bluuebunny/crossref_metadata_2025 huggingface dataset:
Apoptosis with FasL+ cell infiltration in the periphery and thymus of corrected autoimmune mice
Authors: KOBATA T. , TAKASAKI K. , ASAHARA H. , MAI HONG N. , MASUKO‐HONGO K. , KATO T. , HIROSE S. , SHIRAI T. , KAYAGAKI N. , YAGITA H. , OKUMURA K. , NISHIOKA K.
Published: November 2003

Fas (CD95) ligand (L) is a death factor that binds to its receptor, Fas, and induces apoptotic cell death, a crucial process in immunological tolerance. gld (generalized lymphoproliferative disorder) mice, which have a point mutation in the FasL gene, develop spontaneous systemic autoimmune syndromes characterized by hypergammaglobulinaemia and lymphoid hyperplasia owing to accumulation of abnormal B220+ CD3+ cells. Transplantation of wild‐type (wt) bone marrow cells into old gld mice on the same strain background results in normalization of autoimmune syndromes. We characterized the cellular mechanisms (functionally and histologically) of the above phenomena in gld mice after bone marrow transplantation (BMT) to determine the role of apoptosis via Fas/FasL interactions in inducing and maintaining self‐tolerance in vivo. Activated splenocytes from wt and BMT (wt to gld ) mice showed significant cytotoxic activity against Fas‐transfectant cells while those from BMT ( gld to gld ) mice did not. Cells in the thymus, spleen and lymph nodes of gld mice uniformly upregulated Fas expression and were sensitive to Fas‐mediated apoptosis compared with those in wt mice. Cells sensitive to Fas‐mediated apoptosis in gld mice resided not only among abnormal B220+ CD3+ cells but also among conventional lymphocytes. More importantly, histological analysis revealed that cells in the spleen, lymph nodes and thymus frequently underwent apoptosis with infiltration of FasL+ cells in BMT (wt to gld ) mice compared with BMT ( gld to gld ) mice. Our results indicated that apoptosis via Fas/FasL interactions can directly eliminate pathogenic cells responsible for autoimmunity in the periphery and possibly in the thymus in vivo.
Characterization of the bovine ε gene
Authors: MOUSAVI M. , RABBANI H. , HAMMARSTRÖM L.
Published: November 2003

Immunoglobulin E is quantitatively a minor immunoglobulin class in serum, but nevertheless the major class of antibody mediating type I hypersensitivity reactions and hence, type I allergic phenomena. The bovine ε gene is one of the as yet uncharacterized mammalian immunoglobulin genes. We have therefore cloned and determined the cDNA sequence and genomic organization of the gene. It contains four constant region domain‐encoding exons (CH1 to CH4) with a high homology to sheep Cε (87%) and to a lower degree to dog (62%), horse (58%), chimpanzee, orangutan, human (55%), mouse (52%) and rat (52%) Cε genes. Southern blot analysis of bovine genomic DNA, revealed the existence of a single Cε gene with a presence of allelic restriction fragment length polymorphism (RFLP).
Vaccination with recombinant vaccinia viruses protects mice against Mycobacterium tuberculosis infection
Authors: ZHU X. , VENKATAPRASAD N. , IVANYI J. , VORDERMEIER H. M.
Published: November 2003

A number of subunit‐based vaccine candidates have recently begun to erode the exclusive position of Mycobacterium bovis bacillus Calmette–Guérin (BCG), which gives unpredictable and highly variable protection against tuberculosis. In this paper we investigated the protective capacity of the 19 000 MW and 38 000 MW glyco‐lipoproteins of M. tuberculosis expressed by recombinant vaccinia viruses in a mouse Mycobacterium tuberculosis infection model. Both proteins were expressed at high levels by recombinant vaccinia‐infected cells. In addition, two inoculations of C57Bl/6 mice with either recombinant vaccinia virus significantly reduced the bacterial counts in the lungs of M. tuberculosis H37Rv‐infected mice, when compared with the group infected with control virus. This is the first report of protection against tuberculous infection using recombinant vaccinia viruses with results that suggest that secreted glyco‐lipoproteins in conjunction with the vaccinia vector represent suitable candidates for further vaccine‐related studies.
Natural antibodies that react with V‐region peptide epitopes of DNA‐binding antibodies are made by mice with systemic lupus erythematosus as disease develops
Authors: WARD F. J. , KNIES J. E. G. , CUNNINGHAM C. , HARRIS W. J. , STAINES N. A.
Published: November 2003

Cross‐reactive idiotypes (CRI) have been detected on anti‐DNA autoantibodies associated with lesions typical of systemic lupus erythematosus. In order to analyse the antigenic make up of idiotypes on anti‐DNA monoclonal antibodies (mAb) V‐88 (IgG1κ) and F‐423 (IgG3κ), derived respectively from an adult (NZB×NZW)F1 and a fetal MRL/Mp‐lpr/lpr mouse, a set of overlapping hexapeptides representing the VH and VL regions of mAb V‐88 and F‐423 were synthesized and reacted with a range of sera in pepscan enzyme‐linked immunosorbent assays (ELISA) taken from normal and lupus mouse strains. Serum pools were collected both from normal BALB/c and lupus MRL/Mp‐lpr/lpr and (NZB×NZW)F1 mice at 10, 20 and 30 weeks of age and analysed for the presence of spontaneously produced anti‐V‐region peptide IgM and IgG antibodies. IgM antibodies from both the lupus mice reacted with the same V‐region epitopes, and although some epitopes mapped to similar locations in the two mAb, the maps for V‐88 and F‐423 were not identical. In MRL/Mp‐lpr/lpr mice, as lupus disease progressed there was a switch from IgM antibodies to IgG anti‐peptide antibodies whose specificity for the peptide antigens coincided with but was better defined than that of the IgM antibodies. The identified idiotopes were located in both complementary determining regions (CDR) and framework region (FR) regions, indicating that some contribute to CRI shared by other related antibodies, while others were unique to either mAb V‐88 or F‐423. In conclusion, we have dissected and identified a mosaic of antibody V‐region idiotopes that contribute to the idiotype of an anti‐DNA autoantibody and against which autoantibodies are made naturally in lupus disease.
Circulating immune complexes from HIV‐1+ patients induces apoptosis on‘qc normal lymphocytes
Authors: ACEITUNO E. , CASTAÑÓN S. , JIMÉNEZ C. , SUBIRÁ D. , DE GÓRGOLAS M. , FERNÁNDEZ‐GUERRERO M. , ORTÍZ F. , GARCÍA R.
Published: November 2003

