Cancer-associated expression of glycolipid sulfotransferase gene in human renal cell carcinoma cells.

Human renal cell carcinoma (RCC) tissue and a cell line derived therefrom, SMKT-R3, showed markedly increased glycolipid sulfotransferase [cerebroside sulfotransferase (CST); EC 2.8.2.11] activity and accumulated sulfoglycolipids. Recently, we cloned a human CST cDNA from a SMKT-R3 cDNA library (K. Honke et al., J. Biol. Chem., 272: 4864-4868, 1997). In this study, we investigated the expression of the CST gene in seven human RCC lines (SMKT-R1, SMKT-R2, SMKT-R3, SMKT-R4, TOS-1, TOS-2, and ACHN) and their normal counterpart, human renal proximal tubular cells. On Northern blot analysis, a marked increase of CST mRNA was observed in every RCC line, except for ACHN, as compared with normal cells. ACHN cells showed a slightly increased level of CST mRNA. CST activity was correlated with the amount of mRNA. Sulfoglycolipid analysis revealed that expression of lactosylceramide sulfate was correlated with the CST level. Furthermore, we examined the effects of epidermal growth factor (EGF), tetradecanoylphorbol-13-acetate, and genistein, which are known to regulate CST activity in SMKT-R3 cells, on CST-gene expression in various RCC cells. On treatment with EGF, CST mRNA time-dependently increased in accord with its activity in SMKT-R3 cells. Yet, augmentation by EGF was only observed in SMKT-R3. In contrast, a reduction of CST mRNA and activity by tetradecanoylphorbol-13-acetate and genistein was observed in all of the lines examined. Taken together, these findings indicate that in human RCC cells, the CST gene is generally overexpressed via a signaling pathway involving protein kinase-C and tyrosine kinases.

[Simultaneous analysis and detection of pesticides in fresh fruits and vegetables by HPLC and GC].

A method was established for simultaneous determination of pesticide residue in fresh fruits and vegetables by HPLC and GC. CH3CN extraction/NaCl partiton method was used in order to recover hydrophilic pesticide such as acephate, methamidophos. Dimethoate and methamidophos from okra and DDVP from strawberry were detected by GC. On the other hand confirmation method by GC and GC/MS was studied for peaks detected by HPLC with UV and/or FL detector. OPP, TBZ, imazalil chlorpyrifos etc. in citrus fruits were detected by the proposed method.

[Current status and problems of home care for patients with terminal cancer from the viewpoint of local clinics].

On the basis of investigations of 15 patients from our clinic with terminal cancer who were treated by home hospice care, and questionnaires filled out by their caretakers, we examined the current status and problems of the home hospice care system with respect to four phases, namely, the period of preparation for home care (hospitalization period), stable period, terminal period, and the period immediately before death. [Preparation period] The following problems occurred in this phase: introduction of pain management and nutrition management was insufficient; there were only a few cases in which the patient chose home care of his or her own will; and sufficient instructions were not given to caretakers on discharge from the hospital, with respect to medical treatment at home. [Stable period] In two of the four cases in which patients complained of severe pain, the pain was not alleviated because pain management was provided only at the outpatient clinics of the hospital, and collaboration between hospitals and our clinic was insufficient. [Terminal period] Two patients could not be admitted to the hospital upon sudden exacerbation of the condition, suggesting the need to establish a system in which large hospitals can cope with sudden exacerbation of their condition of patients with terminal cancer treated at home. [Period immediately before death] Of the 14 patients who died, 7 died at home and 7 died in the hospital or during transport to the hospital. Three subjects died within a few days after admission. Two of the subjects who died in the hospital or during transport had hoped to stay home until the last moment. Further improvement of the system is necessary in order to meet the needs of terminal cancer patients who wish to die at home. On the basis of the cases taken care of at our clinic, we examined the home care system according classification into three types; hospital-outpatient clinic type; hospital-home care type; and clinic-home care type. An ideal system for the treatment of patients with terminal cancer who hope to stay at home until the last possible moment seems to be the clinic-home care type in which a primary care team that is able to dispatch physicians and nurses, and an around-the-clock support system, are supported by outside organizations and specialists.

