Dietary sardine oil increases erythrocyte membrane fluidity in diabetic patients.

The effects of dietary sardine oil rich in eicosapentaenoic acid, C20:5 (EPA), on erythrocyte membrane fluidity and membrane and plasma lipids were investigated in diabetic and control subjects. Before consumption of this oil, the levels of erythrocyte membrane fluidity were lower in the diabetic subjects, as noted in our previous work (Diabetes 1983; 32:585-91). Decreased membrane polyunsaturated fatty acid contents were evident. Daily consumption of 2700 mg of sardine oil for 8 wk increased erythrocyte membrane fluidity, as determined by electron spin resonance using the 12- or 16-stearic acid label. This increase was seen after 4 wk, and the level remained elevated for 8 wk. Membrane EPA of phospholipid acyl-chains significantly increased after 4 wk and was even more apparent after 8 wk. Membrane-free cholesterol to phospholipid molar ratios significantly decreased after 8 wk. Both the diabetic and normal subjects responded to the sardine oil in the same way. After feeding with sardine oil, there no longer were differences in erythrocyte membrane fluidity between the normal and diabetic subjects. We propose that improvement in membrane fluidity may contribute to the amelioration of altered cell membrane functions in diabetic patients.

Mitogen-activated protein kinase pathway is dispensable for microtubule-active drug-induced Raf-1/Bcl-2 phosphorylation and apoptosis in leukemia cells.

Raf-1 activation and Bcl-2 hyperphosphorylation following treatment with paclitaxel (Taxol) or other microtubule-active drugs is associated with mitotic arrest. Here we show that microtubule-active drugs do not activate the mitogen-activated protein kinase (MAPK) pathway in leukemia cells. PD98059, a MEK inhibitor, and SB202190, a p38 MAP kinase inhibitor, do not abrogate Bcl-2 phosphorylation nor apoptosis. Simultaneously with PARP cleavage, paclitaxel induces cleavage of Bcl-2 protein yielding a potentially pro-apoptotic 22 kDa product. In comparison, the stimulation of Raf-1 by phorbol ester (TPA) activates the MAPK pathway, causes MAPK-dependent p21WAF1/CIP1 induction, Rb dephosphorylation and growth arrest without Bcl-2 phosphorylation or apoptosis. Like TPA, cAMP induces p21WAF1/CIP1 but does not cause Bcl-2 phosphorylation. MEKK1 and Ras, upstream activators of JNK and ERK MAPK, also fail to induce Bcl-2 hyperphosphorylation. Although Lck tyrosine kinase has been recently implicated in Raf-1 activation during mitotic arrest, microtubule-active drugs induce Raf-1/Bcl-2 hyperphosphorylation and apoptosis in a Lck-deficient Jurkat cells. Therefore, microtubule-active drugs induce apoptosis which is associated with Raf-1 and Bcl-2 phosphorylation and Bcl-2 cleavage but is independent of the MAPK pathway. In contrast, TPA-activated MAPK pathway causes p21WAF1/CIP1-dependent growth arrest without apoptosis.

Construction of gene therapy vectors targeting adrenocortical cells: enhancement of activity and specificity with agents modulating the cyclic adenosine 3',5'-monophosphate pathway.

In preliminary studies we demonstrated that the CYP11B1 (11beta-hydroxylase) promoter could direct specific expression of a suicide gene in adrenocortical cancer cells, providing a potentially specific therapeutic option for adrenocortical cancer. In this present study we describe our attempts to enhance the activity of the CYP11B1 promoter while maintaining its specificity for adrenal cells. Using a putative enhancer element from the cholesterol side-chain cleavage (P450scc) gene, the activity of the CYP11B1 promoter in and its specificity for adrenocortical cells were enhanced. Treatment with 8-bromo-cAMP or forskolin resulted in further enhancement. In stably transfected Y-1 cells, in which the herpes simplex virus thymidine kinase (HSV-TK) gene was driven by the CYP11B1 promoter with the P450scc enhancer element, HSV-TK expression and ganciclovir sensitivity were augmented by treatment with 8-bromo-cAMP, forskolin, and ACTH. In summary, we report the construction of a suicide HSV-TK vector with preferential toxicity to adrenocortical cells. We propose that a similar strategy using differentiating agents may be useful in the gene therapy of tumors with unique differentiated properties, including those arising from other endocrine organs.

