Amsp3 may act upstream of Amdnmt3 in female caste differentiation in the honeybee (Apis mellifera).

In honey bees, the process of producing two female castes, including queens and workers, is nutritionally controlled by differential feeding royal jelly to newly emerged larvae. Although they have almost identical genetic blueprints, these castes show striking differences in their morphologies, longevities and reproductive capabilities. DNA methyltransferase 3 (Amdnmt3) gene is involved in the regulatory network for honeybee caste differentiation. Due to the role of two zinc fingers containing transcription factors, SP1 and SP3 in controlling mammalian Dnmts, this study aimed to determine a similar interaction of SPs with Amdnmt3 in the honeybee. We confirmed that the promoter region of Amdnmt3 contained multiple predicted SP1/SP3 binding sites and then investigated the role of AmSP3 in queen-worker differentiation network. We observed that the expression level of Amsp3 was significantly higher in worker larvae than that in queen larvae at 48 h, 84 h and 120 h. Knockdown of Amsp3 expression by RNAi in worker larvae significantly reduced the expression level of Amdnmt3 and caused morphological changes in adult bees towards a queen-like phenotype. However, the expression levels of Amsp3 and Amdnmt3 were repressed by juvenile hormone (JH). Our results suggest that AmSP3 is an important part of the queen-worker differentiation network and supports the role of Amdnmt3 in determining the phenotypic outcome of developing larvae.

Identification of genes associated with litter size combining genomic approaches in Luzhong mutton sheep.

Litter size is one of the most important reproductive traits of sheep, which has pronounced effects on the profit of husbandry enterprises and enthusiasm of breeders. Despite the importance of litter size, the underlying genetic mechanisms have not been entirely elucidated. Therefore, based on a high-density SNP chip, genome-wide comparative analysis was performed between two groups with different fecundity to reveal candidate genes linked to litter size via detection of homozygosity and selection signatures in Luzhong mutton sheep. Consequently, nine promising genes were identified from six runs of homozygosity islands, and functionally linked to reproduction (ACTL7A, ACTL7B, and ELP1), embryonic development (KLF5 and PIBF1), and cell cycle (DACH1, BORA, DIS3, and MZT1). A total of 128 genes were observed under selection, of which HECW1 and HTR1E were related to total lambs born, GABRG3, LRP1B, and MACROD2 to teat number, and AGBL1 to reproductive seasonality. Additionally, the presence of inbreeding depression implies the urgency of reasonable mating system to increase litter size in the present herd. These findings provide a comprehensive insight to the genetic makeup of litter size, and also contribute to implementation of marker-assisted selection in sheep.

Genome-wide association study of body weight and conformation traits in neonatal sheep.

Sheep, an important source of meat, dairy products and wool, play an essential part in the global agricultural economy. Body weight and body conformation are key traits in the sheep industry; however, their underlying genetic mechanisms are poorly understood. In this study, a GWAS was implemented to identify promising genes possibly linked to birth weight (BW) and body conformation traits in neonatal sheep, using a high-throughput chip (630 K). After quality control, 277 individuals and 518 203 variants were analyzed using gemma software in a mixed linear model. A total of 48 genome-wide suggestive SNPs were obtained, of which four were associated with BW, four with withers height (WH), 11 with body length (BL) and 29 with chest girth (CG). In total, 39 genes associated with BW and body conformation traits were identified by aligning to the sheep genome (Ovis aries_v4.0), and most of them were involved in the cell cycle and body development. Promising candidate genes found included the following: FOS like 2 or AP-1 transcription factor subunit (FOSL2) for BW; potassium voltage-gated channel subfamily D member 2 (KCND2) for WH; transmembrane protein 117 (TMEM117), transforming growth factor beta induced (TGFBI), and leukocyte cell-derived chemotaxin 2 (LECT2) for BL; and trafficking kinesin protein 1 (TRAK1) and LOC101102529 for CG. These results provide cues for similar studies aiming at uncovering the genetic mechanisms underlying body development, and marker-assisted selection programs focusing on BW and body conformation traits in sheep.

