bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment with removable partial dentures: a longitudinal study. Part II
Authors: Vanzeveren C. , D'Hoore W. , Bercy P. , Leloup G.
Published: May 2003

summary The aim of this study was to evaluate the effectiveness of an oral rehabilitation by removable partial denture (RPD). Between 1983 and 1994, 629 patients were provided with an RPD at the Dental School of the Université catholique de Louvain. All the RPDs were constructed with a cobalt–chromium framework. All the treatments were provided by dental students under the supervision of clinical instructors. At recall time (1998–2000), 269 patients could not be reached neither by telephone nor by mail and 27 had died. Consequently, 333 patients were called for clinical examination and 254 of these (76·3%) actually attended. For a total of 292 RPDs checked 1893 persistent teeth were listed, with 804 serving as abutments; more than half (57·6%) of these were crowned, for which 51 debondings were recorded. For the period of observation we listed 79 abutments lost (37 at the upper jaw and 42 at the lower jaw), i.e. 9·8% of the total number of abutments. No relation could be established between the type of the abutments (natural or crowned teeth) and the losses observed (Test of Mantel‐Haenszel: P=0·9496). In the upper jaw, the percentage of abutments lost was significantly higher in the presence of free‐end edentulous areas as compared with bounded edentulous areas (Test of Mantel‐Haenszel: P=0·0002); this difference does not appear for the lower jaws (P=0·9558). If we deduct the 25 abutments related with the 11 non‐worn RPDs and the 79 abutments lost, no change becomes apparent for 92·2% of the maxillary abutments and for 85·8% of the mandibular abutments. For the 1089 other teeth, we observed the loss of 40 teeth and the appearance of caries or new fillings for 95 teeth. The fractures of cast clasps represent 3·4%.
Fatigue resistance of removable orthodontic appliance reinforced with glass fibre weave
Authors: Rantala L. I. , Lastumäki T. M. , Peltomäki T. , Vallittu P. K.
Published: May 2003

summary  The aim of this study was to measure the fatigue resistance of fibre‐reinforced composite (FRC) reinforced polymeric parts of a removable orthodontic appliance beside the clasp. The effect of quantity and position of FRC‐reinforcement were investigated. In addition, the influence of water storage on the fatigue properties was determined. The test specimens for eight groups (n=6) were manufactured from autopolymerizing acrylic resin. Polymethylmethacrylate pre‐impregnated woven glass fibre was used as reinforcement of acrylic resin specimens at the region of steel wire clasp. The test specimens of the control group were not reinforced. In the second group, the test specimens were reinforced with one fibre layer (thickness: 0·06 mm) on the tension side, and in the third and fourth group with two fibre layers. Fatigue resistance was measured by applying repeated bending force to the clasp. The highest fatigue resistance values were achieved when the test specimens were fibre‐reinforced with two fibre layers. The lowest fatigue resistance values resulted when the test specimens were not reinforced (P=0·046, anova). Water storage had a tendency to decrease the fatigue resistance in all fibre reinforced test specimen groups. The results suggest that use of the woven polymer pre‐impregnated glass FRC‐reinforcement increases the fracture resistance of orthodontic appliance made of acrylic polymer.
Cytotoxicity of bisphenol A glycidyl methacrylate on cytochrome P450‐producing cells
Authors: Hikage S. , Nakayama K. , Saito T. , Takahashi Y. , Kamataki T. , Suzuki S. , Hongo T. , Sato A.
Published: May 2003

summary  Of the cytochrome P450 (CYP) family of carcinogen‐activating enzymes, CYP3A is the major form found in human livers. The purpose of this study was to investigate the cytotoxic effects of dental resin monomers after being metabolized by CYP3A4 and CYP3A7, using a colony formation assay and a neutral red assay. Specimen wells were plated with transfected cells derived from the Chinese hamster lung at 100 cells well−1. The experimental group consisted of CYP‐producing 3A4‐10 and 3A7‐40 cells, while the control group consisted of non‐CYP‐producing CR‐119 cells. Bisphenol A (BPA) and bisphenol A glycidyl methacrylate (Bis‐GMA) and a positive control (Aflatoxine Bl) were added separately to each well and cultured for 7 days. After cultivation, the number of the colonies was counted and IC50 values were determined. The data were statistically analysed by a Student's t‐test. The resultant of IC50 values indicated that the monomers were not metabolically activated by CYP3A4 or CYP3A7 as compared with the control (P < 0·05). We also confirmed that these monomers act neither as activators nor as inhibitors of CYP3A4 and CYP3A7.
Risk factors for temporomandibular joint pain in patients with disc displacement without reduction – a magnetic resonance imaging study
Authors: Emshoff R. , Brandlmaier I. , Bertram S. , Rudisch A.
Published: May 2003

