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γδT cells are key players in cancer immune surveillance because of their ability to recognize malignant transformed cells, which makes them promising therapeutic tools in the treatment of cancer. However, the biological mechanisms of how γδT-cell receptors (TCRs) interact with their ligands are poorly understood. Within this context, we describe the novel allo-HLA–restricted and CD8α-dependent Vγ5Vδ1TCR. In contrast to the previous assumption of the general allo-HLA reactivity of a minor fraction of γδTCRs, we show that classic anti-HLA–directed, γδTCR-mediated reactivity can selectively act on hematological and solid tumor cells, while not harming healthy tissues in vitro and in vivo. We identified the molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 as the essential determinant for recognition and describe the critical role of CD8 as a coreceptor. We conclude that alloreactive γδT-cell repertoires provide therapeutic opportunities, either within the context of haplotransplantation or as individual γδTCRs for genetic engineering of tumor-reactive T cells.

The objective is to evaluate the efficacy of anthocyanin-rich purple-fleshed sweet potato (PSP) beverage on the serum levels of gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in healthy Caucasians with borderline hepatitis. Forty healthy Caucasians (41–69 years) consumed three bottles of the PSP beverage (177 mg anthocyanins per 125-ml bottle) or placebo (1.3 mg) per day for 8 weeks. Thirty-nine subjects completed the study and two subjects were excluded from statistical analysis. GGT levels in the PSP group on days 15 and 43 were lower (P=0.077 and 0.038, respectively), AST levels in the PSP group on days 29 and 43 were lower (P=0.010 and 0.045, respectively) and ALT level in the PSP group on day 43 was lower (P=0.037) than in the placebo group. The PSP beverage did not induce clinically relevant changes in other blood and clinical chemistry parameters.

An enhancement in the external quantum efficiency (QE) of red phosphorescent organic light‐emitting devices (OLEDs) by using facially encumbered and bulky meso‐aryl substituted PtIIporphyrin complexes is demonstrated. The maximum external QEs of phosphorescent OLEDs doped with the facially non‐encumbered PtIIporphyrin complex1[5,15‐bis[4‐(4,4‐dimethyl‐2,6‐dioxacyclohexyl)phenyl]‐2,8,12,18‐tetrahexyl‐3,7,13,17‐tetramethylporphyrin platinum(II)], the facially encumbered PtIIporphyrin complex2[5,15‐bis(2,6‐dimethoxyphenyl)‐2,8,12,18‐tetrahexyl‐3,7,13,17‐tetramethylporphyrinato platinum(II)], the PtIIporphyrin complex 3that bears bulkier 3,5‐di‐tert‐butylphenyl substituents [5,15‐bis(3,5‐di‐t‐butylphenyl)‐2,8,12,18‐tetrahexyl‐3,7,13,17‐tetramethylporphyrin platinum(II)], and the “doubly‐decamethylene‐strapped” PtIIporphyrin complex 4were 1, 4.2, 7.3, and 8.2 %, respectively. The trend of increasing QE values in the order of 1< 2< 3< 4may be related to facial encumbrance and steric bulkiness of meso‐aryl substituted PtIIporphyrin complexes. Especially, in the case of the PtIIporphyrin 4, it is considered that the “double straps” play an important role in restricting rotational freedom of the meso‐aryl substituents. The triplet excited‐state lifetimes for PtIIporphyrins 1–4in OLEDs at an injection current density of 0.55 mA cm–2were 80, 103, 140, and 152 μs, respectively. We believe that the trend of increasing triplet lifetime in going from 1to 4is correlated with suppressing non‐radiative decay.

