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PubMed1986

Expression of the human immunoglobulin heavy chain gene of the 14q+ chromosome in a t(8;14)-positive Burkitt lymphoma cell line demonstrated in somatic cell hybrids.

A newly isolated Burkitt lymphoma cell line, ROS-1, carrying the specific translocation (8;14) has been studied using somatic cell hybridization techniques. As in other reported Burkitt cell lines, the oncogene c-myc was found to be translocated from the 8q- to the 14q+ chromosome. In contrast to other reports, expression of the mu immunoglobulin (Ig) heavy chain correlated with the presence of the 14q+ derivative and not of its normal homologue. These results indicate that the translocation (8;14) is not necessarily an abortive event for the production of the mu Ig heavy chain by the 14q+ derivative.

Burkitt Lymphoma↗
PubMed1992

Sequences that direct rat tyrosine hydroxylase gene expression.

Investigation of neuroendocrine genes has revealed that transcription is regulated via multiple DNA binding sites, including the cyclic AMP response element (CRE). We show here that for the neuronal and chromaffin-specific gene tyrosine hydroxylase (TH), a 70-bp region (-229 to -160) lacking the CRE is sufficient, in either orientation, to confer levels of chloramphenicol acetyltransferase reporter expression equivalent to or greater than that conferred by 4.8 kb of the rat TH enhancer/promoter region. The 70-bp region contains potential binding sites for AP2, AP1, E2A/MyoD, and POU transcription factors, and functions when linked to the TH promoter, but not when joined to a heterologous RSV promoter. This demonstrates that promoter as well as enhancer elements are important for TH expression. In gel-shift assays, the 70-bp fragment forms a cell type-specific complex with nuclear extracts from TH-expressing cells. which is effectively competed by an oligonucleotide containing AP2, AP1, and E2A/MyoD (E box) sites, but not by one containing the POU site. These data suggest that the AP2, AP1, and/or E box sites may be involved in forming the cell-specific complex. Although it lacks an authentic CRE, the 70-bp region also mediated a twofold transcriptional response to forskolin, equivalent to that found with the endogenous gene. A different region (-60 to -29) bearing a consensus CRE mediated a sixfold increase in transcription in response to forskolin, but only minimally activated basal transcription from the TH promoter in the absence of forskolin.

Animals↗
PubMed1991

Expression of CD45 isoforms by fresh and activated human gamma delta T lymphocytes and natural killer cells.

Naive and primed alpha beta T cells can be distinguished on the basis of their differential expression of CD45RA and CD45RO, respectively. The present study indicates that these CD45-isoforms also identify naive and primed maturational stages of gamma delta T cells and natural killer (NK) cells. In peripheral blood, all V gamma 9-V delta 2 gamma delta T cells reportedly express CD45RO whereas all V delta gamma delta T cells lack CD45RO. Here, we show that these CD45RO- V delta gamma delta T cells all express CD45RA and the CD45RO+ V.9-V delta 2 gamma delta cells lack expression of CD45RA. The V delta T cells acquired CD45RO expression and lost part of their surface CD45RA, following in vitro activation with phytohaemagglutinin or IL-2. Also the CD3-CD16+ NK cells in peripheral blood that are uniformly CD45RA+ CD45RO- completely converted to the CD45RA-CD45RO+ phenotype upon in vitro activation. Moreover, all cloned V.9-V delta 2 and V delta 1 T cells and NK cells express CD45RO and lack expression of CD45RA. Our results strongly suggest that CD45RA and CD45RO are genuine markers for naive and primed lymphocytes that represent distinct differentiation lineages.

Antigens, CD↗
PubMed1990

Cellular expression of lymphocyte function associated antigens and the intercellular adhesion molecule-1 in normal tissue.

