bluuebunny/crossref_metadata_2025 huggingface dataset:
An improved experimental system for determining small folding entropy changes resulting from proline to alanine substitutions
Authors: Street Timothy O. , Bradley Christina Marchetti , Barrick Doug
Published: August 2005

Changes in protein stability can be achieved by making substitutions that increase or decrease the available conformations of the unfolded protein without altering the conformational freedom of the folded protein. Matthews and coworkers (1987) proposed that proline to alanine (P → A) substitution would achieve this type of entropic destabilization. By comparing the Ramachandran area associated with alanine and proline residues, Matthews et al. estimated the unfolding entropy change resulting from P → A substitution to be 4.8 cal mol−1K−1. Although such an entropy difference would produce a substantial free energy change, accurately resolving such free energy changes into entropic and enthalpic components has been difficult. Here, we attempt to quantify the unfolding entropy change produced by P → A substitution by amplifying the effect through multiple substitutions, and by decreasing the uncertainty in determining the unfolding entropy. Variants of a repeat protein, theDrosophilaNotch ankyrin domain, were constructed with a varying number of P → A substitutions at structurally conserved positions. Unfolding entropy values of the variants were determined from free energy measurements taken over a common temperature range using chemical denaturation. Our findings confirm the prediction that increasing the number of proline residues present in similar local environments increases the unfolding entropy. The average value of this increase in unfolding entropy is 7.7 ± 4.2 cal mol−1K−1, which is within error of the value estimated by Matthews et al. (1987).
Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering
Authors: Deuschle Karen , Okumoto Sakiko , Fehr Marcus , Looger Loren L. , Kozhukh Leonid , Frommer Wolf B.
Published: August 2005

A family of genetically‐encoded metabolite sensors has been constructed using bacterial periplasmic binding proteins (PBPs) linearly fused to protein fluorophores. The ligand‐induced conformational change in a PBP allosterically regulates the relative distance and orientation of a fluorescence resonance energy transfer (FRET)‐compatible protein pair. Ligand binding is transduced into a macroscopic FRET observable, providing a reagent for in vitro and in vivo ligand‐measurement and visualization. Sensors with a higher FRET signal change are required to expand the dynamic range and allow visualization of subtle analyte changes under high noise conditions. Various observations suggest that factors other than inter‐fluorophore separation contribute to FRET transfer efficiency and the resulting ligand‐dependent spectral changes. Empirical and rational protein engineering leads to enhanced allosteric linkage between ligand binding and chromophore rearrangement; modifications predicted to decrease chromophore rotational averaging enhance the signal change, emphasizing the importance of the rotational freedom parameter κ2 to FRET efficiency. Tighter allosteric linkage of the PBP and the fluorophores by linker truncation or by insertion of chromophores into the binding protein at rationally designed sites gave rise to sensors with improved signal change. High‐response sensors were obtained with fluorescent proteins attached to the same binding PBP lobe, suggesting that indirect allosteric regulation during the hinge‐bending motion is sufficient to give rise to a FRET response. The optimization of sensors for glucose and glutamate, ligands of great clinical interest, provides a general framework for the manipulation of ligand‐dependent allosteric signal transduction mechanisms.
Studies of helix fraying and solvation using 13C′ isotopomers
Authors: Fesinmeyer R. Matthew , Peterson Eric S. , Dyer R. Brian , Andersen Niels H.
Published: August 2005

Both NMR and IR studies of carbonyl (13C′) isotopomers of designed helices can provide residue‐level details regarding the fractional occurrence and melting behavior of helical ϕ/ψ angles along the sequence of helical peptides, details that cannot be obtained from CD or 1H‐NMR studies. We have studied a classic series of helical models, Ac‐YGG‐(KAXAA)3K‐NH2 (X=A,V), in both aqueous and helix‐favoring media containing fluoroalcohol cosolvents, including a solvent system allowing the observation of cold denaturation. These studies confirmed the strong N‐capping associated with this sequence and revealed more extensive C‐terminal fraying than that calculated using current helicity prediction algorithms. In the X=A series, the central residues are somewhat resistant to thermal melting; it instead occurs predominantly at the frayable C terminus. For the X=V series under cold‐denaturing conditions, the temperature of maximal helicity is not uniform along the sequence and both solvated and nonsolvated helical alanine sites (13C=O stretches at 1592 cm−1 and 1615 cm−1, respectively) are apparent. Correlation between the two spectroscopies employed yielded the intriguing observation that the valine side chain is able to desolvate the i − 4 amide in short monomeric helices. In addition, we report further measurements of the temperature dependence of alanine statistical coil chemical shifts, the temperature dependence of the 13C chemical shift of urea (employed as chemical shift reference), and a useful formula for converting 13C′ shifts into fractional helicities.
Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis
Authors: Pál Gábor , Fong Shun‐Yin , Kossiakoff Anthony A. , Sidhu Sachdev S.
Published: August 2005

Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost‐effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine‐scanning approach was expanded with two additional strategies, serine‐ and homolog‐scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor‐binding sites (site 1) of wild‐type hGH (hGHwt) and of an affinity‐improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine‐ and homolog‐scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild‐type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild‐type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine‐scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine and homolog‐scanning results expand upon and complement the alanine‐scanning results and provide additional data on the robustness of the high affinity receptor‐binding site of hGH.
Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines
Authors: García Javier , Puentes Alvaro , Rodríguez Luis , Ocampo Marisol , Curtidor Hernando , Vera Ricardo , Lopez Ramses , Valbuena John , Cortes Jimena , Vanegas Magnolia , Barrero Carlos , Patarroyo Manuel A. , Urquiza Mauricio , Patarroyo Manuel E.
Published: August 2005

