Long non-coding RNA TTN-AS1 promotes the metastasis in breast cancer by epigenetically activating DGCR8.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Long non-coding RNA TTN-AS1 promotes the metastasis in breast cancer by epigenetically activating DGCR8, by P. Qiu, Y. Dou, L.-Z. Ma, X.-X. Tang, X.-L. Liu, J.-W. Chen, published in Eur Rev Med Pharmacol Sci 2019; 23 (24): 10835-10841-DOI: 10.26355/eurrev_201912_19787-PMID: 31858552" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/19787.

Long non-coding RNA TTN-AS1 promotes the metastasis in breast cancer by epigenetically activating DGCR8.

OBJECTIVE: Breast cancer (BC) is one of the most common fatal cancers. Recent studies have identified the vital roles of long non-coding RNAs (lncRNAs) in the development and progression of BC. This research aimed to investigate the underlying mechanisms of lncRNA TTN-AS1 in the metastasis of BC. PATIENTS AND METHODS: TTN-AS1 expression of tissues was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) in 50 BC patients. Wound healing assay and transwell assay were used to observe the phenotypic alteration of BC cells after knockdown or overexpression of TTN-AS1. Moreover, RT-qPCR and Western blot assay were performed to discover the potential targets of TTN-AS1 in BC. RESULTS: TTN-AS1 expression in BC samples was significantly higher than that of the adjacent tissues. Besides, the migration and invasion of BC cells were markedly inhibited after TTN-AS1 was silenced, while promoted after TTN-AS1 overexpression. In addition, a remarkable decrease of DGCR8 was observed after TTN-AS1 was inhibited in BC cells, while DGCR8 was upregulated after overexpression of TTN-AS1. Furthermore, DGCR8 expression showed significant enhancement in BC tissues and was positively associated with TTN-AS1 level. CONCLUSIONS: Our study uncovered a new oncogene in BC and suggested that TTN-AS1 could enhance BC cell migration and invasion via sponging DGCR8, which provided a novel therapeutic target for the treatment of breast cancer.

Two-beam combined 3.36 J, 100 Hz diode-pumped high beam quality Nd:YAG laser system.

In this paper, we develop a diode-pumped all-solid-state high-energy and high beam quality Nd:YAG laser system. A master oscillator power amplifier structure is used to provide a high pulse energy laser output with a high repetition rate. In order to decrease the amplifier working current so as to reduce the impact of the thermal effect on the beam quality, a beam splitting-amplifying-combining scheme is adopted. The energy extraction efficiency of the laser system is 50.68%. We achieve 3.36 J pulse energy at a 100 Hz repetition rate with a pulse duration of 7.1 ns, a far-field beam spot 1.71 times the diffraction limit, and 1.07% energy stability (RMS).

The status of occupational blood and infectious body fluids exposures in five blood centres in China: a 5-year review.

BACKGROUND: Little information about the occupational exposures to blood and body fluid (BBF) among blood service workers (BSWs) in blood stations in China is available currently. OBJECTIVES: To assess current status of occupational exposure to BBF and assess the knowledge about occupational blood-borne pathogen exposures and universal precaution among BSWs in blood donations in China. To understand the incidence of occupational exposure in five blood centres in China. METHODS: A cross-sectional study was conducted from January 2008 to December 2013. RESULTS: There were a total of 99 BBF exposures reported during the study period. The total incidence of BBF exposures was 4.4 per 100 person-years. Higher rates were observed for persons employed less than five years and persons less than 45 years old. Nurses have the highest percentage (49.5%) of BBF exposures. BBF exposures occurred most commonly during the afternoon (62.7%). Percutaneous injuries were the most common BBF exposures. Most incidents occurred during sharps use (73.4%). The major cause of occupational exposure was that there was no continuous training (48.4%) and improper use of equipment (23.2%). Only 56.6% of BBF exposures had appropriate first aid measures. During this research work, one staff member was reported to have seroconverted to syphilis after BBF exposure. CONCLUSIONS: To reduce BBF exposures, it is urgent to take several effective actions in China, including improved occupational health systems, adequate education, administrative support, increased use of standard precautions, better safety devices/products and work practices.