Construction of gene therapy vectors targeting thyroid cells: enhancement of activity and specificity with histone deacetylase inhibitors and agents modulating the cyclic adenosine 3',5'-monophosphate pathway and demonstration of activity in follicular and anaplastic thyroid carcinoma cells.

Thyroid carcinoma accounts for the majority of deaths from endocrine cancers. Although effective therapies exist for well differentiated tumors, the treatment options for poorly differentiated and anaplastic tumors are much less effective. In the present study we demonstrate that the thyroglobulin (Tg) promoter can be used to direct specific expression of either luciferase or thymidine kinase in thyroid cancer cells. Furthermore, using a putative enhancer element for the Tg gene, the activity of the Tg promoter in and its specificity for thyroid cells were enhanced. In transient transfectants or in stably transfected thyroid carcinoma cells, treatment with the histone deacetylase inhibitors, depsipeptide (FR9012228) and sodium butyrate, alone or in combination with 8-bromo-cAMP, resulted in further enhancement. In experiments in which the herpes simplex virus thymidine kinase (HSV-TK) gene was driven by the Tg promoter and the putative enhancer, HSV-TK expression and ganciclovir sensitivity were augmented. Similar results were obtained in two cell lines derived from a follicular thyroid carcinoma and in two anaplastic thyroid carcinoma cell lines. In summary, we report the construction of a suicide HSV-TK vector with preferential toxicity for thyroid cells. The results in anaplastic thyroid carcinoma cells suggest that it may be of use in the full spectrum of thyroid malignancies.

Napsin A, a member of the aspartic protease family, is abundantly expressed in normal lung and kidney tissue and is expressed in lung adenocarcinomas.

A pair of 35 kDa polypeptides (TAO1/TAO2) are expressed in more than 90% of all primary lung adenocarcinomas but not in other major malignancies. Mass spectrometry of tryptic peptides showed that TAO1/TAO2 is identical to napsin A, a recently described member of the aspartic proteinase family. The site of processing of pronapsin A to the mature form was located. Napsin expression was detected in human lung adenocarcinoma tumors, compatible with the marker nature of TAO1/TAO2 in the diagnosis of primary lung adenocarcinoma. This is important since identification of markers which can distinguish primary lung adenocarcinomas from distant metastases is desirable. Northern blot analysis showed expression of napsin also in normal lung and kidney tissue, and in situ hybridization showed expression in type II alveolar cells of the lung. This protease is concluded to have a specific functional role in the normal alveolar epithelium and is a candidate protease for the proteolytic processing of surfactant precursors.

Reversal of MRP-mediated vincristine resistance in KB cells by buthionine sulfoximine in combination with PAK-104P.

The mechanism of multidrug resistance protein (MRP)-mediated multidrug resistance (MDR) is still unclear. MRP reportedly transports some GSH conjugates. Recently, we demonstrated that a pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4 -(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P), that reversed P-glycoprotein (P-gp)-mediated MDR directly interacted with MRP and completely reversed the vincristine (VCR) resistance in MRP-mediated MDR C-A120 cells. We investigated the reversing effect of PAK-104P in C-A120 cells in combination with buthionine sulfoximine (BSO), another MDR-reversing agent with a different reversing mechanism. In immunoblots, MRP was overexpressed in C-A120 cells. The level of ATP-dependent [3H]VCR uptake was high in membrane vesicles from KB-C2 cells, but low in those from C-A120 and parental KB-3-1 cells. The sensitivity to VCR of C-A120 cells, but not of KB-C2 cells, was considerably increased by 100 microM BSO. VCR accumulation in C-A120 cells, but not in KB-C2 cells, was also enhanced by BSO. BSO did not inhibit ATP-dependent [3H]LTC4 uptake in C-A120 vesicles. The combination of BSO with PAK-104P at their low concentrations resulted in complete reversal of VCR resistance in C-A120 cells. These findings suggested that BSO might not directly interact with MRP and reversed resistance in MRP-mediated MDR cells by reducing the intracellular glutathione (GSH) level that was needed for the transport of drugs by MRP and suggested a role for the combination of drug resistance-modulating agents with different reversing mechanisms in the reversal of MRP-mediated MDR.

