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PubMed1992

Analysis of hepatocellular proliferation: study of archival liver tissue is facilitated by an endogenous marker of DNA replication.

Assessment of liver regeneration with endogenous genes that are expressed during DNA replication is physiological, specific and direct. To determine whether H3 histone messenger RNA expression (which is tightly coupled with DNA synthesis) could be used for this purpose, we initially examined liver regeneration in a mouse model. After partial hepatectomy, RNA transblot studies showed induction of H3 histone messenger RNA expression in regenerating mouse livers. In situ molecular hybridization demonstrated that the overall pattern of H3 histone messenger RNA expression correlated with [3H]thymidine labeling of hepatocytes. After partial hepatectomy, H3 histone messenger RNA expression in hepatocytes peaked at 48 hr (greater than 60 times greater than at 24 hr; p less than 0.001) and then rapidly declined. Although hepatocyte labeling with [3H]thymidine showed similar kinetics of liver regeneration, use of this parameter resulted in overestimation of the proliferative compartment when it was compared with H3 histone messenger RNA expression. Next we determined whether H3 histone messenger RNA expression could be used to study hepatocellular proliferation in archival human material. H3 histone messenger RNA-expressing hepatocytes were identified on in situ hybridization in patients with acute or chronic active hepatitis and active cirrhosis, but not inactive cirrhosis. These studies demonstrate that H3 histone messenger RNA is expressed in a phasic manner during liver regeneration. Use of H3 histone messenger RNA expression to evaluate hepatocellular proliferation should facilitate clinical studies and greatly advance our understanding of the pathophysiology of liver regeneration.

Adolescent↗
PubMed1993

Induction of replicative senescence by 5-azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH-3T3 cells.

To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast-like cells and non-ageing NIH-3T3 cells with 5-azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH-3T3 cultures, 96h exposures to 4 microM 5-azacytidine caused diminished cell proliferation due to cell arrest in the G1 compartments of the second and third cell cycles of serum stimulated cells. The exit from the G0/G1 compartment was not affected. The 5-azacytidine induced cell kinetic disturbances were unstable in NIH-3T3 cultures, such that pre-treated cells reverted to normal cell cycle transit within 2-3 days after termination of treatment. In contrast, 5-azacytidine pre-treated amniotic fluid derived fibroblast-like cell cultures showed persistently elevated G2 phase arrests and delayed G0/G1 phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5-azacytidine exposed cultures showed elevated numbers of G1 phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH-3T3 cells but not in amniotic fluid derived fibroblast-like cells. These contrasting responses to 5-azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non-ageing cell lines.

3T3 Cells↗
PubMed2002

Drosophila damage-specific DNA-binding protein 1 (D-DDB1) is controlled by the DRE/DREF system.

We succeeded in cloning the gene, termed d-ddb1, for a Drosophila homolog of the p127 subunit of the human damage-specific DNA-binding protein, thought to recognize (6-4) photoproducts and related structures. In Drosophila, the gene product (D-DDB1) also appeared to play a role as a repair factor, d-ddb1 knockout Kc cells generated with a RNAi method being sensitive to UV. In addition, UV or methyl methanesulfonate treatment increased d-ddb1 transcripts. However, we found that the gene is controlled by the DRE/DREF system, which is generally responsible for activating the promoters of proliferation-related genes. Moreover, during Drosophila development, the transcription of d-ddb1 changed greatly, with the highest levels in unfertilized eggs, indicating that external injury to DNA is not essential to D-DDB1 function. Interestingly, as with UV irradiation-induced transfer of D-DDB1 to the nucleus from the cytoplasm, during spermatogenesis the protein transiently shifted from one cell compartment to the other. The results indicate that D-DDB1 not only contributes to the DNA repair system, but also has a role in cell proliferation and development.

5' Flanking Region↗
PubMed2006

Citron kinase, a RhoA effector, enhances HIV-1 virion production by modulating exocytosis.

