Superovulation and embryo transfer in Holstein cattle using sexed sperm.

The use of sexed bull sperm in multiple ovulation and embryo transfer (MOET) programs for Holsteins was evaluated for (1) heifers housed at a commercial embryo transfer (ET) facility (Experiments 1 and 2), and (2) heifers and cows on dairy farms (Experiment 3). In Experiment 1, superstimulated heifers were inseminated with 5 x10(6) sexed (X-sorted; n=5) or unsexed (n=5) frozen-thawed sperm from one bull at 12 and 24h after estrus detection. No difference was observed in the rates of transferable embryos (53.4% vs 68.1%), degenerate embryos (24.8% vs 26.6%) and unfertilized ova (21.8% vs 5.3%) between sexed and unsexed sperm, respectively, except for the percent of female transferable embryos diagnosed by embryo sexing (100% vs 49.3%, P<0.0001). In Experiment 2, donors were inseminated twice with 5 x10(6) sexed unfrozen sperm (n=10) or sexed frozen-thawed sperm (n=9). Embryo production rates for both treatments were similar to that observed on a commercial ET facility using unsexed sperm. Pregnancy rates for frozen-thawed embryos were similar for sexed and unsexed sperm (70.4% vs 72.4%, respectively). In Experiment 3, 99 flushes were conducted using sexed frozen-thawed sperm from nine bulls but an overall statistical analysis was not completed because the use of bulls was not balanced. However, for one bull with balanced usage, the rate of transferable embryos was higher in heifers than in cows (P<0.05) inseminated twice with 5 x10(6) sperm/dose (10 x10(6) total). We concluded that the use of sexed frozen-thawed sperm (> or =90% X-sperm biased and 10 x10(6) total sperm) may be economically viable for commercial MOET programs in Holstein heifers.

The terminal oxidase of Photobacterium phosphoreum. A novel cytochrome.

The terminal oxidase of Photobacterium phosphoreum has been purified to the electrophoretically homogeneous state and some of its properties have been studied. The enzyme catalyses oxidation of ascorbate in the presence of phenazine methosulphate or N,N,N',N'-tetramethyl-p-phenylenediamine. The reaction is inhibited by cyanide. Nitrite at comparatively high concentrations inhibits the enzyme, but the enzyme does not catalyse nitrite reduction with ascorbate plus the electron mediator as the electron donor. The enzyme shows the absorption peaks at 632, 565, 534 and 436 nm in the reduced form. It has two kinds of haems: protohaem and haem d. Namely, the enzyme is a 'cytochrome bd'-type oxidase; a novel cytochrome.

Detection of regional lymph node metastases in colon cancer by using RT-PCR for matrix metalloproteinase 7, matrilysin.

Lymph node metastasis is the most important prognostic factor in colon cancer. However, more accurate screening for metastasis than that afforded by conventional pathology remains elusive. We have employed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for a matrix metalloproteinase (MMP), 'matrilysin', because this gene is epithelial-specific and consistently expressed in colorectal cancer cells. The sensitivity of this assay was examined with the matrilysin-producing rectal cancer cell line 'CaR-1'. Matrilysin mRNA was detected in this system when more than 10(4) matrilysin-positive cells existed in a lymph node of ordinary size. Fourteen of 15 (93%) primary colon cancers and none of the surrounding normal tissues expressed matrilysin. All 10 histologically-positive lymph nodes were positive for matrilysin, while of 60 histologically-negative lymph nodes, eight were positive for matrilysin. When the additional sequential sectioning and histological re-examination was performed on five of these eight 'matrilysin-positive, but histologically-negative' lymph nodes, micrometastases were detected in three. Only one of the lymph nodes that were histologically-positive, but negative by matrilysin assay was from a patient with colon cancer in which matrilysin was not detected. In conclusion, RT-PCR assay for matrilysin is a sensitive method for detecting occult metastases in patients with colon cancer, and may complement histologic examination.

Expression of highly polysialylated neural cell adhesion molecule in pancreatic cancer neural invasive lesion.