LncRNA HAGLR accelerates femoral neck fracture healing through negatively regulating miRNA-19a-3p.

OBJECTIVE: This study aims to uncover the function of long non-coding RNA (lncRNA) HAGLR in the healing process of femoral neck fracture and the underlying mechanism. PATIENTS AND METHODS: Expression levels of HAGLR, microRNA-19a-3p (miRNA-19a-3p) and TGFBR2 in fractured femoral neck tissues and adjacent normal tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Regulatory effects of HAGLR on viability, apoptosis, migration, and protein levels of BALP and Osteocalcin in MC3T3-E1 cells were determined. Dual-Luciferase reporter gene assay was conducted to assess the binding in HAGLR/miRNA-19a-3p/TGFBR2. In addition, relative levels of TGFBR2, p-smad2, p-smad3, and RUNX2 in MSCs influenced by HAGLR were detected. RESULTS: HAGLR was downregulated in fractured femoral neck tissues. Knockdown of HAGLR reduced viability and migration, enhanced apoptotic rate, as well as downregulated BALP and Osteocalcin in MC3T3-E1 cells. HAGLR served as miRNA-19a-3p sponge, and miRNA-19a-3p directly targeted 3'-untranslated region (3'-UTR) of TGFBR2. Knockdown of HAGLR downregulated expressions of TGFBR2, p-smad2, p-smad3, and RUNX2 in MC3T3-E1 cells, indicating the inhibited TGF-ß pathway. CONCLUSIONS: LncRNA HAGLR/miRNA-19a-3p/TGFBR2 regulatory loop accelerates the healing process of femoral neck fracture by inhibiting the TGF-ß pathway.

Antiproliferative function of p27kip1 is frequently inhibited in highly malignant Burkitt's lymphoma cells.

Lack of detectable expression of p27kip1 cyclin dependent kinase inhibitor has previously been correlated with high degree of malignancy in human breast, colorectal, gastric and small cell lung carcinomas. Here we demonstrate that an inverse correlation between p27kip1 expression and tumour malignancy also exists in most types of human B cell lymphomas examined. A clear exception was Burkitt's lymphoma (BL), a highly malignant tumour which often expresses high levels of p27kip1. Analysis of p27kip1 derived from Burkitt's lymphoma cell lines expressing high levels of p27kip1, BL40 and BL41, in a cyclin E/cdk2 kinase inhibition assay demonstrated that p27kip1 is not permanently inactivated since heat treatment can restore the inhibitory activity of p27kip1. However, p27kip1 expressed in these two cell lines is largely sequestered in inactive complexes and we have no evidence that c-myc or Epstein-Barr virus are responsible for the sequestration of p27kip1 in these two cell lines although c-myc and EBV are two oncogenic agents often associated with Burkitt's lymphomas. Interestingly, we observed that high level p27kip1 expression often correlated with cyclin D3 overexpression both in vivo and in BL cell lines. The majority of p27kip1 in BL40 cells was complexed with cyclin D3 indicating that overexpressed cyclin D3 may at least be part of the sequestering activity for the inhibitory function of p27kip1. Furthermore, cyclinD3/cdk4 complex could sequester p27kip1 in a cyclin E/cdk2 kinase assay in vitro. Finally, we show that cyclin D3 transfected into an inducible p27kip1 cell line could overcome the G1 arrest mediated by p27kip1. These results argue that in addition to down-regulation of p27kip1 expression, some tumour cells can sequester and tolerate the antiproliferative function of p27kip1. They also suggest a novel role for the overexpression of D-type cyclins as one pathway allowing tumour cells to overcome the antiproliferative function of p27kip1.

Follicular colonization in B-cell lymphoma of mucosa-associated lymphoid tissue.