Exploration of universal cysteines in the binding sites of three opioid receptor subtypes by disulfide-bonding affinity labeling with chemically activated thiol-containing dynorphin A analogs.

A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.

Retrotransposable CR1-like elements in crotalinae snake genomes.

A part of the 3'-flanking region of BP-II gene, which is one of Trimeresurus flavoviridis venom gland phospholopase A2 (PLA2) isozyme genes, has a region homologous to avian chicken repeat 1 (CR1)-element. In the present study, ten CR1-like elements were further identified in T. gramineus venom gland PLA2 isozyme genes, T. flavoviridis PLA2 inhibitor (PLI) genes, and T. flavoviridis and T. gramineus TATA-box binding protein (TBP) genes. Southern blot analysis using a probe for CR1 showed that Crotalinae snake genomes contain a number of CR1-like elements.

Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes.

In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.

Inverse association of serum ascorbic acid level and blood pressure or rate of hypertension in male adults aged 30-39 years.

Correlation analyses between serum ascorbic acid and several risk factors of cerebro- and cardio-vascular diseases were performed on apparently healthy adults (194 persons) aged 30-39 in order to estimate possible functions of ascorbic acid in the prevention of the disease. Serum ascorbic acid had an inverse and the strongest association with systolic blood pressure among the risk factors including blood pressure, total cholesterol, triglyceride, gamma-GTP and obesity. The association was independent of the other variables tested. When the subjects were divided into three different serum ascorbic acid level groups, the prevalence of hypertension (140/90 mmHg and above) was decreased with an increase in the ascorbic acid level. The close relationship of serum ascorbic acid and blood pressure observed in the study suggests that ascorbic acid may have a preventive function against hypertension, or a low ascorbic acid status in hypertensives may promote the further development of arteriosclerosis by the lack of favorable effect of ascorbic acid on lipid metabolism and others.

[Chronic myelogenous leukemia with blastic crisis in which expression of P-glycoprotein was associated with resistance to chemotherapy].

A 55-year-old woman with chronic myelogenous leukemia developed a lymphoid blast crisis (BC) 10 months after diagnosis. By using immunoblotting with a monoclonal antibody against P-glycoprotein (P-gp) C219, her leukemia cells from the first and 3rd crises were shown to be negative for the P-gp, while the cells of the 4th crisis were detected to have a high level of P-gp. This patient did not respond to chemotherapy with several anti-cancer agents in the 4th crisis, although complete remission was achieved in the first, second and third crises after administration of agents including vincristine and prednisolone. Therefore the expression of P-gp in the 4th BC might have been closely related to the resistance to chemotherapy.

Discrimination of a novel type of rat brain delta opioid receptors by enkephalin analog containing structurally constrained cyclopropylphenylalanine (inverted delta Phe).

Four different stereoisomers of cyclopropylphenylalanine (inverted delta Phe) were incorporated into [D-Ala2,Leu5]enkephalin at the position 4. These conformationally restricted enkephalin analogs were evaluated for their binding characteristics to mu and delta opioid receptors in rat brain. A striking finding is that the E-(2R,3S)-isomer binds to a novel class of delta receptors and discriminates this receptor from the ordinary delta receptor. This new type of delta receptor suspected to be a receptor which suppresses the thermal analgesia mediated through mu receptor. The Z-(2R,3R)-isomer was very potent with several times more enhanced affinity to delta receptors than to mu receptors, but could not differentiate the delta receptors. The Z-(2S,3S)-isomer was weak, and E-(2S,3R)-isomer was almost inactive.

