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Modifications of chromatin structure in control of gene expression.

Repetitive sequences in intron and spacer DNA could be sites for binding of chromosomal proteins which maintain chromatin structure and control gene activity. Methylation of DNA guides the binding of acidic nonhistone proteins and maintains the differentiation state during DNA replication. Differentiation inducers modify repressor proteins permitting unfolding of chromatin. Histone H 1 must be removed for gene activity. Phosphorylation of nonhistone proteins probably induces allosteric modifications which permit unfolding of chromatin. Acetylation of nucleosomal histones is necessary to permit passage of RNA polymerase. Deacetylation quickly returns the gene to a normal histone repressed state. Chromosomal RNA attached to nonhistone proteins aids the binding of RNA polymerase to the DNA template. Carcinogens can disrupt normal gene control leading to circumvention of normal cell cycle controls.

Animals↗

Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer.

BACKGROUND: Preclinical studies in animal models have demonstrated tumor regression following intratumoral administration of an adenovirus vector containing wild-type p53 complementary DNA (Ad-p53). Therefore, in a phase I clinical trial, we administered Ad-p53 to 28 patients with non-small-cell lung cancer (NSCLC) whose cancers had progressed on conventional treatments. METHODS: Patients received up to six, monthly intratumoral injections of Ad-p53 by use of computed tomography-guided percutaneous fine-needle injection (23 patients) or bronchoscopy (five patients). The doses ranged from 10(6) plaque-forming units (PFU) to 10(11) PFU. RESULTS: Polymerase chain reaction (PCR) analysis showed the presence of adenovirus vector DNA in 18 (86%) of 21 patients with evaluable posttreatment biopsy specimens; vector-specific p53 messenger RNA was detected by means of reverse transcription-PCR analysis in 12 (46%) of 26 patients. Apoptosis (programmed cell death) was demonstrated by increased terminal deoxynucleotide transferase-mediated biotin uridine triphosphate nick-end labeling (TUNEL) staining in posttreatment biopsy specimens from 11 patients. Vector-related toxicity was minimal (National Cancer Institute's Common Toxicity Criteria: grade 3 = one patient; grade 4 = no patients) in 84 courses of treatment, despite repeated injections (up to six) in 23 patients. Therapeutic activity in 25 evaluable patients included partial responses in two patients (8%) and disease stabilization (range, 2-14 months) in 16 patients (64%); the remaining seven patients (28%) exhibited disease progression. CONCLUSIONS: Repeated intratumoral injections of Ad-p53 appear to be well tolerated, result in transgene expression of wild-type p53, and seem to mediate antitumor activity in a subset of patients with advanced NSCLC.

Adenoviridae↗

Recent progress in the discovery and development of cyclin-dependent kinase inhibitors.

Cyclin-dependent kinases (CDKs) have long been known to be the main facilitators of the cell proliferation cycle. However, they also play important roles in the regulation of the RNA polymerase II transcription cycle. Cancer cells display aberrant cell cycle regulation to gain proliferative advantages and they also appear to have an exaggerated dependence on RNA polymerase II transcriptional activity to sustain pro-survival and antiapoptotic signalling. A picture is now starting to emerge that both the cell-cycle and transcriptional functions of CDKs can be exploited pharmacologically with CDK inhibitors that possess appropriate selectivity profiles. In this article, recent advances into these mechanistic insights and how they can guide clinical development in terms of choice of indication are reviewed, as well as combinations with existing chemotherapies. An overview is also given of recent clinical trial results with the lead CDK inhibitor drug candidates seliciclib (CYC202, (R)-roscovitine; Cyclacel) and alvocidib (flavopiridol; Aventis-NCI), as well as the development of other clinical entries and advanced preclinical compounds. The discussion focuses on oncology, but we point out recent results with CDK inhibitors in virology and nephrology.

Amino Acid Sequence↗

[New methods for diagnosing infection in pregnancy].