Augmented expression of MYC and/or MYCN protein defines highly aggressive MYC-driven neuroblastoma: a Children's Oncology Group study.

BACKGROUND: MYCN amplification with subsequent MYCN protein overexpression is a powerful indicator of poor prognosis of neuroblastoma patients. Little is known regarding the prognostic significance of the homologous MYC protein expression in neuroblastoma. METHODS: Immunostaining for MYCN and MYC protein was performed on 357 undifferentiated/poorly differentiated neuroblastomas. Results were analysed with other prognostic markers. RESULTS: Sixty-seven (19%) tumours were MYCN(+), 38 (11%) were MYC(+), and one(0.3%) had both proteins(+). MYCN(+) tumours and MYC(+) tumours were more likely diagnosed in children>18months with stage4-disease. MYCN(+) tumours were associated with amplified MYCN, Unfavourable Histology (UH), and High-MKI (Mitosis-Karyorrhexis Index). MYC(+) tumours were also frequently UH but not associated with MYCN amplification, and more likely to have low-/intermediate-MKI. Favourable Histology patients without MYC/MYCN expressions exhibited the best survival (N=167, 89.7±5.5% 3-year EFS, 97.0±3.2% 3-year OS), followed by UH patients without MYC/MYCN expressions (N=84, 63.1±13.6% 3-year EFS, 83.5±9.4% 3-year OS). MYCN(+)patients and MYC(+)patients had similar and significantly low (P<0.0001) survivals (46.2±12.0% 3-year EFS, 63.2±12.1% 3-year OS and 43.4±23.1% 3-year EFS, 63.5±19.2% 3-year OS, respectively). Notably, the prognostic impact imparted by MYC expression was independent from other markers. CONCLUSIONS: In this series, ∼30% of neuroblastomas had augmented MYCN or MYC expression with dismal survivals. Prospective study of MYC/MYCN protein expression signature as a new biomarker for high-risk neuroblastomas should be conducted.

Gene expression during induced differentiation of sheep bone marrow mesenchymal stem cells into osteoblasts.

The aim of this study was to observe the dynamic expression of osteopontin, type I collagen, and osteocalcin genes during the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts and confirm whether BMSCs can differentiate and mature into osteoblasts. A healthy, 2-month-old Altay tailed sheep was obtained, and 10 mL bone marrow was extracted from the posterior superior iliac spine of the sheep via puncture. Sheep BMSCs were extracted using a whole bone marrow culture method and cultured for osteogenic induction. Total RNA from the BMSCs was extracted, and the gene expression levels of osteopontin, type I collagen, and osteocalcin at the 1st, 2nd, 3rd, and 4th passages of uninduced and induced BMSCs were detected using reverse transcription-polymerase chain reaction. The surface antigen CD44 was also detected in the BMSCs. After induction by culturing in osteoinductive medium, BMSCs in the 1st, 2nd, 3rd, and 4th passages displayed stage-specific expression of osteopontin, type I collagen, and osteocalcin genes. The positive expression rate of BMSC-specific antigen CD44 in the 4th passage of osteogenic-induced BMSCs was 99.8%. In vitro-cultured sheep BMSCs in osteogenic culture medium gradually differentiated into osteoblasts, which stage-specifically expressed osteoblast-specific genes such as osteopontin, type I collagen, and osteocalcin at the 4th passage after induction. At this passage, these cells had osteoblast functions.

A potent anti-oxidant property: fluorescent recombinant alpha-phycocyanin of Spirulina.