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT‐PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25‐kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20‐amino‐acid‐long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross‐linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.
Rapid and accurate structure determination of coiled‐coil domains using NMR dipolar couplings: Application to cGMP‐dependent protein kinase Iα
Authors: Schnell Jason R. , Zhou Guo‐Ping , Zweckstetter Markus , Rigby Alan C. , Chou James J.
Published: August 2005

Coiled‐coil motifs play essential roles in protein assembly and molecular recognition, and are therefore the targets of many ongoing structural and functional studies. However, owing to the dynamic nature of many of the smaller coiled‐coil domains, crystallization for X‐ray studies is very challenging. Determination of elongated structures using standard NMR approaches is inefficient and usually yields low‐resolution structures due to accumulation of small errors over long distances. Here we describe a solution NMR approach based on residual dipolar couplings (RDCs) for rapid and accurate structure determination of coiled‐coil dimers. Using this approach, we were able to determine the high‐resolution structure of the coiled‐coil domain of cGMP‐dependent protein kinase Iα, a protein of previously unknown structure that is critical for physiological relaxation of vascular smooth muscle. This approach can be extended to solve coiled‐coil structures with higher order assemblies.
A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: Structural determinants of redox potential
Authors: Alagaratnam Sharmini , van Pouderoyen Gertie , Pijning Tjaard , Dijkstra Bauke W. , Cavazzini Davide , Rossi Gian Luigi , Van Dongen Walter M.A.M. , van Mierlo Carlo P.M. , van Berkel Willem J. H. , Canters Gerard W.
Published: August 2005

Flavodoxin II from Azotobacter vinelandii is a “long‐chain” flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three‐dimensional structure determined to a resolution of 2.25 Å by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five‐stranded parallel β‐sheet flanked on either side by α‐helices. An eight‐residue insertion, compared with other long‐chain flavodoxins, forms a short 310 helix preceding the start of the α3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent‐accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.
Hepatitis B Virus Infection and B‐Cell Non‐Hodgkin's Lymphoma in a Hepatitis B Endemic Area: A Case‐control Study
Authors: Kim Jee Hyun , Bang Yung‐Jue , Park Byung Joo , Yoo Taiwoo , Kim Chul Woo , Kim Tae‐You , Heo Dae Seog , Lee Hyo‐Suk , Kim Noe Kyeong
Published: August 2005

Several studies have reported a higher prevalence of chronic hepatitis C virus (HCV) infection in patients with B‐cell non‐Hodgkin's lymphoma and suggested a pathogenic role for HCV, but studies on hepatitis B virus (HBV) infection and non‐Hodgkin's lymphoma are limited. To determine the association between HBV infection and non‐Hodgkin's lymphoma, we performed a case‐control study in Korea, a hepatitis B endemic area. We recruited 222 patients newly diagnosed with non‐Hodgkin's lymphoma at Seoul National University Hospital between January 1997 and December 1998 as cases. Four age‐ and sex‐matched controls were selected for each case, and the control groups comprised of 439 patients with non‐hematological malignancy (control group 1) and 444 subjects with non‐malignant conditions (control group 2). Relative risk of developing non‐Hodgkin's lymphoma among individuals tested positive for hepatitis B surface antigen was calculated after controlling for other potential risk factors of lymphoma, such as smoking, alcohol drinking, transfusion history and HCV infection. Hepatitis B surface antigen was positive in 28 of 222 patients (12.6%) with non‐Hodgkin's lymphoma compared with 32 of 439 (7.3%) in control group 1, and 21 of 444 (4.7%) in control group 2 (P=0.001). The crude odds ratio for B‐cell non‐Hodgkin's lymphoma among the HBV carriers was 2.54 (1.46–4.45) and the adjusted odds ratio was 3.30 (1.69–6.45) by multivariate analysis. The present study suggests that the risk of B‐cell non‐Hodgkin's lymphoma is increased in HBV carriers and warrants further investigation of the possible role of hepatitis B virus in the pathogenesis of B‐cell non‐Hodgkin's lymphoma.
Induction of Intestinal Tumors and Lymphomas in C57BL/6N Mice by a Food‐borne Carcinogen, 2‐Amino‐l‐methyl‐6‐phenylimidazo[4,5‐b]pyridine
Authors: Ochiai Masako , Imai Hiroshi , Sugimura Takashi , Nagao Minako , Nakagama Hitoshi
Published: August 2005