Maternal infections during pregnancy are of special relevance because of the risk of transmission to the fetus. Besides serological methods of diagnosing acute maternal infection, new approaches to assess fetal involvement have been made by invasive procedures, such as ultrasound-guided fetal blood sampling or amniocentesis. The most relevant infections in pregnancy are briefly reviewed with their incidence, consequences of fetal infection, and standard diagnostic procedures. Immunological methods are handicapped by the immaturity of the fetal immune system; direct culture is complicated and time-consuming. The application of molecular biological methods to directly identify the RNA- or DNA-sequences of the infectious agent may be an alternative. Two methods, polymerase-chain-reaction (PCR) and in-situ hybridization (ISH) are explained. Broad clinical use is still hindered by problems in specificity and quantitative accuracy. Nevertheless, successful diagnosis of most of the relevant infections in pregnancy by these molecular biological methods, is published, and discussed in a review of the recent literature.

Cytomegalovirus Infections↗

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.

Enzyme Activation↗

Polymerase chain reaction and in situ hybridization of Epstein-Barr virus in liver biopsy specimens facilitate the diagnosis of EBV hepatitis after liver transplantation.

A nested polymerase chain reaction (nPCR) for Epstein-Barr virus (EBV) DNA, RNA in situ hybridization (EBER-ISH), and immunostaining against the ZEBRA EBV protein for diagnosis of EBV hepatitis were performed on 43 liver biopsy specimens obtained from 18 patients in the 1st year after liver transplantation (LTX). The findings were related to liver histology and results of EBV-nPCR on concomitantly obtained serum samples. EBV DNA was detected in 30% and RNA in 34% of the liver biopsy specimens using nPCR and EBER-ISH, respectively, giving a significant correlation between the two methods (P = 0.003). All but one patient had detectable EBV DNA in serum samples obtained within 1 month of the biopsy. More than 90% of the nPCR and EBER-ISH-positive biopsy specimens were obtained 3 months or less post-LTX. There was no significant difference in EBV genome findings in biopsy specimens with or without lymphocytic-immunoblastic infiltrates, either in nPCR (P = 0.73) or in ISH (P = 0.73). Two of three biopsy specimens with these histological changes suggesting a viral genesis were positive in EBV-nPCR but negative in ISH. Histopathological changes in EBV hepatitis may be nonspecific and masked by other complications. The use of EBV-nPCR and EBER-ISH in liver graft biopsy specimens of heavily immunosuppressed patients may give an early indication of EBV-related disease and can be used to guide therapeutic intervention.

Adolescent↗

Current concepts in antiretroviral therapy failure.

Currently, the goal for the first and second, possibly the third, antiretroviral regimen is the suppression of HIV replication to plasma levels below assay detection (ie, <50 HIV-1 RNA copies/mL). In patients with advanced treatment failure and resistance to numerous drugs, the goal of therapy has been to reduce viral load as much as possible and to maintain immunologic and clinical integrity. With the recent availability of new drugs and new classes of drugs, complete suppression of viral replication may be possible even in late salvage. When designing a regimen for a patient for whom antiretroviral therapy has failed, the regimen should contain at least 3 active drugs, and regimens are best selected with assistance from genotypic and phenotypic drug resistance testing. The selection should also be guided by knowledge of potential pharmacokinetic interactions among drugs. This article summarizes a presentation on antiretroviral failure by Carlos del Rio, MD, at the 8th Annual clinical meeting for Ryan White CARE Act clinicians in New Orleans in June 2005, sponsored by the International AIDS Society-USA and includes more recent data presented at the 13th Conference on Retroviruses and Opportunistic Infections (CROI) in 2006.

Anti-HIV Agents↗

The expression of the receptor-protein tyrosine kinase gene, eck, is highly restricted during early mouse development.

Cell-cell interactions during embryogenesis have been shown to be important for establishing developmental fates in a number of organisms such as Drosophila and Caenorhabditis. One class of genes shown to mediate this process are receptor-protein tyrosine kinases (R-PTKs). To examine whether R-PTKs might participate in similar mechanisms operating in the mammalian embryo, an important prerequisite is to show that these genes are expressed in spatial and temporal patterns consistent with such a role. Here, we analyze the expression of eck, a member of the eph family of R-PTKs, during gastrulation and early organogenesis by in situ hybridization. eck transcripts are first detected in gastrulation stage embryos (6.5-7.5 days post coitum (dpc)) in ectodermal cells adjacent to the distal region of the primitive streak. By the neural plate stage (approximately 7.5 dpc), eck expression becomes restricted to the extreme distal end or node of the primitive streak. After the beginning of somitogenesis (approximately 8.0 dpc), eck expression persists in the node as this structure regresses toward the caudal end of the embryo. In addition, beginning at the mid head fold stage (approximately 7.75 dpc), we observe that eck exhibits a dynamic and spatially restricted expression pattern in the prospective hindbrain region. eck transcripts are initially detected in a 5-cell wide strip of mesodermal cells underlying prospective rhombomere 4 (r4). Subsequently at the beginning of somitogenesis, eck mRNA expression is observed in prospective r4. At the 4--8-somite stage, eck transcripts are observed in r4, mesenchymal cells underlying r4, and surface ectoderm in the vicinity of the developing second branchial arch. By the 10-somite stage, eck mRNA expression in these cells is downregulated. Additionally, at the 5--8-somite stage, eck transcripts are detected initially in the lateral mesenchyme immediately underlying the surface ectoderm adjacent to r5 and r6, and subsequently in surface ectoderm overlying the developing third branchial arch. These data suggest that eck may be involved in cell-cell interactions guiding early hindbrain development.

