H-Ras oncogene counteracts the growth-inhibitory effect of genistein in T24 bladder carcinoma cells.

Among eight human bladder cancer cell lines we examined, only T24 cells were resistant to the growth inhibition effect of genistein, an isoflavone and potent anticancer drug. Since the T24 cell line was the only cell line known to overexpress oncogenic H-Ras(val 12), we investigated the role of H-Ras(val 12) in mediating drug resistance. Herein, we demonstrate that the phenotype of T24 cells could be dramatically reversed and became relatively susceptible to growth inhibition by genistein if the synthesis of H-Ras(val 12) or its downstream effector c-Fos had been suppressed. The inhibition of Ras-mediated signalling with protein kinase inhibitors, such as PD58059 and U0126 which inhibited MEK and ERK, in T24 cells also rendered the identical phenotypic reversion. However, this reversion was not observed when an inhibitor was used to suppress the protein phosphorylation function of PI3 K or PKC. These results suggest that the signal mediated by H-Ras(val 12) is predominantly responsible for the resistance of the cells to the anticancer drug genistein.

Modulation by blood glucose levels of activity and concentration of paraoxonase in young patients with type 1 diabetes mellitus.

Paraoxonase (PON) is a high-density lipoprotein (HDL)-associated esterase, which may prevent the transformation of low-density lipoproteins (LDL) into biologically active, atherogenic particles. PON concentration and activity are affected by PON1 gene polymorphisms and found to be altered in type 2 diabetes patients with retinopathy. We investigated serum PON concentration, in vitro activity and polymorphism at position 54 (L/M, Leu-Met54) in 193 Caucasian adolescents and young adults (88 males, 105 females) with type 1 diabetes mellitus, as well as its relationship to the presence of retinopathy. An inverse linear correlation was found between blood glucose levels and both serum PON concentration (r = -.20, P =.017) and its activity (r = -0.17, P =.037). Patients with elevated blood glucose values (> or =10 mmol/L) had significantly lower levels of both PON concentration (P =.003) and activity (P =.028) than those with lower glucose levels. After adjusting for blood glucose and diabetes duration, PON activity was significantly higher in patients with different stages of retinopathy compared with those without retinopathy (P =.003). The L/L genotype was closely associated with the presence of retinopathy (P <.0001). These data show that young people with type 1 diabetes and the L/L polymorphism at position 54 of PON1 gene are more susceptible to retinal complications. However, the role of serum PON concentration and activity as a possible marker for monitoring late microvascular complications in these patients has to be established.

Role of transglutaminase II in retinoic acid-induced activation of RhoA-associated kinase-2.

Transamidation is a post-translational modification of proteins mediated by tissue transglutaminase II (TGase), a GTP-binding protein, participating in signal transduction pathways as a non-conventional G-protein. Retinoic acid (RA), which is known to have a role in cell differentiation, is a potent activator of TGASE: The activation of TGase results in increased transamidation of RhoA, which is inhibited by monodansylcadaverine (MDC; an inhibitor of transglutaminase activity) and TGaseM (a TGase mutant lacking transglutaminase activity). Transamidated RhoA functions as a constitutively active G-protein, showing increased binding to its downstream target, RhoA-associated kinase-2 (ROCK-2). Upon binding to RhoA, ROCK-2 becomes autophosphorylated and demonstrates stimulated kinase activity. The RA-stimulated interaction between RhoA and ROCK-2 is blocked by MDC and TGaseM, indicating a role for transglutaminase activity in the interaction. Biochemical effects of TGase activation, coupled with the formation of stress fibers and focal adhesion complexes, are proposed to have a significant role in cell differentiation.

Renal cell carcinoma metastasized to the ureteral stump.

A rare case of ureteral stump metastasis 3 months after nephrectomy for a renal cell carcinoma is presented. A 62-year-old female had undergone right radical nephrectomy 3 months earlier because of renal cell carcinoma in our hospital, and she came back due to gross hematuria. Cystoscopy revealed a papillary mass with a vascular pedicle protruding from the right ureteral orifice. Transurethral resection of the bladder tumor over right ureteral orifice was performed and the pathologic result showed clear cell adenocarcinoma, which argued in favor of a metastatic lesion from the previous renal cell carcinoma. Ureterectomy and a bladder cuff excision were then adopted for this patient, but no residual tumor was found over the remaining ureter. Nevertheless, the patient died of cancer 36 months after the event of ureteral stump metastasis.

Bundling of microtubules by motor and tail domains of a kinesin-like calmodulin-binding protein from Arabidopsis: regulation by Ca(2+)/Calmodulin.