AIMS: To express and product a fluorescent antioxidant holo-alpha-phycocyanin (PC) of Spirulina platensis (Sp) with His-tag (rHHPC; recombinant holo-alpha-phycocyaninof Spirulina platensis with His-tag) in 5-l bench scale. METHODS AND RESULTS: A vector harbouring two cassettes was constructed: cpcA along with cpcE-cpcF in one cassette; ho1-pcyA in the other cassette. Lyases CpcE/F of Synechocystis sp. PCC6803 (S6) could catalyse the 82 site Cys in apo-alpha-PC of Sp linking with bilin chromophores, and rHHPC was biosynthesized in Escherichia coli BL21. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0.55 g l(-1) broth in 5-litre bench scale. rHHPC was purified by Ni(2+) affinity column conveniently. The absorbance and the fluorescence emission spectra of rHHPC had lambda(max) at 621 and 650 nm, respectively. The IC(50) values of rHHPC were 277.5 +/- 25.8 microg ml(-1) against hydroxyl radicals and 20.8 +/- 2.2 microg ml(-1) against peroxyl radicals. CONCLUSIONS: Combinational biosynthesis of rHHPC was feasible, and the constant feeding mode was adopted to produce good yields of rHHPC. Fluorescent rHHPC with several unique qualitative and quantitative features was effective on scavenging hydroxyl and peroxyl radicals. SIGNIFICANCE AND IMPACT OF THE STUDY: A potent antioxidant rHHPC was co-expressed, produced and characterized for nutritional and pharmacological values, which would help to develop phycobiliproteins' applications in their fluorescent and biological activities.

Association among EPHB2, TrkA, and MYCN expression in low-stage neuroblastomas.

BACKGROUND: The EPH family is the largest subfamily of receptor protein-tyrosine kinases, consisting of EPHA and EPHB subgroups. Ligands of EPH family receptors are called ephrins, which include ephrin-A and ephrin-B subgroups. We recently found that transcripts encoding the EPHB subgroup (EPHB) and the ephrin-B subgroup (EFNB) were expressed together in neuroblastoma (NB) cell lines. PROCEDURE: In this study, we examined the expression of EPHB and EFNB transcripts in 24 NB specimens representing all clinical stages. We found that several EPHB and EFNB transcripts were expressed together in all NBs examined. RESULTS: Among the transcripts examined, EPHB6 expression was most significantly associated with low stage tumors (stages 1, 2, and 4S; P = 0.0048). TrkA expression was significantly correlated with EPHB6, EFNB2, and EFNB3 expression (P < 0.01 in each case). Taken together, these data indicate that the expression of EPHB6, EFNB2, and EFNB3 may serve as prognostic indicators of favorable NBs. In the low-stage NBs without MYCN amplification, EPHB2 expression was correlated both with MYCN expression and with TrkA expression (P < 0.01 in each case). Moreover, MYCN expression was correlated with TrkA expression (P < 0.01) in the low-stage NBs. CONCLUSIONS: This observation points to the possibility that MYCN expression might contribute to favorable outcome of low-stage NBs.

Prognostic significance of EPHB6, EFNB2, and EFNB3 expressions in neuroblastoma.

BACKGROUND: EPH family receptor tyrosine kinases and their ligand ephrins play pivotal roles in development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6), its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is predictive of favorable disease outcome of neuroblastoma (NB). When combined with TrkA expression, the expression of EPHB6, EFNB2, or EFNB3 predicts more accurately the disease outcome than each of the four variables alone. PROCEDURE: Cox regression and Kaplan-Meier analyses were used to assess the prognostic significance of EPHB6, EFNB2, EFNB3, and TrkA expressions in NB without MYCN amplification. RESULTS: High-level expression of EFNB3 or TrkA predicted favorable NB outcome of NB without MYCN amplification (p < 0.03). As found in the general NB population, EPHB6, EFNB2, or EFNB3 expression in combination with TrkA expression was significantly predictive of the disease outcome of normal MYCN NB (p < 0.01). CONCLUSIONS: EPHB6, EFNB2, and EFNB3 expressions may permit further refinement of the prognostic stratification of NB into favorable and unfavorable groups.

Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma.