The formation of neoplastic B-cell follicles is universally accepted as diagnostic of a follicle centre cell (FCC) lymphoma. Low-grade B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) are characterized by a diffuse infiltrate of cells of uncertain lineage known as "centrocyte-like" cells because of their resemblance to centrocytes (small cleaved cells). Some MALT lymphomas, however, contain numerous follicles and may even have a predominantly follicular appearance. These follicles may be reactive or show immunoglobulin (Ig) light-chain restriction, indicating their neoplastic nature. We have proposed that these neoplastic follicles are not composed of follicle centre cells but result from colonization of reactive follicles by CCL cells. In this study, the immunophenotype and genotype of 10 primary gastrointestinal lymphomas with a follicular component have been determined. One case exhibited the morphological, immunophenotypic, and genotypic features of FCC lymphoma (Ig light-chain restriction, CD10+, KB61 (CDw32)-, Jh, and bcl-2 gene rearrangement). Neoplastic follicles in the remaining nine cases, which showed the features of MALT lymphoma, were of a different phenotype (Ig light-chain restriction, CD10- KB61(CDw32)+), and these lymphomas showed Jh but not bcl-2 gene rearrangement. Taken in conjunction with the morphological features, these findings suggest that in these cases the neoplastic follicles formed as the result of colonization of previously reactive follicles by neoplastic CCL cells. Thus, not all lymphomas containing neoplastic follicles are of FCC origin. Follicular colonization, as seen in low-grade MALT lymphomas, is likely to be a recapitulation of an as yet undescribed normal immunological phenomenon that may involve marginal zone B cells.

Prognostic markers in clinically localised prostate cancer.

The prognostic value of immunohistochemical staining of P53, BCL-2, p27kip1, PSA, AR and MIB-1 was compared with that of established prognostic variables (Gleason score, surgical margins, tumour volume) following radical prostatectomy. Five groups were selected: negative margins with stable serum PSA (n=11), negative margins with rising serum PSA (n=7), positive margins with stable serum PSA (n=7), positive margins with rising serum PSA (6) and patients with micrometastatic disease diagnosed in lymph nodes removed during radical prostatectomy (n=8). Gleason score and tumour volume were of prognostic significance and immunohistochemical staining for MIB-1 and BCL-2 showed added independent prognostic significance in multivariate analysis.

HLA antigen expression in enteropathy associated T cell lymphoma.

AIMS: To investigate the occurrence of abnormal patterns of HLA-ABC and HLA-DR expression in enteropathy associated T cell lymphoma and to relate such abnormalities to the Epstein Barr virus (EBV) status of the tumours. METHODS: Eleven enteropathy associated T cell lymphomas were immunostained with HC10 (HLA-ABC heavy chain) and TAL 1B5 (HLA-DR alpha chain) monoclonal antibodies and polyclonal anti-beta 2 microglobulin (beta 2m, the HLA-ABC light chain) antibodies. In situ hybridisation for EBV using EBER probes was performed on all cases. RESULTS: Tumour cells of two of 11 patients were EBER positive. One of these showed partial, and the other, complete loss of beta 2m. HLA-DR expression was undetectable in both patients. Of the remaining nine EBER negative tumours, two were HLA-ABC heavy chain negative or showed only occasional positive cells and five of nine showed partial or complete loss of the HLA-ABC light chain, beta 2m. Seven of the nine cases were either negative for HLA-DR or showed weak expression in a proportion of tumour cells. CONCLUSIONS: These data show that low or absent HLA-ABC and HLA-DR antigen expression occurs commonly in enteropathy associated T cell lymphoma. These abnormal patterns of HLA expression may be associated with escape from immune attack which, in a minority of patients, could be directed against EBV antigens.

Multiple PCR analyses on trace amounts of DNA extracted from fresh and paraffin wax embedded tissues after random hexamer primer PCR amplification.