2‐Amino‐l‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is the most abundant heterocyclic amine contained in cooked meat and fish. Although PhIP has been demonstrated to induce various types of tumors in rats, lymphomas predominated in mice using the CDF1 strain. To investigate the carcinogenic activity of PhIP on other organs in mice with a different genetic background, PhIP was administered to C57BL/6N mice. After a 40‐week administration of 300 ppm of PhIP in a high‐fat diet followed by continuous feeding with a high fat diet, C57BL/6N mice developed adenomas and adenocarcinomas in the small intestine, the incidences being 52% in males and 68% in females at weeks 95 and 70, respectively. Lymphomas of B‐cell origin also developed in both sexes as frequently as in the CDF1 strain, incidences being 48% in males and 32% in females. Although the incidence in PhIP‐treated female mice did not differ from that in the control mice, lymphomas developed significantly earlier in the PhIP‐treated mice. The present study demonstrated that the intestinal tract is another potential target of PhIP‐induced carcinogenesis in mice, and that the carcinogenic activity of PhIP could be affected by the genetic background of the animals.
Duodenogastric Reflux Increases the Penetration of N‐3H‐Methyl‐N‐nitro‐N‐nitrosoguanidine into the Antral Mucosa of Rats: A Possible Role for Mucosal Erosions and Increased Cell Proliferation in Gastric Carcinogenesis
Authors: Øvrebø Kjell K. , Svanes Knut , Aase Steinar , Grong Ketil , Sørbye Halfdan
Published: August 2005

Duodenogastric reflux is a risk factor for gastric carcinogenesis, but the pathogenesis is not fully understood. We studied the risk of N‐methyl‐N‐nitro‐N‐nitrosoguanidine (MNNG)‐induced carcinogenesis in the antrum of rats with duodenogastric reflux. Duodenal fluid was directed into the stomach through the pylorus (pyloric reflux group) or through a gastrojejunostomy (jejunal reflux group). After twenty‐four weeks, 5‐bromo‐2‐deoxyuridine (BrdU) was injected intravenously and the stomach was exposed to N‐3H‐methyl‐N‐nitro‐N‐nitrosoguanidine (3H‐MNNG). The antral mucosa was examined with immunohistochemistry and autoradiography for identification of proliferating cells (BrdU labelled) and cells at risk of MNNG‐induced carcinogenesis (3H‐MNNG and BrdU‐labelled cells). Duodenogastric reflux increased the number of double‐labelled cells in the antral mucosa from 4.8±0.6 per mm in the control group to 11.3±1.9 in the jejunal reflux group (P<0.05) and 12.7±0.9 in the pyloric reflux group (P<0.05). Mucosal erosions were observed in 15 of 28 annuals with pyloric reflux and the number of double‐labelled cells in the erosion area (4.3±0.7) was higher than in the same area of animals without erosion (1.4±0.5) (P<0.05). Duodenogastric reflux increased the cell proliferation and significantly changed the distance between the surface epithelial lining and the proliferating cells when compared to the controls. These results indicate that duodenogastric reflux increases the penetration of 3H‐MNNG into the antrum mucosa of rats. Increased cell proliferation and erosions increase the number of cells at risk of an initiation process from a penetrating gastric carcinogen.
Telomerase Is Upregulated in Irreversible Preneoplastic Lesions during Bladder Carcinogenesis in Rats
Authors: Shimazui Toru , Ami Yoshihiro , Miyanaga Naoto , Ideyama Yukitaka , Nakahara Takahito , Akaza Hideyuki
Published: August 2005

Multiple occurrence or recurrence after transurethral resection is an important characteristic of superficial bladder tumors. To study bladder carcinogenesis, we focused on detection of telomerase activation, which was investigated in several human cancers, including bladder tumors. We experimentally examined the telomerase activity during bladder carcinogenesis, especially in precancerous lesions, induced by N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) in rats. Male Wistar rats were given 0.05% BBN in water from the age of 8 weeks to 24 weeks. Subgroups were euthanized at 4, 8, 10, 12, 18, and 24 weeks after BBN administration. Using the stretch PCR method, telomerase activity was semiquantified in exfoliated bladder epithelial cells. In addition, telomere length in each subgroup was measured by southern hybridization for the terminal restriction fragment using a (TTAGGG)4 probe. Statistical analyses were performed using analysis of variance and Fisher's PLSD test. Epithelial cells of normal bladder in the control groups and those of diffuse hyperplasia, which was a reversible change at 4 weeks, expressed no telomerase activity. In contrast, telomerase activity significantly increased in the stage after nodular hyperplasia, an irreversible change at 8 weeks, then elevated with carcinogenesis. However, telomere length was still preserved by the 12th week, and was shortened at 18 and 24 weeks. These results suggest that telomerase activation is probably induced independent of telomere shortening during bladder carcinogenesis in the rat, and might be a biological tumor marker of irreversible preneoplastic lesions, which evolve into bladder tumors in the rat.
A Quantitative Evaluation of Active Gelatinolytic Sites in Uterine Endometrioid Adenocarcinoma Using Film in situ Zymography: Association of Stronger Gelatinolysis with Myometrial Invasion
Authors: Zheng Kefei , Nagai Yuichiro , Kishimoto Takashi , Yamazawa Koji , Tate Shinji , Nemori Ryoichi , Hirai Yasuo , Sekiya Souei , Ishikura Hiroshi
Published: August 2005

