Simultaneous production of sphingolipids and ethanol by Kluyveromyces thermotolerans.

Kluyveromyces thermotolerans strain NBRC 1674 was selected for the simultaneous production of sphingolipids and ethanol from beet molasses. The strain gradually synthesized ethanol with fermentation periods and attained a level slightly higher than that of the strain of Saccharomyces cerevisiae usually used for ethanol production. The sphingolipids accumulated in the cells were composed of almost equal amounts of free ceramides and glucosylceramides. The sphingoid bases and fatty acids of the two sphingolipids differed from each other and changed under aerobic and anaerobic growth conditions. Oxygen limitation may cause accumulation of sphinganine by inhibiting sphingolipid desaturases and enhance its proportion in both the sphingolipids.

Rare case of biphasic tumor of the breast with prominent CD34-positive spindle cells.

We report an unusual case of biphasic tumor of the breast with prominent CD34-positive spindle cells. A 53-year-old woman presented with a mass in her right breast. Following an incisional biopsy, a partial mastectomy was done. Histologically, the tumor was a biphasic variant of a malignant spindle cell tumor of the breast. The lack of a leaf-like structure, together with the apparent myoid features of the spindle-shaped tumor cells, made it difficult to distinguish from malignant phyllodes tumor and from myoepithelial carcinoma. Immunohistochemistry revealed the spindle-shaped tumor cells to be myofibroblastic, but not myoepithelial in nature, ultimately categorizing this tumor as a malignant phyllodes tumor with prominent myofibroblastic differentiation.

Interaction between DNA-cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: the role of the neutral helper lipid.

Recent studies have indicated that there are many barriers to successful systemic gene delivery via cationic lipid vectors using the intravenous route. The purpose of this study was to investigate the effect of binding and interaction between erythrocytes, a major constituent of blood cells, and the complexes, in relation to the role of the helper lipid, on the in vivo gene delivery to the lung following intravenous injection. We used three types of cationic lipid vectors, DNA-DOTMA/Chol liposome complexes, DNA-DOTMA liposome complexes, and DNA-DOTMA/DOPE liposome complexes. Although the three types of vectors bind to murine blood cells in vivo and in vitro, DOTMA/Chol and DOTMA complexes with a higher in vivo transfection activity do not induce fusion between erythrocytes, whereas DOTMA/DOPE complexes, a less efficient vector in vivo, induce fusion between the erythrocytes after a short incubation period. Pre-incubation of DOTMA/DOPE complexes with erythrocytes significantly reduced the transfection efficiency while DOTMA/Chol- and DOTMA complexes were more resistant to such treatment. The differences in the physicochemical and structural properties of these complexes could explain the differences in interaction with erythrocytes and subsequent gene expression. Lipids in DOTMA/Chol and DOTMA complexes have a stable lamellar structure. However, lipids in DOTMA/DOPE complexes have a highly curved structure with high fluidity. These results indicate that the interaction with erythrocytes depends on the properties of the cationic lipid vectors and this is an important factor for intravenous gene delivery using cationic lipid vectors.

[A comparative study on the clinical utility of cefozopran and cefpirome against complicated urinary tract infections].

