Some protein kinases are known to be activated by D-erythro-sphingosine (Sph) or N,N-dimethyl-D-erythrosphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser 60 , Ser 59 , or Ser 58 of 14-3-3␤, 14-3-3 , or 14-3-3 , respectively, was termed "sphingosine-dependent protein kinase-1" (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834 -21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase C␦ (PKC␦) based on the following observations. (i) Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an ϳ40-kDa protein band. This fraction had SDK1 activity ϳ50,000-fold higher than that in the initial extract. (ii) This protein had ϳ53% sequence identity to the Ser/Thr kinase domain of PKC␦ based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii) A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the ϳ40-kDa band is the kinase domain of PKC␦. The kinase activity of PKC␦ did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKC␦ did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKC␦ was incubated with caspase-3, which releases the ϳ40-kDa kinase domain. PKC␦ and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.

Background: Orthotopic urinary diversion has become the preferred form of bladder reconstruction after cystectomy. We report on our experience with 66 male patients undergoing this procedure from November 1990 to February 1998. Methods: A neobladder was constructed using an ileal segment with a Hautmann type bladder.Complications were assessed and subdivided into early and late types. Voiding function was evaluated in terms of voiding pattern and continence. Median follow up was 19.5 (range 3.5-87.7) months.Results: There was one (1.5%) perioperative death. The most frequent pouch-related and unrelated early complications were persistent urine leak (7.6%) and prolonged ileus (16.7%), respectively, the majority of cases of which were managed conservatively. Analysis of late complications revealed 6.2% ureteroileal stenosis and 1.5% urethrointestinal stenosis rates, but no case of bladder stone formation. Of the 61 patients in whom voiding function was evaluable, 95.1% achieved excellent daytime continence, while only 67.2% had night-time continence. With regard to posture at voiding, 23 (37.7%) voided in a sitting position. Three of the patients (4.9%) were unable to void and required regular intermittent catheterization. Conclusions: An orthotopic neobladder can be constructed with acceptable morbidity and excellent functional results. We believe that orthotopic urinary diversion offers an attractive alternative to a bladder substitute when cystectomy is required.

Background: Continent urinary reservoirs (CUR) have become one of the major options for patients requiring urinary diversion to improve their quality of life (QOL). To assess whether CUR enhanced postoperative QOL, we surveyed patients with CUR and ileal conduit (IC) using a questionnaire sent by mail. Patients and Methods:The questionnaire consisted of 133 questions that covered physical and mental status, social life, sexual habits and symptoms related to urinary diversions. A total of 172 questionnaires were sent out, and 137 (80%) patients (74 CUR and 63 IC patients) responded. Results: Basic physical conditions were similar in the 2 groups, except for sleeping habits. Regarding social life, however, the CUR group showed better scores in bathing habits and frequency of overnight travel. Parastonial dermatitis was more frequent in the IC group and the patients were more hesitant to show their stoma to others. On the other hand, about half of the patients in the CUR group complained of troublesomeness in self-catheterization, especially at night. Overall, 74% and 41 % of the patients in the CUR and IC group were satisfied with their urinary diversion. When the Kock pouch and Indiana pouch were compared, no statistically significant differences were found in average capacity, maximum capacity, o r frequency of self-catheterization. Conclusions: CUR recipients have enhanced QOL regarding the stoma, travel and sleeping habits as compared to ileal conduit. However, troublesomeness of night time self-catheterization was noted in the CUR group. Individualized selection of the type of urinary diversion with informed consent is essen tia I.

Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N'-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al. (1995) Biochem. Biophys. Res. Commun. 216, 739-747), have been termed "sphingosine-dependent kinases" (SDKs). We showed previously that a purified SDK (termed "SDK1") phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms beta, eta, and zeta but not tau or sigma (Megidish, T., et al. (1998) J. Biol. Chem. 273, 21834-45). In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. (ii) The activation of these SDKs is specific to D-erythro-Sph and its N-methyl derivatives, the effect of L-threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. An SDK separated as fractions "TN31-33" phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed "SDK2". Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin); another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. An SDK activity separated and present in "fraction 42" from Q-Sepharose chromatography specifically phosphorylated GRP105 (or GRP94) and HSP68 but did not phosphorylate PDI or 14-3-3. This SDK is clearly different from other SDKs in its substrate specificity and is tentatively termed "SDK3". Interestingly, substrates of all these SDKs so far identified are molecular chaperones or adapters capable of binding to enzymes and key molecules involved in signal transduction, maintaining tertiary structure of bioactive molecules, or maintaining cellular homeostasis in response to environmental stress. Thus, the essential role of Sph and DMS is to activate molecular chaperones, thereby providing a link to the mechanism by which SDK activity regulates cellular homeostasis and signal transduction.