17ß-Estradiol and 17α-estradiol induce rapid changes in cytoskeletal organization in cultured oligodendrocytes.

Dramatic changes in the cytoskeleton and the morphology of oligodendrocytes (OLs) occur during various stages of the myelination process. OLs in culture produce large membrane sheets containing cytoskeletal veins of microtubules and actin filaments. We recently showed that estrogen receptors (ER) related to ERα/ß were expressed in the membrane sheets of mature OLs in culture. Ligation of these or other membrane ERs in OLs with both 17ß- and 17α-estradiol mediated rapid non-genomic signaling. Here, we show that estrogens also mediate rapid non-genomic remodeling of the cytoskeleton in mature OLs in culture. 17ß-Estradiol caused a rapid loss of microtubules and the actin cytoskeleton in the OL membrane sheets. It also increased phosphorylation of the actin filament-severing protein cofilin, thus inactivating it. Staining for actin barbed ends with rhodamine-actin showed that it decreased the amount of actin barbed ends. 17α-Estradiol, on the other hand, increased the percentage of cells with abundant staining of actin filaments and actin barbed ends, suggesting that it stabilized and/or increased the dynamics of the actin cytoskeleton. The specific ERα and ERß agonists, 4,4',4″-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) and diarylpropionitrile 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN), respectively, also caused the rapid phosphorylation of cofilin. Estrogen-induced phosphorylation of cofilin was inhibited by Y-27632, a specific inhibitor of the Rho-associated protein serine/threonine kinase (ROCK). The Rho/ROCK/cofilin pathway is therefore implicated in actin rearrangement via estrogen ligation of membrane ERs, which may include forms of ERα and ERß. These results indicate a role for estrogens in modulation of the cytoskeleton in mature OLs, and thus in various processes required for myelinogenesis.

cDNA cloning, genomic cloning, and tissue-specific regulation of mouse cerebroside sulfotransferase.

We have isolated a mouse cDNA clone encoding 3'-phosphoadenylylsulfate-galactosylceramide 3'-sulfotransferase (cerebroside sulfotransferase; CST; EC 2.8.2.11) from a kidney cDNA library, using a human CST cDNA clone [Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A. & Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868] as a probe. A recombinant protein of the cloned cDNA showed CST activity. The deduced protein is composed of the same 423 amino acids as human CST and its sequence exhibits 84% identity with that of the human counterpart. Northern-blot analysis and subquantitative reverse transcription-PCR (RT-PCR) analysis showed that the CST gene is preferentially transcribed in stomach, small intestine, brain, kidney, lung, and testis, in that order. To examine differences in transcripts in various tissues, we isolated CST cDNA clones from stomach, small intestine, brain, kidney, and testis by 5'-RACE analysis. We found seven different nucleotide sequences in the 5'-UTR, while the DNA sequences of all the isolated cDNA clones were identical in the coding region. In addition, we isolated CST genomic DNA clones from a mouse genomic library. The clones covered all the 5'-UTR sequences and coding exons including 3'-UTR. RT-PCR analyses of CST mRNAs from various tissues confirmed that CST transcripts are tissue-specifically spliced by alternative use of multiple exons 1. These observations suggest that the tissue-specific expression of the CST gene is explained by alternative usage of multiple 5'-UTR exons flanked with tissue-specific promoters.

Enzymatic sulfation of galactose residue of keratan sulfate by chondroitin 6-sulfotransferase.

