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The fringe tree, Chionanthus retusus (Oleaceae) has been used as a natural remedy that helps a paralytic stroke, dementia, phlegm and malaria. This study was conducted to investigate the antioxidant and anti-aging activities of water extracts obtained from the flesh of C. retusus by reflux extraction at room temperature and 80°C, and pressure heating extraction at 110°C, for an increment of their practical use. The highest extraction yield from the flesh of C. retusus was 67.94% extract by pressure heating extraction at 110°C. The content of total polyphenol compounds was the highest by 72.71 mg/g in 80°C extract by reflux extraction. The highest content of total flavonoid compounds was 7.60 mg/g at 110°C extract. The results of soluble protein and reducing sugar contents showed the highest content of 4.93 mg/g and 46.77 mg/g in 110°C extract, respectively. In the analysis of DPPH radical scavenging activity, all extracts showed an excellent effect of 92% in 1.0 mg/mL concentration. The highest effect of ABTS radical scavenging activity was 88.67% at 110°C extraction in 1.0 mg/mL concentration. Nitrite scavenging ability was the highest as 66.16% at 110°C extract at pH 1.2 and 1.0 mg/mL concentration. The results of SOD-like activity and xanthine oxidase inhibition activities showed the highest effect of 14.95% and 59.45% at 110°C extract in 1.0 mg/mL, respectively. Tyrosinase inhibition activity for whitening effect was the highest of 35.31% at 85°C in 1.0 mg/mL. In the analysis of anti-aging effect, collagenase and elastase inhibition activity showed the highest effects of 37.78% at 110°C and 20.39% at 80°C, respectively. All results of antioxidant activities and anti-aging effects increased along with the concentration increases. These results indicated that the extracts extracted from the flesh of C. retusus at 80°C and 110°C have a large amount of useful ingredients, an excellent antioxidant activity, like as DPPH and ABTS radical scavenging activity, and anti-aging effects to develop functional products than those of 25°C.

© 2019 Elsevier Inc. We previously reported that inducing gamma oscillations with a non-invasive light flicker (gamma entrainment using sensory stimulus or GENUS) impacted pathology in the visual cortex of Alzheimer's disease mouse models. Here, we designed auditory tone stimulation that drove gamma frequency neural activity in auditory cortex (AC) and hippocampal CA1. Seven days of auditory GENUS improved spatial and recognition memory and reduced amyloid in AC and hippocampus of 5XFAD mice. Changes in activation responses were evident in microglia, astrocytes, and vasculature. Auditory GENUS also reduced phosphorylated tau in the P301S tauopathy model. Furthermore, combined auditory and visual GENUS, but not either alone, produced microglial-clustering responses, and decreased amyloid in medial prefrontal cortex. Whole brain analysis using SHIELD revealed widespread reduction of amyloid plaques throughout neocortex after multi-sensory GENUS. Thus, GENUS can be achieved through multiple sensory modalities with wide-ranging effects across multiple brain areas to improve cognitive function. Auditory stimulation combined with light-induced gamma oscillations in the hippocampus CA1 and auditory cortex regions of the brain reduces amyloid levels and improves memory in animal models of Alzheimer's disease.

Neuronal and synaptic loss is characteristic in many neurodegenerative diseases, such as frontotemporal dementia and Alzheimer's disease. Recently, we showed that inducing gamma oscillations with visual stimulation (gamma entrainment using sensory stimuli, or GENUS) reduced amyloid plaques and phosphorylated tau in multiple mouse models. Whether GENUS can affect neurodegeneration or cognitive performance remains unknown. Here, we demonstrate that GENUS can entrain gamma oscillations in the visual cortex, hippocampus, and prefrontal cortex in Tau P301S and CK-p25 mouse models of neurodegeneration. Tau P301S and CK-p25 mice subjected to chronic, daily GENUS from the early stages of neurodegeneration showed a preservation of neuronal and synaptic density across multiple brain areas and modified cognitive performance. Our transcriptomic and phosphoproteomic data suggest that chronic GENUS shifts neurons to a less degenerative state, improving synaptic function, enhancing neuroprotective factors, and reducing DNA damage in neurons while also reducing inflammatory response in microglia.