Neuroblastoma (NB) is a common pediatric tumor that exhibits a wide range of biological and clinical heterogeneity. EPH (erythropoietin-producing hepatoma amplified sequence) family receptor tyrosine kinases and ligand ephrins play pivotal roles in neural and cardiovascular development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6) and its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is associated with low-stage NB (stages 1, 2, and 4S) and high TrkA expression. In this study, we showed that EFNB2 and TrkA expressions were associated with both tumor stage and age, whereas EPHB6 and EFNB3 expressions were solely associated with tumor stage, suggesting that these genes were expressed in distinct subsets of NB. Kaplan-Meier and Cox regression analyses revealed that high-level expression of EPHB6, EFNB2, and EFNB3 predicted favorable NB outcome (P<0.005), and their expression combined with TrkA expression predicted the disease outcome more accurately than each variable alone (P<0.00005). Interestingly, if any one of the four genes (EPHB6, EFNB2, EFNB3, or TrkA) was expressed at high levels in NB, the patient survival was excellent (>90%). To address whether a good disease outcome of NB was a consequence of high-level expression of a "favorable NB gene," we examined the effect of EPHB6 on NB cell lines. Transfection of EPHB6 cDNA into IMR5 and SY5Y expressing little endogenous EPHB6 resulted in inhibition of their clonogenicity in culture. Furthermore, transfection of EPHB6 suppressed the tumorigenicity of SY5Y in a mouse xenograft model, demonstrating that high-level expressions of favorable NB genes, such as EPHB6, can in fact suppress malignant phenotype of unfavorable NB.

High-level expression of EPHB6, EFNB2, and EFNB3 is associated with low tumor stage and high TrkA expression in human neuroblastomas.

Neuroblastoma (NB) is a common pediatric tumor of neural crest origin that is biologically and clinically heterogeneous. EPH family receptor tyrosine kinases and ephrin ligands play fundamental roles in neurodevelopmental processes. Recently, we found that NB cell lines expressed several EPHB and EFNB transcripts, which encode EPHB subgroup receptors and ephrin-B subgroup ligands, respectively. To explore the role of EPHB receptors and ephrin-B ligands in the biology of NB, we examined the expression of EPHB and EFNB transcripts in 47 primary NB specimens. Multiple EPHB and EFNB transcripts were expressed in all of the NB tumors examined, suggesting the involvement of these transcripts in modulating the biological behavior of NB. Higher levels of EPHB6, EFNB2, and EFNB3 expression were found in low-stage tumors (stage 1, 2, and 4S) than in advanced-stage tumors (stage 3 and 4; P = 0.0013, P = 0.0048, and P = 0.027, respectively). Expression of TrkA, a well-established prognostic marker of favorable NB, was positively correlated with EPHB6, EFNB2, and EFNB3 expression (P < 0.0001, P = 0.0019, and P = 0.0001, respectively). MYCN-amplified tumors expressed lower levels of EPHB6, EFNB2, EFNB3, and TrkA transcripts compared to nonamplified tumors (P = 0.0006, P = 0.0023, P = 0.0048, and P = 0.0001, respectively). These data suggest that high-level expression of EPHB6, EFNB2, and EFNB3 is associated with favorable NB and that low-level expression of EPHB6, EFNB2, and EFNB3 correlates with aggressive MYCN-amplified NB. Thus, EPHB6, EFNB2, and EFNB3 may have biological relevance in NB. Further investigation on the biology of these genes may help provide insight into the treatment of NB.

Coexpression of transcripts encoding EPHB receptor protein tyrosine kinases and their ephrin-B ligands in human small cell lung carcinoma.

The EPH family is the largest subfamily of receptor protein tyrosine kinases, consisting of the EPHA and EPHB subgroups. Ephrin-B1, ephrin-B2, and ephrin-B3 are ligands of the EPHB subgroup and are encoded by the EFNB1, EFNB2, and EFNB3 genes, respectively. We have shown previously that EPHB2 transcripts are expressed in six small cell lung carcinoma (SCLC) cell lines. In this study, we examined the expression of EPHB1, EPHB2, EPHB3, EPHB4, and EPHB6 in 4 SCLC tumor specimens and 14 cell lines including 3 cell lines derived from these tumor specimens. To investigate whether potential autocrine loops of EPHB receptors and ephrin-B ligands exist in SCLC, the expression of EFNB1, EFNB2, and EFNB3 was also examined. Our data show that transcripts encoding multiple members of the EPHB subgroup and the ephrin-B subgroup are coexpressed in SCLC cell lines and tumors. These results suggest that the EPHB subgroup receptor kinases may modulate the biological behavior of SCLC through autocrine and/or juxtacrine activation by ephrin-B ligands that are expressed in the same or neighboring cells.