Collagen matrix degradation by malignant tumor cells plays an essential role in the process of tumor invasion and metastasis. The purpose of this study was to detect in situ gelatinase activity in endometrioid adenocarcinomas of the uterine corpus. In order to carry out quantitative evaluation, autoexposure tune (AET) on gelatin‐coated film (film in situ zymography: FIZ) was measured. The gelatinase activity was located primarily within cancers and was prominently suppressed by the addition of a chelating agent to the film. This suggests that matrix metalloproteinases (MMPs) play an important role in the gelatinase activity. The gelatinase activity in the normal endometrium is almost negligible, despite positive immunoreactivity for MMP‐2 and ‐9. Tumor tissues that had invaded more than half of the myometrium showed significantly higher activity than those that had invaded less than half. There was no significant difference in gelatinase activity among tumor stages, grades, vessel invasion or immunoreactivity for MMPs, with the exception that stage 2b cancers showed higher activity than stage la. The study suggested that the level of MMP‐mediated gelatinolysis is an important factor for myometrial invasion in uterine endometrioid adenocarci‐ noma. Thus, a quantitative assessment of active gelatinolysis using FIZ and AET should be a useful tool in evaluating in situ matriolytic activity in local myometrial invasion by uterine endometrioid adenocarcinoma.
High Expression of Human Uroplakin Ia in Urinary Bladder Transitional Cell Carcinoma
Authors: Kageyama Susumu , Yoshiki Tatsuhiro , Isono Takahiro , Tanaka Tsutomu , Kim Choi Jang , Yuasa Takeshi , Okada Yusaku
Published: August 2005

Uroplakins (UPs) Ia, Ib, II, and III are tissue‐specific and differentiation‐dependent transmem‐brane proteins of the urothelium. We assessed the usefulness of human UP Ia as a histological marker by examining its expression in urinary bladder transitional cell carcinoma (TCC). A polyclonal antibody against human UP Ia was raised using a synthesized polypeptide. We applied our antibody to various organ tissues, including urothelium, and observed no crossreactivity. Analysis by RT‐PCR of normal urothelium, TCC and other organ tissues indicated that the human UP Ia gene expression is highly specialized to urothelium, and is conserved in TCC. Using immunohistochemistry, we investigated the expression of UP Ia in TCC from patients who had undergone radical cystectomy and from autopsy cases. Positive staining (10% or more positive cancer cells) was noted in primary lesions from 61 of 63 (96.8%) cystectomy patients. Depending on pathological grade, high expression (50% or more positive cancer cells) was observed in 17 of 18 (94.4%) moderately‐ to well‐differentiated TCC and in 36 of 45 (80.0%) poorly differentiated TCC. With regard to tumor invasion, high expression was noted in 20 of 22 (90.9%) superficial and 33 of 41 (80.5%) muscle‐invasive TCC. Cause‐specific survival rates were 68.6% and 75.0% in high‐ and low‐expression patients, respectively (log‐rank test, P=0.855, mean follow‐up; 65.0 months). In metastases, positive reactions were observed in 13 of 18 (72.2%) lesions. UP Ia may represent a specific histological marker judging from the stable expression, although its value as a prognostic factor remains undetermined.
Blue Light Inhibits the Growth of B16 Melanoma Cells
Authors: Ohara Masayuki , Kawashima Yuzo , Katoh Osamu , Watanabe Hiromitsu
Published: August 2005

Although a number of studies have been carried out to examine the biological effects of radiation and ultraviolet radiation (UV), little is known concerning the effects of visible light. In the present study, exposure of B16 melanoma cells to blue light (wavelength 470 nm, irradiance 5.7 mW/cm2) from a light‐emitting diode (LED) inhibited cell growth in proportion to the period of exposure, with no increase observed in the number of dead cells. The number of B16 melanoma colonies that formed after exposure to blue light for 20 min was only slightly less than that in non‐exposed controls, but the colony size as assessed by the area covered by colonies and cell counts per colony were markedly decreased. The percentages of G0/G1 and G2/M phase cells were markedly increased, with a reduction in S phase cells as determined by flow cytometry after exposure to blue light. Furthermore, analysis of the incorporation of 5–bromo–2′–deoxyuridine (BrdU) into DNA also showed a reduction in the percentage of S phase cells after exposure. These results indicate that blue light exerts cytostatic effects, but not a cytocidal action, on B16 melanoma cells.
Enhanced Anti‐tumor Effect of Trastuzumab in Combination with Cisplatin
Authors: Naruse Ichiro , Fukumoto Hisao , Saijo Nagahiro , Nishio Kazuto
Published: August 2005

The oncogenenic transmembrane tyrosine kinase receptor HER–2/neu is a promising target for treatment of HER–2–overexpressing cancers. The humanized anti‐HER–2/neu antibody Trastuzumab is under clinical evaluation in combination with chemotherapy against breast cancer. The combination of Trastuzumab and cisplatin is expected to be active against HER–2/neu‐expressing tumors. We examined the mechanisms of this combination effect against human solid tumor cells in the presence of human peripheral blood mononuclear cells (PBMCs) using an in vitro MTT assay. The growth‐inhibitory effects of cisplatin (CDDP) on the tumor cells were not significantly affected by Trastuzumab in the absence of effector cells. CDDP alone at a dose of less than 12.5 μM did not affect the viability of PBMCs, as determined by MTT assay, suggesting that PBMCs could exert antibody‐dependent cell‐mediated cytotoxicity (ADCC) at this CDDP concentration. The combination of Trastuzumab and CDDP showed higher cytotoxic effects against the tumor cells in the presence of PBMCs. The CDDP concentration required to inhibit tumor cell growth by 50% was reduced to ∼20% by Trastuzumab in the presence of PBMCs at an effector/target ratio of 10. It may be important to select combined chemotherapeutic agents which do not diminish the ADCC activity of Trastuzumab via PBMCs. Both the expression of HER–2/neu and the ADCC activity may be important determinants of the therapeutic benefit of the Trastuzumab/CDDP combination.
Food Restriction Inhibits the Growth of Intestinal Polyps in Multiple Intestinal Neoplasia Mouse
Authors: Kakuni Masakazu , Morimura Keiichiro , Wanibuchi Hideki , Ogawa Motome , Min Wei , Hayashi Shuji , Fukushima Shoji
Published: August 2005