Aiming at evaluating the utility of cefozopran (CZOP) against complicated urinary tract infections with the velocity of eradication of causal bacteria in early treatment and clinical efficacy by new criteria of UTIs, a comparative study was conducted using cefpirome (CPR) as the control drug. CZOP and CPR were administered by intravenous drip infusion at a dose of 1 g twice daily. The duration of treatment was for 5 days. The study method involved randomized assignment of the subjects to either group CZOP or group CPR. The results were as follows: 1. Of a total of 80 cases treated, 65 (CZOP group--32 cases, CPR group--33 cases) were evaluated for efficacy. 2. The overall clinical efficacy evaluation according to the criteria proposed by Japanese UTI Committee rated the CZOP group as 90.6% (29/32), and the CPR group as 90.9% (30/33), with no significant difference between the 2 groups. Clinical efficacy evaluated by attending physicians rated the CZOP group as 93.8% (30/32) and the CPR group as 90.9% (30/33). There was no significant difference between the 2 groups. 3. The efficacy rates to pyuria on day 2 were 26.7% and 0% for the CZOP group and the CPR group, respectively, indicating a higher efficacy rate for the former (p < 0.05). Those on after treatment were 59.4% and 54.5% for the CZOP group and the CPR group, respectively, with no significant difference between the 2 groups. 4. Regarding the bacteriological effect, the eradication rates of both groups were over 90% on day 1 and after treatment. There was no significant difference between the 2 groups. 5. Side effects occurred in 1 case (2.6%) out of 39 in the CZOP group and in 1 case (2.4%) out of 41 in the CPR group. Laboratory test value fluctuation was noted in 8 (20.5%) of 39 cases in the CZOP group and 11 (26.8%) of 41 cases in the CPR group. There was no significant difference between the 2 groups. The results indicate that CZOP achieves an early efficacy to pyuria, and is as useful as CPR against complicated urinary tract infections.

Assessment of health-related quality of life in renal transplant and hemodialysis patients using the SF-36 health survey.

OBJECTIVES: To determine whether the health-related quality of life (HQOL) for renal transplant patients improved using SF-36 survey scores and to examine which clinical measures after renal transplantation are connected to aspects of their HQOL. METHODS: A total of 117 renal transplant patients and 114 hemodialysis patients, including 49 awaiting transplantation and 65 not awaiting transplantation, were included in this study. The scale scores of the SF-36 survey concerning HQOL were compared between the two groups of patients. The relationships of the clinical episode and complications with the scale scores were examined. RESULTS: The renal transplant patients had significantly higher scores in the physical functioning, bodily pain, general health, and social functioning scales than did the hemodialysis patients. The role-physical functioning, bodily pain, and social functioning scales of the transplant patients were significantly higher than those of the hemodialysis patients not awaiting transplantation. In contrast, the scores, except for that of general health, of the transplant patients were not significantly different from those of the hemodialysis patients awaiting transplantation. Multiple regression analysis demonstrated that the scale scores of physical functioning, general health, and vitality were significantly dependent on the serum level of creatinine in the renal transplant patients (P <0.05). The scores of physical functioning and general health of the patients with a creatinine level >2 mg/dL were significantly lower than those of the patients with 1 mg/dL < creatinine level

Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression.

In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA complexed with the cationic liposomes, which is an important determinant of the optimal mixing ratio in cationic liposome-mediated transfection.

Klinefelter's syndrome in the male infertility clinic.

The clinical features of patients with Klinefelter's syndrome attending a male infertility clinic have been investigated in order to consider their assisted reproduction treatment options. Over 12 years, a total of 148 patients with sterility due to azoospermia had Klinefelter's syndrome. Eight patients were shown by fluorescence in-situ hybridization (FISH) on metaphase spreads to be mosaic (46,XY/47,XXY), and 140 patients showed only 47,XXY. Small testes were observed in 95% of patients and gynaecomastia was seen in 12.4%. Half of the patients showed hypergonadotrophic hypogonadism, while others showed normogonadism (usually hypergonadotrophic). Spermatozoa were observed in semen from one patient with mosaicism and one without. Three-colour FISH revealed hyperploidy in 2.7% and 2.3% of these spermatozoa respectively. Multiple-site testicular biopsies in five recent patients were performed and yielded a specimen with round and elongated spermatids in one patient with 47,XXY karyotype. This sample was cryopreserved for future intracytoplasmic sperm injection. At follow-up, 46% of couples had chosen artificial insemination with donor sperm, and none had chosen adoption. Two patients developed testicular tumours, one a mature teratoma and the other a Leydig cell tumour. Two patients required androgen replacement therapy.

Relationship of acrosin activity to sperm function tests.