summary The purpose of this study was to evaluate whether the magnetic resonance (MR) imaging variables of temporomandibular joint (TMJ) internal derangement, osteoarthrosis and/or effusion may predict the presence of pain in patients with a clinical disorder of an internal derangement type (ID)‐III. The relationship between TMJ ID‐III pain and TMJ internal derangement, osteoarthrosis and effusion was analysed in MR images of 84 TMJs in 42 patients with a clinical unilateral diagnosis of TMJ ID‐III pain. Criteria for including a TMJ ID‐III pain patient were report of orofacial pain referred to the TMJ, with the presence of unilateral TMJ pain during palpation, function and/or unassisted or assisted mandibular opening. Bilateral sagittal and coronal MR images were obtained to establish the presence or absence of TMJ internal derangement, osteoarthrosis and effusion. Using chi‐square analysis for pair‐wise comparison, the data showed a significant relationship between the MR imaging findings of TMJ ID‐III pain and those of internal derangement (P=0·01) and effusion (P=0·00). Of the MR imaging variables considered simultaneously in the multiple logistic regression analysis, osteoarthrosis (P=0·82) and effusion (P=0·08) dropped out as non‐significant in the diagnostic TMJ pain group when compared with the TMJ non‐pain group. The odds ratio that a TMJ with an internal derangement type of disk displacement without reduction might belong to the pain group was strong (2·7:1) and highly significant (P=0·00). Significant increases in risk of TMJ pain occurred with ‘disk displacement without reduction in combination with osteoarthrosis’ (5·2:1) (P=0·00) and/or ‘disk displacement without reduction in combination with osteoarthrosis and effusion’ (6·6:1) (P=0·00). The results suggest that TMJ pain is related to internal derangement, osteoarthrosis and effusion. However, the data re‐emphasize the aspect that these MR imaging variables may not be regarded as the unique and dominant factors in defining TMJ pain instances.
Atomic analysis and knoop hardness measurement of the cavity floor prepared by Er,Cr:YSGG laser irradiation in vitro
Authors: Hossain M. , Nakamura Y. , Tamaki Y. , Yamada Y. , Murakami Y. , Matsumoto K.
Published: May 2003

summary  In the present study, the compositional changes and knoop hardness of the cavity floor prepared by Er,Cr:YSGG laser irradiation was compared with that of the conventional bur cavity. Fifteen laser and 15 bur cavities were cross‐sectioned, and subjected to atomic analysis by SEM‐EDX and knoop hardness test. Statistical analyses were performed using the Mann–Whitney U‐test; a value of P < 0·01 was considered significant. Surface characteristics of the prepared cavities were also investigated by light microscopy and scanning electron microscopy (SEM). The results showed that the quantities of Ca (Ca weight %) and P (P weight %) were increased significantly in the laser cavity floor but no significant differences were found between the Ca/P ratio and knoop hardness number of laser and bur cavities. The SEM observation revealed that the lased cavity surface was irregular and there was also the absence of a smear layer; the orifice of dentinal tubules was exposed. Er,Cr:YSGG laser device is considered as one of the most effective and safe devices for cavity preparation because of its many advantages. This includes easy delivery system, minimal thermal damage to the surrounding tissues, minimal thermal‐induced changes of dental hard tissue compositions, and favourable surface characteristic.
A clinical pilot study of the time‐dependent composition of tooth bleaching systems
Authors: Wille T. , Pesun I. J. , Combe E. C. , Lindquist G. C. , Hodges J. S.
Published: May 2003

summary  This pilot study was undertaken to determine the compositional changes in tooth bleaching materials as a function of time in vivo. Ten patients were recruited and two bleaching systems were used – one a paste and the other a gel. Each material was placed in a custom bleaching tray and worn by each patient for each of four times – 15, 30, 60 and 120 min. The material was collected and chemically analysed for water by Karl Fischer titration and titrated for carbamide peroxide by the US Pharmacopoeia method. The paste material contained 18·66% water as supplied, and after 2 h this rose to between 28·6 and 64·4%. The gel material contained 2·85% water as supplied, and after 2 h this was diluted to between 28·5 and 73·4%. There was considerable difference in saliva uptake by the custom tray between patients. Most water uptake usually occurred within the first 30 min. Peroxide concentrations decreased in an approximately linear manner with time. There was a significant difference between the materials from baseline to 30 min and thereafter (P < 0·0009). This pilot study is an effective technique for chemical evaluation of bleaching materials. The effect of saliva is an important factor to consider, and is one that has hitherto not always been appropriately emphasized.
Galvanic skin response of oral cancer patients during speech
Authors: Nishigawa G. , Natsuaki N. , Maruo Y. , Okamoto M. , Minagi S.
Published: May 2003

summary  Severe speech difficulty is often caused after surgery of an oral cancer. Prosthetic treatment with a removable obturator prosthesis is generally provided for such patients. Although some speech ability is recovered with prosthetic treatment, patients sometimes complain of continued dissatisfaction with their speech. However, it is difficult to evaluate the dissatisfaction. Therefore, a new method for evaluation is desirable. In this study, such a new method using the galvanic skin response as the index for the dissatisfaction of the patient was developed, and its objectivity was investigated. Eleven patients with maxillary bone defects were selected. Prior to the evaluation, improvement of speech with the removable prosthesis was confirmed using the speech intelligibility test and the visual analogue scale. The electrical resistant value at pronunciation was measured with the measuring system composed with the apparatus (galvanic skin response (GSR) measuring apparatus), the personal computer program. The changes for the electrical resistant value after pronunciation were evaluated by calculating the decrease ratio at pronunciation [(the mean electrical resistance before pronunciation − the mean electrical resistance after pronunciation)/the mean electrical resistance before pronunciation]. This decrease ratio at pronunciation was defined as the index of the speech dissatisfaction of the subject. The mean values for the decrease ratio with prosthesis were significantly smaller than the values without prosthesis (P < 0·05). From the results of this study, it is suggested that the measurement of the electrical resistance change of the skin during speech could be a new method for evaluating the speech dissatisfaction of the post‐oral‐cancer patient.
Use of a mandibular advancement device in obstructive sleep apnoea
Authors: Prathibha B. N. , Jagger R. G. , Saunders M. , Smith A. P.
Published: May 2003