A variant transcript encoding an isoform of the human protein tyrosine kinase EPHB2 is generated by alternative splicing and alternative use of polyadenylation signals.

We previously isolated and characterized cDNA clones of DRT (EPHB2), encoding a receptor protein-tyrosine kinase of the EPH family. Northern blot analysis showed that EPHB2 transcripts are expressed in three sizes of approximately 4, 5, and 11 kb, suggesting that these transcripts are generated by alternative splicing and/or alternative use of polyadenylation sites. To explore this possibility, we isolated additional EPHB2 cDNA clones, including clone 5K-1, by re-screening the human fetal brain cDNA library. Nucleotide sequence analysis of clone 5K-1 revealed that it represents a variant transcript of EPHB2 (EPHB2v). Relative to the EPHB2 cDNA sequence previously reported, clone 5K-1 has two coding region deletions of 3 and 93 nucleotides. Nucleotide sequence analyses of EPHB2 genomic DNA fragments corresponding to these deletions suggest that the EPHB2v transcript is generated by alternative splicing. The 3' end of clone 5K-1 contains a polyadenosine stretch preceded by a potential polyadenylation signal, which is not used to generate the EPHB2 transcript. Taken together, these data indicate that EPHB2v is generated by both alternative splicing and alternative use of polyadenylation sites. The EPHB2v protein lacks one arginine residue that resides immediately following the EPHB2 transmembrane domain. In contrast, as a result of the frame shift caused by the 93 nucleotide deletion, the C-terminus of the EPHB2v protein is longer by 70 amino acids than that of EPHB2. We also show that the human neuroblastoma cell line SY5Y and NTera-2N neurons express high levels of EPHB2 and lower levels of EPHB2v. These structural variations found between the EPHB2 and EPHB2v proteins may reflect functional heterogeneity of EPHB2.

cDNA cloning, chromosomal localization, and expression pattern of EPLG8, a new member of the EPLG gene family encoding ligands of EPH-related protein-tyrosine kinase receptors.

By screening a human fetal brain cDNA library under low stringency using cDNA encoding the mouse ligand of Cek5 as a probe, we have isolated a novel cDNA belonging to the EPLG gene family. This family encodes ligands of EPH-related tyrosine kinase receptors. Since the novel gene is the eighth member of the EPLG gene family, it is designated EPLG8. The deduced amino acid sequence of EPLG8 suggests that it encodes a transmembrane protein that is most related to those encoded by EPLG2 and EPLG5. We mapped the EPLG8 gene to human chromosome 17p11.2-p13.1 by PCR screening of human-rodent somatic cell hybrid panels. In the midterm fetus, EPLG8 mRNA is expressed at the highest level in brain, followed by heart, kidney, and lung. In the adult, EPLG8 mRNA expression is restricted to brain. These data suggest that LERK-8, the protein encoded by EPLG8, is important in brain development as well as in its maintenance. Moreover, since levels of EPLG8 expression were particularly high in several forebrain subregions compared to other brain subregions, LERK-8 may play a pivotal role in forebrain function.

ECK, a human EPH-related gene, maps to 1p36.1, a common region of alteration in human cancers.

Mouse eck, a member of the EPH gene family, has been mapped to mouse chromosome 4. The syntenic relationship between this chromosome and human chromosome 1 suggests that the human ECK gene maps to the distal short arm of human chromosome 1 (1p). Since this region is frequently deleted or altered in certain tumors of neuroectodermal origin, it is important to define the specific chromosomal localization of the human ECK gene. PCR screening of a rodent-human somatic cell hybrid panel by ECK-specific primers showed that ECK is indeed localized to human chromosome 1. Additional PCR screening of a regional screening panel for chromosome 1p indicated that ECK is localized to 1p36, distal to FUCA1. Furthermore, fluorescence in situ hybridization analysis with an ECK-specific P1 clone showed that ECK maps proximal to genetic marker D1S228. Taken together, the data suggest that ECK maps to 1p36.1, a region that is frequently deleted in neuroblastoma, melanoma, and other neuroectodermal tumors.