The effect of alloying on the first-order valence transition in Yb 1− x R x InCu 4 (R=Y, La, Ce, Lu) has been studied by magnetization measurements. It has been found that the transition temperature T v increases by substitution of La and Ce for Yb and decreases for the Y and Lu substitutions. In order to reveal the volume contribution to the change of T v , we measured lattice parameters of Yb 1− x R x InCu 4 at room temperature. The lattice parameter increases with x for R=Ce, La, Y and slightly decreases for R=Lu. Using the data of compressibility and the pressure effect on T v for YbInCu 4 we found that the changes of T v by La and Ce alloying are consistent with the volume change. However, both Y and Lu substitutions for Yb lead to a decrease in T v , inconsistent with the volume change. Possible reasons for the decrease of the Kondo temperature below T v by Y and Lu alloying are discussed.

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal approximately 500-bp region, leaving 3' overhangs. This result is consistent with observations in telomerase-negative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.

The mouse telomerase holoenzyme, which synthesizes telomeric DNA de novo, is a ribonucleoprotein complex that includes the mouse telomerase RNA component (mTERC), mouse telomerase-associated protein (mTEP1) and mouse telomerase reverse transcriptase (mTERT). To determine the role of telomerase in urethane-induced lung tumorigenesis in A/J mice we examined telomerase activity and the expression of each telomerase subunit in 20 tumor samples, harvested at 16, 28, 40 and 50 weeks after urethane treatment. The telomeric repeat amplification protocol assay showed that statistically significant telomerase activation occurred both early and late in tumorigenesis. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that mRNA expression levels of mTEP1 and mTERT were up-regulated during tumor progression, while mTERC expression was not significantly different between tumors and normal lung. We further examined mTEP1 protein expression in normal lung tissue and lung tumors; western blot analysis showed preferential expression of mTEP1 protein in lung tumors compared with normal lung and immunohistochemistry revealed that a majority of the adenoma cells were positively stained in the nucleus, whereas only a few of the adjacent normal alveolar cells were immunoreactive. In addition, we investigated DNAs of the 20 tumor samples by single strand conformation polymorphism and sequencing analyses to examine whether alterations of the p53 gene in exons 5-8 were associated with telomerase activity. Although we found one nonsense, two missense, two silent and one simultaneous double mutation at different codons in six late stage tumors, there was no apparent correlation between telomerase activity and p53 mutations. Collectively, these results suggest that mTEP1 as well as mTERT may be involved in the regulation of telomerase activity and that telomerase activation may contribute to lung tumorigenesis in A/J mice independently of p53 gene alterations.

Oxidative stress in the colon is associated with the incidence of colon cancer. In situ, the suppression of oxidative stress in the colon would be an effective form of prevention of the cancer. In this study we investigated the transit of the radical scavenging activity of milk products through the hamster intestinal tract. Two types of skim milk products were prepared by Maillard reaction and then lactic acid fermentation. Heat treatment enhanced the radical scavenging activity for 2,2- diphenyl-1-picrylhydrazyl radical of skim milk. The activity was further increased by fermentation with Lactobacillus caseistrain Shirota. Normal hamsters were fed these milk products for 14 d. For potential radical scavenging activity per unit dry weight of feces and cecal content, the groups ranked in the order of fermented product-fed hamstersheated product-fed hamsterscontrol hamsters, reflecting the order of the potential of the corresponding diets. Approximately 12% of the activity of the heated and the fermented product diets passed through the gastrointestinal tract. These results suggest that some of the radical scavenging activity generated by food processing reached the colon in nonabsorbable products.