To evaluate the effects of prostaglandin analogs (PGAs) on cell viability and apoptosis in cultured astrocytes obtained from the lamina cribrosa (LC) of the human optic nerve head (ONH).Astrocytes were cultured from LC samples obtained from human donor ONH and treated with three kinds of acid form of PGAs: latanoprost (LAT-A), tafluprost (TAF-A), and bimatoprost (BIM-A) (0.1, 1, 10, 50 and 100 ug/mL). Cell viability was assessed using the WST-1 assay. Cell apoptosis was measured using the deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay. Apoptotic protein expression was evaluated using western blot analysis.ONH astrocytes expressed FP receptor in western blot analysis. In the presence of 0.1 ug/mL of LAT-A, BIM-A, and TAF-A, the cell viability was 85%, 85% and 82%, respectively. WST-1 assay revealed about 50% of cell viability following treatment with 50 ug/mL of all PGAs. After exposing astrocytes to 10 ug/mL of each PGA for 24 hours, apoptotic cells were stained in TUNEL assay. Western blot analysis revealed that the PGAs up-regulated Bax (pro-apoptotic protein) and down-regulated Bcl-xL (anti-apoptotic protein) in the astrocytes.PGAs affected cell viability in cultured astrocytes obtained from human ONH LC. PGA treatment may induce apoptosis in ONH astrocytes.

A genetic variation of Lepista nuda and two genus Lepista species (L. irina and L. sordida) were analyzed by random amplified polymorphic DNA (RAPD) and internal transcribed spacer (ITS) sequence analysis. In the resulting RAPD analysis, 22 out of 40 random primers amplified polymorphic RAPD fragment patterns, the amplified bands were 355, and DNA fragment sizes were 200-400bp. Intraspecific genetic dissimilarity of the 10 L. nuda strains were calculated to range from 0% to 21.60%, L. sordida from 16.93% to 24.82%, L. irina were 20.62% to 25.54%, and intraspecific genetic dissimilarity of L. sordida and L. irina was 23.49%. The 673 base pairs were sequenced during the analysis of the ITS I and II region; six L. nuda strains intraspecific genetic dissimilarities ranged from 1.58% to 11.47%, L. nuda and L. sordida from 3.83% to 12.88%, L. nuda and L. irina from 7.11% to 15.61%, and intraspecific genetic variation between L. sordida and L. irina was 4.79%. The findings showed that RAPD and ITS sequencing could be used for developing molecular genetic markers and screening of unidentified genus Lepista species.

In the present study, the proximate composition, sugar, minerals, total phenolic and flavonoid compounds, and amino acids in Calystegia japonica (C. japonica) were measured to determine if it can be used as a nutritional and functional material for the development of valuable foods. The mean crude protein, fat, and ash contents of the leaves were 5.75, 2.46, and 7.77%, respectively. The soluble-protein contents of the leaves and roots were 146.78 and 33.67 mg%, respectively. The reducing-sugar and free-sugar contents of the leaves were 682.70 and 166.00 mg%, respectively, and those of the roots were 2,934.89 and 37.70 mg%. The mineral content of the leaves was 3,122.13 mg%, and that of the roots was 1,540.85 mg%. The three elements Ca, K, and Mg were very rich in all their parts, with minerals accounting for 96-99% of their total mineral contents. The total phenolic compound of the leaves was 3,028.89 mg%, and the total flavonoid compound was 382.67 mg%. The phenolic and flavonoid compounds in the leaves were more than 7.6 times those in the roots. The free-amino acid levels in the leaves and roots were 2,467.15 and 1,334.81 mg%, respectively. The results of the comparison of the leaves and roots of C. japonica showed that the leaves had a rich proximate composition consisting of minerals, total phenolic and flavonoid compounds, and amino acid. This suggests that C. japonica leaves are potentially useful sources of functional and favorite foods and nutraceuticals.

In this study, we assessed the free radical scavenging and xanthine oxidase inhibitory activities of extracts isolated from the dried roots and stems (including leaves) of Angelica dahurica. The irritation response from these extracts was also assessed to determine potential cosmetic use. Both sources of A. dahurica extracts exhibited radical scavenging properties to different extents. The free radical scavenging potency (EC 50 ) of the stems (including leaves) of A. dahurica was 243.33 ㎍/mL, which is significantly lower (p＜0.01) than that observed of the roots (1,161.79 ㎍/mL). The inhibition values (EC 50 ) of xanthine oxidase were as follows: 435.19 ㎍/mL (roots) and 434.66 ㎍/mL (stems). We noted no significant differences between the two plant parts with regard to ability to inhibit xanthine oxidase activity. After the application of A. dahurica extracts to rabbits for skin and eye irritation tests, no negative effects were observed; therefore, the extracts are considered to be non-irritating to the skin and eye.