A detailed immunohistological analysis of normal tissues for the distribution of lymphocyte function-associated antigens (LFA) and the intercellular adhesion molecule-1 (ICAM-1) showed several hitherto unrecognised patterns of LFA-3 and ICAM-1 expression. The widespread, but not ubiquitous, distribution of LFA-3 contrasted with the more restricted distribution of ICAM-1. Among epithelial cells, all tissues which were ICAM-1 positive were also LFA-3 positive with the single exception that thymic cortical epithelium, in contrast to previous reports, expressed only ICAM-1. It was striking that LFA-3 molecules were absent in some tissues which are considered to be sites of immunological privilege (such as brain and testis), suggesting an additional mechanism by which these microenvironments maintain immunological autonomy. Furthermore, the unexpected finding that LFA-3 is strongly expressed on intercalated discs of cardiac muscle may possibly be related to a non-immune function, or indicate a structurally similar epitope expressed by an unrelated molecule within this tissue.

Antigens, Surface↗
PubMed1990

Cloning, structural analysis, and expression of the human fast twitch skeletal muscle troponin C gene.

The gene encoding human fast skeletal muscle troponin C (TnC) was cloned, mapped, and sequenced. The locations of intron positions in this gene were compared to those in the related genes for mouse slow skeletal TnC and vertebrate and nonvertebrate calmodulins. We detected strikingly similar purine-rich DNA sequences on the coding strand in the basal promoter of the genes for fast and slow troponin C and chicken calmodulin II which may represent conserved regulatory elements in genes of the vertebrate troponin C/calmodulin gene family. We mapped the transcriptional start site of the gene and analyzed the expression of TnC test genes in the myogenic cell lines C2, L8, and H9c2(2-1) and in the human fibroblast line HuT12. Constructs comprising 4.7 or 6.2 kilobase pairs of 5'-flanking sequence (including the genuine transcriptional start site) upstream of the chloramphenicol acetyltransferase gene as reporter expressed the hybrid gene in C2 cells but not in nonmuscle cells. Surprisingly, no expression was found in cell lines L8 and H9c2(2-1) despite the fact that all three muscle cell lines vigorously express the endogenous TnC fast mRNA after differentiation. The discrepancy between the expression of endogenous genes and the test gene in these cell lines indicates different requirements for regulatory elements in different myogenic cells.

Amino Acid Sequence↗
PubMed1983

Expression and partial DNA sequence of the chicken beta H-globin gene.

We have determined the DNA sequence of the 3' end of the putative beta H-globin gene of chickens and used an S1 nuclease protection assay to analyze transcription of this gene. Appearance of mRNA corresponding to the putative beta H-globin gene in the erythroid cells of developing chicken embryos coincides with the reported expression of the beta H-globin protein, a constitutent of hemoglobin H which is a minor component in the definitive cells of late embryos and newly hatched chickens, thereby confirming the tentative identification of this gene as beta H. Analysis of the DNA sequence of the 3'-flanking region of the beta H gene revealed the presence of a complex array of direct repeats. A part of this "repeat cluster" is homologous to an inserted element in the large intron of the goat beta A- and beta C-globin genes.

Animals↗
PubMedDate not supplied

Cytochrome P-450 in the brain. Potential evolutionary and therapeutic relevance of localization of drug-metabolizing enzymes.

The cytochrome P-4502D6 enzyme is reportedly expressed in the brain. It was hypothesized that brain P-450 may serve to diminish exposure and toxicity from exogenous substances by enhancing their elimination from the central nervous system (CNS). To test this, a physiologic-based kinetic model was developed to simulate drug concentrations in brain and blood in the presence and absence of CNS metabolism. The amount of cytochrome P-450 in the brain was set at 0.25% of hepatic levels to reflect the small amounts of enzyme reportedly present in the CNS. Simulations were performed for low, intermediate, and high clearance drugs, assuming the presence or absence of brain P-450 enzymes. Enzyme localization was simulated by systematically reducing the volume into which the enzyme was expressed. No difference could be detected in drug concentrations in the blood, regardless of whether enzyme was present and/or localized in brain tissue. Marked differences, however, were observed in steady-state tissue drug levels that were highly dependent on the presence or absence of the brain enzyme, its degree of localization, and its efficiency for its substrate. These results suggest that large intersubject variability in CNS response to some drugs could reflect interpatient differences in CNS drug metabolism.

