalize elution times so that they are less dependent on minor alterations in the elution conditions, we have devised a new scale for elution time, which we have named a reversed-phase scale. The elution times on the reversed-phase scale (the R values) are read from a conversion curve constructed using the elution times of eight selected standard PA-sugar chains. The partial elution times on the reversed-phase scale of 22 monosaccharide residues were calculated from the R values of 93 PA-sugar chains. The R values obtained by summing the partial elution times of all the component monosaccharide residues became much closer to the R values obtained from the reversed-phase scale, compared to the results obtained using the previous method. In addition, the R values were less influenced by minor change in the elution conditions. These features of the new scale allow more accurate structural assignment of sugar chains.

Fertilization using male germ line-cells.

In recent years, favorable results have been achieved in patients suffering from azoospermia by microinsemination of spermatozoa taken from their testes. Microinsemination is being introduced in the treatment of patients who have no spermatozoa in their testes via their spermatid and spermatocyte. There are still doubts relating to immature male germ line-cells, such as whether they have, oocyte activating factors, the level of stability of DNA of cell nuclei, and the differences in chromosome numbers. The relatively few cases of gestation using the human spermatid treatment may be due to embryological problems resulting from the instability of nuclear DNA and the insufficiency of oocyte activating factors, which are the result of imperfect microinjection techniques. Improvements in techniques for the clinical application of spermatid and secondary spermatocyte, as well as the collection of basic data to confirm embryological safety are therefore necessary.

Wide use of skin-lightening soap may cause mercury poisoning in Kenya.

In a previous study, we speculated that some of the high mercury levels observed in head hair from a total of 14 subjects who resided around Lake Victoria, Tanzania, might be attributable to the habitual use of toilet soap containing considerable amounts of mercury (Harada et al. Sci Total Environ 1999;227:249-256). In August 1998, the current study was conducted to investigate if such mercury-containing soap was also available in the surroundings of Lake Victoria, Kenya, and if so, its toxic effects. A total of nine goldminers, 44 fishermen and their families, and 12 residents of Kisumu City, Kenya, volunteered for the study. Fourteen types of toilet soap were collected in Kisumu. Total mercury content was very significantly higher than in European-made soap (0.47-1.7%, as mercury iodide) compared with Kenya-made soap (0.41 x 10(-4)-6.2 x 10(-4)%). Indeed, all the subjects with a high hair mercury level (> 36.1 ppm) had made habitual use of European-made soap, accompanied by various symptoms, such as tremor, lassitude, vertigo, neurosthenia, and black and white blots, suggesting inorganic-mercury poisoning. On the other hand, any subject who had used soap other than the European-made soap, did not exceed a mercury level of 10 ppm in hair that is well within normal limits (Harada et al. Sci Total Environ 1999:227:249-256). The findings obtained suggest that the mercury-containing soap must be barred from circulation without delay, and that the residents' health in addition to the environmental pollution in Lake Victoria (Kenya as well as Tanzania) should be kept under close observation.

Monitoring of mercury pollution in Tanzania: relation between head hair mercury and health.

Through 1996 into 1997, a spot investigation on mercury contamination was conducted three times in Tanzania, especially around the Lake Victoria. A total of 150 goldminers, 103 fishermen and their families, and 19 residents of Mwanza City volunteered for the current study. A high total mercury level of 48.3 ppm (near to 50 ppm, a critical level of Minamata disease) and over in the head hair was observed in six goldminers (highest value, 953 ppm), four fishermen and their families (highest value, 416 ppm), and four Mwanza people (highest value, 474 ppm). With the exception of these 14 subjects, however, each mean total mercury level was well within the normal range (below 10 ppm). Out of the goldminers examined, 14 cases were diagnosed as a mild form of inorganic-mercury poisoning according to their clinical symptoms (such as polyneuropathy mercurialis, neuroasthemia, or tremor mercurialis) and the low ratio of methylmercury to total mercury, whereas neither inorganic-mercury poisoning nor methylmercury poisoning (Minamata disease) was noted in the fishermen and their families or in the Mwanza people. In addition, some subjects who showed a high total mercury level made habitual use of toilet soap containing much mercury. The findings obtained suggest that the mercury pollution in Tanzania is not very serious, however, it should be observed continuously.