RhoGTPases play important roles in the regulation of protein transport and membrane recycling. Little is known, however, about how RhoGTPases affect HIV-1 virion production, which is dependent on the endosomal sorting pathway. We report that ectopic expression of citron kinase (citron-K), a RhoA effector, preferentially enhances HIV-1 virion production. Depletion of endogenous citron-K inhibits HIV-1 virion production. Citron-N, which lacks the kinase domain, also enhances HIV-1 virion production. The leucine zipper, Rho-binding and zinc finger domains of citron-N are necessary for the enhancement activity. Citron-K also enhances murine leukemia virion production and the HIV-1 late domain is not required for the citron-K-mediated enhancement. Ectopic expression of citron-K leads to the formation of cytoplasmic structures containing citron-K and HIV-1 Gag proteins. HIV-1 and citron-K cooperatively enhance acidic endosome and lysosome compartments. Finally, citron-K promotes exocytosis of microvesicles or exosomes that co-purify with HIV-1 virions. We conclude that citron-K enhances HIV-1 virion production by stimulating the endosomal compartments and exocytosis.

Animals↗
PubMed2006

Requirement of the N-terminal region of orthobunyavirus nonstructural protein NSm for virus assembly and morphogenesis.

The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.

Amino Acid Sequence↗
PubMed2005

The effect of lateral cortex disruption and repair on the stability of the medial opening wedge high tibial osteotomy.

BACKGROUND: Medial opening wedge high tibial osteotomy is gaining popularity as a treatment option for medial compartment degenerative disease in the young, active patient. One of the potential technical pitfalls of this procedure is inadvertent disruption of the lateral tibial cortex during distraction at the osteotomy site. PURPOSE: (1) To investigate the effect of lateral cortex disruption on stability during medial opening wedge high tibial osteotomy and (2) to evaluate 3 different methods of repair of the disrupted lateral cortex. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 50 validated replicate tibias were evaluated in a medial opening wedge high tibial osteotomy model. Specimens were divided into 5 groups: (1) control, or intact lateral cortex (n = 10); (2) disrupted lateral cortex (n = 10); (3) lateral cortex repaired with 1 staple (n = 10); (4) lateral cortex repaired with 2 staples (n = 10); and (5) lateral cortex repaired with a periarticular plate and screws (n = 10). Specimens were placed in compression and torsion under physiologic loads, and stiffness and micromotion were calculated. RESULTS: Disruption of the lateral cortex resulted in a 58% reduction in axial stiffness and a 68% reduction in torsional stiffness compared to control specimens (P < .05). Disruption of the lateral cortex also resulted in increased micromotion at the osteotomy site. All 3 methods of repair of the lateral cortex restored stiffness and micromotion values to those of the control group (P < .05). CONCLUSIONS: Instability at the osteotomy site may contribute to the high rate of delayed union or nonunion associated with medial opening wedge high tibial osteotomy. Repair of the lateral tibial cortex by each of these techniques restored stability to the osteotomy site in this replicate tibia model and might be effective in clinical use. However, more studies are needed to further explore the relationship between lateral cortex disruption and patient outcomes in the clinical setting before definitive conclusions can be drawn.

Biomechanical Phenomena↗
PubMed2002

Gene discovery within the planctomycete division of the domain Bacteria using sequence tags from genomic DNA libraries.

BACKGROUND: The planctomycetes comprise a distinct group of the domain Bacteria, forming a separate division by phylogenetic analysis. The organization of their cells into membrane-defined compartments including membrane-bounded nucleoids, their budding reproduction and complete absence of peptidoglycan distinguish them from most other Bacteria. A random sequencing approach was applied to the genomes of two planctomycete species, Gemmata obscuriglobus and Pirellula marina, to discover genes relevant to their cell biology and physiology. RESULTS: Genes with a wide variety of functions were identified in G. obscuriglobus and Pi. marina, including those of metabolism and biosynthesis, transport, regulation, translation and DNA replication, consistent with established phenotypic characters for these species. The genes sequenced were predominantly homologous to those in members of other divisions of the Bacteria, but there were also matches with nuclear genomic genes of the domain Eukarya, genes that may have appeared in the planctomycetes via horizontal gene transfer events. Significant among these matches are those with two genes atypical for Bacteria and with significant cell-biology implications - integrin alpha-V and inter-alpha-trypsin inhibitor protein - with homologs in G. obscuriglobus and Pi. marina respectively. CONCLUSIONS: The random-sequence-tag approach applied here to G. obscuriglobus and Pi. marina is the first report of gene recovery and analysis from members of the planctomycetes using genome-based methods. Gene homologs identified were predominantly similar to genes of Bacteria, but some significant best matches to genes from Eukarya suggest that lateral gene transfer events between domains may have involved this division at some time during its evolution.