Metabolic dependence of the fluidity of intact erythrocyte membrane.

The motion of spin labeled phosphatidylcholine embedded in the lipid bilayer of erythrocyte membrane increased in association with metabolic ATP-depletion. The fluidity change was reversed by subsequent ATP-replenishment. However, levels of cellular 1,2-diacylglycerol, GSH or intracellular Ca2+ contributed little to the fluidity change. Temperature dependence of the fluidity indicated the disappearance of inflection points in ATP-depleted cells, thereby suggesting alterations in protein-lipid interactions. The fluidity change was also reproduced with oxidizing agents which cross-link spectrin. We suggest that the lipid phase state of membrane is maintained by protein-lipid interactions which depend on the metabolic state of the cells, particularly ATP levels.

Patterns of pelvic invasion are prognostic in the treatment of locally recurrent rectal cancer.

BACKGROUND: Local recurrence of rectal cancer after curative resection remains a difficult clinical problem. The aim of this study was to elucidate prognostic risk factors after resection of recurrent cancer. METHODS: Between January 1983 and December 1999, 83 patients with locally recurrent rectal cancer were studied retrospectively for survival benefit by re-resection. Sixty patients underwent resection for recurrent cancer, including total pelvic exenteration in 30 patients and sacrectomy in 23 patients. The extent of locally recurrent tumour was classified by the pattern of pelvic invasion as follows: localized, sacral invasion and lateral invasion. RESULTS: Multivariate analysis showed that the pattern of pelvic invasion was a significant prognostic factor which independently influenced survival after resection of recurrent cancer (P < 0.001). The 5-year survival rates were 38 per cent in the localized type (n = 27), 10 per cent in the sacral invasive type (n = 16) and zero in the lateral invasive type (n = 17). CONCLUSION: Resection for locally recurrent rectal cancer is potentially curative in patients with localized or sacral invasive patterns of recurrence. Alternatives should be explored in patients with recurrence involving the lateral pelvic wall.

Inhibitors of transcription, proteasome inhibitors, and DNA-damaging drugs differentially affect feedback of p53 degradation.

Mutations of the p53 gene are the most common abnormalities in human cancer. In contrast to mutant p53, wild-type (wt) p53 protein is present at low levels due to rapid degradation by proteasome. We demonstrated that wt p53 protein stabilization following DNA damage or proteasome inhibition did not abolish the wild-type conformation. DNA damage did not cause accumulation of ubiquitinated forms of wt p53, suggesting abrogation of ubiquitination. Consistent with this, the E6 oncoprotein which targets p53 for ubiquitination abolished stabilization of p53 protein by DNA-damaging drugs but not by proteasome inhibitors. In contrast to the effects on wt p53, inhibitors of proteolysis downregulated mutant p53. Regulation of p53 levels can be explained by a feedback mechanism where wt p53 transcriptionally induces "sensor" proteins (Mdm-2, as an example) and these, in turn, target p53 for degradation. Like p53, Mdm-2 is degraded by proteasome. Therefore, inhibition of proteasome caused accumulation of Mdm-2, leading to degradation of mutant p53 by the remaining proteolytic activity of the cell. We propose that inhibition of transcription should increase wt p53 protein due to inhibition of Mdm-2 synthesis. An inhibitor of transcription, alpha-amanitin, dramatically induced wt p53 protein, whereas Mdm-2 protein was downregulated. Moreover, alpha-amanitin increased p53 protein levels in E6-transfected cells. Although inhibitors of transcription, such as actinomycin D, also damage DNA, reduction of Mdm-2 or other putative "sensor" proteins may contribute to their p53-stabilizing activity. Similarly, antimetabolites augment accumulation of wt p53 due to interference with RNA synthesis.