Neurotropism of pancreatic cancer is one of the hypotheses explaining neural invasion, which is one of the characteristics of pancreatic cancer. In these studies, we immunohistochemically examined neural cell adhesion molecules (NCAM), homophilic adhesion molecules expressed on the nerve cells, as a factor of neurotropism, in 15 pancreatic cancer operatively obtained, especially in neural invasive lesions. We also investigated the role of polysialic acid (PSA), which is attached to NCAM and related to the malignant potential of cancers. NCAM was detected in 66.7% of pancreatic cancers, and in all 9 cases with massive perineural invasion. In neural invasive lesions, however, there were perineurium and endoneurium, which do not express NCAM, between the cancer and nerve cells. PSA was also detected in the pancreatic cancers expressing NCAM. Moreover, PSA expression was stronger in the perineural invasive lesions than in the main tumor and was related to the cancer cell proliferation investigated by Ki-67 staining. It is unlikely therefore, that NCAM plays an important role in neurotropism. However, the NCAM expressed on the pancreatic cancer was attached to PSA, which itself plays an important role in the malignant potential of this disease.

Luminescence and respiratory activities of Photobacterium phosphoreum. II. Control by monovalent cations.

Luminescent activity of spheroplasts of the cells of Photobacterium phosphoreum was stimulated by Rb+ and K+ and inhibited by Na+ in the medium. Opposite effects of these ions were observed on the rate of O2 consumption of the spheroplasts through the cytochrome and luciferase electron transfer systems. In vitro activities of NADH-FMN reductase and luciferase were only slightly stimulated by Rb+, K+, and Na+, while Na+ exhibited significant activation of the NADH-oxidizing activity of the cells. The redox level of cytochrome b in the spheroplasts during steady-state respiration in an Na+ medium was more reduced, while that of NAD(P) was more oxidized, than those in an Rb+ or K+ medium. Na+ activates the cytochrome electron transfer system at a point between NADH and cytochrome b, and thus has a stimulative effect on cellular O2 consumption. Rb+ and K+ do not show similar activation, but the cellular NAD(P) was brought to a more reduced level, so that in vivo luminescence is stimulated in an Rb+ or K+ medium.

Luminescence and respiratory activities of Photobacterium phosphoreum. Competition for cellular reducing power.

Changes in the in vivo luminescence, respiratory activities, contents of cytochromes, extractable luciferase and NAD(P)H-FMN reductase during growth of the wild (bright) strain of Photobacterium phosphoreum and its dim mutant were determined. The intensity of the in vivo luminescence per cell increased 10 times in the wild strain and 750 times in the dim strain during logarithmic growth, while the contents of luciferase and NAD(P)H-FMN reductase remained almost constant. It is suggested that a characteristic change in the mode of competition of the luminescence reaction system with another electron transfer chain involving cytochromes for NAD(P)H take place during the growth of this bacterium.

Segmental mediolytic arteritis [correction of arteries]: a case report with review of the literature.

Segmental mediolytic arteritis is a very rare vascular disease which causes sudden intraabdominal hemorrhage. The disease is characterized by degeneration of the arterial media, followed by aneurysmal dilatation and rupture of the involved artery. Up to now, only 13 cases have been reported, and this unique disease is not fully recognized among general pathologists and physicians. Here, we present a case of segmental mediolytic arteritis involving the propria hepatic artery, which resulted in intraabdominal hemorrhage, and consequently hypovolemic circulatory disturbance. Histologically, the rupture focus showed degeneration and desquamation of the intima and media with fibrin-like material covering the exposed adventitia. Inflammatory infiltrates were only noted in the rupture focus as a secondary reactive change. Other than the rupture focus, there were two foci showing similar findings. This disease has rarely been reported and is seldom recognized as a cause of arterial rupture. In cases of sudden intraabdominal hemorrhage, segmental mediolytic arteritis should be considered as a possible cause in addition to atherosclerotic and mycotic aneurysm, traumatic injury and vasculitis syndromes.

High-level expression, purification, and some properties of a recombinant cephalosporin-C deacetylase.