Isolated immune complexes from sera of 49 out of 67 human immunodeficiency virus‐1‐positive (HIV‐1+) patients (CIC–HIV+), composed of anti‐HIV–HIV‐Ag, could induce apoptosis on normal phytohaemagglutinin (PHA)‐activated lymphocytes. DNA degradation was detected by propidium iodide staining. This activity is directed against CD4+ lymphocytes as demonstrated by double binding of CIC–HIV+ and anti‐CD4 on apoptotic cells. Expression of Fas antigen is prior to apoptotic phenomena. CIC–HIV+ apoptosis inducers belong mainly to asymptomatic HIV‐infected patients, indicating that immune complexes from these patients can destroy CD4+ lymphocytes.
Administration of anti‐IL‐12 antibody in vivo inhibits rejection of a rat histiocytoma and suppresses cytokine response in a tumour‐bearing host
Authors: LAKSHMI RAO K. , VARALAKSHMI CH. , MUBARAK ALI A. , KHAR* A.
Published: November 2003

Activated macrophages are the major producers of heterodimeric cytokine interleukin‐12 (IL‐12). Earlier evidence suggested that early rejection of AK‐5 tumours is mediated by IL‐12 through interferon‐γ (IFN‐γ) production, involving activation of natural killer (NK) cells and upregulation of T‐helper 1 (Th1)‐type cytokine response. Injection of anti‐IL‐12 antibody into AK‐5 tumour‐bearing animals resulted in a large number of changes in the host immune response towards the tumour. These animals showed diminished NK‐mediated antibody‐dependent cell‐mediated cellular cytotoxicity (ADCC) activity, down‐regulation of Th1‐type cytokine response, decreased anti‐tumour antibody response ultimately leading to either delay or inhibition of the tumour‐regression process. There was also increased production of IL‐10 in the animals that had received anti‐IL‐12 antibody thereby resulting in the down‐regulation of IL‐2 and IFN‐γ production. IL‐12 plays a major role in the activation of the different immune parameters responsible for early rejection of AK‐5 tumour. We also studied the activation status of macrophages from tumour‐transplanted animals and their ability to produce IL‐12. Monocytes/macrophages from antibody‐injected animals were less active and produced lower quantities of IL‐12, IL‐1β and tumour necrosis factor‐α (TNF‐α) as compared with the macrophages from AK‐5 tumour‐bearing animals.
Characterization of adhesive interactions between mast cells and laminin isoforms: evidence of a principal role for α6 integrin
Authors: VLIAGOFTIS H. , METCALFE D. D.
Published: November 2003

Mast cells are known to adhere to laminin, although there is limited information on the characteristics of this event. To further examine this adhesive interaction, we thus determined the adherence of murine mast cell lines and primary bone marrow cultured mast cells (BMCMC) to murine laminin (mLN), human placental laminin‐1 (hLN), merosin (laminin‐2) and various laminin fragments, concentrating on activating stimuli, the involvement of integrins, and the effect on mast cell activation. Murine mast cells were found to adhere to both mLN and hLN and to merosin, not only following exposure to phorbol 12‐myristate 13‐acetate (PMA), but also after FcεRI aggregation or addition of stem cell factor (SCF). Adhesion to laminin was partially inhibited by soluble E8 and PA22‐2, both fragments of laminin that promote mast‐cell adhesion when bound on surfaces. Mast‐cell lines and BMCMC consistently expressed high levels of α6 integrin. Antibody to α6 blocked spontaneous and inhibited activated mast‐cell adhesion to hLN, and inhibited mast‐cell adhesion to mLN and its fragment E8. Mast‐cell adhesion to both laminin isoforms increased FcεRI‐mediated mast‐cell secretion. These observations demonstrate that mast‐cell attachment to laminin is promoted by physiological stimuli, is mediated principally by α6 integrin, and results in enhanced cell activation.
Production of antibody to hepatitis B surface antigen (anti‐HBs) by murine hepatitis B virus carriers: neonatal tolerance versus antigen presentation by dendritic cells
Authors: KUROSE K. , AKBAR SK. MD. F. , YAMAMOTO K. , ONJI M.
Published: November 2003