The effect of food restriction (FR) on spontaneous intestinal carcinogenesis in multiple intestinal neoplasia (Min) mice was examined. Thirty male Min mice were allotted to ad libitum feeding control and 20% FR groups from six weeks of age until the end of the 13‐week experimental period. Although the total number of visible intestinal polyps in the FR group was not significantly different from the control group value, a significant decrease in large‐sized polyps (>2 mm) and an increase in small‐sized polyps (>2 mm) were observed in the distal small intestine. In this segment, the percentage of apoptotic cells counted in intestinal polyps in the FR group was significantly higher than in the control group, the percentage of proliferating cell nuclear antigen (PCNA)‐positive cells not being significantly different. These results indicate that the FR may inhibit the growth of intestinal polyps in the Min mouse, and that apoptosis contributed in part to the inhibitory effect.
Gene Expression for Suppressors of Telomerase Activity (Telomeric‐repeat Binding Factors) in Breast Cancer
Authors: Saito Kaori , Yagihashi Atsuhito , Nasu Shunichi , Izawa Yoko , Nakamura Masashi , Kobayashi Daisuke , Tsuji Naoki , Watanabe Naoki
Published: August 2005

Mechanisms regulating telomerase activity and telomere length remain incompletely understood in human breast cancer. We therefore studied gene expression for telomeric‐repeat binding factors (TRFs) in relation to telomerase activity, telomere length, and clinicopathologic factors in human breast cancer. Telomerase activity was detected in 65.8% of 38 breast cancers, but none of 16 non‐cancerous samples. Terminal restriction fragments were longer in noncancerous than in cancerous tissues, but not significantly. Among 8 patients with both cancer and paired noncancerous tissue available for terminal restriction fragments length assay, terminal restriction fragments were shorter in cancers than in paired noncancerous samples in all but one. Significantly more mRNA encoding TRF1 and 2 was detected in noncancerous than in cancer tissues. Additionally, expression of TRF1 and 2 mRNA was significantly higher in cancers without detectable telomerase activity than in cancers showing activity. Expression of these genes tended to show a negative correlation with terminal restriction fragments length, but this was not statistically significant. No correlation was seen between TRF1 or 2 mRNA expression, and clinicopathologic factors except for TRF1 with respect to tumor size and progesterone receptor status. In addition to reactivation of telomerase activity, escape from negative regulation of this activity is needed to maintain telomere length during cell proliferation in breast cancer. Genes encoding telomerase inhibitors might be of value in gene therapy against human breast cancer.
Mutations of p53, c‐kit, K‐ras, and β‐Catenin Gene in Non‐Hodgkin's Lymphoma of Adrenal Gland
Authors: Nakatsuka Shin‐ichi , Hongyo Tadashi , Syaifudin Mukh , Nomura Taisei , Shingu Norihisa , Aozasa Katsuyuki
Published: August 2005

Malignant lymphoma of the adrenal gland is a rare disease, usually with diffuse large cell morphology and B‐cell immunophenotype, and often associated with Epstein‐Barr virus infection. In this study, mutations of p53, c‐kit, K‐ras, and β‐catenin gene were analyzed in 17 cases (13 males and four females with ages ranging from 25 to 84 years) of such lymphomas by polymerase chain reaction‐single strand conformation polymorphism followed by direct sequencing. Selected exons in each gene, representing hot spots, were analyzed. All 44 mutations detected were single‐nucleotide substitutions and 33 were missense mutations. Nineteen mutations were detected in exon 5 and/or 7 of the p53 gene in nine of 17 cases (52.9%) and 21 in exon 11 and/or 17 of the c‐kit gene in 10 of 14 cases (71.4%). Bilateral adrenal lesions in one case who had not received any adjuvant therapy showed different mutational patterns of the p53 and c‐kit genes, suggesting different clonal evolution of lymphoma between the left and right sides. Mutation at codon 13 of the K‐ras gene was detected in one of 14 cases (7.1%), and in exon 3 of the β‐catenin gene in three of 12 cases (25%). All but one mutation were transition mutations, indicating that some endogenous mutagens act in lymphomagenesis in the adrenal gland. Our results suggest that p53 and c‐kit gene mutations might play a role in adrenal lymphomagenesis.
Multiplexed Analysis of Post‐PCR Fluorescence‐labeled Microsatellite Alleles and Statistical Evaluation of Their Imbalance in Brain Tumors
Authors: Yoshimoto Koji , Iwaki Toru , Inamura Takanori , Fukui Masashi , Tahira Tomoko , Hayashi Kenshi
Published: August 2005

Detection of the loss of chromosomal regions in cancerous tissues has diagnostic and prognostic relevance, and the development of a reliable and cost‐effective technique for this is clinically important. Here we present an efficient technique for quantitative detection of microsatellite alleles, using a post‐PCR fluorescence‐labeling procedure and multiplexed analysis. We also present a new statistical method for the interpretation of the data that permits reliable and sensitive evaluation of the allelic status of sampled DNA. A high‐resolution analysis of allelic imbalance on chromosomes 1p, 10 and 19q in 28 glioma samples of various types using this method revealed that allelic imbalances are more frequent than have been reported, suggesting the diagnostic value of this method in examining the genetic profiles of gliomas.
Prognostic Significance of Matrix Metalloproteinase‐7 (MMP‐7) Expression at the Invasive Front in Gastric Carcinoma
Authors: Liu Xiu Ping , Kawauchi Shigeto , Oga Atsunori , Tsushimi Kureo , Tsushimi Mutsuo , Furuya Tomoko , Sasaki Kohsuke
Published: August 2005