To maximize the expression of the cephalosporin-C deacetylase (CAH) gene isolated from Bacillus subtilis SHS 0133 in Escherichia coli, a series of expression plasmids was constructed with various spacings between the Shine-Dalgarno sequence and the ATG initiation codon. As the most efficient expression plasmid, we selected pCAH431, which has the trp promoter, a replication origin derived from pAT153, and a spacing of 13 nucleotides. E. coli JM103 with pCAH431 produced 4.9 g of CAH per liter on cultivation at 37 degrees C for 20 h in a 30-l jar fermentor. Since the amount of CAH reached about 70% of the total protein in the soluble fraction of the cells, and CAH was recovered from the cell extracts in an active form, the CAH was purified easily to homogeneity by only one column chromatography step. Twenty grams of 7-aminocephalosporanic acid was completely converted to deacetyl-7-aminocephalosporanic acid, a starting material for cefcapene pivoxil hydrochloride, by 12 mg of the purified enzyme without significant appearance of by-products. Thus, our expression and purification system has made the industrial production of CAH possible.

Segmental pericholangial fibrosis: a peculiar benign fibrosing disease at the hepatic hilum.

The authors report on a 9-year-old child who underwent surgery to remove a tumor of the hepatic hilum with preoperative radiographic studies suggestive of malignancy, but whose surgical specimens showed a peculiar fibrosing disease. The lesion was localized to the bifurcation of the hepatic duct, where the bile duct wall and the surrounding tissue was markedly fibrotic. No malignant cells or epithelial destruction were seen. The patient's postoperative course was uneventful, and he is without any sign of recurrence 2 years after surgery. Because the histological features of this case do not correspond to any established disease, including primary sclerosing cholangitis, the authors believe it represents a new entity, segmental pericholangial fibrosis. Local resection resulted in a good outcome. A review of the literature disclosed a few similar cases with a benign clinical course.

[Hepatic resection for advanced carcinoma of the gallbladder].

Hepatic resection for advanced carcinoma of the gallbladder must be decided upon based on the modes of cancer spread to the liver. The cystic vein through the liver bed is considered an important route of liver metastasis, because liver metastases of gallbladder carcinoma are found frequently around the liver bed. About 70% of early metastatic foci demonstrated microscopically occur in segments 4a and 5. Resection of segments 4a and 5 is considered to be an adequate range of hepatectomy for patients with subserosal invasion, because early metastatic foci are detected not only in patients with direct invasion of the liver but also in those without direct invasion. For patients with direct liver invasion, various degrees of hepatic resection are needed to comply with the depth of direct invasion. It is necessary to achieve negative surgical margins 2 cm from the tumor. Because cancer cells extend along the Glissonian sheath in patients with hilar invasion, extended right hepatectomy with caudate lobectomy is required in these patients. A future problem is to establish the safety of extended hepatectomy in these patients.

[Mode of spreading and biological behavior in bile duct carcinoma].

There are three macroscopic types of hepatic bile duct carcinoma, such as papillary (P-), nodular (N-) and diffuse (D-) type. Immunohistochemical studies demonstrated that P-type expressed cadherin and catenin higher than N- and D-types. The expressions of both cadherin and catenin were found stronger in pap and tub1 than tub2. The nuclear area of cancer cell, correlated with both labeling index of Ki-67 and aberrant accumulation of p53, was significantly larger in the subserosal layer than in the mucosal layer. These may explain the differences in the biological behavior between P- and N, D-types. P-type grows within the mucosal layer, while N- and d-type are more invasive, developing into the subserosal layer. Our clinical data also demonstrates the poor prognosis of N-, D-type of hepatic bile duct carcinoma. On these basis of the biological malignancy of N, D-type, it is critical to remain the surgical margin free from cancer cells to cure this type of hepatic bile duct carcinoma.

Differential expression of Tenomodulin and Chondromodulin-1 at the insertion site of the tendon reflects a phenotypic transition of the resident cells.