AIM: To establish a simple and reliable polymerase chain reaction (PCR) methodology for random amplification of whole genomic DNA from limited histopathological samples. METHODS: Trace amounts of genomic DNA extracted from fresh tissue and individual lymphoid follicles microdissected from archival paraffin wax tissue sections were amplified using a two-phase PCR protocol with random hexamers as primers (RP-PCR). The randomly amplified DNA samples were used as templates for specific PCR amplifications. To check the fidelity of the RP-PCR, products of the specific PCR amplifications were further analysed by single stranded conformation polymorphism (SSCP) or sequencing. RESULTS: Using a minute fraction of RP-PCR template pool, multiple PCR analyses, including those for beta globin gene, p53 gene (exon 5-6, exon 7, exon 8-9 and exon 7-9), and rearranged immunoglobulin heavy chain gene fragments (VH framework 3 to JH and VH framework 2 to JH) were successfully performed. No artefactual mutations were identified in the products of these specific PCR reactions by SSCP or sequencing when compared with the products from the original DNA. CONCLUSION: This method is simple and reliable, and permits multiple genetic analyses when only a limited amount of tissue is available.

The polymerase chain reaction in the demonstration of monoclonality in T cell lymphomas.

AIMS: To evaluate polymerase chain reaction (PCR) amplification of T cell receptor (TCR) beta and gamma chain genes as a means of demonstrating monoclonality in T cell lymphomas using histological samples; to compare the performance of PCR with Southern blot analysis. METHODS: TCR-beta, TCR-gamma and immunoglobulin heavy chain (IGH) genes were analysed using PCR in 55 cases of T cell lymphoma (28 frozen tissue and 27 paraffin wax embedded samples), diagnosed using morphological and immunohistochemical criteria. The 28 frozen samples were subjected to Southern blot analysis using TCR-beta, TCR-gamma and IGH gene probes. Twenty five B cell lymphomas and 21 non-neoplastic lymphoid tissue samples were used as controls. RESULTS: Using TCR-beta PCR, monoclonality was detected in 24 (44%) of 55 T cell lymphomas compared with 43 (78%) of 55 using TCR-gamma PCR and in 82% with both techniques. Five (9%) of 55 T cell lymphomas were IGH PCR positive. None of the non-neoplastic lymphoid control samples were PCR positive. All B cell lymphomas showed a polyclonal pattern with TCR-beta PCR while a single B cell lymphoma was positive using TCR-gamma primers. With TCR-beta PCR, a monoclonal result was seen in 12 (43%) of 28 frozen samples of T cell lymphoma, compared with 23 (82%) of 28 using Southern blot analysis. With TCR-gamma PCR, 19 (68%) of 28 frozen tissue samples were positive, compared with 26 (93%) of 28 using Southern blot analysis. A single case showed IGH rearrangement by Southern blot analysis. CONCLUSION: TCR-gamma PCR should be the method of choice for analysis of clonality in paraffin wax embedded sections of lymphoproliferative lesions, as TCR-beta PCR has a high false negative rate. Southern blot analysis remains the most successful technique when sufficient fresh tissue samples and resources are available.

Epstein-Barr virus associated lymphoproliferative disorder with fatal involvement of the gastrointestinal tract in an infant.

A case of fatal Epstein-Barr virus (EBV) associated lymphoproliferative disorder is reported in an 11 month old female. Heavy infiltrates of CD20 + and EBV EBER mRNA expressing lymphoid blasts were found to cause a series of ulcers along the entire length of the gastrointestinal tract and there was an ileal perforation. Similar infiltrates were also found in lymph nodes, spleen, and liver. Although blood phenotypic analysis performed shortly before her death revealed a severe decrease in T lymphocytes, neither the patient nor other members of her family had a history of primary or secondary immunodeficiency. EBV infection is common in children. However, such a fatal infection of the virus has not apparently been described previously in infants without pre-established immunodeficiency.

Cytogenetic study of B-cell lymphoma of mucosa-associated lymphoid tissue.