We have previously found that the purified chondroitin 6-sulfotransferase (C6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine in chondroitin, catalyzed the sulfation of keratan sulfate, and that both the C6ST activity and the keratan sulfate sulfotransferase (KSST) activity were expressed in COS-7 cells when C6ST cDNA was transfected. In this report we describe some properties of the KSST activity contained in the purified C6ST, and characterize the sulfated products formed from keratan sulfate and partially desulfated keratan sulfate. Optimal pH, requirement for cationic activators, and Km value for PAPS of the KSST activity were very similar to those of the C6ST activity. 35S-Labeled glycosaminoglycans formed from keratan sulfate and partially desulfated keratan sulfate were N-deacetylated by treatment with hydrazine/hydrazine sulfate and then cleaved with HNO2 at pH 4, and the resulting products were reduced with NaB3H4. Analysis of the degradation products with paper chromatography and high performance liquid chromatography provided evidence that C6ST transferred sulfate to position 6 of galactose residue which was glycosidically linked to N-acetylglucosamine 6-sulfate residue or to N-acetylglucosamine residue. Northern blot analysis using poly (A)+ RNA from 12-d-old chick embryos indicated that the message of C6ST was expressed not only in the cartilage but also in the cornea in which keratan sulfate is actively synthesized.

Molecular cloning and expression of cDNA encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase.

We have isolated a cDNA clone encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11). Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from human renal cancer cell cDNA by the polymerase chain reaction method. The amplified cDNA fragment was then used as probe to screen a human renal cancer cell cDNA library. The isolated cDNA clone contained an open reading frame encoding 423 amino acids including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. There is no significant homology between this sequence and either the sulfotransferases cloned to date or other known proteins. Northern blot analysis demonstrated that a 1.9-kilobase mRNA was unique to renal cancer cells. When the cDNA was inserted into the expression vector pSVK3 and transfected into COS-1 cells, galactosylceramide sulfotransferase activity in the transfected cells increased from 8- to 16-fold over that of controls, and the enzyme product, sulfatide, was expressed on the transformed cells.

Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line.

Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.

[Studies on the identification of sulfadimidine in pork by high performance liquid chromatography with photodiode array detector and gas chromatograph-mass spectrometry].

Sulfadimidine (SDD) was detected in pork imported from Mexico by HPLC with gradient elution system. UV spectrum of the peak was measured and confirmed by photodiode array detector, moreover identified by GC/MS after methylation with diazomethane. Content of SDD in the sample was 0.1 ppm and detection limit of SDD by the proposed method was 0.02 ppm. Application of this method to CLP, SMR, SMM, SDM and SQX was studied, and satisfactory results were obtained.

Changes in phenytoin concentrations in blood and cerebrospinal fluid caused by direct hemoperfusion in a patient intoxicated with phenytoin.

We performed direct hemoperfusion (DHP) 5 times on a patient with consciousness disorder and phenytoin intoxication. We then measured the phenytoin concentrations in her cerebrospinal fluid (CSF) and blood at various times. After the first DHP session, consciousness began to improve, and it normalized after the fourth DHP session when the blood concentration of phenytoin had decreased from 54.0 microg/ml to 16.5 microg/ml. The average plasma phenytoin elimination rate of DHP was 18.0% over 120-180 min. The concentration of phenytoin in the CSF decreased as that in the blood was lowered by DHP. The average reduction rate of phenytoin in the CSF after a DHP session was 23.7%, which was similar to the rate of elimination from the blood. The CSF/blood phenytoin ratio was 0.17, and no marked changes were detected before or after a DHP session.

Pure Rotational Spectra of SO: Rare Isotopomers in the 80-GHz to 1.1-THz Region

Pure rotational spectra of rare isotopomers of sulfur monoxide, SO, have been recorded with the Cologne Terahertz Spectrometer, Germany, and the millimeter- and submillimeter-wave spectrometer at Nobeyama, Japan. In total, 176 new transitions have been measured in the X3Sigma- electronic ground state, including the first laboratory detection of the rare isotopomer 36SO. New lines are also reported for 33SO and S17O in their vibrational ground states, and for 33SO and S18O in the first excited vibrational state. A simultaneous fit of 451 transitions has led to an improved set of isotopically invariant parameters for rotation and fine structure. Hyperfine structure constants for 33SO and S17O have been obtained also from the global fit, including first values for the magnetic nuclear spin-rotation interaction. These are compared to other molecules. The isotopically invariant parameters allow precise frequency predictions for the submillimeter-wave region far beyond 1 THz for all SO isotopomers, of importance to astrophysical applica- tions.