Three bark extracts of Albizia julibrissin were prepared using water (AW), 70% (v/v) ethanol (AE), and hot water (AHW). Organic solvent fractions were analyzed for total flavonoids and polyphenols, antioxidant activities, and inhibitory activities against xanthine oxidase. Total flavonoid and polyphenol contents of the AHW extract were 8.57 mg/g and 108.67 mg/g, respectively. The SOD-like activities of all extracts, assayed at 1.0 mg/mL, were 10.46-16.73%. The nitrite-scavenging ability of the AHW extract, assayed at pH 1.2, was 60.82%, and the value was /mL. The electron-donating ability of the AHW extract, at 0.3 mg/mL, was 92.30%; the values of the AW and AHW extracts were /mL and /mL, respectively; thus higher than that of ascorbic acid (/mL). Xanthine oxidase inhibition by the AHW extract, at 1.0 mg/mL, was 94.05%. These results indicate that the AHW of A. julibrissin has potential as a natural antioxidant, for addition to foods and nutraceuticals.

This study was conducted to analyze components of the fruit and stem of Vitex rotundifolia as part of a study on the nutritional and functional materials, and the development of a functional food. General components of V. rotundifolia fruit are moisture 12.92%, carbohydrate 78.67%, crude protein 3.22%, crude fat 1.73% and 3.46% crude ash. V. rotundifolia stem was moisture 11.30%, carbohydrate 80.87%, crude protein 4.78%, crude fat 0.64 % and 2.41% crude ash. The content of V. rotundifolia reducing sugar was 646.07 mg% (fruit) and 1,547.97 mg% (stem). The total amount of free sugar was fruit 5.66 mg% and stem 90.79 mg%. The content of soluble protein was 3,268.12 mg% in fruit and 4,927.55 mg% in stem. The polyphenol compound content was 608.06 mg% and 808.06 mg%, respectively. Total amount of hydrolyzed amino acid of V. rotundifolia fruit and stem were 3,095.75 mg% and 2,135.84 mg%, while that of free amino acid of fruit and stem were 79.99 mg%, 81.20 mg%, among which cysteine (2,010.82 mg%) was the highest in the V. rotundifolia fruit. In the results of mineral analysis, the content of K was the highest in fruit (2,184.00 mg%) and stem (1,469.20 mg%).

Water extracts from root, stem and leaf of Ailanthus altissima were utilized to determine antioxidant properties such as electron donating ability (EDA), nitrite scavenging ability, superoxide dismutase (SOD)-like activity, and the inhibitory activities of xanthine oxidase (XO) and tyrosinase. The EDA of root extract was the highest as 77.33% at 0.5 mg/mL concentration and that of stem extract was 70.01% at 1.0 mg/mL. The nitrite scavenging ability of leaf extract revealed the highest effect as 95.18% at pH 1.2, 1.0 mg/mL while those of stem and root extracts were 55.17% and 33.33%, respectively. The leaf extract showed the highest SOD like activity as 26.77% at 1.0 mg/mL, the measurement of root extract was 3.82% and that of stem extract was not effective. All kinds of extracts had strong inhibitory activities on XO of over 92% at 1.0 mg/mL. The highest activity on tyrosinase inhibition was obtained from leaf extract of 16.33% at 2.0 mg/mL. The results indicated that among the three extracts, the leaf extract has a strong and extensive antioxidant activity.

In the present study, we analyzed the antioxidant, antiplatelet aggregation, anti-inflammatory, and tyrosinase inhibitory activities of a variety of solvent extracts of Elaeagnus multiflora Thunb. (Family Elaeagnaceae). Among the solvent extracts of E. multiflora, the ethyl acetate extract (EE) exhibited the highest 1,1-diphenyl-2-picrylhydrazyl radical and xanthine oxidase inhibition activity, as well as the greatest tyrosinase inhibition activity. Only the chloroform extract (CE) inhibited platelet aggregation, and that was a weak effect with 19.29% inhibition at 250 microg/mL, as compared to controls. The CE was also the most potent inhibitor of nitric oxide production among the tested fractions, with almost 100% inhibition at 500 microg/mL. We also detected 2,4-bis(1,1-dimethylethyl)phenol in the CE and EE, via a gas chromatography-mass spectrometry assay. In conclusion, we found that E. multiflora Thunb. has antioxidant and antiplatelet aggregation effects to some extent, and its CE and EE possess potent inhibitory effects on nitric oxide production and tyrosinase activity.