Amino Acid Sequence↗

Lead-catalyzed cleavage of yeast tRNAPhe mutants.

Yeast tRNA(Phe) lacking modified nucleotides undergoes lead-catalyzed cleavage between nucleotides U17 and G18 at a rate very similar to that of its fully modified counterpart. The rates of cleavage for 28 tRNA(Phe) mutants were determined to define the structural requirements of this reaction. The cleavage rate was found to be very dependent on the identity and correct positioning of the two lead-coordinating pyrimidines defined by X-ray crystallography. Nucleotide changes that disrupted the tertiary interactions of tRNAPhe reduced the rate of cleavage even when they were distant from the lead binding pocket. However, nucleotide changes designed to maintain tertiary interactions showed normal rates of cleavage, thereby making the reaction of a useful probe for tRNA(Phe) structure. Certain mutants resulted in the enhancement of cleavage at a "cryptic" site at C48. The sequences of Escherichia coli tRNA(Phe) and yeast tRNA(Arg) were altered such that they acquired the ability to cleave at U17, confirming our understanding of the structural requirements for cleavage. This mutagenic analysis of the lead cleavage domain provides a useful guide for similar analysis of autocatalytic self-cleavage reactions.

Base Sequence↗

Design, biological activity and NMR-solution structure of a DNA analogue of yeast tRNA(Phe) anticodon domain.

Design of biologically active DNA analogues of the yeast tRNA(Phe) anticodon domain, tDNAPheAC, required the introduction of a d(m5C)-dependent, Mg(2+)-induced structural transition and the d(m1G) disruption of an intra-loop dC.dG base pair. The modifications were introduced at residues corresponding to m5C-40 and wybutosine-37 in tRNA(Phe). Modified tDNAPheAC inhibited translation by 50% at a tDNAPheAC:ribosome ratio of 8:1. The molecule's structure has been determined by NMR spectroscopy and restrained molecular dynamics with an overall r.m.s.d. of 2.8 A and 1.7 A in the stem, and is similar to the tRNA(Phe) anticodon domain in conformation and dimensions. The tDNAPheAC structure may provide a guide for the design of translation inhibitors as potential therapeutic agents.

Anticodon↗

Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P.

The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited.

Base Sequence↗

Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus ("Streptococcus milleri group") are of different clinical importance and are not equally associated with abscess.

Difficulties in distinguishing organisms of the "Streptococcus milleri group" (SMG; Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus), have caused ambiguity in determining their pathogenic potential. We reviewed 118 cases in which SMG isolates had been identified using 16S rDNA sequence. S. constellatus and S. anginosus were isolated far more frequently than was S. intermedius. Nearly all isolates of S. intermedius and most isolates of S. constellatus, but only 19% of those of S. anginosus, were associated with abscess. Our findings suggest that speciation of the SMG may guide diagnostic evaluation, give insight into the possible role of coinfecting organisms, and help assess the need to search for occult abscess.

Abscess↗

Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases.

The primary sequence of human DNA polymerase alpha deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage phi 19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain, whereas a potential DNA primase interaction domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DNA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.

Amino Acid Sequence↗

Mapping Sub-National Respiratory Virus Circulation in Cambodia Using Metatranscriptomic Sequencing: A Multi-Center Hospital-Based Surveillance Study.

