A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance

Considerable interest has been focused on telomerase because of its potential use in assays for cancer diagnosis, and for anti-telomerase drugs as a strategy for cancer chemotherapy. A number of assays based on the polymerase chain reaction (PCR) have been developed for evaluation of telomerase activity. To overcome the disadvantages of the conventional telomerase assay [telomeric repeat amplification protocol (TRAP)] related to PCR artifacts and troublesome post-PCR procedures, we have developed a telomeric repeat elongation (TRE) assay which directly measures telomerase activity as the telomeric elongation rate by biosensor technology using surface plasmon resonance (SPR). 5′-Biotinylated oligomers containing telomeric repeats were immobilized on streptavidin-pretreated dextran sensor surfaces in situ using the BIACORE apparatus. Subsequently, the oligomers associated with the telomerase extracts were elongated in the BIACORE apparatus. The rate of TRE was calculated by measuring the SPR signals. We examined elongation rates by the TRE assay in 18 cancer and three normal human fibroblast cell lines, and 12 human primary carcinomas and matching normal tissues. The elongation rates increased in a concentration- and time-dependent manner. Those of cancer cells were two to 10 times higher than fibroblast cell lines and normal tissues. Telomerase activities and its inhibitory effects of anti-telomerase agents as measured by both the TRE and TRAP assays showed a good correlation. Our assay allows precise quantitative comparison of a wide range of human cells from somatic cells to carcinoma cells. TRE assay is suitable for practical use in the assessment of telomerase activity in preclinical and clinical trials of telomerase-based therapies, because of its reproducibility, rapidity and simplicity.     

Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 120 mg. kg-1. day-1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6-5.5 microM) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 microM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.     

Free-energy landscape of a chameleon sequence in explicit water and its inherent alpha/beta bifacial property

A sequence in yeast MATalpha2/MCM1/DNA complex that folds into alpha-helix or beta-hairpin depending on the surroundings has been known as "chameleon" sequence. We obtained the free-energy landscape of this sequence by using a generalized-ensemble method, multicanonical molecular dynamics simulation, to sample the conformational space. The system was expressed with an all-atom model in explicit water, and the initial conformation for the simulation was a random one. The free-energy landscape demonstrated that this sequence inherently has an ability to form either alpha or beta structure: The conformational distribution in the landscape consisted of two alpha-helical clusters with different packing patterns of hydrophobic residues, and four beta-hairpin clusters with different strand-strand interaction patterns. Narrow pathways connecting the clusters were found, and analysis on the pathways showed that a compact structure formed at the N-terminal root of the chameleon sequence controls the cluster-cluster transitions. The free-energy landscape indicates that a small conditional change induces alpha-beta transitions. Additional unfolding simulations done with replacing amino acids showed that the chameleon sequence has an advantage to form an alpha-helix. Current study may be useful to understand the mechanism of diseases resulting from abnormal chain folding, such as amyloid disease.     

The dendritic cell-specific chemokine,dendritic cell-derived CC chemokine 1, enhances protective cell-mediated immunity to murine malaria

Cell-mediated immunity plays a crucial role in the control of many infectious diseases, necessitating the need for adjuvants that can augment cellular immune responses elicited by vaccines. It is well established that protection against one such disease, malaria, requires strong CD8(+) T cell responses targeted against the liver stages of the causative agent, Plasmodium spp. In this report we show that the dendritic cell-specific chemokine, dendritic cell-derived CC chemokine 1 (DC-CK1), which is produced in humans and acts on naive lymphocytes, can enhance Ag-specific CD8(+) T cell responses when coadministered with either irradiated Plasmodium yoelii sporozoites or a recombinant adenovirus expressing the P. yoelii circumsporozoite protein in mice. We further show that these enhanced T cell responses result in increased protection to malaria in immunized mice challenged with live P. yoelii sporozoites, revealing an adjuvant activity for DC-CK1. DC-CK1 appears to act preferentially on naive mouse lymphocytes, and its adjuvant effect requires IL-12, but not IFN-gamma or CD40. Overall, our results show for the first time an in vivo role for DC-CK1 in the establishment of primary T cell responses and indicate the potential of this chemokine as an adjuvant for vaccines against malaria as well as other diseases in which cellular immune responses are important.     

Cellular distribution and bioactivity of the key steroidogenic enzyme,cytochrome P450side chain cleavage,in sensory neural pathways

Neurosteroids are steroids produced within the nervous system. Based on behavioural responses evoked in animals by synthetic steroid injections, several studies suggested neurosteroid involvement in important neurophysiological processes. These observations should be correlated only to neuroactive effects of the injected steroids. Neurosteroids mostly control the CNS activity through allosteric modulation of neurotransmitter receptors within concentration ranges used by neurotransmitters themselves. Therefore, neurosteroid production within pathways controlling a neurophysiological process is necessary to consider neurosteroid involvement in that process. Because of the increasing speculation about pain modulation by neurosteroids based on pharmacological observations, we decided to clarify the situation by investigating neurosteroidogenesis occurrence in sensory pathways, particularly in nociceptive structures. We studied the presence and activity of cytochrome P450side chain cleavage (P450scc) in rat pain pathways. P450scc-immunoreactive cells were localized in dorsal root ganglia (DRG), spinal cord (SC) dorsal horn, nociceptive supraspinal nuclei (SSN) and somatosensory cortex. Incubation of DRG, SSN or SC tissue homogenates with [3H]cholesterol yielded the formation of radioactive metabolites including [3H]pregnenolone of which the synthesis was reduced in presence of aminogluthetimide, a P450scc inhibitor. These first neuroanatomical and neurochemical results demonstrate the occurrence of neurosteroidogenesis in nociceptive pathways and strongly suggest that neurosteroids may control pain mechanisms.     

Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28gamma (11S regulator gamma) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected. Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted.     

Ovarian dynamics and their associations with peripheral concentrations of gonadotropins,ovarian steroids,and inhibin during the estrous cycle in goats

Ovarian changes determined by daily transrectal ultrasound and its relationship with FSH, LH, estradiol-17beta, progesterone, and inhibin were investigated in six goats for three consecutive interovulatory intervals. Estrous cycles were synchronized using two injections of prostaglandin F2alpha analogue 11 days apart. All follicles 3 mm or greater in diameter and corpora lutea were measured daily. A follicular wave was defined as one or more follicles growing to 5 mm or greater in diameter. The day that the follicles reached 3 mm in diameter was defined as the day of wave emergence, and the first wave after ovulation was defined as wave 1. During the interovulatory interval (mean +/- SEM, 21.3 +/- 0.4 days; n = 18), follicular waves emerged at 0.3 +/- 0.5, 6.5 +/- 0.2, and 12.1 +/- 0.4 days for wave 1, wave 2, and wave 3, respectively, in goats with three waves of follicular development and at -0.6 +/- 0.3, 4.7 +/- 0.2, 9.4 +/- 0.5, and 13.4 +/- 0.5 days for wave 1, wave 2, wave 3, and wave 4, respectively, in goats with four waves of follicular development (Day 0 = the day of ovulation). The mean diameter of the largest follicle of the ovulatory wave was significantly larger than those of the largest follicles of the other waves. Corpora lutea could be identified ultrasonically at Day 3 postovulation and attained 12.1 +/- 0.3 mm in diameter on Day 8. Transient increases in plasma concentrations of FSH were detected around the day of follicular wave emergence. The level of FSH was negatively correlated with that of inhibin. These results demonstrated that follicular waves occurred in goats and that the predominant follicular wave pattern was four waves with ovulation from wave 4. These results also suggested that the emergence of follicular waves was closely associated with increased secretion of FSH.     

Characterization of rat follistatin-related gene: effects of estrous cycle stage and pregnancy on its messenger RNA expression in rat reproductive tissues

Follistatin-related gene (FLRG) was first identified as a target of a chromosomal translocation in a human B-cell leukemia. Because FLRG protein binds to activins and bone morphogenetic proteins, FLRG is postulated to be a regulator of these growth factors. However, physiological aspects of FLRG are unclear. To elucidate the physiology of FLRG, we examined expression of FLRG in reproductive tissues of the rat. FLRG mRNA was abundantly expressed in the placenta. FLRG mRNA was also expressed in the ovary, uterus, testis, lung, adrenal gland, pituitary, kidney, small intestine, and heart. During the second half of pregnancy, expression of FLRG in the placenta continuously increased, whereas follistatin mRNA levels decreased from Day 12 to Day 14 and remained low thereafter. FLRG was also expressed in decidua. Levels of decidual FLRG mRNA remained low from Day 12 to Day 16 and then noticeably increased until Day 20. In contrast, follistatin mRNA was highly expressed in the decidua on Day 12, continuously decreased until Day 16, and then remained at relatively low levels thereafter. During the rat estrous cycle, levels of ovarian FLRG mRNA fluctuated diurnally, with highest levels during daytime, and did not change relative to the day of the estrous cycle. The present results suggest that FLRG may play a role in the regulation of reproductive events.     

The Complete Primary Structure of Molluscan Shell Protein 1 (MSP-1), an Acidic Glycoprotein in the Shell Matrix of the Scallop Patinopecten yessoensis

The complete primary structure of MSP-1, a major water-soluble glycoprotein in the foliated calcite shell layer of the scallop Patinopecten yessoensis, is reported. The full-length complementary DNA for MSP-1 isolated by polymerase chain reaction contained a sequence for a signal peptide of 20 amino acids followed by a polypeptide of 820 amino acids with calculated molecular mass of 74.5 kDa. The deduced amino acid sequence of MSP-1 includes a high proportion of Ser (32%), Gly (25%), and Asp (20%), and the predicted isoelectric point is 3.2; in these respects, MSP-1 is a typical acidic glycoprotein of mineralized tissues. A repeated modular structure characterizes MSP-1, with a sequence unit between 158 and 177 amino acids in length being repeated 4 times in tandem in the middle part of the protein. The repeated unit comprises 3 modules (SG, D, and K domains), each having a distinct amino acid composition and sequence. The SG domain is almost exclusively composed of Ser and Gly residues. The D domain is rich in Asp residues, potential N-glycosylation and phosphorylation sites. The K domain is rich in Gly residues and has a core of basic residues. The Asp residues are arranged more or less regularly in the D domains, exhibiting some repeated motifs such as Asp-Gly-Ser-Asp and Asp-Ser-Asp. Further, the 4 D domains indicate remarkable overall sequence similarities to each other. These observations suggest that the regular arrangements of COO⁻ groups in the D domain side chains may be important for specific control of crystal growth. Received September 19, 2000; accepted February 9, 2001