The inability of hepatitis B virus (HBV) transgenic mice, which express abundant hepatitis B surface antigen (HBsAg) in sera from the neonatal period onwards, to produce antibody to HBsAg (anti‐HBs) is considered to be due to defective function of lymphocytes. The defective function is thought to result from neonatal tolerance because antigenic challenge during the neonatal period is considered to be a tolerogenic event rather than an immunogenic one. However, a series of mixed culture experiments in vitro showed that lymphocytes taken from transgenic mice that had been injected with HBsAg in complete Freund’s adjuvant (CFA) constitutively produced anti‐HBs when cultured with dendritic cells from age‐, sex‐ and major histocompatibility complex (MHC)‐matched normal mice, but not when cultured with dendritic cells from transgenic mice. The expression of major histocompatibility complex (MHC) class II and B 7.2 (CD86) antigens on dendritic cells was significantly lower in transgenic mice compared with the same from the normal mice (P<0·05). Treatment of transgenic mice with interferon‐γ (IFN‐γ) resulted in up‐regulation of MHC class II on dendritic cells, and lymphocytes from HBsAg‐injected transgenic mice produced anti‐HBs in vitro when cultured with dendritic cells from IFN‐γ‐treated transgenic mice, but not when cultured with the dendritic cells from untreated transgenic mice. These experiments have shown that defective function of antigen‐presenting cells (APC), not immunogenic tolerance, is responsible for the inability of murine HBV‐carriers to produce anti‐HBs. Production of anti‐HBs by lymphocytes from HBsAg‐injected transgenic mice in the presence of dendritic cells that express higher levels of MHC class II and CD86 antigens has inspired optimism that a more effective vaccine therapy can be developed for chronic HBV‐carriers, injecting vaccine containing HBsAg with modulator(s) of APC function of dendritic cells.
Linkage of protein kinase C‐β activation and intracellular interleukin‐2 accumulation in human naive CD4 T cells
Authors: HASSAN J. , RAINSFORD E. , REEN D. J.
Published: November 2003

A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long‐term cellular functions. Protein kinase C‐β (PKC‐β) has been reported to be involved in interleukin‐2 (IL‐2) synthesis in T lymphocytes. In this study, the role of PKC‐β in intracellular accumulation of IL‐2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (α and βI) Go6976, and one which inhibits both classical and non‐classical isotypes, GF109203X, caused a complete block in cytoplasmic IL‐2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL‐2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC‐β translocation. Flow cytometric assessments of PKC‐β and its isoforms βI and βII correlated with Western blotting analysis and these results were further supported by the use of PKC‐β‐positive (HUT 78) and ‐negative (BW5147) T‐cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC‐β is critical for accumulation of intracellular IL‐2. The influence of CsA in reducing but not blocking IL‐2 synthesis is discussed. PMA‐induced down‐regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non‐classical PKC isoforms.
Fcγ receptor‐dependent clearance is enhanced following lipopolysaccharide in vivo treatment
Authors: PALERMO M. S. , ALVES ROSA F. , FERNÁNDEZ ALONSO G. , ISTURIZ M. A.
Published: November 2003

Lipopolysaccharides (LPS) occupy centre stage in the pathogenesis of gram‐negative sepsis. Although LPS are potent stimulators of the mononuclear phagocyte system (MPS), their effects on immune complex (IC)‐specific clearance have not yet been reported. In order to evaluate this issue, we examined the MPS function after LPS treatment by measuring intravascular removal rate of syngeneic erythocytes sensitized with specific immunoglobulin G (IgG) (EA). Our findings showed that LPS, directly or through the release of endogenous cytokines, enhance Fcγ receptor (FcγR)‐dependent clearance. The EA uptake by liver, spleen and bone marrow was significantly increased leading to an effective clearance of immune complexes. Splenic antibody‐dependent cellular cytotoxicity (ADCC), an in vitro indicator of FcγR functionality, was also increased after in vivo LPS treatment. However, cytometric studies showed that endotoxin did not modify FcγR expression on splenocytes, but markedly enhanced the expression of CD11b/CD18 (Mac‐1), an adhesion molecule closely related to FcγR activity. We conclude that LPS enhance FcγR‐dependent effector functions and suggest that this effect is mediated through alterations in adhesion molecules.
Antimetastatic effect of NK1+ T cells on experimental haematogenous tumour metastases in the liver and lungs of mice
Authors: SEKI S. , HASHIMOTO W. , OGASAWARA K. , SATOH M. , WATANABE H. , HABU Y. , HIRAIDE H. , TAKEDA K.
Published: November 2003

Depletion of both natural killer 1.1+ (NK1+) intermediate αβ T‐cell receptor (int T) cells and NK cells by in vivo treatment with anti‐NK1 antibody greatly increased hepatic metastases of intravenously injected EL4 cells as well as pulmonary metastases of 3LL cells in C57BL/6 mice. However, depletion of NK cells alone by anti‐asialo GM1 (AGM1) antibody treatment did not increase the metastases in either organ. Interleukin‐12 (IL‐12) administration into mice induced strong cytotoxicities of NK cell‐depleted liver and lung mononuclear cells (MNC) comparable to those without NK‐cell depletion and inhibited metastases in either organ. In contrast, in both NK cell‐ and NK1+ int T‐cell‐depleted mice, IL‐12 could not induce cytotoxic activity of liver and lung MNC and metastases in both organs increased with or without IL‐12 treatment. These results confirmed the fact that NK1+ int T cells are more potent antitumour effectors than NK cells against experimental haematogenous tumour metastases.
Adoptive transfer of allergen‐specific CD4+ T cells induces airway inflammation and hyperresponsiveness in Brown–Norway rats
Authors: HACZKU A. , MACARY P. , HUANG T.‐J. , TSUKAGOSHI H. , BARNES P. J. , KAY A. B. , KEMENY D. M. , CHUNG K. F. , MOQBEL R.
Published: November 2003