To evaluate the clinicopathological significance of matrix metalloproteinase‐7 (MMP‐7) expression in gastric carcinoma, we investigated immunohistochemically MMP‐7 expression in 214 gastric carcinomas, and examined its relations with the clinicopathologic parameters including patient prognosis. MMP‐7 expressed predominantly in cancer cells, and MMP‐7‐positive tumor cells were preferentially found in deeply invading nests, especially at the invasive front. The mean MMP‐7 labeling index (LI) at the invasive front was significantly higher in tumors invading or penetrating the muscularis propria and in stages II‐IV than within the submucosal layer and in stage I, respectively (P<0.001). Statistical analysis revealed that MMP‐7 LI at the invasive front was related to lymph node metastasis, vascular invasion, and lymphatic permeation, when all 214 cases were examined as one group (P<0.05 for all), and the cases with high MMP‐7 expression at the invasive front showed significantly more unfavorable prognosis as compared with that of low MMP‐7 expression tumors (P<0.01). Multivariate analysis revealed that TNM stage and MMP‐7 expression status at the invasive front were independent prognostic factors (P=0.0017, relative risk (RR)=3.12; P=0.0019, RR=2.67, respectively). Our findings indicated that expression of MMP‐7 at the invasive front is closely associated with local invasiveness, and might be a reliable prognostic marker for patients with gastric carcinoma.
VCP (p97) Regulates NFKB Signaling Pathway, Which Is Important for Metastasis of Osteosarcoma Cell Line
Authors: Asai Tatsuya , Tomita Yasuhiko , Nakatsuka Shin‐ichi , Hoshida Yoshihiko , Myoui Akira , Yoshikawa Hideki , Aozasa Katsuyuki
Published: August 2005

In order to identify genes associated with metastasis, suppression subtractive hybridization (SSH) was performed using murine osteosarcoma cell line Dunn and its subline with higher metastatic potential, LM8. SSH revealed expression of the gene encoding valosin‐containing protein (VCP; also known as p97) to be constitutively activated in LM8 cells, but it declined in Dunn cells when the cells became confluent. Because VCP is known to be involved in the ubiquitination process of Inhibitor‐αBα (IαBα), an inhibitor of nuclear factor‐αB (NFαB), whether VCP influences NFαB activation or not was examined by using VCP‐transfected Dunn cells (Dunn/VCPs). When stimulated with tumor necrosis factor‐α (TNFα), Dunn/VCPs showed constantly activated NFαB, although in the original Dunn cells and control vector transfectant (Dunn/Dunn‐c) NFαB activation ceased when the cells became confluent. Western immunoblot analysis showed an increase of phosphorylated IαBα (p‐IKBα) in the cytoplasm of confluent Dunn/Dunn‐c cells compared to that of Dunn/VCPs. Therefore, decrease of p‐IKBα degrading activity might be responsible for the decrease in NFKB activation. In vitro apoptosis assay demonstrated increased apoptosis rates of Dunn/Dunn‐c cells after TNFα stimulation compared to those of Dunn/VCPs and LM8 cells. In vivo metastasis assay showed increased incidences of metastatic events in Dunn/VCP‐1 inoculated male C3H mice compared to those in Dunn/Dunn‐c inoculated mice. These findings suggested that VCP expression plays an important role in the metastatic process. Anti‐apoptotic potential in these cells owing to constant NFKB activation via efficient cytoplasmic p‐IKBα degrading activity may explain the increased metastatic potential of these cells.
Evaluation of an Indicator for Lymph Node Metastasis of Esophageal Squamous Cell Carcinoma Invading the Submucosal Layer
Authors: Nakajima Yasuaki , Nagai Kagami , Miyake Satoshi , Ohashi Kenichi , Kawano Tatsuyuki , Iwai Takehisa
Published: August 2005

Lymph node metastasis is a major prognostic factor for esophageal squamous cell carcinoma (ESCC). In recent years, endoscopic mucosal resection (EMR) has been developed with excellent results for the treatment of the superficial ESCC. To make the EMR treatment successful, it is important to establish a good indicator to identify ESCC patients at a high risk of lymph node metastasis. In this study, we examined clinicopathological and immunohistochemical factors to investigate the factors involved in lymph node metastasis of ESCC invading to the submucosal layer (sm‐ESCC). Surgical specimens from 84 sm‐ESCC patients were examined. Among 84 sm‐ESCC patients, 33 (39.3%) had lymph node metastases. Clinicopathologically, tumor depth, lymphatic invasion and blood vessel invasion showed significant correlations with lymph node metastasis by univariate analysis. Tumor depth and lymphatic invasion showed significant correlations by multivariate analysis of these factors. Immunohistochemically, P53 accumulation was observed in 45 cases (53.6%), cyclin D1 overexpression in 25 (29.8%), and pRB in 65 (77.4%). P53 accumulation, cyclin D1 overexpression and MIB‐1 Labeling Index were significantly associated with lymph node metastasis by univariate analysis, and P53 accumulation showed a significant correlation with lymph node metastasis by multivariate analysis. Among tumor depth, lymphatic invasion and P53 accumulation, tumor depth and lymphatic invasion were significantly correlated with lymph node metastasis (P=0.0023 and P=0.0092, respectively) by multivariate analysis. These data suggest that tumor depth and lymphatic invasion can be considered as good indicators for lymph node metastasis among patients with sm‐ESCC. In addition, P53 accumulation could be helpful to identify the patients who need additional treatment after EMR.
Selective Expansion of Human Natural Killer Cells from Peripheral Blood Mononuclear Cells by the Cell Line, HFWT
Authors: Harada Hideki , Saijo Kaoru , Watanabe Satoru , Tsuboi Koji , Nose Tadao , Ishiwata Isamu , Ohno Tadao
Published: August 2005