Kinesin-like calmodulin-binding protein (KCBP), a novel kinesin-like protein from plants, is unique among kinesins and kinesin-like proteins in having a calmodulin-binding domain adjacent to its motor domain. KCBP localizes to mitotic microtubule (MT) arrays including the preprophase band, the spindle apparatus, and the phragmoplast, suggesting a role for KCBP in establishing these MT arrays by bundling MTs. To determine if KCBP bundles MTs, we expressed C-terminal motor and N-terminal tail domains of KCBP, and used the purified proteins in MT bundling assays. The 1.5 C protein with the motor and calmodulin-binding domains induced MT bundling. The 1.5 C-induced bundles were dissociated in the presence of Ca(2+)/calmodulin. Similar results were obtained with a 1.4 C protein, which lacks much of the coiled-coil region present in 1.5 C protein and does not form dimers. The N-terminal tail of KCBP, which contains an ATP-independent MT binding site, is also capable of bundling MTs. These results, together with the KCBP localization data, suggest the involvement of KCBP in establishing mitotic MT arrays during different stages of cell division and that Ca(2+)/calmodulin regulates the formation of these MT arrays.

An aldose reductase intragenic polymorphism associated with diabetic retinopathy.

The polyol pathway has been considered important in the development of diabetes long-term complications. Diabetic patients with microvascular disease have increased gene expression and enzyme activity, which may be due to variants in the aldose reductase gene. An association of an intragenic BamHI polymorphic site with diabetic retinopathy and nephropathy has been suggested, but the BamHI site has not been confirmed. In the current study, long template PCR-RFLP and DNA sequencing were used to ascertain its existence. A single substitution of A to C at 95th nucleotide of intron 8 was identified. This polymorphism was investigated in 164 adolescents with type 1 diabetes in whom diabetic retinopathy was assessed by stereoscopic retinal photography. Both the wild haplotype B and homozygote BB were significantly more common in the adolescents with retinopathy than in those without retinopathy (P = 0.018 and 0.002, respectively). We also confirmed an association between a previously described (CA)n repeat sequence and retinopathy in these adolescents (P < 0.0005). However, there was no association between the two polymorphisms.

Tumor angiogenesis and metastasis: correlation in invasive renal cell carcinoma.

BACKGROUND: Experience suggests that tumor growth is dependent on angiogenesis. The intensity of angiogenesis in human cancer is reported to be predictive of the probability of metastasis in many types of cancer. The aims of this study were 1) to determine the relationship of microvessel density (MVD) in renal cell carcinoma to pathologic stage, and 2) to evaluate the role of MVD in metastasis. METHODS: Paraffin-embedded tumor specimens were reviewed from 34 unselected patients with RCC who had undergone surgery from 1986 to 1990 at Taichung Veterans General Hospital. The pathology findings and clinical records were reviewed to note relationships between pathologic stage and whether or not metastasis had occurred. Specimens were studied from 16 cases (eight Stage I cancers, five Stage II and three Stage III) without metastasis and from 18 cases (two Stage I, six Stage II, six Stage III and four Stage IV) in which metastasis later developed. Microvessels were highlighted by immunostaining endothelial cells for factor VIII-related antigen. Microvessels were counted in a x-400 field (0.1885 mm2/field) in the most active areas of neovascularization. RESULTS: The 16 patients without metastasis have survived for between 65 and 136 months (mean, 94.5 months), up to the present time. Of the 18 patients with metastasis, 15 died and three survived, with mean survivals of 42.8 months (range, 12-99 months). Mean overall MVD was 99.6 vessels; mean MVD was 98.5, 96.2, 109.3 and 90.0 in Stages I, II, III and IV tumors, respectively. Mean MVD was 99.3 in patients without metastasis and 99.9 in patients with metastasis. CONCLUSIONS: MVD does not correlate with pathologic stage and is of no prognostic significance in renal cell carcinoma.

A variant of paraoxonase (PON1) gene is associated with diabetic retinopathy in IDDM.