We analyzed the contents of polyphenol compounds, the antioxidant activity and the physiological activity to investigate the functional effects of extracts from Angelica dahurica by the reflux water extraction (RW), reflux ethanol extraction (RE) and pressure heating water extraction (PW). The content of phenolic compounds of PW was the highest at 156.30 ㎎/g, and those of RW and RE were 31.69 ㎎/g and 26.34 ㎎/g, respectively. The electron donating ability (EDA) were in the range of 30.56%～52.74% and superoxide dismutase (SOD) like activity were 10.96%～23.24% at 1,000 ㎍/mL. The nitrite scavenging ability of PW at pH 1.2 was 61.47%, higher than those of RW (16.81%) and RE (17.78%). The xanthine oxidase inhibitory were 90.91% and tyrosinase inhibitory rate of RE was the highest (51.71%) at the concentration of 5,000 ㎍/mL. All extracts were increased with increments of the extract concentrations.

This study was carried out to analyze the components of the stem of bush clover, Lespedeza bicolor to obtain basic data on the studies of the physiological function and a possibility of utilizing L. bicolor. General components of L. bicolor were 82.35 carbohydrate, 13.38 moisture, 2.80 crude protein, 0.86 crude ash and 0.61 crude fat. The content of reducing sugar was 147.58 mg/100 g. The total amount of free sugar was 52.4 mg/100 g that was composed of 31.0 mg/100 g fructose, 16.7 mg/100 g glucose and 4.7 mg/100 g sucrose. In the results of mineral analysis, the content of Ca was the highest (472.97 mg/100 g) and followed by 206.70mg/100 g of K. The total amount of hydrolyzed amino acid was 148.95 mg/100 g that consist of 31.74 mg/100g of essential amino acid and 117.24 mg/100 g of non-essential amino acid. Total free amino acids were contained 106.39 mg/100 g that was composed of 8.41 mg/100 g essential amino acids and 97.98 mg/100 g the non-essential, and proline (62.92 mg/100 g) was the highest that account for 59.8 of total free amino acids. Total content of amino acid derivatives was 30.01 mg/100g and that of -aminoisobutyric acid was the highest as 12.57 mg/100 g among them.

This study was investigated to analyze the effect of extracts from the Lepista nuda, on the antioxidant activity to form a part of studies on the functional materials of L. nuda. Antioxidant activity of L. nuda extracts was evaluated by measuring the electron-donating ability (EDA), the superoxide dismutase (SOD)-like activity, and the nitrite-scavenging ability. The EDAs of water and ethanol extracts at the concentration of 1,000 ppm by the rotary heating method from L. nuda were and , respectively and those of water and ethanol extracts by microwave-assisted method were and , respectively. The measurements of SOD-like activity were in the range of at 1,000 ppm. EDA and SOD were increased with the concentrations of extracts. The nitrite-scavenging ability at the concentration of 1,000 ppm was the highest and at pH 1.2, and was decreased with an increment of pH value. These results indicated that microwave­assisted water extract from L. nuda showed the highest activities on the EDA and nitrate-scavenging ability, while the rotary heating ethanol extract had the highest effect on the SOD-like activity.

This study was carried out to analyze the soil properties of Lepista nuda habitats to form a part of the studies on the characteristics of L. nuda. The soil samples collected from seven studied areas were investigated for soil properties such as soil moisture, soil pH, and the contents of organic and inorganic matters. The content of soil moisture was 29.7% and the content of organic matter was 32.1%. Total nitrogen was 0.74% and soil pH was 4.75, which was ranged from 3.6 to 5.20. In the content of inorganic matter, the content of Fe was highest as 1,024 ppm, K 183 ppm, Mn 21.9 ppm, Ca 2.02 ppm, Zn 1.46 ppm and Mg was lowest as 0.51 ppm.