Biological Evolution↗
PubMed1991

Growth and cell cycle regulation of mRNA levels in GH3 cells.

In the rat anterior pituitary gland, estrogen increases both prolactin (PRL) mRNA levels and stimulates the proliferation of PRL-producing cells. The temporal sequence of these events suggests that PRL gene expression may be coordinated with cell proliferation. We investigated the relationship between cell cycle progression and the accumulation of the PRL mRNA, as well as several other mRNAs, in the rat pituitary tumor GH3 cell line. Serum-deprived cells progressed from G0 to S phase in 20-24 h following serum stimulation. During this time, beta-actin mRNA levels increased 7-fold in 5 h, then returned to basal levels prior to the beginning of S phase. Histone H1 mRNA levels increased approximately 3-fold as cells entered S phase. These data are consistent with the cell cycle-dependent regulation of beta-actin and histone H1 gene expression reported for other cell types. Glucocorticoid receptor mRNA levels were barely detectable in serum-deprived cells but rapidly increased 3- to 5-fold following serum stimulation. This increase resulted in glucocorticoid receptor mRNA levels that were equivalent to those seen in cells maintained in serum-containing medium, suggesting that serum factors regulate glucocorticoid receptor gene expression. In contrast to these changes in gene expression, the levels of PRL and growth hormone (GH) mRNAs gradually increased 2-fold while the cells progressed through G1 phase. Similarly, in cells synchronized to progress through S and G2 phases following aphidicolin treatment, histone H1 gene expression showed a specific increase in S phase cells, whereas PRL and GH mRNA levels changed little with cell cycle progression. These results indicate that the levels of PRL and GH mRNAs are not regulated in a cell cycle-dependent manner. When changes in estrogen responsiveness were determined during the cell cycle, we found that estradiol treatment was capable of increasing PRL mRNA accumulation independent of cell cycle progression and cell cycle distribution in synchronized GH3 cells. These results support the hypothesis that the hormonal regulation of PRL gene expression is not significantly affected by cell growth.

Actins↗
PubMed2026

Maternal redd1 mRNA decline triggers mTORC1 activation during the blastula-gastrula transition in zebrafish embryos.

During early metazoan development, maternal mRNAs and proteins stored in the egg sustain initial cellular functions. After the blastula stage, developmental control shifts to zygotic gene expression, and maternal transcripts are progressively degraded. Although mTORC1 is a central regulator of global mRNA translation and cell growth, its role in controlling maternal mRNA translation prior to gastrulation remains poorly understood. In zebrafish embryos, the mTORC1 inhibitor redd1 is abundantly expressed after fertilization but decreases following the maternal-to-zygotic transition (MZT), inversely correlating with mTORC1 activity. Overexpression of redd1 suppresses mTORC1, impairs gastrulation, and reduces translation of 5'TOP mRNAs and key regulatory genes, underscoring the necessity of relieving mTORC1 inhibition after the blastula stage. To investigate redd1 translation under conditions of low mTORC1 activity, we injected reporter mRNAs containing its 5' and 3' UTRs. The 3'UTR promoted polyadenylation and enhanced translation, while both UTRs enabled efficient reporter expression despite mTORC1 suppression, indicating that redd1 mRNA is translated independently of canonical mTORC1 pathways. Similarly, maternal mRNAs such as nanog, myca, pou5f3, and ccnb1, as well as the early zygotic transcript dharma, are translated through mTORC1-independent mechanisms. Together, these findings reveal a transient phase of mTORC1 suppression in early zebrafish embryos and demonstrate that select maternal and zygotic mRNAs bypass this regulation to ensure proper developmental progression.

Animals↗
PubMed1989

Cloning, sequence and expression in Escherichia coli of cDNA for ovine pregrowth hormone.

cDNA prepared from mRNA from ovine anterior pituitary glands was cloned in Escherichia coli and the sequence of a clone encoding the full coding sequence of ovine pregrowth hormone (preGH) determined. The predicted sequence for ovine GH agrees with that determined previously on the protein, except that residue 99 is asparagine rather than aspartic acid. The cDNA sequence also accords with one of the two genomic sequences for the ovine GH gene that have been reported. Expression plasmids using trp and lac promoters were constructed which allowed expression at low levels of ovine preGH in E. coli, as detected by immunoblotting and immunoassay.