Serum paraoxonase is a glycoprotein which binds to high-density lipoproteins (HDL) and may prevent oxidation of LDL by hydrolyzing lipid peroxides. Two polymorphisms identified in the paraoxonase gene (Met-Leu 54 and Gln-Arg 192) have been associated with cardiovascular disease. Oxidative low-density lipoprotein (LDL) is also toxic to retinal capillary endothelial cells and pericytes, so that mildly modified LDL may contribute to the development of diabetic retinopathy. To investigate the potential significance of these polymorphisms in the pathogenesis of diabetic retinopathy in IDDM, 80 patients with diabetic retinopathy and 119 controls without diabetic retinopathy were investigated in the current project. The allelic frequency of leucine 54 (L) was significantly higher in the group with retinopathy than without retinopathy (73% vs. 57%, p < 0.001). The genotype L/L was strongly associated with the development of diabetic retinopathy (p < 0.001), but a similar association was not found with Gln-Arg 192. Leucine 54 is a risk factor for diabetic retinopathy.

Incidental renal cell carcinoma: pathologic features and survival rate.

BACKGROUND: The aim of this study was to compare outcomes in patients with symptomatic and incidental renal cell carcinoma (RCC). METHODS: From October 1982 to December 1996, 200 patients with RCC were enrolled in this study. Their medical records were reviewed for symptoms, tumor stage, and lymph node and metastatic status. Symptomatic and incidental RCCs were compared by the overall survival rate of patients. The survival rate was determined by the Kaplan Meier method. Log rank testing was used to analyze the statistical difference in the survival period between both groups. RESULTS: The proportion of incidental RCC was 21% (42/200). The majority of cases (73.8%) were diagnosed primarily by abdominal ultrasonography. Incidental RCCs were smaller in size than symptomatic RCCs (5.1 +/- 2.0 cm vs. 7.5 +/- 1.8 cm, p = 0.001). Incidental RCCs were of a lower stage, and patients with incidental RCCs had significantly longer overall survival rates than those with symptomatic RCCs (p < 0.001). CONCLUSIONS: Ultrasonography is a useful tool for the detection of incidental RCC. Improvement in the overall survival rate of incidental RCC patients suggests that when these tumors are identified earlier, treatment results are better.

A ground-based study for a shuttle BRIC experiment on gravity effects on gene expression.

A BRIC (Biological Research In a Canister) experiment to investigate the effects of reduced gravity at the molecular level using Arabidopsis has been initiated. In preparation for a space flight experiment, a series of ground-based studies were conducted. Results from these studies indicate that: 1) up to 20,000 seeds can be germinated on a 100 mm diameter Petri plate, 2) nylon membrane is the best surface for recovery of plant material after freezing, 3) depending on the age of the seedlings at the time of freezing, 20 to 40 g of tissue can be obtained from Petri plates that fit in a single canister; 4) tissue from one canister yields adequate amounts of RNA to perform differential display to isolate gravity-regulated genes. Our results indicate that the proposed BRIC experiment is feasible and can provide valuable information on the possible effects of microgravity on gene regulation.

Interaction of Arabidopsis kinesin-like calmodulin-binding protein with tubulin subunits: modulation by Ca(2+)-calmodulin.

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified novel kinesin-like protein that appears to be unique to and ubiquitous in plants. KCBP is distinct from all other known KLPs in having a calmodulin-binding domain adjacent to its motor domain. We have used different regions of KCBP to study its interaction with tubulin subunits and the regulation of this interaction by Ca(2+)-calmodulin. The results show that the carboxy-terminal part of the KCBP, with or without calmodulin-binding domain, binds to tubulin subunits and this binding is sensitive to nucleotides. In the presence of Ca(2+)-calmodulin the motor with calmodulin-binding domain does not bind to tubulin. This Ca(2+)-calmodulin modulation is abolished in the presence of antibodies specific to the calmodulin-binding domain of KCBP. Similar binding studies with the carboxy-terminal part of KCBP lacking the calmodulin-binding domain show no effect of Ca(2+)-calmodulin. These results indicate that Ca(2+)-calmodulin modulates the interaction of KCBP with tubulin subunits and this modulation is due to the calmodulin-binding domain in the KCBP. Calcium-dependent calmodulin modulation of KCBP interaction with tubulin suggests regulation of KCBP function by calcium, the first such regulation of a kinesin heavy chain among all the known kinesin-like proteins.

Intracellular glutathione content of urothelial cancer in correlation to chemotherapy response.

We evaluated the possible correlation between intracellular glutathione (GSH) and drug sensitivity of urothelial cancer. Tissue GSH content of surgical specimens from 20 patients with urothelial cancer was assayed with high performance liquid chromatography (HPLC). GSH levels of cancer tissue (7.887 +/- 6.176 microM/mg protein) were significantly higher than GSH levels of normal mucosa (1.345 +/- 1.252 microM/mg). All patients having measurable lesions were then treated with methotrexate, epirubicin and cisplatin (MEC). These patients were classified into three groups according to clinical response criteria. GSH content in cancer tissue from four patients with complete response was 0.804 +/- 1.183 microM/mg protein. However, the cancer cells from patients with partial response and non-response contained a significantly higher level of GSH (6.295 +/- 2.459 (n = 8) and 12.955 +/- 6.141 microM/mg protein (n = 8), respectively). Intracellular glutathione content may play an important role in intrinsic resistance of urothelial cancer to MEC chemotherapy. It might be potentially used to predict drug sensitivity in urothelial cancer patients before starting chemotherapy.