To develop the new type of salt-fermented seafoods, the salt-fermented oysters in olive oil (product SO) were manufactured, and food components and quality characteristics of product SO were examined. The optimum processing condition for product SO is as follows. The raw oyster with no shell was washed off with 3% saline solution. Then dewatered, and dipped in the brine-salting solution made up with saturated saline solution and oyster sauce (2 : 1 v/v) mixture added 1% sodium erythorbic acid and 0.2% polyphosphate. After salt-fermentation it ripened by brine salting at 51 for 15 days. Then dried at 15 for 4 hours with cool-air, and packed in No. 3B hexahedron type can. Finally, poured with olive oil and seamed it by double-seamer. The moisture, crude protein, crude ash and volatile basic nitrogen contents of the product SO were 61.6%, 12.0%, 16.3% and 34.3 mg/100 g, respectively. In taste-active components of the product SO, total amount of free amino acids is 2,335.4 mg/100 g and it has increased by 50% overall during salt-fermentation 15 day. Taurine, glutamic acid, proline, glycine, alanine, -alanine and lysine were detected as principal free amino acids. The contents of inorganic ions were rich in Na and K ion, while the amounts of nucleotide and its related compounds and other bases except betaine were small. From the results of this research, the product SO had a superior organoleptic qualities compared with conventional oyster product, and could be reserved in good conditions for storage 90 days at room temperature.

To analyze chromosome inversions of Drosophila immigrans, wild flies were captured from large vineyards located in the suburbs of Yecheon and Gyeongsan from October 1999 to 2001. With the egg samples obtained singly at each of the 799 females of D. immigrans, cytological examinations were carried out for the type and frequency of inversions. Two types of different inversions were found only in the second chromosome. The inversions detected were known to be the cosmopolitan inversion “A” and “B”. The mean frequency of inversion A was estimated to be 0.074 in Yecheon and 0.066 in Gyeongsan and that of B was to be 0.026 in Yecheon and 0.021 in Gyeongsan, respectively. In the frequency ratio, inversion A was significantly higher than that of B. The present populations of D. immigrans showed subtle differences from other Korean populations in inversion frequencies. To account for the local variations observed in inversion frequencies, several hypotheses are discussed such as founding event or selective force.

To find if a difference in telomerase or survivin expression exists between non-neoplastic tissues and hydatidiform moles, and explore expression of those proteins in normal placental development, post-term gestation, and preeclampsia.Formalin-fixed placental tissues were selected from collections of the Department of Pathology at the University of Colorado. Five specimens of each trimester, five each of preeclamptic and post-term placentas, and 23 molar pregnancies were selected. The telomerase catalytic protein hTERT was localized in placental tissues using the catalyzed signal amplification system, and survivin was localized by conventional immunoperoxidase method. Staining was graded on a scale of zero to 4.hTERT staining was detected in sections of 42 of 48 specimens (23 of 23 hydatidiform moles, 19 of 25 non-neoplastic placental tissues). The intensity of staining for hTERT was higher in hydatidiform moles (mean 3.3, median 3) compared with levels in non-neoplastic placental tissues (mean 0.92, median 1) (P.001). Survivin was detected in 39 of 48 specimens (22 of 23 hydatidiform moles, 17 of 25 non-neoplastic placental tissues). Compared with non-neoplastic tissues (mean 0.88, median 1), survivin levels were elevated in hydatidiform moles (mean 1.35, median 1) (P =.031).Survivin and telomerase were increased in hydatidiform moles, suggesting that regulation of apoptosis and stabilization of telomere length might be involved in neoplastic transformation of the placenta. The patterns of expression observed for survivin and telomerase in non-neoplastic placental tissues suggest that the control of apoptosis and stabilization of telomeric DNA might also be involved in normal gestational development.

Background Shortening of telomeres (specialized structures at the ends of chromosomes) beyond a certain length may signal a cell to stop dividing and to enter senescence. A ribonucleoprotein enzyme, telomerase, is a key component in maintaining telomere length. Because the majority of cancers express telomerase but most normal somatic tissues do not, we measured the level of telomerase expression in primary breast cancer specimens for correlation with traditional prognostic indicators and disease outcome. Methods Telomerase activity was measured in frozen human breast cancer specimens by use of the Telomeric Repeat Amplification Protocol (TRAP) assay. The level of telomerase activity was expressed as total product generated (TPG) and was corrected for specimen cellularity by expressing it as a ratio of TPG to the sample's 28S ribosomal RNA content. Results A preliminary study of 150 breast cancer specimens demonstrated that telomerase activity correlated with the fraction of cells in S phase of the cell cycle (r(sp) = .23). In a larger prognostic study of 398 tumors from patients with lymph node-positive breast cancer, telomerase expression correlated with S-phase fraction, progesterone receptor level, DNA ploidy, and lymph node status. After correcting for sample cellularity, increasing TPG levels were associated with decreased disease-free survival (P = .041) and overall survival (P = .009) of the patients. The telomerase activity level remained strongly predictive of death (P = .027) and marginally predictive of disease recurrence (P = .08) after adjustment for other prognostic factors. All P values are two-sided. Conclusions Telomerase activity in human breast cancers is associated with a more aggressive tumor phenotype in patients.