Susceptibility to oxidation of copper-induced plasma lipoproteins from Japanese eel: protective effect of vitellogenin on the oxidation of very low density lipoprotein.

The susceptibility to oxidation of copper-induced plasma lipoproteins from Japanese eel Anguilla japonica was examined with the guidance of thiobarbituric acid-reactive substances (TBARS). The TBARS values of copper-induced plasma lipoproteins increased with increasing the lipid-to-apolipoprotein ratios and very low density lipoprotein (VLDL) exhibited the highest TBARS value. On the other hand, vitellogenin, estrogen-induced precursor of egg yolk proteins, was resistant to copper-induced oxidation and seemed to chelate low concentrations of copper ion. Vitellogenin also protected the copper-induced oxidation of VLDL because of its antioxidant function. Vitellogenin seemed to serve as transition metals-binding lipoprotein by which free-radical reactions in the oocytes were extensively depressed.

Eight cases of gastric tumors with calcification.

Gastric tumors with calcification have been considered to be relatively rare. We treated 8 cases of tumors involving calcification, 4 cases of gastric carcinomas and 4 other cases of submucosal tumors with ulceration. These cases are reported along with a review of the cases reported in Japan.

Efficient injection of bull spermatozoa into oocytes using a Piezo-driven pipette.

Recently, mouse and human offspring have been successfully obtained from embryos developed after intracytoplasmic sperm injection(ICSI), using a Piezo micromanipulator. In this study, the Piezo-ICSI procedure was used with in vitro matured bovine oocytes known to be difficult to fertilize microsurgically. The efficacy of Piezo-ICSI versus conventional ICSI was examined after oocytes were activated and fertilized with or without calcium ionophore (A23187) exposure. In conventional ICSI, the rate of fertilization was 19% (11/59) with A23187 and 5% (2/38) without it. However, when the Piezo-ICSI procedure was performed, the fertilization rate was 72% (47/65) with A23187 and 72% (28/39) without it. The rate of oocyte survival after microinjection was nearly similar for both methods. We suggest that the bovine oocyte is successfully activated and fertilized when an immobilized spermatozoon is injected exactly into the ooplasm through the oolemma, perforated easily by the pulsation of the Piezo. Moreover, an activating procedure such as exposure of oocytes to A23187 is not necessary, because the so-called sperm factor (oocyte activating substances) is incorporated into the ooplasm along with a spermatozoon. In this respect, the Piezo-ICSI was more efficient than the conventional ICSI method for fertilizing and thus obtaining more bovine embryos.

Accumulation of germanium in the tissues of a long-term user of germanium preparation died of acute renal failure.

Acute renal failure developed in a patient accompanied by systemic manifestations such as myopathy and skin rash. The patient, a middle aged house wife, had been taking 600 mg of germanium (Ge) preparation daily for 18 months as an elixir. The main component of the preparation was GeO2 and some organic compound was also present. Histological study of the kidney post mortem showed foamy cell transformation of glomerular epithelia, degeneration of tubular epithelia with red blood cell casts and urate crystals, and a mild proliferation of mesangial matrix. Analysis of the tissue content of Ge, prompted by her history, revealed an increased accumulation of the metal. As compared to a non-user died of liver cirrhosis, the concentration of the metal was higher particularly in the spleen (183X), thyroid gland (175X), psoas muscle (93X), jejunum (76X), and renal cortex (69X). So far, neither accumulation of Ge in humal tissue nor systemic toxicity of the Ge in human has been reported. The relevance of massive accumulation of Ge to the renal failure as well as to other systemic manifestations the patient presented remains to be clarified.