The insertion site of the tendon to the skeletal element is hypovascular and is one of the most common sites of dysfunction in the musculoskeletal system. However, the resident cells have been poorly defined due to a lack of a specific marker for tenocytes. We previously reported that Tenomodulin (Tnmd) and Chondromodulin-1 (Chm1) are homologous angiogenesis inhibitors and predominantly expressed in the avascular region of tendons and cartilage, respectively. In this study, we analyzed the expression of Tnmd, Chm1, alpha 1 chain of the type I collagen (Col1a1) and alpha 1 chain of the type II collagen (Col2a1) at the insertion site of the Achilles, patellar, or rotator cuff tendons of 1-week-old rabbits by in situ hybridization analysis. Tnmd was co-expressed with Col1a1 in tenocytes of these tendons, while Chm1 and Col2a1 were detected in chondrocytes of the hyaline cartilage. Interestingly, the cell population between Tnmd/Col1a1 positive tenocytes and Chm1/Col2a1 positive chondrocytes expressed Col1a1 but none of the other markers (Tnmd, Chm1, and Col2a1). Red blood cells were exclusively present at the interface between the tendon substance and cartilage in the insertion site of the Achilles tendon. Lack of Tnmd and Chm1 in this newly characterized cell population may allow the transitional zone between the poorly vascularized tendon and cartilage to establish the unique vascular pattern for blood supply.

Photoaddressed spatial light modulator using transmissive and highly photosensitive amorphous-silicon carbide film.

A novel photoaddressed spatial light modulator with a highly photosensitive hydrogenated amorphoussilicon carbide photoreceptor and a ferroelectric liquid-crystal layer has been developed that operates in a transmission mode for visible light. The hydrogenated amorphous-silicon carbide photoreceptor shows a high photosensitivity comparable with that of hydrogenated amorphous silicon, which is prepared by a plasma chemical vapor deposition method with helium dilution of the source gases SiH(4) and C(2)H(2) An image is input to the photoacdressed spatial light modulator by blue or green light, and an output is read out by red light as a transmitted image. The photoaddressed spatial light modulator exhibits a response time of ~50 µs and a contrast ratio of 30:1 under a write light (λ = 565 nm) of 1.5 mW/cm(2) intensity and a resolution of 90 line pairs/mm.

Syntheses based on the tautomerism of purine and pyrimidine derivatives.

Synthetic applications of the tautomerism based on purine and pyrimidine derivatives were investigated. Particularly, chloropyrimidine derivatives, which were reactive enough to go back to the more stable lactam form, were used for dehydration and desulfhydration. Alkoxypyrimidines were thermally cleaved to give olefins or esters, together with the lactim to lactam transformation. The extrusion of alkylthio group from thioalkyl purines or pyrimidines took place under bacic conditions to give olefin, indicating another tautomeric transformation.

Gene cloning, nucleotide sequence, and expression of a cephalosporin-C deacetylase from Bacillus subtilis.

The gene encoding a cephalosporin-C deacetylase (CAH) from Bacillus subtilis SHS 0133 was cloned and sequenced. The nucleotide sequence contained an open reading frame encoding a polypeptide consisting of 318 amino acids, the molecular weight of which was in good agreement with the value obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence contained the common sequence Gly-X-Ser-X-Gly found in many esterases, lipases, and serine proteases. This indicates that CAH is a serine enzyme. A possible promoter sequence which is very similar to the consensus sequences of -35 and -10 regions recognized by B. subtilis RNA polymerase utilizing sigma factor H was found in the 5'-flanking region of the CAH structural gene. Two repeated A+T-rich blocks consisting of 24 bp were also found in the upstream region of the initiation codon. We constructed a series of expression plasmids by inserting the CAH gene into Escherichia coli ATG vectors. The degree of CAH gene expression depended on promoters and vector plasmids, which have different replication origins. The expressed CAH protein was an active form in the soluble fraction obtained after cell disruption. The highest expression level was accomplished with an expression plasmid, pCAH400, which has the trp promoter and the replication origin derived from pAT153. In the fermentation using a 30-liter jar fermentor, the transformant E. coli JM103(pCAH400) produced 440 U of CAH per ml of culture during a 24-h incubation. This value corresponded to 2.1 g of CAH protein in 1 liter of culture broth.

Optical neural network using vector-feature extraction.