The recently described B-cell lymphomas arising in mucosa-associated lymphoid tissue (MALT) form a distinct clinico-pathologic group of non-Hodgkin's lymphoma, and therefore would be expected to be characterized by a recurrent chromosomal aberration. We have analyzed the cytogenetics of 23 cases of MALT lymphomas arising in the stomach, small intestine, lung, and lacrimal gland. In each case the presence of an abnormal clonal cell population was confirmed by the identification of rearranged bands when digested tumor DNA was hybridized with a probe to the joining region of the immunoglobulin heavy chain gene. Metaphase spreads were obtained in 14 cases, of which 9 cases showed an abnormal karyotype. Although no unifying aberration was detected, rearrangements of chromosome 1p, and numerical abnormalities of chromosomes 3 and 7, may play a role in the genesis of these tumors.

Cellular H- and M-type lactate dehydrogenase (LDH) isoenzymes and tumour diagnosis--an immunohistochemical assessment.

Cellular H- and M-type lactate dehydrogenase (LDH) isoenzymes were determined immunohistochemically in a wide range of normal, inflammatory, benign neoplastic, and malignant tissues to assess the possible value of the isoenzymes in tumour diagnosis. Both types of LDH were detected almost ubiquitously in normal tissues. Increased staining intensity of LDH, especially M-type LDH, was demonstrated in most types of malignant tumours studied. Similar increased staining intensity of the isoenzymes was also noted to parallel the proliferative activity of the cells in non-neoplastic diseases and benign neoplasms. The results suggest that changes of cellular H- and M-type LDH isoenzymes are not specific for any type of malignant tumours. The increase in cellular H- and M-type LDH isoenzymes appears to be related to proliferation of cells in general.

The polymerase chain reaction in histopathology.

Thanks to the advent of the polymerase chain reaction (PCR) molecular genetic study of histological samples is now a relatively straightforward task and the vast histopathology archives are now open to molecular analysis. In this review we outline technical aspects of PCR analysis of histological material and evaluate its application to the diagnosis and study of genetic, infectious and neoplastic disease. In addition, we describe a number of newly developed methods for the correlation of PCR analysis with histology, which will aid the understanding of the molecular basis of pathological processes.

A genotypic study of low grade B-cell lymphomas, including lymphomas of mucosa associated lymphoid tissue (MALT).

Genotypic analysis has led to the implication of certain oncogenes in the pathogenesis of specific groups of non-Hodgkin's lymphoma. Rearrangements of c-myc are associated with Burkitt's lymphoma and of bcl-2 with centroblastic/centrocytic lymphoma. Rearrangement of bcl-1 has yet to be associated with a specific group of lymphoma. In this study DNA from 62 cases of low grade B-cell lymphoma, classified using the Kiel classification, were analysed by Southern blotting and hybridization with probes to bcl-1, bcl-2, and c-myc. Rearrangements of bcl-2 were found in a proportion of centroblastic/centrocytic lymphoma comparable to other published studies. Rearrangement of c-myc was not found in any case studied. Bcl-1 rearrangement was found in 2/9 cases of B-CLL, and 3/6 cases of centrocytic lymphoma. This incidence of bcl-1 rearrangement in centrocytic lymphoma suggests that it is a characteristic change. No rearrangement of bcl-1, bcl-2 or c-myc was found in any case of lymphoma of mucosa associated lymphoid tissue (MALT), providing further evidence that, in spite of having a similar morphology to some other groups of low grade B-cell lymphoma, lymphomas of MALT comprise a distinct entity.

Clonality analysis of defined B-cell populations in archival tissue sections using microdissection and the polymerase chain reaction.

A simple microdissection technique involving the use of a drawn-out glass pipette was developed for isolation of defined cell subsets from tissue sections. Using this technique and the polymerase chain reaction (PCR), clonally rearranged immunoglobulin (Ig) heavy chain genes were reliably amplified in single neoplastic follicles or few hundreds of tumour cells isolated from archival haematoxylin and eosin or immunostained sections of B-cell lymphomas. A polyclonal nature was consistently demonstrated in reactive lymphoid follicles or interfollicular reactive B-cells within the same lymphoma sections. Microdissection of lymphoma cells from within foci of chronic inflammation improved the resolution of tumour-specific PCR products by reducing amplification of background polyclonal B-cell sequences. The combination of microdissection and PCR techniques, therefore, provides an important tool for the investigation of B-cell lymphomas and also allows simple and specific access for other molecular genetic analyses of different cell subsets on tissue sections.