Identification of epitope recognized by an anti-c-myc monoclonal antibody that cross-reacts with E. coli sigma factor using phage display libraries.

BACKGROUND: During the epitope mapping of monoclonal antibodies specific for myc proteins, two E. coli proteins cross-reactive with an anti-c-myc monoclonal antibody (MYC-X-5/1) were identified. One of the proteins is approximately 90 kDa and the other is over 150 kDa in apparent molecular mass. The molecular masses of these cross-reactive proteins suggested that they may be subunits of E. coli RNA polymerase. OBJECTIVES: We have investigated whether or not the proteins cross-reactive with MYC-X-5/1 are subunits of E. coli RNA polymerase. In addition, we have attempted to determine the epitope of MYC-X-5/1. STUDY DESIGN: The reactivity of MYC-X-5/1 antibody was tested against highly purified E. coli RNA polymerase holo-enzyme preparations and the cell lysate made from E. coli carrying a multi-copy plasmid with an insert of the rpoD gene, the structural gene for the E. coli sigma subunit. The epitope of MYC-X-5/1 was determined by use of phage display of random peptide libraries. RESULTS: On immunoblotting assays, MYC-X-5/1 reacted with the 90-kDa protein in the E. coli RNA polymerase preparations and with the 90-kDa protein over-expressed in E. coli carrying the plasmid with the rpoD insert. In addition, we have deduced the epitope of the MYC-X-5/1 antibody to be residues 235-245 of the human c-myc protein. A highly similar sequence to this was also identified in residues 62-72 of the sigma subunit of E. coli RNA polymerase. CONCLUSION: These data demonstrated that the 90-kDa protein cross-reactive with MYC-X-5/1 is the sigma subunit of E. coli RNA polymerase. Furthermore, this study shows that random peptide libraries displayed on filamentous phage are useful tools for epitope mapping and defining cross-reactivities of monoclonal antibodies.

Molecular characterization and chromosomal localization of DRT (EPHT3): a developmentally regulated human protein-tyrosine kinase gene of the EPH family.

By screening a human fetal brain cDNA expression library using a monoclonal antiphosphotyrosine antibody and by 5' RACE procedures, we have isolated overlapping cDNAs encoding a receptor-type tyrosine kinase belonging to the EPH family, DRT (Developmentally Regulated EPH-related Tyrosine kinase gene). The DRT gene is expressed in three different size transcripts (i.e. 4, 5 and 11 kb). DRT transcripts are expressed in human brain and several other tissues, including heart, lung, kidney, placenta, pancreas, liver and skeletal muscle, but the 11 kb DRT transcript is preferentially expressed in fetal brain. Steady-state levels of DRT mRNA in several tissues, including brain, heart, lung and kidney, are greater in the midterm fetus than those in the adult. DRT transcripts are detectable at low levels in a human teratocarcinoma cell line (NTera-2), but its expression is greatly increased after the NTera-2 cells are induced to become postmitotic neurons (NTera-2N) by retinoic acid treatment. These data suggest that DRT plays a part in human neurogenesis. A large number of tumor cell lines derived from neuroectoderm express DRT transcripts, including 12 neuroblastomas, two medulloblastomas, one primitive neuroectodermal tumor and six small cell lung carcinomas (SCLC). Interestingly, several neuroblastoma cell lines with 1p deletion and one SCLC cell line express DRT transcripts of aberrant size (i.e. 3, 6 and 8 kb) in addition to those found in normal tissues. We mapped the DRT gene to human chromosome 1p35-1p36.1 by PCR screening of human-rodent somatic cell hybrid panels and by fluorescence in situ hybridization. As the distal end of chromosome 1p is often deleted in neuroblastomas and altered in some cases in SCLCs, these chromosomal abnormalities may have resulted in the generation of aberrant size transcripts. Thus, the DRT gene may play a part in neuroblastoma and SCLC tumorigenesis.

cDNA cloning, molecular characterization, and chromosomal localization of NET(EPHT2), a human EPH-related receptor protein-tyrosine kinase gene preferentially expressed in brain.