Characterization of a glucose-repressible ADP-ribosylation factor 3 (ARF3) from Saccharomyces cerevisiae.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin, and are believed to participate in vesicular transport in both exocytic and endocytic pathways. Based on size, phylogenetic analysis, amino acid sequence, and gene structure, mammalian ARFs fall into three classes (class I, ARFs 1, 2, 3; class II, ARFs 4, 5; class III, ARF6). Two ARF genes (yARF1, yARF2) are known in Saccharomyces cerevisiae and believed to participate in vesicular trafficking in the Golgi system; the double deletion mutant is not viable. A third yeast ARF (yARF3) cDNA has been cloned by polymerase chain reaction-based procedures. It contains an open reading frame of 549 bases encoding a protein of 183 amino acids, with a deduced amino acid sequence more identical (60%) to that of the class III mammalian ARF than to those of the other two classes (52-56%). The yARF3 protein, however, reacted poorly with antibodies against any of the three classes of mammalian ARFs. In the presence of GTP, recombinant yARF3 protein stimulated cholera toxin-catalyzed auto-ADP-ribosylation. yARF3 gene transcription, similar to that of yARF2, was repressed by glucose. As yARF3 was not essential for cell viability and was not required for endoplasmic reticulum to Golgi protein transport, it may provide an opportunity to define an ARF function in another kind of vesicular trafficking.

Characterization of class II and class III ADP-ribosylation factor genes and proteins in Drosophila melanogaster.

ADP-ribosylation factors (ARFs) are ubiquitous approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and are involved in intracellular vesicular transport. Based on size, phylogenetic analysis, amino acid identity, and gene structure, mammalian ARFs fall into three classes (class I, ARF1, -2, and -3; class II, ARF4 and -5; class III, ARF6). A class I ARF had been identified in Drosophila melanogaster. To search for ARFs of other classes in Drosophila, polymerase chain reaction-based techniques were used, resulting in cloning of Drosophila ARF (dARF) II and dARF III with deduced amino acid sequences similar to those of class II and class III mammalian ARFs, respectively. The three Drosophila ARF genes map to different chromosomes and the coding regions have different splicing sites. dARF II mRNA, like ARF I mRNA, is fairly uniformly distributed throughout adult flies, whereas dARF III mRNA is significantly more abundant in heads than in legs or bodies. Recombinant dARF II and dARF III have biochemical and immunological properties similar to those of human ARF5 (hARF5) and hARF6, respectively. These observations are consistent with the conclusion that the three classes of ARFs are present in non-mammalian as well as mammalian species.

Unusual pressure dependence of the lateral motion of pyrene-labeled phosphatidylcholine in bipolar lipid vesicles.

The lateral mobility of a pyrene-labeled phosphatidylcholine probe in liposomes containing archaebacterial bipolar lipids has been studied isothermally as a function of pressure. The pressure-dependence of the probe mobility, R, is found to be slightly positive or zero in the temperature range of 17 - 48 degrees C. At temperatures > 48 degrees C, R becomes negative and decreases with temperature. The data indicate that lateral mobility only becomes appreciable at high temperatures. In addition, the R values obtained with other lipid membranes are much lower than that obtained with bipolar liposomes, implying that the membranes of archaebacterial liposomes are laterally immobile, as compared to other lipid membranes.

Effect of long-term alcohol ingestion on the femoral head of rabbit.