High consumption of soybean and soybean-related products is hypothesized to contribute to protection against breast cancer. Soybean is a rich source of genistein, a putative cancer chemopreventive agent. Fermented soy milk (FSM), which is made of soy milk fermented with the Bifidobacterium breve strain Yakult, contains larger amounts of the isoflavone aglycones genistein and daidzein than unfermented soy milk. In the present study, we examined the effects of FSM and its component isoflavone mixture (genistein:daidzein 4:1) on 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats. Starting at 7 weeks of age, female Sprague-Dawley rats were given PhIP at a dose of 85 mg/kg body wt by intragastric administration four times a week for 2 weeks. They were fed control high fat basal diet or experimental high fat diet containing 10% FSM or 0.02 or 0.04% isoflavone mixture during and after carcinogen exposure. The incidences (percentage of rats with tumors) of mammary gland tumors were 71% in the control diet group, 51% in the FSM group and 68 and 61% in the groups treated with isoflavone mixture at 0.02 and 0.04%, respectively. Mammary tumor multiplicities (number of tumors per rat) were 1.2 +/- 0.2 for 10% FSM, 2.2 +/- 0.4 for 0.02% isoflavone mixture and 1.5 +/- 0.3 for 0.04% isoflavone mixture, being clearly smaller than the control diet value (2.6 +/- 0.5). Furthermore, feeding of FSM and the isoflavone mixture at both doses reduced the sizes of mammary tumors. Since the amounts of isoflavones in 10% FSM are approximately equivalent to those in the 0.02% isoflavone mixture, the chemopreventive activity of FSM could be partly attributable to the presence of isoflavones such as genistein and daidzein.

The purpose of this study was to clarify the mechanical properties of 42 brands of nickel-titanium alloy orthodontic wires from 9 manufacturers by conducting three-point bending tests under uniform testing conditions. Manufacturers included A-Company, Hoya Medical, Lancer, Ormco, Rocky Mountain, Sankin, Tomy (GAC), TP, and 3M/Unitek. Cobalt-chrome, and titanium-molybdenum alloy wires were also tested as a reference for comparison of force levels. All reported data were recorded during the unloading process to simulate the force that a wire exerts on a tooth as it is moved into the dental arch from a position of malocclusion. The following results were obtained for the nickel-titanium wires tested. (1) Among the 0.016 inch round wires tested under a maximum deflection of 1.5 mm, the difference between the smallest (Copper nickel-titanium 35) and the largest (Aline) load values was 136 g. For the 0.016 x 0.022 inch rectangular wires tested, the difference between the smallest (Copper nickel-titanium 40) and the largest (Aline) load values was 337 g. (2) The change in load between 1.5 and 0.5 mm of deflection was examined to clarify the superelastic properties of the wires tested. For the 0.016 inch wires, 17 wire brands produced a load difference of less than 100 g, and two brands produced a difference of at least 100 g (Aline and Titanal = 100 g). For the 0.016 x 0.022 inch wires, 15 brands produced a load difference of less than 100 g, and eight brands produced a difference of over 100 g. The smallest and largest load differences were 3 g (Copper nickel-titanium 35) and 200 g (Aline). (3) The majority of the samples with a smaller load difference between deflections of 1.5 mm and 0.5 mm in the unloading process were found among super-elastic wires, while samples with a larger load difference were predominantly found among work-hardened wires. Compared with cobalt-chrome and TMA wires, nickel-titanium alloy wires exert significantly less force. However, the amount of force varies greatly from brand to brand. Consequently, when using nickel-titanium alloy wires, brands must be selected carefully by taking into consideration the severity of the malocclusion and the stage of orthodontic treatment in each case. It is the intent of this study to offer clinicians an unbiased guide for the selection of appropriate nickel-titanium alloy wires. (Am J Orthod Dentofacial Orthop 1999;115:390-5)

p53 is currently considered to be a tumor suppressor gene product, and its alterations are suggested to be involved in several human malignancies. Here we show evidence of the possible involvement of p53 gene mutations in lymphoid leukemias studied by reverse transcriptase- polymerase chain reaction, single strand conformation polymorphism analysis, and nucleotide sequencing. Fourteen patients with various leukemias were examined and two with acute lymphoblastic leukemia and one with Waldenstrom's macroglobulinemia were identified to have mutations in the coding region of the p53 gene. These mutations included point mutation, triplet deletion, and single nucleotide insertion. Furthermore, expression of the wild-type p53 mRNA was not detected in the samples from these three patients. In one of them, chromosome 17p was deleted, suggesting the absence of the nonmutated p53 gene, whereas in the other two patients, chromosome 17p seemed to be intact by cytogenetic analysis. Our results suggest that alterations of the p53 gene may have a role in the genesis of some leukemias.