BACKGROUND: Genomic surveillance can guide early detection of and response to emerging epidemics. Metatranscriptomic sequencing was used to investigate sub-national respiratory virus circulation in Cambodia from 2020 to 2023. METHODS: Nasopharyngeal swabs were collected from individuals aged 2&#x2009;months to 65&#x2009;years with influenza-like illness in four Cambodian hospitals. Metatranscriptomic data were generated by short-read RNA sequencing. Bernoulli space-time scan statistics were used to identify temporal virus clusters. Bayesian inference of phylogenetic trees was used to compute divergence times for temporally clustered, highly represented viruses (influenza A/H3N2 and B, Betacoronavirus 1, respiratory syncytial virus [RSV] A and B), and publicly available global influenza virus genomes. RESULTS: Of 1093 individuals, 499 (45.7%) had detectable respiratory viruses belonging to 68 distinct species. Moderate (N&#x2009;>&#x2009;20) discrete time-clusters were noted of RSV-A (37 cases), Betacoronavirus 1 (21 cases), RSV-B (22 cases), and A/H3N2 (30 cases). The posterior median of time to most recent common ancestor ranged from 0.71&#x2009;years (95% HPD 0.38-1.10) for Betacoronavirus 1 and 1.31&#x2009;years (95% HPD 0.60-3.20) for A/H3N2, to 2.75&#x2009;years (1.82-4.26) for RSV-A and 4.79&#x2009;years (2.39-7.74) for RSV-B. A/H3N2 and influenza B virus genomes mapped to clades 3C.2a1b.2a.2a and Victoria 1A.3a.2, respectively, and inter-mixed with concurrent global strains. CONCLUSIONS: Multiple respiratory viruses circulated at a sub-national level in Cambodia from 2020 to 2023 despite pandemic disruptions. Influenza virus population diversity decreased during the height of lockdown but recovered in mid-2022. Re-emerging influenza strains were distinct from historically circulating strains and clustered with contemporaneous global variants, suggesting multiple external introductions.

Humans↗

The first complete mitochondrial genome of Strigea falconis (Digenea: Strigeidae) reveals six tandemly repeated trnE-containing units and provides mt evidence for the non-monophyly of the family Strigeidae.

BACKGROUND: Phylogenetic relationships among members in the order Diplostomida remain contentious, with mitochondrial (mt) and nuclear genomic data often yielding conflicting topologies. A major limitation is the availability of only a few mt genomes from the type genus Strigea, hindering a robust test of the monophyly of the family Strigeidae and the order Diplostomida. RESULTS: The mt genome of S. falconis was completely sequenced for the first time, which was a circular molecule of 16,872&#xa0;bp in length, encoding the typical set of 36 mt genes and six duplicate tRNA-Glu genes. Notably, there were seven identical and consecutive tandem repeat units each consist of a 169&#xa0;bp non-coding region followed by a trnE gene in the newly assembled genome. Phylogenomic analyses based on concatenated predicted amino acid sequences of 12 proteins robustly placed S. falconis in the same clade as Apharyngostrigea pipientis. Crucially, the family Strigeidae was not recovered as monophyletic. Instead, two species within Strigeidae, Cardiocephaloides medioconiger and Cotylurus marcogliesei, clustered with representatives of Diplostomidae, providing mt evidence for the paraphyly of Strigeidae under the current sampling. CONCLUSIONS: The newly sequenced mt genome of S. falconis reveals a previously unreported six-copy tandem repeat of trnE-containing units among currently available diplostomoid mt genomes. Phylogenetic analyses based on mt protein-coding genes provide additional mt evidence that the family Strigeidae was not recovered as monophyletic under the present taxon sampling. However, because mt genomes represent a single maternally inherited linkage group, broader taxon sampling, independent nuclear phylogenomic data, and explicit sensitivity analyses will be required to confirm these relationships and guide any formal systematic revision.

Animals↗

Uncommitted Xenopus blastula cells can be directed to uniform muscle gene expression by gradient interpretation and a community effect.