Detection of varicella-zoster virus genome in the vitreous humor from two patients with acute retinal necrosis; lacking or having a PstI cleavage site.

Using polymerase chain reaction, we detected the varicella-zoster virus genome in the vitreous humor of two patients with clinically diagnosed acute retinal necrosis. One of the two cases was thought to be caused by infection with a varicella-zoster virus lacking a PstI cleavage site. We could not find any clinical differences between the two substrains. The presence of a PstI cleavage site on the varicella-zoster virus genome might not be associated with the occurrence of acute retinal necrosis.

Evaluation of fungal DNA sequence using polymerase chain reaction in infection-free vitreous humor.

We evaluated the vitreous bodies from 14 clinically nonfungal infectious eyes to establish a baseline for the presence of a fungal genome using polymerase chain reaction (PCR). The vitreous samples were all PCR negative, except for one eye with chronic uveitis. Blood samples examined were all PCR negative. We concluded that fungal DNA sequences could not be amplified in vitreous specimens obtained from patients with clinically diagnosed proliferative diabetic retinopathy, macular hole, or epiretinal membrane.

Radiographical evaluation of bone maturation in Japanese black beef cattle.

Epiphyseal ossification in the radius, ulna, metacarpus, proximal phalanx and tuber calcaneus was examined radiographically in Japanese Black beef cattle. The grade of standard ossification was assessed monthly for each epiphysis. Bone maturations could be divided into 8 grades for the distal radius and distal ulna, 7 grades for the distal metacarpus, 5 grades for the proximal phalanx, and 8 grades for the tuber calcaneus, respectively. The closure of the epiphyseal line completed at the earliest (12-14 months of age) on the proximal phalanx, and at the latest (35-37 months of age) on the distal ulna. Changes in gradings were steep at 0 to 5 months of age but became almost constant after 10 months of age in all the epiphyses. There were no significant differences in bone maturation between the cattle with different sex and breeding conditions.

Precise measurements of the gamma-ray emission probabilities of 186Re and 188Re.

Gamma-ray emission probabilities of 186Re and 188Re are important in nuclear medicine, and have been precisely measured by 4pibeta-gamma coincidence using a live-timed two-dimensional data-acquisition system. The gamma-ray emission probabilities of the 137.2 keV for 186Re and 155.1 keV for 188Re were 0.09487+/-0.00029 and 0.15425+/-0.00072, respectively.

[MRI findings in studies of distal myopathy with rimmed vacuoles].

A case of distal myopathy with rimmed vacuoles was studied with MRI, which showed a characteristic distribution of the affected muscles. A 41-year-old man who presented a slowly progressive weakness in his lower legs starting 11 years previously was admitted to our hospital of further investigation. Neurological examinations showed muscular wasting and weakness in the neck flexors, the flexors of the forearm, the flexors and adductors of the thigh and the extensors of the lower legs. Needle electromyography showed a myopathic pattern. Muscle biopsy revealed a variation in fiber size, an increase in internal nuclei, fatty infiltration and scattered rimmed vacuoles in a histochemical study. Electron microscopy revealed that rimmed vacuoles contained numerous lamellar bodies and glycogen particles. T1 and T2 weighted MRI showed high signals in the m. adductor of thigh m. biceps femoris, m. semimembranosus, m. semitendinosus, m. tibialis anterior, m. tibialis posterior, m. extensor digitorum longus, m. extensor digitorum brevis, m. peroneus, and m. gastrocnemius. There were three merits for the application of MRI to distal myopathy, (1) easy detection of the affected muscles as fatty change is expressed with a high signal intensity by MRI, (2) no affection by the presence of bones in MRI, and (3) the possibility to have a transverse section and a sagittal and coronal section in MRI. In this case MRI was very useful to detect the affected muscles and to observe the progress.