Following allergen exposure, sensitized Brown–Norway rats develop airway hyperresponsiveness (AHR) and eosinophilic inflammation together with an increase in activated T cells (CD25+) in the airways. We tested the hypothesis that CD4+ T cells are involved directly in the acquisition of AHR. Spleen T cells from animals that were injected intraperitoneally on three consecutive days with ovalbumin/Al(OH)3, showed a dose‐dependent proliferative response in vitro to ovalbumin, but not to bovine serum albumin, as measured by []>
3H]thymidine uptake. For total T‐cell transfer, spleen cells obtained from donor rats 4 days after sensitization were depleted of adherent cells by a nylon wool column separation. CD4+ and CD8+ T cells were purified by immunomagnetic beads cell separation. Recipient naive rats were injected intravenously with 50×106 total T cells, 20×106 and 5×106 CD4+cells, and 5×106 CD8+ cells, and were exposed to ovalbumin aerosol 24 hr afterwards. After a further 24 hr, airway responsiveness to acetylcholine (ACh) was measured and provocative concentration (PC) values (PC100, PC200 andPC300) (the ACh concentration needed to achieve 100, 200 and 300% increase in lung resistance above baseline) were calculated. Airway responsiveness was significantly increased in recipients of sensitized total T cells compared with recipients of cells from saline‐injected donor rats (P<0·05). There were significantly increased eosinophil major basic protein (MBP)+ cell counts/mm2 in airway submucosal tissue in the hyperreactive rats and a significant correlation was found between the number of MBP+ cells and PC100 (r = 0·75; P<0·03) in recipients of sensitized total T cells. Purified CD4+ T cells from sensitized donors induced AHR in naive recipients (P<0·05), while sensitized CD8+ and naive CD4+ cells failed to do so. Our data indicate that T cells may induce AHR through an eosinophilic airway inflammation and that CD4+ T cells may have a direct effect in this process in Brown–Norway rats.
Effect of B‐cell receptor engagement on CD40‐stimulated B cells
Authors: SCHILIZZI B. M. , BOONSTRA R. , THE T. H. , DE LEIJ L. F. M. H.
Published: November 2003

Activation of human B cells in vitro either by cross‐linking of surface immunoglobulins (sIg) or by triggering CD40 antigen, in the presence of interleukin‐10 (IL‐10) and interleukin‐2 (IL‐2), may result in high levels of immunoglobulin secretion in vitro. We studied the combined effects of ligation of the B‐cell receptor (BCR) and CD40 [

with anti‐CD40 monoclonal antibody (mAb)] on B‐cell proliferation and production of human immunoglobulin. For this purpose highly purified splenic B cells were cultured with various combinations of anti‐CD40 and IL‐10/IL‐2 or IL‐4 in the presence of CD32‐transfected L cells. Simultaneous cross‐linking of the BCR was achieved by mAb held on CD32–L cells or Staphylococcus aureus (SA). We found that dual BCR and CD40 ligation with IL‐10/IL‐2 leads to reduced immunoglobulin G (IgG) secretion compared with B cells stimulated with either anti‐CD40 and IL‐10/IL‐2, or compared with B cells stimulated with SA or anti‐BCR mAb and IL‐10/IL‐2. Dual BCR and CD40 ligation with anti‐immunoglobulin mAb (anti‐κ+anti‐λ light chains) but not with SA induced a similar reduction in IgM production. The reduced immunoglobulin secretion found during dual ligation is accompanied by increased proliferation. This was independent of cytokine stimulation but SA/CD40‐induced proliferation was increased in the presence of IL‐10/IL‐2, although not with IL‐4. The combination anti‐κ and anti‐λ with anti‐CD40 showed a long‐term suppression of IgG and IgM production (at least 14 days), while anti‐κ or anti‐λ alone, or SA, allowed a moderate recovery of immunoglobulin production by day 14. These results suggest that simultaneous B‐cell antigen receptor cross‐linking and CD40 engagement via CD40L on T cells induces strong initial proliferation. This may be followed later by antibody production depending on the strength of the BCR signal and the presence of the appropriate cytokines.
Structuration theory and new technology: analysing organizationally situated computer‐aided design (CAD)
Authors: Brooks Laurence
Published: November 2003

It is becoming generally established that modern research and writings need to acknowledge the organizational context that surrounds the use of a technology (see Rachel & Woolgar, 1995; Westrup, 1996). However, previous efforts have been criticized for their failure to accumulate consistent research findings or develop coherent theoretical frameworks (Markus & Robey, 1988). Part of the problem is that a wide diversity of perspectives is adopted by organizational researchers, and fundamental issues behind these perspectives have yet to be properly examined and resolved (see Burrell & Morgan, 1979); this paper adopts the perspective that some resolution of these issues is possible, or at least should be sought. One approach is to adopt the theoretical framework, built on Giddens' structuration theory (Giddens, 1990), as presented by Orlikowski and Robey (1991), and to use it to analyse a new technology case, that of computer‐aided design/draughting.
Cloning and sequence of a gene for a homologue of the C subunit of the V‐ATPase from the salivary gland of the tickAmblyomma americanum(L.)
Authors: McSwain J. L. , Luo C. , DeSilva G. A. , Palmer M. J. , Tucker J. S. , Sauer J. R. , Essenberg R. C.
Published: November 2003

A 1084 base pair partial cDNA showing similarity to the C subunit of the vacuolar ATPase (V‐ATPase) was isolated on a clone from a cDNA library made from salivary glands from 3‐day‐old feeding adult

Amblyomma americanum

(L.) female ticks. The 5′ end was completed using primer extension and the two pieces joined to form a complete cDNA of 1373 bp. This mRNA is expressed in embryos and the salivary glands of unfed adults and adult females at all stages of feeding. Specific inhibitors of the V‐ATPase decrease the rate of dopamine‐stimulated secretion of isolated salivary glands, but not as much as ouabain, an inhibitor of the Na+, K+ ATPase, indicating that a V‐ATPase may participate in the mechanism of salivary fluid secretion in