Amino Acid Sequence↗
PubMed2026

A bireporter recombinant SARS-CoV-2 Omicron BA.5 for in vitro and in vivo studies.

The continuous emergence of variants of concern (VoCs) represents a significant challenge to effectively control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although FDA-approved vaccines and antivirals have been successfully developed and implemented for the prophylactic and therapeutic intervention of SARS-CoV-2 infection, recent VoCs could escape protection garnered by previous vaccine and antiviral approaches. Determining the efficacy of prophylactics and/or therapeutics against recent VoCs will assist in efficiently controlling currently circulating SARS-CoV-2 strains. We used our previously described bacterial artificial chromosome-based reverse genetics approach for Omicron BA.5 to generate a recombinant SARS-CoV-2 BA.5 encoding a fusion of ZsGreen to Nanoluciferase (rBA.5 ZsG-Nluc) from the locus of the viral nucleocapsid (N) protein separated by the porcine teschovirus-1 2A proteolytic cleavage site. The rBA.5 ZsG-Nluc replicates to levels comparable to recombinant BA.5 wild type (rBA.5 WT) and expresses high levels of ZsG and Nluc in cultured cells. This facilitates tracking viral infection and the identification of antivirals and neutralizing antibodies with EC50 and NT50 values, respectively, similar to those obtained with rBA.5 WT. Importantly, in Keratin-18 human angiotensin-converting enzyme-2 mice, rBA.5 ZsG-Nluc retains the same pathogenicity and ability to replicate in the lungs of infected mice as rBA.5 WT. Using rBA.5 ZsG-Nluc, we detected Nluc activity systemically and Nluc and ZsG expression in the lungs of infected mice using an in vivo imaging system. Our results demonstrate the feasibility of using rBA.5 ZsG-Nluc to track viral infections and identify prophylactics and therapeutics against recent SARS-CoV-2 VoCs in vitro, ex vivo, and in vivo.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of the coronavirus disease 2019 pandemic, is continually evolving to escape immunity acquired by previous natural infections or vaccinations. Moreover, recent SARS-CoV-2 variants of concern (VoCs) have acquired antiviral-resistant mutations to FDA-approved drugs. The emergence of these VoCs highlights the importance of identifying new prophylactics and therapeutics against currently circulating SARS-CoV-2 strains. We generated a recombinant bireporter Omicron BA.5 SARS-CoV-2 (rBA.5 ZsG-Nluc) that expresses reporter proteins, which are useful for cellular and whole animal studies, and has similar viral replication and pathogenicity to a wild-type recombinant Omicron BA.5 SARS-CoV-2. In Keratin-18 human angiotensin-converting enzyme-2 mice, rBA.5 ZsG-Nluc infection can be tracked systemically or in the lungs of infected mice using an in vivo imaging system. We establish a proof-of-concept platform of rBA.5 ZsG-Nluc in combination with an ancestral SARS-CoV-2 strain expressing mCherry to simultaneously identify antivirals and neutralizing antibodies against original and recent SARS-CoV-2 strains.

SARS-CoV-2↗
PubMed2026

Development of a rapid antiviral screening assay based on GFP reporter virus of bovine enterovirus.

