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Some effects of growth conditions on steady state and heat shock induced htpG gene expression in continuous cultures of Escherichia coli.

Most of the data concerning heat shock gene expression reported in the literature are derived from batch culture experiments under substrate and nutrient sufficient conditions. Here, the effects of dilution rate and medium composition on the steady state and heat shock induced htpG gene expression have been investigated in continuous cultures of Escherichia coli, using a chromosomal htpG-lacZ gene fusion. During steady state growth temperature dependent patterns of the relative htpG expression were found to be largely similar, irrespective of the growth condition. However, nitrogen-limited growth resulted in a markedly reduced specific steady state htpG expression as compared to growth under carbon limitation or in complex medium, correlating qualitatively with the total cellular protein content. During heat shock, tight temperature controlled expression was evident. While the relative heat shock induced expression was largely identical at various dilution rates in a given growth medium, significantly different response patterns were observed in the three growth media at any given dilution rate. From these results a clearly temperature regulated htpG expression during both, steady and transient state growth in continuous culture is evident, which is further significantly affected by the growth condition used.

Culture Media

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

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

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

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

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

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

A 5'-upstream region of a bovine keratin 6 gene confers tissue-specific expression and hyperproliferation-related induction in transgenic mice.

Keratins, the constituents of epithelial intermediate filaments, are precisely regulated in a tissue- and development-specific manner, although little is known about the molecular mechanisms underlying this regulation. The expression pattern of keratin 6 is particularly complex, since besides being constitutively expressed in hair follicles and in suprabasal cells of a variety of internal stratified epithelia, it is induced in epidermis in both natural and artificially caused hyperproliferative situations. Therefore, the regulatory sequences controlling keratin 6 gene activity are particularly suitable for target gene expression in a tissue-specific manner. More interestingly, they can be skin-induced in transgenic animals or in gene therapy protocols, particularly those addressing epidermal hyperproliferative disorders. To delimit the regions containing these regulatory elements, different parts of the bovine keratin 6 gene linked to a beta-galactosidase reporter gene have been assayed in transgenic mice. A 9-kbp fragment from the 5' upstream region was able to provide both suprabasal tissue-specific and inducible reporter expression.

Animals

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

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

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

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

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

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

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

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

Immunohistochemical detection of tumor necrosis factor-alpha, other cytokines and adhesion molecules in human livers with alcoholic hepatitis.

This immunohistochemical study was designed to investigate the possible contribution to and topographical distribution of some important cytokines, such as tumour necrosis factor alpha (TNF alpha) and interleukins, in acute alcoholic hepatitis. The well-known inductive capacity of these cytokines with respect to the expression and/or up-regulation of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1), was a further point to be studied. Moreover, the proposed induction of adhesion molecules might also be associated with the activation and attraction of a special population of inflammatory cells characteristic for alcoholic hepatitis. Frozen liver samples from patients who died with signs of acute alcoholic hepatitis were evaluated using the alkaline phosphatase anti-alkaline phosphatase immunostaining technique and also single and double indirect immunofluorescence. In acute alcoholic hepatitis TNF alpha could be detected predominantly in ballooned hepatocytes, which often contained alcoholic hyalin (Mallory bodies). Moreover, TNF alpha showed a co-distribution with ICAM-1 expressed in the membranes of hepatocytes and with the occurrence of CD11b positive polymorphonuclear leukocytes (neutrophils) suggesting a possible major role of the beta 2-integrin Mac-1 as a ligand for ICAM-1. No induction of ELAM-1 could be found. In alcoholic hepatitis cytokines may be responsible for the induction of the adhesion molecule ICAM-1 on hepatocytic membranes and activate a defined population of inflammatory cells, thus contributing to the characteristic histological picture of acute alcoholic hepatitis with its concentration of neutrophils especially in areas with ballooned Mallory body-containing hepatocytes. Our results are in line with clinical findings showing high levels of TNF alpha and interleukin-1 in sera of patients with alcoholic hepatitis and with the already reported expression of ICAM-1 on hepatocytes.

Antigens, CD

Regulation of substance P is similar to that of vasoactive intestinal peptide after axotomy or explantation of the rat superior cervical ganglion.

The regulation of the expression of substance P (SP) in the rat superior cervical ganglion was compared to that of vasoactive intestinal peptide (VIP) in vivo after axotomy and in vitro after explantation. Previous studies have demonstrated that both neuropeptides increase after explantation, depolarization, and decentralization; however, whereas VIP expression increases after postganglionic axotomy, SP expression reportedly does not. To compare the effect of axotomy on these two peptides directly, the content of both was determined in individual ganglia at various times after surgery. The level of VIP-like immunoreactivity (IR) is increased at 2 days, reaches a peak at 6 days, and then declines by 14 days to approximately half its peak value. The level of SP-IR also increases 2 days after axotomy, but returns to control values by day 6. The increase in SP-IR is accompanied by an increase in beta-preprotachykinin mRNA, suggesting that the elevation in SP content is due, at least in part, to enhanced peptide synthesis. Immunocytochemical localization of SP-IR revealed the presence of immunoreactive principal neurons in axotomized, but not in sham-operated ganglia. Similarities in the regulation of these two neuropeptides were also investigated in organ culture by examining the effects of dexamethasone and interleukin-1 beta on VIP content, since the former has been shown to prevent the increase in SP in culture, while the latter has been found to enhance this increase (Kessler, Adler, Bell, et al., 1983, Neuroscience 9:309-321; Freidin and Kessler, 1991, Proc. Natl. Acad. Sci. USA 88:3200-3203; Hart, Shadiack, and Jonakait, 1991, J. Neurosci. Res. 29:282-291). As with SP expression, dexamethasone reduces the increase in VIP expression, while interleukin-1 beta increases it. Thus, both in vivo and in vitro, similar changes in VIP and SP expression are observed following a number of experimental manipulations, suggesting that expression of the two peptides is regulated by qualitatively similar mechanisms in sympathetic neurons.

Animals