A. americanum

, but the volume of saliva secreted is more dependent on an active Na+, K+ ATPase.
Rapid cloning of insect transposon insertion junctions using ‘universal’ PCR
Authors: Beeman  , Stauth D. M.
Published: November 2003

Very highly degenerate primers with short specific 3′ anchor sequences and 5′ adaptors were used in conjunction with nested specific primers to amplify large numbers of unknown insertion junctions of the insect retrotransposon

Woot

, using genomic DNA as template for the polymerase chain reaction (PCR). This technique, sometimes referred to as universal PCR, is a powerful method for molecular characterization of transposon insertions into genomes, and more generally for short‐distance chromosome walking through unknown DNA. Twenty‐four unique insertion junctions were cloned and sequenced from two strains of

Tribolium castaneum

and one strain of

T. freemani

. Inspection of these sequences revealed that integration of the

Woot

retrotransposon is cued by the insertion target motif, GTAC, in both species.
Preferential codon usage and two types of repetitive motifs in the fibroin gene of the Chinese oak silkworm,Antheraea pernyi
Authors: Yukuhiro K. , Kanda T. , Tamura T.
Published: November 2003

In this paper we describe the peculiar structures and preferential codon usage found in wild silkworm fibroin genes. We determined a 1350 bp nucleotide sequence from the Chinese oak silkworm,

Antheraea pernyi

. The deduced amino acid sequence was partitioned into thirteen polyalanine‐containing repetitive motifs, which was one of the characteristics of

Antheraea

fibroins. Eleven of these arrays can be classified into two types of motifs depending on difference in amino acid sequences following polyalanine. Repetitive motifs structurally similar to those of

A. pernyi

were detected in a homologue of the Japanese oak silkworm,

Antheraea yamamai

. The most remarkable feature of this study was preferential codon usage, especially seen in alanine synonymous codons within both homologues of

Antheraea

: isocodon GCA most frequently occurred in alanine isocodons. In contrast, GCU isocodon was the most abundant in

Bombyx mori

fibroin heavy chain that lacks polyalanine arrays. This result strongly suggests different modes of selective constraint between the two types of fibroin gene. The similar finding that GCA isocodon was most frequent in two dragline silk sequences of the spider,

Nephila clavipes

, is consistent with our results because of the repetitive polyalanine‐containing arrays seen in spider dragline silk.
Generation of clonal diversity by sexual reproduction in the greenbug, Schizaphis graminum
Authors: Shufran K. A. , Peters D. C. , Webster J. A.
Published: November 2003

In the United States, the greenbug, Schizaphis graminum (Rondani), reproduces primarily by apomictic parthenogenesis. Although a periodic sexual cycle exists, the extent to which it occurs naturally and its influence on the genetic variability of greenbug populations is unclear. Length variation in the intergenic spacer (IGS) of the rRNA cistron in the greenbug indicates that populations are made up of many genetically distinct clones. Previous laboratory studies have shown the stability of the IGS within parthenogenetic clones. By inducing the sexual reproductive cycle of the greenbug, we conducted both intra‐ and inter‐clone matings and studied the inheritance of the IGS in the offspring. In both mating schemes, rearrangements in the IGS were apparent. IGS diversity found among the offspring could be attributed to unequal cross‐over and probably other molecular drive events during meiosis. Periodic sexual reproduction is a primary mechanism for the generation and maintenance of genetic variability in greenbug populations, and explains the level of clonal diversity found in previous studies.
The white gene of the tephritid fruit fly Bactrocera tryoni is characterized by a long untranslated 5′ leader and a 12 kb first intron
Authors: Bennett C. L. , Frommer M.
Published: November 2003

A 300 bp fragment from exon 6 of the white gene of Bactrocera tryoni was used to screen a B. tryoni genomic library. One positive (∼14 kb) insert contained exons 2–6 of white by nucleotide and amino acid sequence similarity to the white genes of D. melanogaster (O'Hare et al., 1984; Pepling & Mount, 1990). Lucilia cuprina (Garcia et al., 1996). Ceratitis capitata (Zwiebel et al., 1995) and Anopheles gambiae (Besansky et al., 1995). A white 5′ cDNA fragment containing exons 1, 2 and part of exon 3 was amplified, cloned and sequenced. An inverse PCR fragment of genomic DNA was generated, containing the exon 1 coding region plus ∼2.1 kb of upstream sequence, encompassing the putative promoter of the gene. Exon 1 was found to be 728 bp long, encoding the first twenty‐five amino acids. The full length of intron 1 was shown to be 12 kb (amplified using long PCR protocols), up to 3 times the length of the longest white intron 1 isolated to date.
Molecular identification of sympatric chromosomal forms of Anopheles gambiae and further evidence of their reproductive isolation
Authors: Favia G. , della Torre A. , Bagayoko M. , Lanfrancotti A. , Sagnon N'F. , Touré Y. T. , Coluzzi M.
Published: November 2003

Three chromosomal forms of Anopheles gambiae s.s., designated as Bamako, Mopti and Savanna, were studied for diagnostic PCR assays based on the analysis of the X‐linked ribosomal DNA (rDNA). The study was performed on a 1.3 kb fragment containing part of the 28S coding region and part of the intergenic spacer region. The amplified material was cut with fourteen restriction enzymes to detect Restriction Fragment Length Polymorphisms (RFLPs). The enzymes Tru9I and HhaI produced patterns of DNA bands which differentiated Mopti from Savanna and Bamako; moreover, a distinct ‘hybrid’ pattern was recognized in the F1 female progeny from the cross of Mopti with either one of the other two chromosomal forms. The diagnostic significance of the PCR‐RFLP assay was verified on 203 karyotyped females from field samples collected in two villages in Mali and one village in Burkina Faso. Agreement was observed between the chromosomal and the molecular identifications. No ‘hybrid’ molecular patterns were detected even among carriers of rare heterokaryotypes hypothetically produced by crosses between Mopti and Savanna. The results confirm previous observations indicating barriers to gene flow within An. gambiae s.s. and supporting the specific status of the taxonomic units proposed on cytogenetic ground.
Efficacy, cleaning ability and safety of different devices for gutta‐percha removal in root canal retreatment
Authors: HÜLSMANN M. , STOTZ S.
Published: November 2003