Human cDNA clones encoding the UUAG-binding heterogeneous nuclear ribonucleoprotein (hnRNP) D0 protein have been isolated and expressed. The protein has two RNA-binding domains (RBDs) in the middle part of the protein and an RGG box, a region rich in glycine and arginine residues, in the C-terminal part ("2xRBD-Gly" structure). The hnRNP A1, A2/B1, and D0 proteins, all possess common features of the 2xRBD-Gly structure and binding specificity toward RNA. Together, they form a subfamily of RBD class RNA binding proteins (the 2xRBD-Gly family). One of the structural characteristics shared by these proteins is the presence of several isoforms presumably resulting from alternative splicing. Filter binding assays, using the recombinant hnRNP D0 proteins that have one of the two RBDs, indicated that one RBD specifically binds to the UUAG sequence. However, two isoforms with or without a 19-amino acid insertion at the N-terminal RBD showed different preference toward mutant RNA substrates. The 19-amino acid insertion is located in the N-terminal end of the first RBD. This result establishes the participation of the N terminus of RBD in determining the sequence specificity of binding. A similar insertion was also reported with the hnRNP A2/B1 proteins. Thus, it might be possible that this type of insertion with the 2xRBD-Gly type RNA binding proteins plays a role in "fine tuning" the specificity of RNA binding. RBD is supposed to bind with RNA in general and sequence-specific manners. These two discernible binding modes are proposed to be performed by different regions of the RBD. A structural model of these two binding sites is presented.

The p53 gene is currently considered to function as a tumor-suppressor gene in various human malignancies. In hematologic malignancies, alterations in the p53 gene have been shown in some human leukemias and lymphomas. Although mutations in the p53 gene are infrequent in acute myelogenous leukemia (AML) patients, we show in this report that alterations in the p53 gene are frequent in myeloid leukemia cell lines. We studied alterations of the p53 gene in nine human myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and direct sequencing. Expression of the p53 gene was not detected at all by RT-PCR in two of the nine cell lines. In these two cell lines, Southern blot analysis showed gross rearrangements and deletions in both of the p53 alleles. Six of the nine cell lines were found to express only mutant p53 mRNA by RT-PCR/SSCP analysis and direct sequencing, and wild-type p53 mRNA was not detected. Two of the mutant p53 mRNAs were shown to be products of abnormal splicing events induced by intronic point mutations. Taken together, eight of nine human myeloid leukemia cell lines expressed no or an undetectable amount of wild-type p53 mRNA. Three of the eight cell lines were growth factor- dependent. Our results suggest that inactivation of the p53 gene may be a common feature in myeloid leukemia cell lines and may play an important role in the establishment of these cell lines.

Rat c-raf was found to be activated in a transformant obtained with DNA of a hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline. This activated c-raf was cloned in a cosmid vector and actively transforming clones were obtained. Comparison of the restriction maps of this activated c-raf and cloned normal rat c-raf revealed a recombination in the 5'-terminal region of the activated form of this gene. The recombined DNA was shown to be actively transcribed and possibly to form a fused mRNA with c-raf, which is slightly smaller than normal c-raf mRNA. Since this recombination was not detected in the original tumor by Southern blot analysis, it presumably occurred during transfection.

The yeast Y' element is a highly polymorphic repetitive sequence present in the subtelomeric regions of many yeast telomeres. The Y' element is classed as either Y'-L or Y'-S, depending on its length. It has been reported that survivors arising from telomerase-deficient yeast mutants compensate for telomere loss by the amplification of Y' elements. The total Saccharomyces cerevisiae genome DNA data base was searched for Y' elements, and 11 Y'-Ls and eight Y'-Ss were identified. As reported previously, many of the sequences were found to contain long open reading frames which potentially encode helicase. We examined the expression of the Y' elements in telomerase-deficient Deltatlc1 survivors, in which the TLC1 gene encoding the yeast telomerase template RNA had been disrupted, and found that the Y' element is highly expressed in the survivors, but not in the wild-type cells. Moreover, we demonstrated that the survivors produce a Y'-encoded protein designated as Y'-Help1 (Y'-helicase protein 1), and that this protein possesses helicase activity. Therefore, we suggest that the Y' element has a novel and potentially important role in trans, in addition to the well characterized role in cis, in telomerase-independent telomere maintenance in yeast.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin fibroblasts, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas 21 other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis.