In recent years, bovine enterovirus (BEV) has been increasingly associated with diarrhea in cattle in China, posing new challenges for disease control in the cattle industry. However, the mechanisms underlying BEV pathogenesis and virulence remain poorly understood. Infectious cDNA clones provide a powerful tool for dissecting viral replication and pathogenic mechanisms. In this study, we generated a full-length infectious cDNA clone of the BEV-F isolate HB19-1. Three overlapping fragments spanning the complete viral genome were amplified by RT-PCR and assembled downstream of a cytomegalovirus (CMV) promoter placed immediately upstream of the 5' untranslated region (5'UTR). To establish a reporter virus system, the green fluorescent protein (GFP) gene was inserted between the 5'UTR and the N terminus of VP4, followed by a 2A cleavage sequence (IKTAG) at the C terminus of GFP. The recombinant rHB19-GFP virus was successfully rescued. Growth curve analysis demonstrated that rHB19-GFP exhibited slower replication kinetics at early time points relative to the parental HB19-1 virus, with no significant difference in their peak viral titers. This GFP-expressing reporter virus enables convenient monitoring of BEV replication and provides a useful platform for antiviral screening. Using this system, we found that 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) inhibited BEV replication, suggesting its potential as an antiviral candidate. Overall, the rHB19-GFP infectious clone developed here offers a practical tool for studying BEV biology and for identifying antiviral compounds against BEV.

Animals↗
PubMed1991

Promoter and genotype dependent transient expression of a reporter gene in plant protoplasts.

For analysis of expression of three different plant promoters such as CaMV 35S, rbc S and mas, compact plasmid vectors were constructed by use of beta-glucuronidase (GUS) gene and nos termination signal. The plasmid molecules were introduced into tobacco and tomato protoplasts by using the Mg2+/PEG transformation protocol described by Negrutiu et al. The transient assays revealed maximum expression two days after DNA uptake. The comparative studies show the following order of promoters mas, CaMV 35S, rbc S as far as the activity is concerned. We also detected genotype-dependent promoter activity in the case of tomato.

DNA↗
PubMed1992

CD4 is expressed on murine pluripotent hematopoietic stem cells.

We show here for the first time that pluripotent hematopoietic stem cells express the CD4 antigen. CD4+ cells isolated from mouse marrow repopulated all hematopoietic lineages in both the long-term repopulation assay and the competitive repopulation assay. This finding indicates that the CD4+ population contains primitive stem cells with extensive repopulation capacity. Interestingly, the CD4- population had significant life-sparing activity, even though this population was depleted of long-term repopulating stem cells when compared with CD4+ cells. The majority of the cells that respond to the stroma in Whitlock-Witte cultures with B-cell differentiation were recovered in the CD4- population. Thus, this bone marrow (BM)-derived B-cell precursor lacks CD4, which is in contrast to myeloid precursors and thymus-derived lymphoid precursors that reportedly express CD4. We show further that the CD4 molecule expressed on BM cells is similar in molecular weight and epitope makeup to the CD4 antigen found on thymocytes. Detection of CD4 on BM cells is dependent on using high concentrations of antibodies. Thus, it is not surprising that expression of CD4 on pluripotent stem cells has been missed previously. Taken together, our data suggest that the CD4 molecule may play an important role in lineage definition in early hematopoietic differentiation.

Animals↗
PubMed1991

Molecular events involved in transcriptional activation of heat shock genes become progressively refractory to heat stimulation during aging of human diploid fibroblasts.

We examined the induction, by heat shock, of heat shock transcription factor (HSTF) DNA-binding and hsp 70 gene promoter activities during aging of the IMR-90 human diploid fibroblasts. Cells with population doubling level (PDL) ranging from 15-48 were heat shocked at temperatures of 39, 42, and 45 degrees C for various time periods; the binding of HSTF to its consensus DNA was determined by gel retardation assay and the promoter activity of the human hsp 70 gene was analyzed by transient expression of reporter gene activity. We observed that the induction of HSE-binding activity was inversely related to the PDL of the cells used. Importantly, as cells progress through their life span, a higher temperature and a longer period of heat shock were needed to evoke an optimal increase in HSE-binding activity. A substantial and rapid (within 30 min) increase in HSE-binding activity was observed when PDL 20 cells were heat shocked at 39, 42, or 45 degrees C. However, PDL 35 cells did not respond to 39 degrees C, and PDL 48 cells responded slowly to heat shock at 45 degrees C, but not 39 or 42 degrees C. Experiments on the heat induced increase in hsp 70 promoter driven reporter gene expression provided similar information on the age-dependent decrease in transcriptional activation of hsps. These results were further corroborated by quantitation of the abundance of mRNA of hsp 70. Analysis of the cAMP induced expression of the rat somatostatin promoter driven CAT gene provided evidence that the decrease in transcriptional activation of hsps in aging diploid cells was not a reflection of a generalized dysfunction of signal transduction. We conclude that functional changes in the heat shock response occur before cells lose their capacity to replicate, and we suggest that these changes are likely to have a central role in the expression of the aging phenotype.