Expression of the multidrug resistance-associated protein (MRP) gene in urothelial carcinomas.

The intrinsic or acquired resistance of urothelial cancer to chemotherapy is one major obstacle to successful treatment. Generally, the expression level of P-glycoprotein in urothelial cancer is low, so we accordingly investigated the expression of multidrug resistance-associated protein (MRP). We examined the expression of MRP mRNA by means of slot-blotting samples of 11 renal pelvic and/or ureteral tumors, 33 bladder tumors, one lung metastasis from a ureter tumor, 7 non-cancerous urothelia from patients with transitional-cell carcinoma (TCC) and one urothelium from a patient with renal-cell carcinoma (RCC). We also estimated, by Southern blotting, whether or not the MRP gene was amplified in clinical specimens that overexpressed MRP mRNA. MRP was detected immunohistochemically using a polyclonal antibody against MRP. In all, 5 of 11 renal pelvic and/or ureter tumors (45.5%), 17 of 33 bladder tumors (51.5%) and 4 of 7 non-cancerous urothelia of TCC patients (57.1%) expressed more than 2-fold the MRP mRNA levels of drug-sensitive human KB cells. There was no significant difference in the MRP mRNA level between primary and recurrent tumors. Low-grade urothelial carcinomas (G1 and G2 TCCs) expressed significantly higher levels of MRP mRNA than the high-grade G3 TCC. The MRP gene was not amplified in urothelial carcinomas, irrespective of their expression levels of MRP mRNA. Immunohistochemically, MRP was located mainly on the plasma membrane, but also detected on the cytoplasm of cancer cells. MRP may be one mechanism responsible for intrinsic drug resistance in low-grade urothelial cancer.

Expression of the multidrug-resistance-associated protein (MRP) gene in human colorectal, gastric and non-small-cell lung carcinomas.

MRP has been identified as another multidrug-resistance (MDR) gene and may be involved in an alternative MDR mechanism in some solid tumors. We investigated the expression of MRP mRNA in multidrug-resistance KB sublines (KB-8-5, KB-C2, C-A40 and C-A120), human non-small-cell lung carcinomas (NSCLC), gastric and colorectal carcinomas, and compared it with that in drug-sensitive human KB cells. MRP gene expression was elevated in 8 of 9 (89%) squamous-cell carcinomas of the lung. Furthermore, MRP expression in 4 squamous-cell carcinomas (L13, 18, 19 and 20) was more than 3.6 times higher than in KB-3-1 cells, and the average MRP mRNA expression level of all squamous-cell carcinomas was significantly higher than that of adenocarcinoma of the lung and of colorectal and gastric carcinomas. These results suggested that the MRP is responsible, at least in part, for drug resistance in some squamous-cell carcinomas of the lung.

Clinical studies on dissolution of gallstones using ursodeoxycholic acid.

Ursodeoxycholic acid (UDCA), 7beta hydroxy epimer of chenodeoxycholic acid (CDCA), has been used as a choleretica for 20 years in Japan. Recent report showing increased excretion of UDCA in bile after CDCA administration may suggest the possibility that UDCA has similar effects to CDCA on bile cholesterol unsaturation and on gallstone dissolution. The present paper describes the clinical usefulness of UDCA for gallstone patients during the past two years. Seventy-four gallstone patients with functioning gall-bladders, 19 men and 55 women with a mean age of 48 years, have been treated for 6 months or more. UDCA, supplied in tablets (Ursosan), was given 450 mg per day. The disappearance or the reduction of stone size or number, or both (dissolution effect) was recognized in 32 out of 74 patients (43%). In case of radiolucent stones, the overall effective rate was estimated for 24 of 46 patients (52%). There may be no significant difference in dissolution effect between CDCA and UDCA treatment, however, the merit of UDCA treatment seems to have its few side effects.