The purpose of this study was to evaluate the efficacy, cleaning ability and safety of five different devices and techniques to remove gutta‐percha root canal fillings. One hundred and twenty extracted single‐rooted anterior and premolar teeth were enlarged to ISO size 35 and obturated with laterally condensed gutta‐percha using AH 26 as the sealer. Removal of gutta‐percha was performed with the following devices and techniques: (a) Gates–Glidden and Hedstrom files, (b) only Hedstrom files, (c) Hedstrom files and chloroform, (d) the Endotec and Hedstrom files, and (e) the XGP drill and Hedstrom files. The following data were recorded: time taken to reach the desired working length, time required for the removal of the gutta‐percha, and the amount of material extruded apically. The teeth were split longitudinally and photographed. Cleanliness of the root canal walls was scored using the projected slides with a total magnification of approximately 70 X. The fastest technique to reach the working length was using the XGP drill (e), followed by the Gates–Glidden drills (a), Hedstrom files and chloroform (c), and the Endotec device (d).The use of Hedstrom files (b) without any additional support proved to be most time‐consuming. Differences were Statistically significant (U‐test, P > 0.05) between the rotary devices and the manual techniques. Time for complete removal of gutta‐percha was again shortest with the XGP drills (e), followed by the Gates‐Glidden burs (a), the Endotec device (d), Hedstrom files with chloroform (c), and Hedstrom files alone (b). The XGP burs and the Gates‐Glidden drills worked significantly faster than the other techniques. The amount of debris and filling material extruded apically in most cases did not exceed 0.1 mg. No significant differences could be detected between the groups (U‐test, P > 0.05). Root canal cleanliness proved best following the use of Hedstrom files without additional support (b) and the Gates‐Glidden drills (a), followed by Hedstrom files in combination with chloroform (c), the XGP‐gutta‐percha remover (e), and the Endotec device (d). When using the XGP two instrument fractures occurred in the apical parts of the root canals preventing further instrumentation to the apical foramen. When using the Gates‐Glidden burs four instrument fractures occurred, but all fragments could be removed with forceps immediately. The results suggest that the XGP gutta‐percha remover and the Gates‐Glidden drills are efficient and time saving devices to remove gutta‐percha but include a certain risk of instrument breakage and may leave some filling material inside the root canal. The best root canal cleanliness was achieved with Hedstrom files alone which, on the other hand, were shown to be the most time‐consuming.
TNF‐α release in monocytes after exposure to calcium hydroxide treated Escherichia coli LPS
Authors: BARTHEL C. R. , LEVIN L. G. , REISNER H. M. , TROPE M.
Published: November 2003

Lipopolysaccharide (LPS), a cell wall component of Gram negative anaerobic bacteria, has been implicated in the pathogenesis of periapical disease resulting from infected root canals. Calcium hydroxide [Ca(OH)2] has been shown to be an effective medicament in such infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)2 may also be the result of direct inactivation of LPS. The aim of this study was to investigate whether the toxic potential of an Escherichia coli LPS could be reduced or eliminated by Ca(OH)2. Four concentrations of E. coli LPS ranging from 1–1000 ng/ml sterile water were incubated in duplicate either with 25 mg Ca(OH)2 or sterile water alone. Controls consisted of Ca(OH)2 without LPS or sterile water only. Monocytes were collected from peripheral blood by centriiuging through a gradient and plated to a specific density. Adherent monocytes were incubated for 4 days at 37° with 5% CO2 in M199 medium with 10% autologous serum. The different LPS solutions were added to the wells on day 5. After 4 h the supernatants were collected and quantitatively assayed for TNF‐α using a commercial ELISA kit. Statistical analysis was performed with ANOVA. Results indicated that Ca(OH)2 is able to eliminate the ability of an E. coli LPS to stimulate TNF‐α production in peripheral blood monocytes (P<0.0001).
Shaping ability of NT Engine and McXim rotary nickel–titanium instruments in simulated root canals.Part 2
Authors: THOMPSON S. A. , DUMMER P. M. H.
Published: November 2003

The aim of this laboratory‐based study was to determine the shaping ability of NT Engine and McXim nickel‐titanium rotary instruments in simulated root canals. A total of 40 canals with four different shapes in terms of angle and position of curve were prepared with NT Engine and McXim instruments, using the technique recommended by the manufacturer. Part 2 of this report describes the efficacy of the instruments in terms of prevalence of canal aberrations, the amount and direction of canal transportation and overall postoperative shape. Pre‐ and postoperative images of the canals were taken using a video camera attached to a computer with image analysis software. The pre‐ and postoperative views were superimposed to highlight the amount and position of material removed during preparation. No zips, elbows, perforations or danger zones were created during preparation. Forty‐two per cent of canals had ledges on the outer aspect of the curve, the majority of which (16 out of 17) occurred in canals with short acute curves. There were significant differences (P<0.001) between canal shapes in terms of the incidence of ledges. There were highly significant differences (P<0.001) between the canal shapes in total canal width at specific points along the canal length and in the amount of resin removed from the inner and outer aspects of the curve. The direction of canal transportation at the endpoint of preparation was most frequently towards the outer aspect of the curve, especially in canals with 40° curves. At the beginning of the curve, transportation in the majority of canals was towards the inner aspect of the curve. Mean absolute transportation was less than 0.03 mm throughout the curve and towards the endpoint, with significant differences between canal shapes occurring at the apex (P<0.05) and at the beginning of the curve (P<0.001). Under the conditions of this study, NT Engine and McXim rotary nickel–titanium instruments created no aberrations other than ledges and produced only minimal transportation. The overall shape of canals was good.
Undergraduate endodontic teaching in the United Kingdom: an update
Authors: QUALTROUGH A. J. E. , DUMMER P. M. H.
Published: November 2003