An anchorage‐dependent Wilnis tumor cell line HFWT was found to stimulate selective and remarkable expansion of human natural killer (NK) cells from human peripheral blood mononuclear cells (PBMC). After PBMC of healthy donors were cultured on irradiated HFWT cells for 10–21 days, the lymphocytes expanded 58‐ to 401‐fold. This NK cell expansion required direct contact of PBMC with live, but not fixed, HFWT cells. The PBMC from an end‐stage brain tumor patient also expanded 156‐fold, whereas those cultured with irradiated NK‐sensitive K562 grew only 30.5‐fold. CD16+CD56+ NK cells accounted for more than 70% of the population expanded on HFWT cells. No essential difference in expression of NK receptors was observed in the expanded NK cells on HFWT and K562 and without feeder cells. The expanded NK cells killed not only fresh HFWT cells but, unexpectedly, also MHC class I‐expressing autologous brain tumor cells at an effector/target ratio of 4 for 24 h. These results will contribute to the development of a large‐scale preparation method for human NK cells, which will aid studies of NK cell biology and possible treatment of brain tumors.
The Roles of JNK1 and Stat3 in the Response of Head and Neck Cancer Cell Lines to Combined Treatment with All trans‐retinoic Acid and 5‐Fluorouracil
Authors: Masuda Muneyuki , Toh Satoshi , Koike Koji , Kuratomi Yuichiro , Suzui Masumi , Deguchi Atsuko , Komiyama Sohtaro , Weinstein I. Bernard
Published: August 2005

We have used a combination of vitamin A (all‐trans‐retinyl palmitate), 5‐fluorouracil (5‐FU) and radiation to treat human head and neck squamous cell carcinoma (HNSCC). This chemoradiother‐ apy is called “FAR therapy” In this study we examined the effects of all‐trans‐retinoic acid (ATRA), the active metabolite of vitamin A, and ATRA plus 5‐FU on two HNSCC cell lines (YCU‐ N861 and YCU‐H891) to gain insight into the molecular mechanisms of FAR therapy. ATRA at 1 μM (the order of concentration found in HNSCC tumors treated with FAR therapy) inhibited cell proliferation and caused Gl cell cycle arrest in both cell lines. This was associated with a decrease in cyclin D1, an increase in p27Kipl and a reduction in the hyperphosphorylated form of retinoblastoma protein (pRB). With YCU‐N861 cells, ATRA also caused a decrease in Bcl‐2 and Bcl‐XL and an increase in Bax. Both ATRA and 5‐FU activated c‐Jun N‐terminal kinase (JNK) 1 and the combination of both agents resulted in additive or synergistic activation of JNK1, and also enhanced the induction of apoptosis. The YCU‐H891 cells, in which the epidermal growth factor receptor (EGFR)‐signal transducer and activator of transcription 3 (Stat3) pathway is constitutively activated, were more resistant to treatments with ATRA, 5‐FU and the combination of both agents than YCU‐N861 cells. A dominant negative Stat3 construct strongly enhanced the cellular sensitivity of this cell line to 5‐FU but not to ATRA. In addition there is evidence that activation of Stat3 is associated with cellular resistance to radiation in HNSCC. Therefore, the addition to FAR therapy of agents that inhibit activation of the Stat3 pathway may enhance the clinical response of patients with HNSCC to FAR therapy.
Expression of Thymidine Phosphorylase in Primary Human Renal Cell Carcinoma by ELISA Method
Authors: Kinsui Hidetoshi , Ueda Takeshi , Suzuki Hiroyoshi , Isaka Shigeo , Sekita Nobuyuki , Komiya Akira , Tobe Toyofusa , Yamanishi Tomonori , Akakura Koichiro , Ichikawa Tomohiko , Igarashi Tatsuo , Ito Haruo
Published: August 2005

Thymidine phosphorylase (TP) expression in 100 paired samples of renal cell carcinoma (RCC) and normal adjacent tissue was analyzed by an ELISA method. We also investigated whether TP expression correlates with clinicopathological findings and clinical outcomes of these patients. Median TP expression was 9‐fold (range, 0.5–56) higher in primary tumor than in non‐cancerous renal tissue (P<0.0001). There was a significant difference with respect to tumor venous invasion. TP expression was significantly higher in patients with such venous invasion than in those without (P=0.018). However, there was no correlation between TP level and other clinicopathological findings and the survival curves. These results suggest that ELISA is useful for evaluating TP expression of human RCC and may provide a novel approach to therapy for patients with RCC.
A bioinformatic tool for analysis of EST transcript abundance during infection‐related development by Magnaporthe grisea
Authors: SOANES DARREN M. , TALBOT NICHOLAS J.
Published: August 2005