summary  The present study investigated the effectiveness of an intra‐oral mandibular advancement device in the treatment of patients with obstructive sleep apnoea (OSA) who could not tolerate or who had failed to comply with continuous positive airway pressure (CPAP). Thirty‐five patients diagnosed by sleep study as suffering from OSA, who had either been unable to tolerate or had been non‐compliant with CPAP treatment, were included in the study. The subjects completed an Epworth sleep questionnaire. The subjects then had an oral appliance made. After using the appliance for 3 months, the patients repeated the questionnaires and had a repeat sleep study performed with the oral appliance in situ. Thirty‐one subjects completed the investigation. Mean AHI pre‐ and post‐study were 26·64 and 24·06, respectively (P > 0·05). Mean Epworth scores pre‐ and post‐study were 16·32 and 14·64, respectively (P > 0·05). Those patients with a pre‐study AHI < 20 (n = 23), however, did significantly better with the appliance (P < 0·0001). Those patients with a pre‐study AHI > 20 did not benefit from this device (P > 0·05). The main problems encountered were initial jaw discomfort in 18 patients and dry mouth in 11 patients (both of which improved with continued usage). It was concluded that the type of appliance used in this study can be recommended for those with mild OSA who are unable to tolerate CPAP.
Interleukin‐8 Production in Tuberculous Pleurisy: Role of Mesothelial Cells Stimulated by Cytokine Network Involving Tumour Necrosis Factor‐α and Interleukin‐1β
Authors: Park J. S. , Kim Y. S. , Jee Y. K. , Myong N. H. , Lee K. Y.
Published: May 2003

Interleukin‐8 (IL‐8) plays an important role in the host immune response to Mycobacterium tuberculosis by recruiting inflammatory cells to the site of infection. Here, we investigated the role of pleural macrophages and mesothelial cells in the production of IL‐8 in tuberculous pleurisy. Large concentrations of IL‐8 were detected in tuberculous pleural effusions, but not in pleural effusions associated with congestive heart failure (CHF). Tuberculous pleural macrophages and M. tuberculosis‐infected CHF pleural macrophages produced large concentrations of IL‐8. When immunohistochemistry was performed on pleural tissues, antigenic IL‐8 was detected in the mesothelial cells lining the tuberculous pleura. Direct stimulation of cultured CHF pleural mesothelial cells with M. tuberculosis induced IL‐8 secretion. However, conditioned media from M. tuberculosis‐infected pleural macrophages (CoMTB) induced greater mesothelial cell IL‐8 secretion. Tumour necrosis factor‐α (TNF‐α) and IL‐1β induced mesothelial cell IL‐8 mRNA expression, and neutralizing anti‐TNF‐α antibody and IL‐1 receptor antagonist nearly completely obliterated CoMTB‐induced mesothelial cell IL‐8 mRNA expression and protein secretion. These findings demonstrate that both pleural macrophages and mesothelial cells produce IL‐8 in tuberculous pleurisy, and cytokines produced by M. tuberculosis‐infected macrophages mediate mesothelial cell IL‐8 production.
Cellular Mechanism of Thymic Involution
Authors: Li L. , Hsu H.‐C. , William G. E. , Stockard C. R. , Ho K.‐J. , Lott P. , Yang P.‐A. , Zhang H.‐G. , Mountz J. D.
Published: May 2003

Involution of the thymus and alterations in the development of thymocytes are the most prominent features of age‐related immune senescence. We have carried out a comparative analysis of thymocyte and stroma in rapid thymic involution DBA/2 (D2) strain of mice compared with slow involution C57BL/6 (B6) strain of mice. Analysis of mice at 15 months of age suggested an age‐related decrease in the thymocyte cell count, a block in the development of T cells and cortical involution in D2 mice compared with 3‐month‐old mice. TUNEL (terminal‐deoxynucleotidyl‐transferase‐mediated dUTP–digoxigenin nick end labelling) staining and fluorescence‐activated cell sorter (FACS) analysis showed that there was a significant increase in apoptotic cells in the cortex region of thymus in 15‐month‐old D2 mice compared with the same aged B6 mice. The thymocyte proliferation rate, as assessed by bromodeoxyuridine (BrdU) staining and [3H]‐thymidine incorporation assay, was lower in 3‐month‐old D2 mice compared with the same age B6 mice. Immunohistochemical staining showed that the arrangement of MTS (mouse thymus stromal)‐10+ epithelial cells and MTS‐16+ connective tissue staining pattern had become disorganized in 15‐month‐old D2 mice but remained intact in B6 mice of the same age. These results suggest that, in D2 mice, both the thymocytes and stromal cells exhibit age‐related defects, and that the genetic background of mice plays an important role in determining age‐related alterations in thymic involution.
Comparison of Immunoglobulin Heavy Chain Rearrangements Between Peripheral and Glandular B Cells in a Patient with Primary Sjögren's Syndrome
Authors: Hansen A. , Jacobi A. , Pruss A. , Kaufmann O. , Scholze J. , Lipsky P. E. , Dörner T.
Published: May 2003