In a previous study, activated rat c-raf was detected by an NIH 3T3 cell transfection assay, and a rearrangement was demonstrated in the 5' half of the sequence of the gene. In the present study, the cDNAs of normal and activated rat c-raf were analyzed. Results showed that the activated c-raf gene is transcribed to produce a fused mRNA, in which the 5' half of the sequence is replaced by an unknown rat sequence. This mRNA codes a fused c-raf protein. The normal and activated c-raf cDNAs were each connected to the long terminal repeat of Rous sarcoma virus and transfected into NIH 3T3 cells. Only the activated form had transforming activity. We conclude that the rearrangement is responsible for the activation of c-raf.

HeLa cell nuclear proteins that bind to single-stranded d(TTAGGG)n, the human telomeric DNA repeat, were identified and purified by a gel retardation assay. Immunological data and peptide sequencing experiments indicated that the purified proteins were identical or closely related to the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2-B1, D, and E and to nucleolin. These proteins bound to RNA oligonucleotides having r(UUAGGG) repeats more tightly than to DNA of the same sequence. The binding was sequence specific, as point mutation of any of the first 4 bases [r(UUAG)] abolished it. The fraction containing D and E hnRNPs was shown to bind specifically to a synthetic oligoribonucleotide having the 3' splice site sequence of the human beta-globin intervening sequence 1, which includes the sequence UUAGG. Proteins in this fraction were further identified by two-dimensional gel electrophoresis as D01, D02, D1*, and E0; intriguingly, these members of the hnRNP D and E groups are nuclear proteins that are not stably associated with hnRNP complexes. These studies establish the binding specificities of these D and E hnRNPs. Furthermore, they suggest the possibility that these hnRNPs could perhaps bind to chromosome telomeres, in addition to having a role in pre-mRNA metabolism.

Human platelet-derived endothelial cell growth factor (hPD-ECGF) is a novel angiogenic factor which stimulates endothelial cell growth in vitro and promotes angiogenesis in vivo. We report here the cloning and sequencing of the gene for hPD-ECGF and its flanking regions. This gene is composed of 10 exons dispersed over a 4.3-kb region. Its promoter lacks a TATA box and a CCAAT box, structures characteristic of eukaryotic promoters. Instead, six copies of potential Sp1-binding sites (GGGCGG or CCGCCC) were clustered just upstream of the transcription start sites. Southern blot analysis using genomic DNAs from several vertebrates suggested that the gene for PD-ECGF is conserved phylogenetically among vertebrates. The gene for hPD-ECGF was localized to chromosome 22 by analysis of a panel of human-rodent somatic cell hybrid lines.

Using in situ hybridization and a panel of human × rodent somatic cell hybrids, which discriminates between four different regions of human chromosome 22, we have localized the gene for human platelet-derived endothelial cell growth factor (ECGFl) to 22q13, placing ECGFl distal to the PDGFB locus at 22q12.3→q13.1.

A peptide was obtained from culture filtrates of a bacterium which was newly isolated and tentatively named Bacillus cereus SW. The peptide was composed of Asx, Ser, Glx, Leu, Tyr, Pro, and an unknown amino acid in a ratio of 2:1:1:1:1:1:1, but, unless hydrolyzed with HCI, it was ninhydrin reaction negative. The peptide effectively inhibited the growth of all fungi and yeasts so far examined, whereas it inhibited none of the bacteria tested.

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