Information regarding the levels of mRNA transcript abundance under different conditions, or in specific tissue types, can be obtained by analysis of the frequency of EST sequences in randomly sequenced cDNA libraries. Here we report a bioinformatics tool, which provides a means of identifying genes that are differentially expressed during pathogenesis‐related development by the rice blast fungus Magnaporthe grisea. A total of 31 534 M. grisea ESTs were obtained from dbEST at NCBI, clustered into 8821 unique sequences (unisequences) and manually annotated. Transcript profiles were then calculated for 958 unigenes identified from eight different cDNA libraries. The data were integrated into the Consortium for Functional Genomics of Microbial Eukaryotes (COGEME) database (http://cogeme.ex.ac.uk/) and a web‐based front end was designed to allow users to access and interrogate the generated datasets.
A gene with domains related to transcription regulation is required for pathogenicity in Colletotrichum acutatum causing Key lime anthracnose
Authors: CHEN HUI‐QIN , CAO LIHUA , DEKKERS KATHERINE L. , ROLLINS JEFFREY A. , KO NAN J. , TIMMER L. W. , CHUNG KUANG‐REN
Published: August 2005

Colletotrichum acutatum causes Key lime anthracnose (KLA) and postbloom fruit drop (PFD) of citrus. We utilized restriction enzyme‐mediated integration (REMI) mutagenesis to produce six non‐pathogenic mutants from a KLA isolate after screening 1064 transformants on detached Key lime leaves. Subsequently, a gene designated KLAP1 (Key Lime Anthracnose Pathogenicity) was identified from one of the mutants and was demonstrated genetically to be required for pathogenicity to Key lime leaves. The predicted polypeptide encoded by KLAP1 contains a cAMP and cGMP‐dependent protein kinase phosphorylation site, and two RGD (Arg–Gly–Asp) cell attachment sequences, a bipartite nuclear targeting sequence, a fungal G‐protein alpha subunit signature, a putative metal‐binding zinc finger (Cys2His2) and a putative HMG‐I/Y (‘high mobility group’ non‐histone chromatin protein encoding genes) DNA‐binding domain (A+T hook), suggesting that KLAP1 may function as a transcription activator in C. acutatum. Sequences homologous to KLAP1 were detected in most C. acutatum isolates examined, and similarity was found in several classes of fungi, animals, plants and bacteria, indicating that KLAP1 is a putative, uncharacterized, conserved transcription activator in fungi. Targeted gene disruption of KLAP1 yielded mutants that were blocked in the penetration stage and were completely defective in pathogenicity on Key lime leaves, but remained pathogenic to flower petals. Complementation of a klap1‐null mutant with a full‐length KLAP1 gene clone restored complete ability to incite lesions on Key lime. The results indicate that KLAP1 is an important pathogenicity factor in C. acutatum.
Microbial avirulence determinants: guided missiles or antigenic flak?
Authors: SKAMNIOTI PARASKEVI , RIDOUT CHRISTOPHER J.
Published: August 2005

Avirulence (avr) determinants are incompatibility factors which elicit host plant defence responses in a gene‐for‐gene manner. They are produced by fungi, bacteria and viruses, and their recognition by resistance genes has been extensively studied for decades. But why should a microbe keep a molecule that allows it to be recognized? One argument is that avr genes perform some essential function and must be kept despite giving the pathogen away. Many bacterial avr determinants have been shown to be effectors, which contribute to virulence and aggressiveness. If this were always the case, mutants lacking these essential molecules would be at a serious disadvantage. Some disadvantage has been shown for a small number, but for the majority there is no effect on virulence. This has been explained by functional redundancy for bacterial and fungal avr determinants, with other molecules compensating for the deletion of these essential genes. However, this argument is counter‐intuitive because by definition these individual genes are no longer essential; so why keep them? With increasing numbers of avr genes being identified, efforts to elucidate their function are increasing. In this review, we take stock of the accumulating literature, and consider what the real function of avr determinants might be.
Metabolic and stress adaptation by Mycosphaerella graminicola during sporulation in its host revealed through microarray transcription profiling
Authors: KEON JOHN , RUDD JASON JAMES , ANTONIW JOHN , SKINNER WENDY , HARGREAVES JOHN , HAMMOND‐KOSACK KIM
Published: August 2005

Pathogenic microbes must successfully adapt to the host environment, acquiring nutrients and tolerating immune/defence responses. Studies on host–pathogen interactions at the transcriptome level have predominantly investigated host responses. Here we present a broad‐scale transcriptional analysis on a fungal pathogen during sporulation within its host environment. Septoria leaf blotch is an important fungal disease of cultivated wheat and is caused by the ascomycete fungus Septoria tritici (teleomorph Mycosphaerella graminicola). A cDNA microarray containing 2563 unigenes was generated and then used to compare fungal nutrition and development in vitro under nutrient‐rich and nutrient‐limiting conditions and in vivo at a late stage of plant infection. The data obtained provided clear insights into metabolic adaptation in all three conditions and an elevated stress adaptation/tolerance specifically in the host environment. We conclude that asexual sporulation of M. graminicola during the late stage of plant infection occurs in a rich nutritional environment involving adaptation to stresses imposed in part by the presence of reactive oxygen species.