Myoepithelial sialadenitis (MESA) of the major salivary glands is a characteristic feature of primary Sjögren's syndrome (pSS). To delineate systemic and organ‐specific influences on B cells in a patient with pSS and benign MESA, individual B cells were simultaneously obtained from the peripheral blood and inflamed parotid gland. Immunoglobulin variable heavy chain (VH) rearrangements in single sorted CD19+ B cells were subsequently amplified, sequenced and analysed. Despite the presence of two clonal expansions using VH1‐08 and VH2‐70 segments, respectively, the majority of glandular B cells were polyclonal, resembling the VH gene usage and mutational pattern of the corresponding blood population. However, striking differences were observed in the proportion of cells expressing mutated VH rearrangements (blood, 28.9% versus parotid, 80.4%; P < 0.0001). Moreover, the glandular productive VH rearrangements differed significantly from their blood counterparts by a higher mutational frequency (P < 0.0001), shorter CDR3 lengths (P = 0.001) and a less frequent usage of JH6 (P = 0.0292), indicating an accumulation of memory B cells in the inflamed parotid. Thus, both preferential influx/homing of memory B cells and local proliferation may contribute to the pattern of benign MESA in pSS. Notably, one of the glandular clonal rearrangements (using VH1‐08) was also detected in the patient's peripheral repertoire.
The Cholera Toxin B Subunit is a Mucosal Adjuvant for Oral Tolerance Induction in Type 1 Diabetes
Authors: Bregenholt S. , Wang M. , Wolfe T. , Hughes A. , Bærentzen L. , Dyrberg T. , Von Herrath M. G. , Petersen J. S.
Published: May 2003

When conjugated to various proteins, the nontoxic B‐chain of cholera toxin (CTB) significantly increases the ability of these proteins to induce immunological tolerance after oral administration. Here, we investigated if a nonconjugated form of CTB enhances the induction of immune tolerance after oral insulin administration. Induction of immunological tolerance was studied after oral administration of insulin preparations in three mouse models; an insulin/ovalbumin coimmunization model, a model of virus‐induced diabetes in transgenic RIP‐LCMV‐NP mice and in nonobese diabetic (NOD) mice serving as a model of spontaneous diabetes. In the immunization model, we demonstrate that mixing with CTB increases the tolerogenic potential of insulin, approximately 10 fold. Titration of the CTB concentration in this system revealed that an insulin : CTB ratio of 100 : 1 was optimal for the induction of bystander suppression. Further studies revealed that this insulin : CTB ratio also was optimal for the prevention of diabetes in a virus‐induced, transgenic diabetes model. In addition, the administration of this optimal insulin–CTB preparation significantly prevented the onset of diabetes in old NOD mice with established islet infiltration. The data presented here demonstrate that CTB, even in its unconjugated form, functions as a mucosal adjuvant, increasing the specific tolerogenic effect of oral insulin.
Regulator T Cells: Specific for Antigen and/or Antigen Receptors?
Authors: Rubin B. , Diaz de Durana Y. , Li N. , Sercarz E. E.
Published: May 2003

Adaptive immune responses are regulated by many different molecular and cellular effectors. Regulator T cells are coming to their rights again, and these T cells seem to have ordinary α/β T‐cell receptors (TCRs) and to develop in the thymus. Autoimmune responses are tightly regulated by such regulatory T cells, a phenomenon which is beneficial to the host in autoimmune situations. However, the regulation of autoimmune responses to tumour cells is harmful to the host, as this regulation delays the defence against the outgrowth of neoblastic cells. In the present review, we discuss whether regulatory T cells are specific for antigen and/or for antigen receptors. Our interest in these phenomena comes from the findings that T cells produce many more TCR‐α and TCR‐β chains than are necessary for surface membrane expression of TCR‐αβ heterodimers with CD3 complexes. Excess TCR chains are degraded by the proteasomes, and TCR peptides thus become available to the assembly pathway of major histocompatibility complex class I molecules. Consequently, do T cells express two different identification markers on the cell membrane, the TCR‐αβ clonotype for recognition by B‐cell receptors and clonotypic TCR‐αβ peptides for recognition by T cells?
Quantification of Cytokine Gene Expression Using an Economical Real‐Time Polymerase Chain Reaction Method Based on SYBR® Green I
Authors: Ramos‐Payán R. , Aguilar‐Medina M. , Estrada‐Parra S. , González‐y‐Merchand J. A. , Favila‐Castillo L. , Monroy‐Ostria A. , Estrada‐Garcia I. C. E.
Published: May 2003

Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme‐linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low‐cost reagents for real‐time PCR. This was achieved by adapting a conventional RT‐PCR protocol to the quantitative real‐time format, by the addition of the SYBR® Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12‐myristate 12‐acetate (PMA)–ionomycin. The results using this technique were compared with those obtained with the well‐established gene array method. We conclude that the use of the SYBR® Green I reagent during real‐time RT‐PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post‐PCR manipulation.
Interferon‐γ Receptor‐1 Gene Polymorphism in Tuberculosis Patients from Croatia
Authors: Fraser D. A. , Bulat‐Kardum L. , Knezevic J. , Babarovic P. , Matakovic‐Mileusnic N. , Dellacasagrande J. , Matanic D. , Pavelic J. , Beg‐Zec Z. , Dembic Z.
Published: May 2003