Acrosin activity of spermatozoa from infertile patients and fertile volunteers was measured. Acrosin activity of spermatozoa from asthenozoospermic patients and patients with unexplained infertility was lower than that from fertile volunteers. We utilize the zona-free hamster egg-sperm penetration test to select candidates for conventional in vitro fertilization among unexplained infertile patients. The zona-free hamster egg sperm penetration test, however, requires several hours and special equipment which are not used in the clinical setting. It is preferable that other sperm function tests or a combination of them can replace this test. Thus three distinct tests of sperm function, namely, acrosin activity, Penetrak test, hypo-osmotic swelling test, were compared with the hamster test, using spermatozoa from patients with unexplained infertility. A combination of the Penetrak test and measurement of acrosin activity could predict the results of the zona-free hamster egg sperm penetration test with 88.2% accuracy. Thus, the hamster test should be done when either Penetrak test or measurement of acrosin activity showed abnormal values.

Tamsulosin and chlormadinone for the treatment of benign prostatic hyperplasia. The Kobe University YM617 Study Group.

The recent introduction of selective alpha-adrenoceptor blockers adds a further therapeutic option for the treatment of benign prostatic hyperplasia (BPH). Tamsulosin, a selective alpha 1-blocker, has proved effective in relieving irritative and obstructive symptoms caused by BPH. To investigate whether the combination of tamsulosin with the anti-androgenic drug chlormadinone is of further therapeutic benefit, 80 patients randomly received tamsulosin 0.2 mg daily, chlormadinone 50 mg daily or a combination of tamsulosin 0.2 mg and chlormadinone 50 mg daily for 16 weeks. Greater improvement in subjective symptoms of BPH was obtained with either tamsulosin alone or in combination with chlormadinone than with chlormadinone alone. However, the greatest improvement in objective uroflowmetric data was obtained with chlormadinone in combination with tamsulosin. Thus, the combination of tamsulosin with chlormadinone appears to be more beneficial than either of these agents used as monotherapy. Further investigation is required to fully evaluate the therapeutic effects of this combination. After the trial period one-third of the chlormadinone and tamsulosin/chlormadinone-treated patients needed no further treatment due to the satisfactory relief of symptoms. At 12 months follow-up, however, one-fourth of the patients had undergone transurethral resection of the prostate (TUR-P) regardless of medication. This suggests a limitation of the medical treatment of BPH.

Crystallization and preliminary X-ray analysis of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans.

Three crystalline forms of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans were obtained by hanging-drop vapor-diffusion methods. One of them, which had crystallized under slightly milder conditions than the others, was suitable for X-ray analysis. Its asymmetric unit contains one dimeric molecule and the solvent content is higher than in other protein crystals. The crystal structure was solved in a preliminary manner by the molecular-replacement technique.

Crystal structure of a pyrimidine dimer-specific excision repair enzyme from bacteriophage T4: refinement at 1.45 A and X-ray analysis of the three active site mutants.

Crystallographic study of bacteriophage T4 endonuclease V, which is involved in the initial step of the pyrimidine dimer-specific excision repair pathway, has been carried out with respect to the wild-type and three different mutant enzymes. This enzyme catalyzes the cleavage of the N-glycosyl bond at the 5'-side of the pyrimidine dimer, and subsequently incises the phosphodiester bond at the apyrimidinic site through a beta-elimination reaction. The structure of the wild-type enzyme refined at 1.45 A resolution reveals the detailed molecular architecture. The enzyme is composed of a single compact domain classified as an all-alpha structure. The molecule is stabilized mainly by three hydrophobic cores, two of which include many aromatic side-chain interactions. The structure has a unique folding motif, where the amino-terminal segment penetrates between two major alpha-helices and prevents their direct contact, and it is incompatible with the close-packing category of helices for protein folding. The concave surface, covered with many positive charges, implies an interface for DNA binding. The glycosylase catalytic center, which comprises Glu23 and the surrounding basic residues Arg3, Arg22 and Arg26, lie in this basic surface. The crystal structures of the three active-site mutants, in which Glu23 was replaced by Gln(E23Q) and Asp (E23D), respectively, and Arg3 by Gln (R3Q), have been determined at atomic resolution. The backbone structures of the E23Q and R3Q mutants were almost identical with that of the wild-type, while the E23D mutation induces a small, but significant, change in the backbone structure, such as an increase of the central kink of the H1 helix at Pro25. In the catalytic center of the glycosylase, however, these three mutations do not generate notable movements of protein atoms, except for significant shifts of some bound water molecules. Thus, the structural differences between the wild-type and each mutant are confined to the remarkably small region around their replaced chemical groups. Combined with the biochemical studies and the difference circular dichroism measurements, these results allow us to conclude that the negatively charged carboxyl group of Glu23 is essential for the cleavage of the N-glycosyl bond, and that the positively charged guanidino group of Arg3 is crucial to bind the substrate, a DNA duplex containing a pyrimidine dimer. The amino terminal alpha-amino group is located at a position approximately 4.4 A away from the carboxyl group of Glu23. These structural features are generally consistent with the reaction scheme proposed by Dodson and co-workers.