Twenty New Zealand rabbits were chosen for this study (5 for controls and 15 for the alcohol group). In the alcohol group, each rabbit was fed with rice wine (2 g/kg/day alcohol) for 1 year. Bone marrow pressure of the femoral basal neck, erythrocyte sedimentation rate (ESR), liver functions, uric acid, triglyceride, cholesterol, amylase, and blood alcohol level were measured at 0 day, 6 months and 1 year. Finally, the rabbits were sacrificed at the end of the study. The livers and femoral heads were examined for pathology. The bone volume and fat cell size of the femoral head were measured with a computerized "Bone Scanner". The results (1 year comparison) were a significantly higher level of cholesterol in the alcohol group (72 +/- 40 mg% vs 26 +/- 8 mg%, p less than 0.05) and greater bone marrow pressure (27.71 +/- 8.97 mmHg vs 18.20 +/- 4.09 mmHg, p less than 0.05), and no significant difference in triglyceride, uric acid and liver function, ESR, etc., when using Student's 2-tailed t-test. Liver changes (fatty liver) occurred in 3 animals in the alcohol group. The effect of alcohol on the trabecular bone volume was not significant (51.5 +/- 4.6 vol.% vs 50.0 +/- 5.2 vol.%), but the average fat cell size of the femoral head was significantly larger than that for normal controls (2,745 +/- 605 mu2 vs 2,185 +/- 458 mu2, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic motions of 1,6-diphenyl-1,3,5-hexatriene in interdigitated C(18):C(10)phosphatidylcholine bilayers.

This study investigates the dynamic behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C(18):C(10)phosphatidylcholine [C(18):C(10)PC] bilayers. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated bilayers above the transition temperature. The rotation of DPH in C(18):C(10)PC has been described in terms of the thermal coefficient of rotation using the modified Y-plot method which takes into account the limiting anisotropy value. During the phase transition of C(18):C(10)PC, DPH exhibits a thermal coefficient b2M = 0.41 - 0.51 degrees C-1 which is similar to the b2M values obtained with noninterdigitated phosphatidylcholine bilayers. Differential polarized phase-modulation fluorometry has also been employed to study the dynamic behavior of DPH in C(18):C(10)PC in real time. The data show that DPH contains considerable motion in the highly ordered mixed interdigitated bilayers. The DPH motion steadily increases with an increase in temperature as shown by the rotational correlation time, and the wobbling diffusion constant. However, the limiting anisotropy, the order parameter, and the width of the lifetime distribution undergo an abrupt decrease, and a corresponding abrupt increase in the cone angle, at approximately 16 degrees C. This temperature range is near the onset temperature of the phase transition as determined by differential scanning calorimetry. The rotational parameters show strong hysteresis on heating and cooling. All the rotational parameters derived from DPH fluorescence in mixed interdigitated C(18):C(10)PC exhibit magnitudes similar to those obtained from non interdigitated gel phases of symmetric diacylphosphatidylcholines. These results, combined with those obtained with dehydroergosterol (Kao, Y. L., P. L.-G. Chong, and C. Huang. 1990. Biochemistry. 29:1315-1322), suggest that considerable rotational mobility of small molecules can be sustained in an intramolecularly highly ordered interdigitated lipid matrix, implying that the membrane maintains a fluid environment around membrane perturbants even when the lipid matrix is extensively interdigitated.

Time-resolved fluorometric and differential scanning calorimetric investigation of dehydroergosterol in 1-stearoyl-2-caprylphosphatidylcholine bilayers.

Thermal and dynamic properties of dehydroergosterol (DHE) in 1-stearoyl-2-capryl-sn-glycero-3-phosphocholine [C(18):C(10)PC] have been studied by differential scanning calorimetry (DSC) and multifrequency phase-modulation fluorometry. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form highly ordered mixed interdigitated bilayers below the maximal transition temperature, Tm, and partially interdigitated bilayers above Tm. This lipid system is thus unique in assessing the interactions between sterols and interdigitated lipid bilayers. DHE is a fluorescent analogue of cholesterol shown in previous studies to behave like cholesterol in noninterdigitated symmetric diacylphosphatidylcholines. DSC data show that DHE exhibits similar characteristics to cholesterol [Chong & Choate (1989) Biophys. J. 55, 551-556] in C(18):C(10)PC bilayers. DHE abolishes the phase transition of C(18):C(10)PC at 27 mol % compared to 25 mol % for cholesterol and decreases Tm, the onset temperature (To), and the completion temperature (Tc), at a similar rate to cholesterol at about -0.25 degrees C per mole percent DHE. Fluorescence data show that the rotational motion of DHE can be described by a hindered anisotropic model. In the gel state of C(18):C(10)PC, the rotational correlation of DHE decreases monotonically with increasing DHE content up to 24 mol %, suggesting that DHE causes a disordering/spacing effect on the packing of mixed interdigitated C(18):C(10)PC bilayers. The rotational correlation time undergoes an abrupt increase from 24 to 27 mol % DHE. Abrupt changes in the DSC parameters were also observed in the neighborhood of 27 mol %, suggesting that major reorganization takes place around this concentration.(ABSTRACT TRUNCATED AT 250 WORDS)