Recent studies have indicated that the interleukin‐12/interferon‐γ (IFN‐γ) axis is important in mycobacterial infection susceptibility. Using an intronic (CA)n polymorphic microsatellite marker within the IFN‐γ receptor‐1 (IFNGR1) gene, we have compared the allelic frequencies of this marker in hospitalized tuberculosis patients (n = 120) with that of controls (n = 87) from Rijeka, Croatia. We identified 13 (CA)n alleles in the tuberculosis patients, whereas only 10 were found in the controls. A significant difference between one allelic marker and the control group was observed (P = 0.02, 95% confidence interval 0.14–0.94), suggesting a possible protective association. In contrast, several other allelic markers showed a trend towards association with the disease. We also found a trend towards an increased frequency in homozygosity of one allelic marker in patients (11.7%) as compared with controls (4.6%). We conclude that there is no evidence for disease association of the IFNGR1 gene marker in Mendelian‐type (single‐allele) inheritance. However, our results also suggest that unidentified allelic variations in the IFNGR1 gene might elevate or decrease the risk in this ethnic population, as a part of the multigenic predisposition to tuberculosis.
Cloning, Sequencing and Expression of Immunoglobulin Variable Regions of Murine Monoclonal Antibodies Specific for the P1.7 and P1.16 PorA Protein Loops of Neisseria meningitidis
Authors: Ihle Ø. , Beckstrøm K. J. , Michaelsen T. E.
Published: May 2003

The P1.7 and P1.16 epitopes on the PorA protein on the outer membrane of Neisseria meningitidis can induce protective antibodies upon vaccination. Structural analysis of antibodies to these targets can give information on the immune response induced by these epitopes and can reveal any structural similarities among the antibodies. To do so, we have isolated the immunoglobulin (Ig) variable genes from four mouse hybridomas expressing antibodies against the P1.7 and P1.16 epitopes. These V genes were successfully expressed as functional chimeric (ch) mouse/human IgG1 antibodies by subcloning them into expression vectors containing the constant genes of human heavy and light chains. Sequencing the two sets of V genes against P1.16 revealed a high degree of homology, similar to that previously published for P1.7 V genes. The close homology allowed us to interchange heavy and light chains between antibodies in some instances to construct new antibodies that bind the original antigen. This study demonstrates that the immune response in mice against the meningococcal PorA protein epitopes P1.7 as well as P1.16 is limited to few and very similar germline genes, and therefore the P1.7‐ and P1.16‐specific antibodies share high degree of similarities amongst each other. These V genes were used to construct chimeric antibodies with conserved antigen‐binding activity.
Analysis of T‐Cell Receptor Beta Chain Variable Gene Segment Usage in Healthy Adult Responders and Nonresponders to Recombinant Hepatitis B Vaccine
Authors: Soroosh P. , Shokri F. , Azizi M. , Jeddi‐Tehrani M.
Published: May 2003

One to 10 per cent of healthy adult individuals do not produce protective levels of anti‐hepatitis B surface (HBs) antibodies, following a standard vaccination protocol. Hole in HBs antigen (Ag)‐specific T‐cell repertoire is amongst the possible defects, which may lead to humoral unresponsiveness and is the main objective of this study. We analysed TcR BV (T‐cell receptor beta chain variable) gene usage in T lymphocytes from nine healthy adult responders and six nonresponders to recombinant HB vaccine, before and after booster vaccination. CD4+ and CD8+ T‐cell populations were isolated from peripheral blood mononuclear cells by magnetic beads, and the expression of TcR BV genes in each population was investigated by reverse transcription polymerase chain reaction and hybridization with specific probe. When the usage of each TcR BV gene within CD4+ and CD8+ T cells of the responders was compared with that of nonresponders, statistically significant difference (P < 0.01) was noted for BV5S2‐3 gene family in CD4+ T cells of nonresponders. Furthermore, individual vaccinees were shown to overexpress several TcR BV genes. To characterize the T‐cell repertoire and determine their clonal nature, analysis of CDR3 length polymorphism was performed. Our results show that T‐cell response to HBsAg is generally oligoclonal and involves multiple BV families. Furthermore, overexpressed individual TcR BV genes and CDR3 length distributions in response to HBsAg are subject‐dependent. In conclusion, our results are not in line with the notion that defective TcR repertoire may be an explanation for unresponsiveness to recombinant HBsAg vaccine.
The Possibility of B‐Cell‐Dependent T‐Cell Development
Authors: Keshavarzi S. , Rietz C. , Simões S. , Shih S. , Platt J. L. , Wong J. , Wabl M. , Cascalho M.
Published: May 2003

The development of T cells is thought to be independent of B cells. However, defects in cell‐mediated immunity in individuals with B‐cell deficiency suggest the contrary. To test whether B cells affect T‐lymphocyte development, we constructed mice with a monoclonal T‐cell compartment (MT) and monoclonal B‐ and T‐cell compartments (MBTs). In these mice, the T cells expressed a DO 11.10 transgenic (DO‐T) cell receptor restricted to major histocompatibility complex (MHC) class IId. While CD4+ DO‐T lymphocytes are rare in transgenic H‐2b MT mice, we found that in H‐2b MBT mice under the influence of B cells, DO‐T lymphocytes mature into large numbers of CD4+ peripheral T cells. H‐2b MBT mice have more CD4+ thymocytes than H‐2b MT mice. These data are consistent with the view that B cells play some role in thymocyte development.
T‐Cell Receptor Excisional Circles, Telomere Length, Proliferation and Apoptosis in Peripheral Blood Mononuclear Cells of Human Immunodeficiency Virus‐Infected Individuals after 18 Months of Treatment Induced Viral Suppression
Authors: Aladdin H. , Katzenstein T. , Dreves A.‐M , Ryder L. , Gerstoft J. , Skinhøj P. , Pedersen B. K. , Ullum H.
Published: May 2003