Amino Acids↗
PubMed1996

Test system for exposing fish to resuspended, contaminated sediment.

We describe a new test system for exposing fish to resuspended sediments and associated contaminants. Test sediments were resuspended by revolving test chambers on rotating shafts driven by an electric motor. The timing, speed, and duration of test-chamber revolution were controlled by a rheostat and electronic timer. Each chamber held 45 litres of water and accommodated about 49 g of test fish. The system described had three water baths, each holding six test chambers. We illustrate the performance of this system with results from a 28-day test in which juvenile bluegills Lepomis macrochirus were exposed to resuspended, riverine sediments differing in texture and cadmium content. The test had one sediment-free control and five sediment treatments, with three replicates (chambers) per treatment and 25 fish per replicate. Two-thirds (30 litres) of the test water and sediment in each chamber was renewed weekly. The mean concentration of total suspended solids (TSS) did not vary among treatments; the grand-mean TSS in the five sediment treatments was 975 mg litre(-1), similar to the target TSS of 1000 mg litre(-1). At the end of the test, an average of 50% of the introduced cadmium was associated with the suspended sediment compartment, whereas the filtered (0.45 microm) water contained 0.4% and bluegills 1.8% of the cadmium.

Journal Article↗
PubMed1999

The immunoevasive function encoded by the mouse cytomegalovirus gene m152 protects the virus against T cell control in vivo.

Cytomegaloviruses encode numerous functions that inhibit antigen presentation in the major histocompatibility complex (MHC) class I pathway in vitro. One example is the mouse cytomegalovirus (MCMV) glycoprotein gp40, encoded by the m152 gene, which selectively retains murine but not human MHC class I complexes in the endoplasmic reticulum-Golgi intermediate compartment/cis-Golgi compartment (Ziegler, H., R. Thäle, P. Lucin, W. Muranyi, T. Flohr, H. Hengel, H. Farrell, W. Rawlinson, and U.H. Koszinowski. 1997. Immunity. 6:57-66). To investigate the in vivo significance of this gene function during MCMV infection of the natural host, we constructed recombinants of MCMV in which the m152 gene was deleted, as were the corresponding virus revertants. We report on the following findings: Deletion of the m152 gene has no effect on virus replication in cell culture, whereas after infection of mice, the m152-deficient virus replicates to significantly lower virus titers. This attenuating effect is lifted by reinsertion of the gene into the mutant. Mutants and revertants grow to the same titer in animals deprived of the function targeted by the viral gene function, namely in mice deficient in beta2-microglobulin, mice deficient in the CD8 molecule, and mice depleted of T cells. Upon adoptive transfer of naive lymphocytes into infected mice, the absence of the m152 gene function sensitizes the virus to primary lymphocyte control. These results prove that MHC-reactive functions protect CMVs against attack by CD8(+) T lymphocytes in vivo.

3T3 Cells↗
PubMed2004

Spread of hepatitis B viruses in vitro requires extracellular progeny and may be codetermined by polarized egress.

Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells.

Animals↗
PubMed1986

Dopaminergic substrates of cocaine-induced place conditioning.

Recently, Spyraki et al. (Brain Research, 253 (1982) 195-203) reported that cocaine-induced place preference conditioning was unaffected by blockade of central dopamine (DA) or norepinephrine function. In addition, systemic injections of the local anesthetic procaine produced place preference conditioning. The present study was undertaken to further evaluate the possible role of DA in cocaine-induced place conditioning. In Expt. 1, a partial replication of Spyraki et al., systemic cocaine (5.0 mg/kg, i.p.) produced significant place conditioning that was not disrupted with the DA antagonist pimozide (1.0 mg/kg, i.p.). In Expt. 2, cocaine was microinjected unilaterally into the lateral ventricles to eliminate peripheral local anesthesia. Cocaine (50.0 micrograms, i.c.v.) produced place conditioning and pretreatment with pimozide (1.0 mg/kg, i.p.) disrupted the effect. In Expt. 3, place conditioning was not observed when cocaine presentations (50.0 micrograms, i.c.v.) were paired with both compartments. The substrates of cocaine-induced place conditioning were further investigated in Expt 4: Procaine (250 micrograms, i.c.v.) did not produce place conditioning whereas the DA agonist bromocriptine (50.0 micrograms, i.c.v.) did. Results suggest the involvement of central DA in cocaine-induced place conditioning.