Cellular immortality is believed to be a critical step in tumorigenesis. As an important component of the telomere maintenance mechanism, the activation of the enzyme telomerase is tightly associated with cellular immortality and cancer. Telomerase expression is detected in a majority of tumours, but is absent in most somatic tissues and correlates to clinical outcome in a number of cancer types. Telomerase expression is associated with the stage of differentiation but not necessarily with the rate of cell proliferation. Data also indicate that inhibition or absence of telomerase may result in cell crisis in cancer cells and tumour regression in cancer patients. These results suggest that cancer therapy based on telomerase inhibition could be a more effective and safer treatment for cancer, as well as provide a more accurate means for diagnosing and predicting clinical outcome in cancer. Complete understanding of the role of telomerase in tumorigenesis through well-designed clinical studies will have a significant clinical impact on the treatment and diagnosis of cancer, c 1997 Elsevier Science Ltd. All rights reserved.

The association of human telomerase activity with an indefinite replicative capacity of cells in vitro and advanced tumors in vivo is gaining wide support. The increasing interest in studying various aspects of telomerase expression in cancer required the development of a sensitive and reliable protocol for the extraction and detection of telomerase activity in cell culture material, and from small tissue samples obtained from biopsy, surgical reaction of tumors, and autopsy. Recently a novel procedure for the extraction and detection of telomerase activity was developed (Science 1994; 266: 2011-2015) which resulted in an estimated 10 4 fold improvement in detectability compared with previous methods. The described procedures not only dramatically increased sensitivity but also allowed fast and efficient detec- tion of telomerase activity in a large number of samples. A number of technical aspects which are of critical importance for reproducibility and relia- bility of this assay using clinical material are addressed in this report. In addition, new methods to perform telomerase assays without the use of radioisotopes are described.

Two new species belonging to the genera Microdrosophila and Drosophila, collected from Daegwallyeong and Mt. Halla in Korea are described with illustrations of the male genitalia. Microdros ophila (Microdros ophila) gang‐wonensis n. sp. seems close to M. (M.) purpurata Okada, 1956, but the new species has different phallic organ. Drosophila (Drosophila) taekjuni is close to D. (D.) brevitabula Zhang and Toda, 1992, in form, but differs in the shape of aedeagus. Eight species of the genus Microdrosophila and five species in the D. immigrans species‐group of genus Drosophila, including the new species are known to be distributed in Korea.

Maintenance of chromosomal telomeres is necessary for continued cell growth, and this is carried out in germline tissues by telomerase. In contrast to most somatic tissues, many tumours have telomerase activity. The RNA component of human telomerase (hTR) was measured by Northern analyses of 150 primary untreated neuroblastomas and compared with clinical stage at diagnosis. hTR expression > 33 (relative to cell line control = 100) was seen in 41% of all tumours and the frequency of hTR > 33 increased with stage of disease. Expression of hTR may be involved in progression of neuroblastoma.

The telomeric repeat amplification protocol (TRAP) assay has been used to test telomerase activity in numerous cancer specimens. We describe primers, controls and quantification methods for the TRAP assay to accurately measure the level of telomerase activity in clinical samples. The assay is reliable and reproducible in routine analyses and can be used to estimate the processivity of telomerase activity.

Telomerase has been detected by telomerase repeat amplification protocol (TRAP) assay in cervical dysplasia and squamous cell carcinoma but not in most normal cervical tissues. In the present study, the cellular localization of the protein catalytic subunit of telomerase (hTERT) and the RNA component (hTR) were investigated by a sensitive immunohistochemical technique and by in situ hybridization, respectively. hTERT protein was detected in all diagnostic categories of cervical specimens. hTERT was localized predominantly to the lower suprabasal levels of normal squamous mucosa but was detected throughout virtually all levels of the lesional epithelium in low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs), and squamous cell carcinoma (SCC). Telomerase expression correlated with hTERT detection in SCC and HSIL but was not detected by TRAP assay in most samples of normal mucosa or LSIL. The distribution of hTR correlated with the localization of hTERT in HSIL and SCC but was restricted to the basal and suprabasal cell layers in normal mucosa and LSIL.