This study evaluated the effect of highly active antiretroviral therapy (HAART)‐induced viral suppression on T‐cell receptor excisional circles (TRECs), telomere length, proliferative responses and spontaneous as well as phytohaemagglutinin (PHA)‐stimulated lymphocyte apoptosis in 27 human immunodeficiency virus (HIV)‐infected individuals followed for 18 months during HAART. Our results show that HAART significantly increased the level of TRECs in CD4+ cells (P = 0.003) after 18 months of almost continuously suppressed HIV‐RNA levels. Lymphocyte proliferative responses and apoptosis levels in patients were significantly lower and significantly higher, respectively, compared with healthy controls. The proliferative response and apoptosis levels did not change during follow up. Changes in telomere length were observed in CD4+ and in CD8+ T cells. The study demonstrated that HAART induces normal TREC levels in the CD4+ T‐cell pool. However, the other perturbed functions in T cells indicate that immune reconstitution is incomplete and may need longer viral suppression.
Increased Brain‐Derived Neurotrophic Factor Expression in White Blood Cells of Relapsing–Remitting Multiple Sclerosis Patients
Authors: Gielen A. , Khademi M. , Muhallab S. , Olsson T. , Piehl F.
Published: May 2003

Central nervous system (CNS)‐autoreactive immune responses can exert neuroprotective effects, possibly mediated via the release of neurotrophic factors from infiltrating leucocytes. Herein, we analysed neurotrophin and cytokine mRNA levels using TaqMan polymerase chain reaction in unstimulated peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients in remission and controls. We demonstrate that mRNA for brain‐derived neurotrophic factor (BDNF), but not neurotrophin‐3 or nerve growth factor (NGF), is readily detectable in PBMC and that levels in MS are increased by approximately 60% compared with patients with other neurological diseases or healthy subjects. These results provide additional evidence that a potentially neuroprotective facet of autoimmune inflammation is present in MS.
Characterization of surface n‐alkanes and fatty acids of the epiphytic lichen Xanthoria parietina, its photobiont a green alga Trebouxia sp., and its mycobiont, from the Jerusalem hills
Authors: Torres A. , Dor I. , Rotem J. , Srebnik M. , Dembitsky V. M.
Published: May 2003

Surface alkanes and fatty acids from the thalli of the lichen Xanthoria parietina, its photobiont Trebouxia sp., and its mycobiont were analysed by GC‐MS. The green alga Trebouxia sp. synthesized mainly unsaturated fatty acids such as (Z,Z,Z)‐9,12,15‐18 : 3 (Z,Z)‐9,12‐18 : 2 and (Z)‐9‐18 : 1, and light alkanes C8‐C15 (up to 83% of total n‐alkanes). However, the mycobiont contained mainly saturated fatty acids such as hexadecanoic (16 : 0) and octadecanoic acid (18 : 0), and also very long‐chain n‐alkanes C22–C34. Dehydroabietic acid was found in both lichen and mycobiont. The occurrence of different amounts of n‐alkanes and fatty acids in the photobionts and mycobionts of X. parietina was shown for the first time. Lichens collected from different locations in the Jerusalem hills contained n‐alkanes ranging in concentration from 187 to 211 mg·(g dry wt)−1; n‐alkane concentrations in the photobiont and mycobiont were 17–24 and 215–262 mg·(g dry wt)−1, respectively.
Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae
Authors: Griessler Richard , Schwarz Alexandra , Mucha Jan , Nidetzky Bernd
Published: May 2003

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic α1,4‐glucosyltransferases that are active as dimers of two identical, pyridoxal 5′‐phosphate‐containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100‐fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure‐function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate‐dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit‐to‐subunit contact region made these residues primary candidates for site‐directed mutagenesis. Individual Arg→Ala mutants were purified and characterized using time‐dependent denaturation assays in urea and at 45 °C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild‐type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20‐fold greater in the wild‐type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.
Dissociation of DNA polymerase α‐primase complex during meiosis in Coprinus cinereus
Authors: Namekawa Satoshi , Hamada Fumika , Sawado Tomoyuki , Ishii Satomi , Nara Takayuki , Ishizaki Takashi , Ohuchi Takashi , Arai Takao , Sakaguchi Kengo
Published: May 2003

Previously, the activity of DNA polymerase α was found in the meiotic prophase I including non‐S phase stages, in the basidiomycetes, Coprinus cinereus. To study DNA polymerase α during meiosis, we cloned cDNAs for the C. cinereus DNA polymerase α catalytic subunit (p140) and C. cinereus primase small subunit (p48). Northern analysis indicated that both p140 and p48 are expressed not only at S phase but also during the leptotene/zygotene stages of meiotic prophase I. In situ immuno‐staining of cells at meiotic prophase I revealed a sub population of p48 that does not colocalize with p140 in nuclei. We also purified the pol α‐primase complex from meiotic cells by column chromatography and characterized its biochemical properties. We found a subpopulation of primase that was separated from the pol α‐primase complex by phosphocellulose column chromatography. Glycerol gradient density sedimentation results indicated that the amount of intact pol α‐primase complex in crude extract is reduced, and that a smaller complex appears upon meiotic development. These results suggest that the form of the DNA polymerase α‐primase complex is altered during meiotic development.
Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N‐terminus of HIV‐1 Tat indicate at least two inhibitor binding sites
Authors: Lorey Susan , Stöckel‐Maschek Angela , Faust Jürgen , Brandt Wolfgang , Stiebitz Beate , Gorrell Mark D. , Kähne Thilo , Mrestani‐Klaus Carmen , Wrenger Sabine , Reinhold Dirk , Ansorge Siegfried , Neubert Klaus
Published: May 2003