Anecdotal evidence and scientific surveys suggest that the quality of root canal treatment within the General Dental Services is often less than ideal. Six years ago, it was reported that endodontic teaching in the United Kingdom was given a low priority and that the time devoted to the laboratory practice of root canal treatment should be increased. In the following year, the European Endodontic Society (ESE) published guidelines for the teaching of Endodontology and made recommendations as to what should be included in the undergraduate curriculum. Shortly afterwards, the ESE published quality guidelines for endodontic treatment. The aim of this project was to determine the current pattern of undergraduate endodontic teaching within the dental schools of the UK. Data were gathered by questionnaire from all 14 undergraduate dental schools and supplemented with further detailed enquiries where necessary. The results were compared with that reported following an identical survey carried out previously. Although the ESE curriculum guidelines for endodontic teaching provide an overall framework for teaching, the results of this study confirmed that a number of topics were either not included or were covered only briefly. However, the average time devoted to the preclinical instruction of root canal treatment in the first clinical year had increased from 14 to 24 h, with additional time provided in subsequent years by the majority of schools. In most instances, staff teaching endodontology in the UK have no specialist training.
Influence of fibronectin on the motility of human spermatozoa
Authors: WENNEMUTH G. , SCHIEMANN P. J. , KRAUSE W. , GRESSNER A. M. , AUMÜLLER G.
Published: November 2003

The objective of the present study was to scrutinize the concentration of seminal fibronectin and the potential effects of exogenous fibronectin on human sperm motility. In addition, variability in the localization of fibronectin on human spermatozoa from andrological patients was studied, at both the light and electron microscopic levels. A total of 58 freshly ejaculated semen samples from patients attending for infertility treatment were submitted to sperm motility analysis and ELISA quantification of seminal plasma and cell‐bound fibronectin. Immunofluorescence and immunoelectron microscopy revealed a relatively broad distribution pattern of fibronectin immunoreactivity on sperm heads and testicular spermatids. Addition of a fibronectin antiserum to vital spermatozoa in vitro at a moderate dilution (1:50) resulted in a significant increase in sperm motility. Purified plasma fibronectin, added at various concentrations to a preparation of live spermatozoa, was found to inhibit sperm motility in a dose‐dependent manner. At concentrations from 0.18 to 0.5 mg fibronectin per ml ejaculate, no motile spermatozoa were recorded. Seminal plasma fibronectin ranged between 0.8 and 1000 μg/ml in infertility patients. There was a significant inverse correlation between sperm motility and seminal fibronectin in patients with oligo‐astheno‐teratozoospermia. In a preliminary study in patients with varicocele or hypogonadism, no such correlation was found.
The acrosome reaction response assay: a new way to identify patients in whom pentoxifylline would improve the human follicular fluid‐induced acrosome reaction
Authors: BRIGGILER C. I. MARÍN , CALVO L. , LANCUBA S. , DELCOURT S. , GOGORZA S. , TRICÁRICO J. M. , TESTA R. , CAMEO M. A. S.
Published: November 2003

The effect of pentoxifylline on the spontaneous and follicular fluid (hFF)‐induced acrosome reaction (AR) was studied in an attempt to identify those patients who would benefit from the use of pentoxifylline as an AR‐stimulating agent. Pentoxifylline (1 mg/ml) produced no significant increase over spontaneous AR at any of the time points studied (60 min: 6.2% ± 2.0 spontaneous AR vs 7.4% ± 2.0 pentoxifylline AR; 120 min: 7.1% ± 1.6 spontaneous vs 9.0% ± 2.5 pentoxifylline; 180 min: 7.0% ± 1.8 spontaneous vs 10.9% ± 4.0 pentoxifylline, n = 6). On the other hand, hFF produced a significant increase in AR (6.9% spontaneous AR vs 11.2% hFF AR, p < 0.001, n = 39) and this effect was enhanced by pre‐incubation with pentoxifylline (15.0% pentoxifylline + hFF AR vs 11.2% hFF AR, p < 0.001). There were no differences in percentage AR induced by hFF or pentoxifylline + hFF between patients with normal (n = 16) and abnormal (n = 25) semen samples. Individual analysis of the effects of hFF and pentoxifylline + hFF on AR showed great variability between patients and enabled us to classify them into four categories: inducible‐responder (15%), non‐inducible‐responder (23%), inducible‐non‐responder (41%) and non‐inducible‐non‐responder (21%). An induction index (II) (induction produced by hFF divided by spontaneous AR) and a response index (RI) (induction produced by pentoxifylline + hFF divided by hFF) were calculated for each sample. Patients previously classified as inducible exhibited an II > 0.30 and patients previously classified as responders showed an RI > 0.26. There was no correlation between II and RI values (Spearman, r = −0.185, p = 1).