Structural composition of hammerhead ribozymes.

Eleven kinds of hammerhead ribozymes were designed and synthesized to investigate the structural composition of the complexes and to find suitable crystallization conditions, the substrate chains having been modified to prevent hydrolysis. Electrophoresis patterns indicated that the two strands except for the substrate chain form a binary complex and that they form a ternary complex when mixed with the substrate chain. Both complexes were crystallized. The crystal of the binary complex belongs to a Laue symmetry of 32 (space group of P321, P3(1)21, or P3(2)21) with cell dimensions of a = b = 53.4 and c = 59.4 A. The volume per one nucleotide allows the asymmetric unit to contain one binary complex. Our results suggest that the catalytic part forms a rigid ribozyme structure which induces a scissile reaction when the substrate is bound, in a similar manner to an enzyme protein.

Müllerian duct cyst with 46, XYq-.

We report a case of a müllerian duct cyst associated with a 46, XYq- chromosome anomaly. Müllerian duct cyst is a consequence of the abnormal regression of paramesonephric derivative. In the past many different manifestations have been associated with the 46, XYq-, anomaly. We discuss the possibility that a müllerian duct cyst may be one clinical manifestation of 46, XYq-.

The significance of gonadotropin-releasing hormone test for predicting fertility after varicocelectomy.

To predict the surgical outcome in 30 infertile men with varicocele, endocrinologic evaluation by the GnRH test was performed before and 6 months after the high ligation of the left internal spermatic vein. Semen analysis was performed and the levels of basal T and PRL were examined preoperatively and 6 months postoperatively. In the pregnancy group, the preoperative excessive response of LH to the GnRH test decreased significantly after surgery. Significant reduction in the LH response is considered to be a valuable parameter for predicting fertility.

Müllerian duct cyst with 46, XYq-.

We report a case of a müllerian duct cyst associated with a 46, XYq- chromosome anomaly. Müllerian duct cyst is a consequence of the abnormal regression of paramesonephric derivative. In the past many different manifestations have been associated with the 46, XYq-, anomaly. We discuss the possibility that a müllerian duct cyst may be one clinical manifestation of 46, XYq-.

[Ultrastructure of immotile spermatozoa obtained from infertile male patients].

We sometimes experienced infertile patients whose sperms had no motility but were not stained by Eosin Y. In this paper we report five cases of so-called "immotile spermatozoa". The ultrastructure of sperm tails was examined by transmission electrone microscope (TEM). These cases were selected from the out-patient population who attended infertility clinic of our department. The semen analyses showed that all the cases had sperm motility below 1% and more than 90% of the spermatozoa were proven alive. Family history revealed that one case had an infertile sibling. None of them had situs inversus, bronchiectasis and chronic sinusitis which are classic trias of Kartagener's syndrome. They had no symptoms of upper respiratory tract infection which was caused by the abnormality in the flagella of the respiratory tract. The TEM pictures of sperm tails showed partial deletion of inner dynein arms in two cases, lack of central microtubular doublets (so-called 9 + 0) in two cases and disarrangement of microtubular doublets in one case. For the treatment of these cases there is no effective means but AID. However, the rapid progress of IVF-ET techniques and a report that the spermatozoa from Kartagener's syndrome had showed penetration into eggs encouraged us to think the micromanipulation of spermatozoa with IVF-ET as a hopeful option of the treatment in the near future.