Dipeptidyl peptidase IV (DP IV, CD26) plays an essential role in the activation and proliferation of lymphocytes, which is shown by the immunosuppressive effects of synthetic DP IV inhibitors. Similarly, both human immunodeficiency virus‐1 (HIV‐1) Tat protein and the N‐terminal peptide Tat(1–9) inhibit DP IV activity and T cell proliferation. Therefore, the N‐terminal amino acid sequence of HIV‐1 Tat is important for the inhibition of DP IV. Recently, we characterized the thromboxane A2 receptor peptide TXA2‐R(1–9), bearing the N‐terminal MWP sequence motif, as a potent DP IV inhibitor possibly playing a functional role during antigen presentation by inhibiting T cell‐expressed DP IV [Wrenger, S., Faust, J., Mrestani‐Klaus, C., Fengler, A., Stöckel‐Maschek, A., Lorey, S., Kähne, T., Brandt, W., Neubert, K., Ansorge, S. & Reinhold, D. (2000) J. Biol. Chem.275, 22180–22186]. Here, we demonstrate that amino acid substitutions at different positions of Tat(1–9) can result in a change of the inhibition type. Certain Tat(1–9)‐related peptides are found to be competitive, and others linear mixed‐type or parabolic mixed‐type inhibitors indicating different inhibitor binding sites on DP IV, at the active site and out of the active site. The parabolic mixed‐type mechanism, attributed to both non‐mutually exclusive inhibitor binding sites of the enzyme, is described in detail. From the kinetic investigations and molecular modeling experiments, possible interactions of the oligopeptides with specified amino acids of DP IV are suggested. These findings give new insights for the development of more potent and specific peptide‐based DP IV inhibitors. Such inhibitors could be useful for the treatment of autoimmune and inflammatory diseases.
A unique variant of streptococcal group O‐antigen (C‐polysaccharide) that lacks phosphocholine
Authors: Bergström Niklas , Jansson Per‐Erik , Kilian Mogens , Skov Sørensen Uffe B.
Published: May 2003

Streptococcus mitis strain SK598, which represents a subgroup of biovar 1, possesses a unique variant of the C‐polysaccharide found in the cell wall of all strains of Streptococcus pneumoniae and in some strains of S. mitis. This new variant lacks the choline methyl groups in contrast to the previously characterized forms of C‐polysaccharide, which all contain one or two choline residues per repeat. The following structure of the repeating unit of the SK598 polysaccharide was established:

where AAT is 2‐acetamido‐4‐amino‐2,4,6‐trideoxy‐d‐galactose.

This structure is identical to the double choline‐substituted form of C‐polysaccharide, except that it is substituted with ethanolamine instead of choline. This extends the number of recognized C‐polysaccharide variants to four.
Comparison of the solution structures of angiotensin I & II
Authors: Spyroulias Georgios A. , Nikolakopoulou Panagiota , Tzakos Andreas , Gerothanassis Ioannis P. , Magafa Vassiliki , Manessi‐Zoupa Evy , Cordopatis Paul
Published: May 2003

Conformational analysis of angiotensin I (AI) and II (AII) peptides has been performed through 2D 1H‐NMR spectroscopy in dimethylsulfoxide and 2,2,2‐trifluoroethanol/H2O. The solution structural models of AI and AII have been determined in dimethylsulfoxide using NOE distance and 3JHNHα coupling constants. Finally, the AI family of models resulting from restrained energy minimization (REM) refinement, exhibits pairwise rmsd values for the family ensemble 0.26 ± 0.13 Å, 1.05 ± 0.23 Å, for backbone and heavy atoms, respectively, and the distance penalty function is calculated at 0.075 ± 0.006 Å2. Comparable results have been afforded for AII ensemble (rmsd values 0.30 ± 0.22 Å, 1.38 ± 0.48 Å for backbone and heavy atoms, respectively; distance penalty function is 0.029 ± 0.003 Å2). The two peptides demonstrate similar N‐terminal and different C‐terminal conformation as a consequence of the presence/absence of the His9‐Leu10 dipeptide, which plays an important role in the different biological function of the two peptides. Other conformational variations focused on the side‐chain orientation of aromatic residues, which constitute a biologically relevant hydrophobic core and whose inter‐residue contacts are strong in dimethylsulfoxide and are retained even in mixed organic‐aqueous media. Detailed analysis of the peptide structural features attempts to elucidate the conformational role of the C‐terminal dipeptide to the different binding affinity of AI and AII towards the AT1 receptor and sets the basis for understanding the factors that might govern free‐ or bound‐depended AII structural differentiation.
Phosphorylation of NF‐κB proteins by cyclic GMP‐dependent kinase
Authors: He Bin , F. Weber Georg
Published: May 2003