Vulvar intraepithelial neoplasias (VINs) are potentially premalignant lesions of the squamous mucosa. The immunohistochemical distribution of the catalytic protein subunit of telomerase (hTERT) and the patterns of X chromosome inactivation were investigated as markers of neoplasia in samples from a patient with multifocal and diffuse VIN. hTERT nuclear staining in VIN correlated with squamous maturation and the degree of nuclear atypia. Normal mucosa revealed faint nuclear staining of parabasal cells and lower intermediate layer squamous cells. Monoclonal composition was demonstrated in 0 of 3 samples of VIN1, 2 of 3 samples of VIN2, and 13 of 13 samples of VIN3. The patterns of X chromosome inactivation indicated intramucosal extension and multifocal origin of individual lesions. Five samples of histologically normal vulvar squamous epithelium revealed a random pattern of X chromosome inactivation, consistent with polyclonal composition. All 19 samples from 9 lesions contained human papillomavirus (HPV)-16 sequences. Neither mutations in the p53 tumor suppressor gene or K-ras oncogenes nor loss of heterozygosity at 7 chromosomal loci were detected in any of the 19 samples of VIN. These results demonstrate that HPV-associated VIN may result from multifocal and diffuse 2-dimensional intraepithelial expansion of an immortalized monoclonal cell population.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA poly-merases cannot fully replicate the ends of linear molecules1–4. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours5,6. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division7–9. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation10. In addition to the human telomerase RNA component (hTR; ref. 11), TP1/TLP1 (refs 12,13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme14, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (SpTrt!) telomerases15,17, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues17,18. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

The progressive shortening of the ends of chromosomes (telomeres) during cell division may serve as a mitotic clock for replicative senescence. Telomerase, a ribonucleoprotein which synthesizes telomeric DNA and maintains telomere length, is absent from most normal somatic cells but is expressed in immortal cells. Low levels of telomerase activity have been detected in peripheral blood mononuclear cells (PBMC) and hematopoietic cells and an increase in telomerase activity during T cell activation has recently been reported. In this study, we show that the increase in telomerase activity during T cell activation was transient and did not prevent the loss of telomeres in long-term T cell cultures. Analysis of the mechanism of telomerase induction showed that the increase in telomerase activity was accompanied by an increase in the levels of hTR, the RNA component of human telomerase. Moreover, telomerase induction occurred in the presence of aphidicolin, indicating that DNA synthesis was not required. Increased telomerase expression was observed when PBMC were activated with phorbol myristate acetate (PMA) and ionomycin, indicating that it was independent of early transmembrane signals. It was, however, linked to the T cell signal transduction pathway, as inhibiting protein kinase C with bisindolylmaleimide prevented the increase in telomerase activity.

The loss of telomeric DNA may serve as a mitotic clock which signals cell senescence and exit from cell cycle. Telomerase, and enzyme which synthesizes telomeric repeats de novo, is required to maintain telomere lengths. In humans, significant telomerase activity has been found in cells with essentially unlimited replicative potential such as reproductive cells in ovaries and testes, immortal cell lines and cancer tissues, but not in most normal somatic cells or tissues. We have now examined telomerase expression in subpopulations of hematopoietic cells from adult human bone marrow using a sensitive polymerase chain reaction-based telomeric repeat amplification protocol. Telomerase activity was found at low levels in the highly enriched primitive hematopoietic cells (CD34+CD71loCD45RAlo) and was increased transiently when these cells were cultured in the presence of a mixture of cytokines. In contrast, the early progenitors (CD34+CD71+) expressed telomerase activity at a higher level which was subsequently downregulated in response to cytokines. Telomerase activity remained low in the more mature CD34-cells upon exposure to cytokines. Taken together, our results suggest that telomerase is expressed at a basal level in all hematopoietic cell populations examined, is induced in a primitive subset of hematopoietic progenitor cells and is downregulated upon further proliferation and differentiation of these cells. We have previously observed telomere shortening in cytokine-stimulated primitive hematopoietic cells. The low and transient activation of telomerase activity described here thus appears insufficient to maintain telomere lengths in cultured hematopoietic cells.

Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.