Base Sequence↗
PubMed1992

Specific distribution of an epididymal 50 kDa protein revealed by an anti-Mos protein monoclonal antibody.

An anti-Mos protein monoclonal antibody, 4A6, was used to investigate the distribution of the antigen in the epididymis, in which the c-mos gene is reportedly expressed. The 4A6-reactive antigen was found on the basement membrane and luminal surface of the epithelial cells in the caput epididymis of BALB/c male mice as well as in the proximal corpus epididymis, the cauda epididymis, and the vas deferens. The 4A6 antigen was also found on the luminal surface of the epithelial cells in the epididymis of male germ cell-deficient C57BL/6J-Wv/Wv mice. This confirmed that the 4A6 antigen does not derive entirely from the testicular c-Mos protein but is synthesized in the epididymis. Western blot analysis revealed that the molecular weight of the epididymal 4A6 antigen was 50 kDa, which is unusually high for the c-Mos protein. With its specific distribution in the epididymis, the protein should play a specific role in functions of the epididymis.

Animals↗
PubMed2026

Site-specific gene integration by recombinase-mediated cassette exchange in anhydrobiotic Pv11 cells.

Pv11 cells, derived from Polypedilum vanderplanki, uniquely tolerate complete desiccation (anhydrobiosis). Although a CRISPR/Cas9-based precise integration method (CRIS-PITCh) has been developed for Pv11 cells, a CRISPR-free strategy that fixes both the genomic locus and transgene copy number has not yet been established. Here, we implement recombinase-mediated cassette exchange (RMCE) in Pv11 cells and generate master cell lines that retain anhydrobiosis following genetic engineering. We first evaluated the activity of multiple site-specific recombinases in Pv11 cells using a transient two-plasmid reporter assay. Flp, Cre, and Bxb1 recombinases all excised a test cassette and activated a green fluorescent protein (GFP) reporter, whereas phiC31 integrase mediated recombination at the DNA sequence level but did not induce reporter expression under our construct configuration. To enable genomic RMCE, we inserted an FRT/FRT3-landing pad (LP) into a previously identified genomic safe-harbor locus using CRIS-PITCh and isolated clonal master cell lines by single-cell sorting. Using the established master line, Flp-based RMCE achieved site-specific cassette exchange at the LP, producing HaloTag fluorescence and drug resistance upon successful exchange. In addition, the expected post-exchange sequence was confirmed by sequencing. We further established an all-in-one RMCE vector combining the Flp recombinase and donor cassette on a single plasmid. Together, these results demonstrate locus-defined, single-copy transgene integration in anhydrobiotic Pv11 cells via RMCE and provide a standardized, CRISPR-free workflow for routine genetic manipulation in this unique cell line. This workflow facilitates both fundamental research and applied biotechnological applications using desiccation-tolerant cells.

Anhydrobiosis↗
PubMed1989

A comparison between different models of the relation between recorded intradental nerve impulse activity and reported pain in man.

In this study three different models of the relation between intradental nerve impulse activity (INA) and pain report, expressed by finger-span (FS), in man were investigated. The techniques were (i) a previously described mathematical model, (ii) the relation between the Areas of INA and FS and (iii) the relation between the Peaks of INA and FS. Their usefulness in 533 clinical experiments on 15 subjects were evaluated by means of a statistical analysis of the variability and accuracy of the models. It was shown that the mathematical model was the most accurate and stable method of the three different methods investigated. This was indicated by, among other things, a significantly smaller variability for the mathematical model within experimental sessions than between sessions. The two other methods were found to be of less scientific value since they had an undesired high variability within sessions, and also showed contradicting results during control conditions.

Adult↗