The effect of dietary zinc and prothymosin alpha on cellular immune responses of RF/J mice.

Mice of the RF/J strain on a normal diet are defective in some aspects of cellular immunity, as evidenced by their susceptibility to infection with Candida albicans, their failure to release detectable quantities of circulating migration inhibitory factor (MIF) in vivo, and the presence of a low rate of phagocytosis and killing by peritoneal macrophages. When the mice were fed a high-zinc diet (300 ppm) for 4 weeks and then treated daily with 160 ng prothymosin alpha, an increase occurred in resistance to infection with C. albicans, in the capacity to release MIF in vivo into the circulation and in the capacity of peritoneal macrophages to engulf (phagocytose) and kill cells of C. krusei. In addition, the number of spleen lymphocytes producing antibody to a T-dependent antigen was significantly increased in the mice fed a high-zinc diet and inoculated daily with prothymosin alpha.

Adaptational response in transcription factors during development of myocardial hypertrophy.

Cardiac hypertrophy is characterized, among others, by the molecular events which selectively activate the expression of genes for contractile proteins within individual myocardial cells. As such, myosin light chain 2 (MLC-2), which is upregulated in the hypertrophic state in both rat and human, serves as a marker for hypertrophy. In an attempt to investigate the gene regulatory mechanisms of this phenomenon, we tested the hypothesis that certain transcription factors are directly involved in the development of cardiac hypertrophy by demonstrating the presence of cardiac tissue-specific regulatory elements in the 5'-flanking region of the MLC-2 promoter and testing them in the gel mobility shift assay for their binding activity to nuclear proteins from hypertrophied and normal cardiac tissue. In nuclear extracts from the ventricular tissues of the spontaneously hypertensive rat (SHR), distinctive changes in two families of activator proteins, the A/T-rich DNA-binding transcription factors, myocyte enhancer factor (MEF-2) and CArG-binding factor, manifested in a developmentally dictated manner paralleling the evolution of cardiac hypertrophy in these animals. Extracts isolated from brains and skeletal muscle tissues from the same animals did not exhibit the changes in binding activity. Also, the changes were not apparent when a distal negative regulatory element (CSS), which confers cardiac-specific expression, was tested in gel mobility shift assays. The ubiquitous TATA-binding proteins remained unchanged in comparing SHR with the control strain WKY in the same assay. These data support the notion that the expression of specific transcription factors is modulated in response to hypertrophy related signals which execute changes at the gene level effecting the enrichment of certain contractile proteins in an effort discrete and estranged from the basal transcription machinery.

Production of monoclonal antibodies to lactate dehydrogenase (LDH) isoenzymes for immunohistochemical study on fixed tissue section.

Purified lactate dehydrogenase (LDH) isoenzyme 1 (H or B subunits) and isoenzyme 5 (M or A subunits) were used to prepare monoclonal antibodies (MAb) suitable for immunohistochemical detection on formalin fixed paraffin-embedded tissue sections. In the initial fusions, screening of the antibodies was based on enzyme linked immunosorbent assay (ELISA) against the immunogens. None of the antibodies obtained was satisfactory. There were various problems related to specificity, crossreactivity, affinity and also the properties of the monoclonal antibody itself. Using a combined system involving more than one method for screening, two suitable monoclonal antibodies, MAb65 (to H-type LDH) and MAb25 (to M-type LDH) were selected. Both antibodies reacted specifically with corresponding LDH isoenzymes as shown in a series of tests. Their reactivity in sections of formalin fixed paraffin-embedded tissue indicated that both antibodies are suitable reagents for immunohistochemical studies.