The transcription factor NF‐κB is activated in cellular stress responses. This requires rapid regulation of its function, which is accomplished, in part, by various modes of phosphorylation. Even though diverse DNA binding subunits of NF‐κB proteins may transactivate from distinct recognition sequences, the differential regulation of transcription from the large number of NF‐κB responsive sites in various gene promoters and enhancers has been incompletely understood. The cyclic GMP‐dependent kinase (PKG) is an important mediator of signal transduction that may induce gene expression through cAMP response element binding protein (CREB) and through other, yet undefined, mechanisms. We have previously characterized a signal transduction pathway that leads to activation‐induced cell death in T‐lymphocytes and involves the activation of PKG. Here we demonstrate that the NF‐κB proteins p65, p49 (also called p52), and p50 are specific substrates for this kinase. PKG dose‐dependently increases the transactivating activity of p65 from the NF‐κB consensus sequence. It also mediates dose‐dependently an increase in transcriptional activity by p49 or p50 from a unique CCAAT/enhance binding protein (C/EBP)‐associated NF‐κB site, but not from the consensus site. Phosphorylation of p65, p50, or p49 does not alter their subcellular distribution. Because the release of cytosolic p65/p50 heterodimers into the nucleus is by itself insufficient to differentiate all the numerous NF‐κB promoter sequences, phosphorylation of the DNA‐binding subunits reveals a form of differential regulation of NF‐κB activity and it implies a novel pathway for PKG‐induced gene transcription. These observations may bear on mechanisms of programmed cell death in T‐lymphocytes. They may also be relevant to ongoing efforts to induce cancer cell apoptosis through activation of PKG.
The mystery of nonclassical protein secretion
Authors: Nickel Walter
Published: May 2003

Most of the examples of protein translocation across a membrane (such as the import of classical secretory proteins into the endoplasmic reticulum, import of proteins into mitochondria and peroxisomes, as well as protein import into and export from the nucleus), are understood in great detail. In striking contrast, the phenomenon of unconventional protein secretion (also known as nonclassical protein export or ER/Golgi‐independent protein secretion) from eukaryotic cells was discovered more than 10 years ago and yet the molecular mechanism and the molecular identity of machinery components that mediate this process remain elusive. This problem appears to be even more complex as several lines of evidence indicate that various kinds of mechanistically distinct nonclassical export routes may exist. In most cases these secretory mechanisms are gated in a tightly controlled fashion. This review aims to provide a comprehensive overview of our current knowledge as a basis for the development of new experimental strategies designed to unravel the molecular machineries mediating ER/Golgi‐independent protein secretion. Beyond solving a fundamental problem in current cell biology, the molecular analysis of these processes is of major biomedical importance as these export routes are taken by proteins such as angiogenic growth factors, inflammatory cytokines, components of the extracellular matrix which regulate cell differentiation, proliferation and apoptosis, viral proteins, and parasite surface proteins potentially involved in host infection.
An alternative mechanism of product chain‐length determination in type III geranylgeranyl diphosphate synthase
Authors: Hemmi Hisashi , Noike Motoyoshi , Nakayama Toru , Nishino Tokuzo
Published: May 2003

(All‐E) prenyl diphosphate synthases catalyze the consecutive condensation of isopentenyl diphosphates with allylic prenyl diphosphates, producing products with various chain‐lengths that are unique for each enzyme. Some short‐chain (all‐E) prenyl diphosphate synthases, i.e. farnesyl diphosphate synthases and geranylgeranyl diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain‐length of the product. However, among these enzymes, which are classified into several types based on the possessive patterns of such characteristics, type III geranylgeranyl diphosphate synthases, which consist of enzymes from eukaryotes (excepting plants), lack these features. In this study, we report that mutagenesis at the second position before the conserved G(Q/E) motif, which is distant from the well‐studied region, affects the chain‐length of the product for a type III geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae. This clearly suggests that a novel mechanism is operative in the product determination for this type of enzyme. We also show herein that mutagenesis at the corresponding position of an archaeal medium‐chain enzyme also alters its product specificity. These results provide valuable information on the molecular evolution of (all‐E) prenyl diphosphate synthases.
The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes
Authors: Béven Laure , Castano Sabine , Dufourcq Jean , Wieslander Åke , Wróblewski Henri
Published: May 2003

Peptides composed of leucyl and lysyl residues (‘LK peptides’) with different compositions and sequences were compared for their antibacterial activities using cell wall‐less bacteria of the class Mollicutes (acholeplasmas, mycoplasmas and spiroplasmas) as targets. The antibacterial activity of the amphipathic α‐helical peptides varied with their size, 15 residues being the optimal length, independent of the membrane hydrophobic core thickness and the amount of cholesterol. The 15‐residue ideally amphipathic α helix with a +5 positive net charge (KLLKLLLKLLLKLLK) had the strongest antibacterial activity, similar to that of melittin. In contrast, scrambled peptides devoid of amphipathy and the less hydrophobic β‐sheeted peptides [(LK)nK], even those 15‐residue long, were far less potent than the helical ones. Furthermore, the growth inhibitory activity of the peptides was correlated with their ability to abolish membrane potential. These data are fully consistent with a predominantly flat orientation of LK peptides at the lipid/water interface and strongly supports that these peptides and probably the linear polycationic amphipathic defence peptides act on bacterial membranes in four main steps according to the ‘carpet’ model: (a) interfacial partitioning with accumulation of monomers on the target membrane (limiting step); (b) peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer (as already shown with liposomes and erythrocytes); (c) plasma membrane permeabilization/depolarization via a detergent‐like effect; and (d) rapid bacterial cell death if the extent of depolarization is maintained above a critical threshold.