By screening a human fetal brain cDNA expression library using a monoclonal anti-phosphotyrosine antibody, we have isolated a cDNA clone encoding a receptor type protein-tyrosine kinase belonging to the EPH family, NET (neuronally expressed EPH-related tyrosine kinase). NET shows 87% homology in nucleotide sequence and 99% homology in the deduced amino acid sequence to rat elk, suggesting that NET is the human homologue of elk. The NET gene is mapped to human chromosome 3q21-q23 by PCR screening of a human-rodent somatic cell hybrid panel and by fluorescence in situ hybridization. Examination of NET mRNA expression in several human tissues has shown that the NET gene is expressed preferentially in brain as a 5-kb transcript. Steady-state levels of NET mRNA in human brain are greater in the midterm fetus than in the adult. Lower levels of NET mRNA are found in fetal kidney and adult skeletal muscle. The expression pattern of NET mRNA thus differs from that of elk, suggesting that these two gene products may perform distinct roles in human and rat. NET transcripts are detected in human NTera-2 teratocarcinoma cells after retinoic acid-induced neuronal differentiation. Several human tumor cell lines derived from neuroectoderm including primitive neuroectodermal tumor, small cell lung carcinoma, and neuroblastoma also express NET transcripts. Since the NET mRNA expression in human brain is developmentally regulated and is induced during neuronal differentiation, NET potentially plays important roles in human neurogenesis.

An alternative view of T-cell receptor-MHC interaction: T-cell receptor binds transversally to the alpha-helices of the MHC molecule.

We have attempted to elucidate the relative orientation of the T-cell receptor (TCR) to the major histocompatibility complex (MHC)-antigen complex during antigen recognition, using the T-cell response to B10.A (I-Ek) and B10.A(5R) (I-Eb) mice to the 1-23(H) peptide derived from glycoprotein D of the herpes simplex virus. The 1-23(H)-specific T-cells derived from both B10.A and B10.A(5R) mice use the same set of V alpha genes and a different array of V beta genes. The CDR1s of these TCR beta chains share residues at particular positions. The CDR2s of the TCR beta chains have a negative charge, which correlates with I-Eb reactivity and with the positively charged polymorphic residues residing at the C-terminal end of the alpha-helix of the I-Eb beta chain of the class II molecule. Taken together, the data suggest that the TCR beta chain interacts with both the alpha and beta chains of the MHC class II molecule, as does the TCR alpha chain.

T cells from unprimed mice respond to the self antigen heme, in a class II restricted manner, at a frequency similar to alloresponses.

Heme is a non-protein autoantigen which stimulates potent proliferative responses by T cells from unprimed mice of some strains. These studies show that T cells responding to heme in primary responses are predominantly CD4+, classically I-A restricted, and use diverse TCR characterized by the expression of distinct V, D and J gene segments. These characteristics distinguish heme from superantigens and mitogens which exhibit degenerate MHC restriction and, in the case of superantigens, restricted V gene usage. Using limiting dilution analysis these studies also show that the potent primary response of H-2s mice reflects a high frequency (0.26-0.45%) of heme responsive T cells in the periphery, comparable to the frequency of alloresponsive T cells reported by others in primary mixed lymphocyte reactions. In contrast, heme responsive T cells occur at -10-fold lower frequency in unprimed H-2d mice (0.03%). To determine the antigen recognized by heme reactive T cells, the mass spectra of peptides eluted from the high responder haplotype, I-A(s), were examined. These indicated a markedly different molecular weight distribution of peptides isolated from cells grown in the presence of heme, compared with those from cells grown in its absence. This suggests that heme mediates the expansion of diverse T cells in the peripheral repertoire by a mechanism similar to that for allogeneic responses in which the profile of naturally processed peptides bound to the MHC class II molecule is changed.