Our results show that pentoxifylline is an enhancer of the induced AR only in some patients. We propose an `AR response assay' to identify responder patients and thus enable rational use of this drug.
Involvement of the adrenal medulla in ejaculatory reactions in the dog
Authors: KIHARA K. , SATO K. , OSHIMA H.
Published: November 2003

To explore the peripheral signal controlling ejaculatory reactions, contraction of the seminal tract and seminal emission were monitored in the dog during electrical stimulation of the lumbar and greater splanchnic nerves in the presence or absence of sympathetic pathways connecting the lumbar sympathetic trunk to the seminal tract including the vas deferens, prostate and bladder neck. Electrical stimulation of the lumbar splanchnic nerve caused seminal emission, elevation of intraluminal pressure of the vas deferens and bladder neck, and contraction of the prostate without elevation of blood pressure. Transection of all peripheral sympathetic nerve pathways to the seminal tract completely blocked these responses. Electrical stimulation of the greater splanchnic nerve caused a marked elevation of blood pressure as well as the responses described above in both the presence and absence of peripheral sympathetic nerve pathways to the seminal tract. However, clamping the adrenal veins bilaterally blocked all of the above responses and declamping immediately reversed the block. Serum levels of epinephrine, norepinephrine and dopamine were significantly increased by electrical stimulation of the greater splanchnic nerve, while cortisol levels remained unchanged. Furthermore, intravenous administration of epinephrine (5 μg/kg) caused responses similar to those elicited by stimulating the greater splanchnic nerve. Dogs in which all sympathetic pathways to the seminal tract had been transected chronically showed retrograde ejaculation during manual penile stimulation. The above results indicate possible involvement of the greater splanchnic nerve and adrenal medulla in ejaculatory reactions in the dog.
Frequently subnormal semen profiles of normal volunteers recruited over 17 years
Authors: LEMCKE B. , BEHRE H. M. , NIESCHLAG E.
Published: November 2003

In order to establish semen profiles for men in the general population, we analysed the semen parameters of 187 men attending the Institute of Reproductive Medicine between 1977 and 1993 as volunteers for clinical studies. If several ejaculates were obtained from the volunteer, only the first ejaculate was used for analysis. More than half of the ejaculates of these healthy men showed at least one abnormal parameter, so that only 46% of the volunteers could be classified as being `normozoospermic' according to WHO guidelines. Aside from a decrease in the proportion of motile spermatozoa, no change in semen parameters could be detected with advancing age of the volunteers. No negative influence of the presence of a varicocele, the grade of varicocele or a history of maldescended testis on semen parameters was detected. The serum levels of FSH, LH and testosterone showed no significant age‐related change over the period of investigation. Follow‐up analysis of the fertility of the volunteers revealed a proportion of unwanted barrenness similar to or even lower than that in the general population. The analysis demonstrates that the limits of normality for semen parameters may require redefinition.
Apoptosis as a mechanism of germ cell loss in elderly men
Authors: BRINKWORTH M. H. , WEINBAUER G. F. , BERGMANN M. , NIESCHLAG E.
Published: November 2003

In comparison with other mammals, human spermatogenesis is known to be an inefficient process, of which germ cell degeneration is a normal part. This study was performed to determine the mechanism of cell death in the testes of elderly men. Testes from 20 patients undergoing orchidectomy for prostate cancer were fixed, sectioned and processed for the detection of apoptosis using the in situ end‐labelling technique. In addition, the formation of DNA ladders, a hallmark of apoptosis, was also investigated. Occurrence of apoptosis was not confined to a particular germ cell population but comprised all types of germ cells. Sertoli cell apoptosis was not encountered. The numbers of degenerating germ cells were determined per standard reference area, but no significant relationship was found between the mean values and age or testis weight. Analysis of the median values for germ cell death per reference area suggested that apoptosis occurs in clusters within the testis but is a rare occurrence outside these areas. It is concluded that spontaneous apoptosis can mediate germ cell death in a variety of cell types in the aged human testis.
Stage‐specific degeneration of germ cells in the seminiferous tubules of non‐obese diabetic mice
Authors: SAINIO‐PÖLLÄNEN S. , HENRIKSÉN K. , PARVINEN M. , SIMELL O. , PÖLLÄNEN P.
Published: November 2003

The cause of fertility problems in insulin‐dependent diabetes is largely unknown. To evaluate the role of autoimmunity‐associated phenomena in the testis as a possible cause of the derangement in spermatogenesis, the stage‐specific apoptosis of germ cells in the insulitis phase of pre‐diabetes was quantified in the testes of non‐obese diabetic (NOD) mice. The seminiferous epithelium of normal BALB/c and NOD mice contained cells positive for in‐situ end‐labelling (ISEL) of DNA. ISEL‐positive germ cells formed clusters in the seminiferous epithelium of the NOD mice in marked contrast to the seminiferous epithelium of the BALB/c mice, which contained only individual cells positive for ISEL. ISEL‐positive cells were present in the basal and luminal compartments of the epithelium. Ultrastructural analysis and demonstration of externalized phosphatidyl serine confirmed that the cells were undergoing apoptosis. The ultrastructurally apoptotic cells included spermatogonia, spermatocytes and spermatids. In cytological squash preparations of segments of seminiferous tubules from NOD mice aged 17–20 weeks, the number of ISEL‐positive cells/mm tubule was significantly lower in segments at stages I–II of the seminiferous epithelial wave but higher at stages III–IV in comparison to BALB/c mice. The numbers of ISEL‐positive cells/mm tubule in the other stages were similar in the two strains of mice. Analysis of 32P‐3′ ‐end labelled DNA from the testes showed that the BALB/c mice had relatively more DNA fragmentation than did the NOD mice. These data suggest that autoimmune insulitis in the NOD mice is associated with increased amounts and abnormal stage distribution of apoptosis in the seminiferous epithelium, resulting in derangement of spermatogenesis.