The animal cap cells of Xenopus blastulae behave as multi-potent stem cells in so far as they can differentiate along many unrelated pathways according to the kind and amount of signal factor that they experience. At first, animal cap cells activate early zygotic genes across a broad range of TGFbeta concentrations; soon after this, they activate later genes more intensely and over a narrow concentration range. Here we show that uncommitted blastula cells can be directed, by the sequential influence of a particular concentration of a TGFbeta morphogen and an FGF-mediated community effect, to form a homogeneous single cell type. As a result of these two signalling processes, an entire population of animal cap cells can be converted, in the absence of other signals, to a uniform population of one tissue type. Mesoderm cells that experience a particular concentration of activin increase their XMyoD expression by 10-fold and become distinct from neighbouring cells that received lower or higher concentrations of activin. The signalling processes that we employ here may be important in normal development and useful in guiding stem cell differentiation.

Activins↗

[Clinical significance of MDR gene expression in malignant pleural effusion and ascites and solid tumors].

OBJECTIVE: To discuss the guiding significance of multidrug resistance (MDR) gene expression for monitoring the clinical effectiveness of chemotherapy upon cancer, and to analyze the MDR1 and glutnthione-s-transferase(GST-pi) mRNA expression rates in primary breast cancer, and their correlation with the clinical outcome and prognosis. METHODS: The mRNA expression levels of MDR1 in 16 cases with malignant pleural effusion and ascites and 102 cases of solid tumors were measured using semiquantitative RT-PCR assay. The MDR1 and GST-pi mRNA expression rates in 84 cases of breast cancer, out of the 102 cases of solid tumors, were also studied in the same way. RESULTS: (1) An average coincidence rate of 84.5% was found between the mRNA expression level of MDR1 and the clinical response to chemotherapy in the 16 cases with malignant pleural effusion and ascites and 18 cases of solid tumors. (2) The positive rate of MDR1 mRNA expression in primary breast cancer tissue was 79.7%, with the medium and high levels as high as 33.3%, while the positive rate of GST-pi mRNA expression was 33.3%, with the medium and high expression levels being as low as 4.8%. There was a highly significant difference between the two groups (P < 0.01). The positive rates of MDR1 mRNA in the group having received chemotherapy before surgery and the group not receiving chemotherapy before surgery were 87.8% and 68.6% respectively, showing a lymph nodes and in the group without metastases in axillary lymph nodes were 89.1% and 68.4% respectively, indicating a significant difference (P < 0.05). The positive rates of GST-pi mRNA in the group suffering distant metastases and the group not suffering distant metastases were 71.4% and 20.6% respectively, showing a significant difference (P < 0.05). The GST-pi mRNA expression rates in the estrogen receptor (ER)- and progesterone receptor (PR)-positive groups were higher than those in the ER- and PR-negative groups, indicating a significant difference (P < 0.05) . CONCLUSION: The mRNA expression level of MDR1 has a relatively high correlation with the clinical response to chemotherapy, and thus can be used to monitor the effectiveness of chemotherapy. MDR1 is the main drug resistance mechanism in breast cancer. Chemotherapy before surgery induces MDR1 mRNA expression. MDR1 e xpression and GST-pi mRNA expression serve as a significant prognostic indicator for breast cancer, thus having guiding significance for assessing prognosis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Interferon and TLR genes, but not endogenous bornavirus-like elements, limit BoDV1 replication after intracerebral infection.

Borna disease virus 1 (BoDV1) is a disease-causing agent in some livestock and, as has recently been shown, in humans. What constitutes a protective immune response to BoDV1 is unclear. Previous studies found that endogenous bornavirus-like nucleoprotein elements (EBLNs) present in mammalian genomes produce piRNAs antisense to BoDV1 nucleoprotein mRNAs. As a known function of piRNAs is to restrict transposons via RNA interference, it has been hypothesized that EBLN-derived piRNAs may restrict BoDV1. Here we used EBLN knockout (KO) and other KO mice to test genetic factors potentially involved in antiviral immunity to BoDV1. In previous reports, BoDV1 replication was higher in mice deficient in interferon gamma, and we confirmed a role for this cytokine in BoDV1 restriction at 12 weeks post infection using mice lacking its receptor. We show that BoDV1 replicates to higher levels in the brain of mice without Toll-like receptor 7 (TLR7), suggesting a role for this innate immune receptor in BoDV1 immunity. In contrast, mice lacking piRNA-producing EBLNs were no more susceptible to BoDV1 infection than wild-type under the infection conditions used here. We thus expand the genetic evidence implicating specific conventional immune pathways in BoDV1 control and conclude that EBLN-derived piRNA-guided antiviral silencing, if it occurs, is relatively less impactful in intracerebral infection of neonates.

Animals↗