An optical neural network model and a key device for its optical implementation are proposed. The model, named the vector-feature-extracting optical neural network, can correctly recognize hand-written letters and can easily be implemented in optics. The key device, named a feature-extracting optical neuron device, can selectively extract specific line segments included in an optical input pattern. In this paper the structure and recognition process of the vector-feature-extracting optical neural network are shown in detail. The function of the feature-extracting optical neuron device is experimentally shown. In addition, the optical system based on the vector-feature-extracting optical neural network is described.

Denitrification, a novel type of respiratory metabolism in fungal mitochondrion.

Subcellular localization and coupling to ATP synthesis were investigated with respect to the denitrifying systems of two fungi, Fusarium oxysporum and Cylindrocarpon tonkinense. Dissimilatory nitrate reductase of F. oxysporum or nitrite reductase of C. tonkinense could be detected in the mitochondrial fraction prepared from denitrifying cells of each fungus. Fluorescence immunolocalization, cofractionation with mitochondrial marker enzymes, and cytochromes provided evidence that the denitrifying enzymes are co-purified with mitochondria. Respiratory substrates such as malate plus pyruvate, succinate, and formate were effective donors of electrons to these activities in the mitochondrial fractions. Moreover, nitrite and nitrate reduction were shown to be coupled to the synthesis of ATP with energy yields (P:NO3- or P:2e ratios) of 0.88 to 1.4, depending upon whether malate/pyruvate or succinate were provided as substrates. Nitrate or nitrite reductase activity was inhibited by inhibitors such as rotenone, antimycin A, and thenoyltrifluoroacetone. Thus, fungal denitrification activities are localized to mitochondria and are coupled to the synthesis of ATP. The existence of these novel respiration systems are discussed with regard to the origin and evolution of mitochondria.

Identification of a component that induces flowering of Lemna among the reaction products of alpha-ketol linolenic acid (FIF) and norepinephrine.

A stress-induced fatty acid [FIF; 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] incubated with (-)-norepinephrine (NE) strongly induces flower formation in Lemna paucicostata [Yokoyama et al. (2000), Plant Cell Physiol. 41: 110). The increase of flower-inducing activity was well correlated with the decrease in FIF in the incubation mixture, and the reaction proceeded rapidly at higher pH. We detected small amounts of many active components in the mixture after incubation by HPLC analysis. In this study, two major components, named FN1 and FN2, of the reaction mixture were isolated, and their absolute stereostructures were determined. FN1 showed a strong flower-inducing activity and was identified as a tricyclic alpha-ketol fatty acid, 9(R)-11-[(2'R,8'R,10'S,11'S)-2',8'-dihydroxy-7'-oxo-11'-[(Z)-2-pentenyl]-9'-oxa-4'-azatricyclo[6.3.1.0(1.5)]dodec- 5'en-10'-yl]-9-hydroxy-10-oxoundecanoic acid [corrected]. FN2, the C-9 epimer of FN1, showed no flower-inducing activity. The absolute stereostructure of FIF was also determined by a modification of Mosher's method. The 9-hydroxyl group was found to be predominantly 9R, with an enantiomeric excess of 40% (70% 9R and 30% 9S). FN1 was derived from 9R-type FIF and FN2 from 9S-type FIF. Various catecholamines and related substances were investigated for the ability to develop flower-inducing activity upon incubation with FIF. The essential structures were catechol and ethylamine groups (dopamine).

Stress-induced factor involved in flower formation of Lemna is an alpha-ketol derivative of linolenic acid.

A stress-induced substance(s) (factor C) incubated with norepinephrine (NE) has strong flower-inducing activity in Lemna paucicostata. We isolated an essential component (FIF) of factor C, and clarified its chemical structure as 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid, an alpha-ketol derivative of linolenic acid, which is formed via 9-hydroperoxy linolenic acid. Synthesized FIF showed flower-inducing activity after incubation with NE (factor C activity) equivalent to that formed in the stressed Lemna. Jasmonic acid and 13-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoic acid (12,13-alpha-ketol linolenic acid), both of which are formed via 13-hydroperoxide of linolenic acid and all other derivatives of FIF synthesized by chemical and enzymatic processes failed to show the factor C activity. These results suggest that the molecular structure of FIF is very specific for the factor C activity.