Animals↗
PubMed2005

3-D physical models of amitosis (cytokinesis).

Based on Newton's laws, extended Coulomb's law and published biological data, we develop our 3-D physical models of natural and normal amitosis (cytokinesis), for prokaryotes (bacterial cells) in M phase. We propose following hypotheses: Chromosome rings exclusion: No normally and naturally replicated chromosome rings (RCR) can occupy the same prokaryote, a bacterial cell. The RCR produce spontaneous and strong electromagnetic fields (EMF), that can be alternated environmentally, in protoplasm and cortex. The EMF is approximately a repulsive quasi-static electric (slowly variant and mostly electric) field (EF). The EF forces between the RCR are strong enough, and orderly accumulate contractile proteins that divide the procaryotes in the cell cortex of division plane or directly split the cell compartment envelope longitudinally. The radial component of the EF forces could also make furrows or cleavages of procaryotes. The EF distribution controls the protoplasm partition and completes the amitosis (cytokinesis). After the cytokinesis, the spontaneous and strong EF disappear because the net charge accumulation becomes weak, in the protoplasm. The exclusion is because the two sets of informative objects (RCR) have identical DNA codes information and they are electro magnetically identical, therefore they repulse from each other. We also compare divisions among eukaryotes, prokaryotes, mitochondria and chloroplasts and propose our hypothesis: The principles of our models are applied to divisions of mitochondria and chloroplasts of eucaryotes too because these division mechanisms are closer than others in a view of physics. Though we develop our model using 1 division plane (i.e., 1 cell is divided into 2 cells) as an example, the principle of our model is applied to the cases with multiple division planes (i.e., 1 cell is divided into multiple cells) too.

Bacteria↗
PubMed2006

AML1 deletion in adult mice causes splenomegaly and lymphomas.

AML1 (RUNX1) encodes a DNA-binding subunit of the CBF transcription factor family and is required for the establishment of definitive hematopoiesis. AML1 is one of the most frequently mutated genes associated with human acute leukemia, suggesting that genetic alterations of the gene contribute to leukemogenesis. Here, we report the analysis of mice carrying conditional AML1 knockout alleles that were inactivated using the Cre/loxP system. AML1 was deleted in adult mice by inducing Cre activity to replicate AML1 deletions found in human MDS, familial platelet disorder and rare de novo human AML. At a latency of 2 months after induction, the thymus was reduced in size and frequently populated by immature double negative thymocytes, indicating defective T-lymphocyte maturation, resulting in lymphatic diseases with 50% penetrance, including atypical hyperplasia and thymic lymphoma. Metastatic lymphomas to the liver and the meninges were observed. Mice also developed splenomegaly with an expansion of the myeloid compartment. Increased Howell-Jolly body counts indicated splenic hypofunction. Thrombocytopenia occurred due to immaturity of mini-megakaryocytes in the bone marrow. Together with mild lymphocytopenia in the peripheral blood and increased fractions of immature cells in the bone marrow, AML1 deficient mice display features of a myelodysplastic syndrome, suggesting a preleukemic state.

Animals↗
PubMed2006

Stem cell niches in the adult mouse heart.

Cardiac stem cells (CSCs) have been identified in the adult heart, but the microenvironment that protects the slow-cycling, undifferentiated, and self-renewing CSCs remains to be determined. We report that the myocardium possesses interstitial structures with the architectural organization of stem cell niches that harbor long-term BrdU-retaining cells. The recognition of long-term label-retaining cells provides functional evidence of resident CSCs in the myocardium, indicating that the heart is an organ regulated by a stem cell compartment. Cardiac niches contain CSCs and lineage-committed cells, which are connected to supporting cells represented by myocytes and fibroblasts. Connexins and cadherins form gap and adherens junctions at the interface of CSCs-lineage-committed cells and supporting cells. The undifferentiated state of CSCs is coupled with the expression of alpha(4)-integrin, which colocalizes with the alpha(2)-chain of laminin and fibronectin. CSCs divide symmetrically and asymmetrically, but asymmetric division predominates, and the replicating CSC gives rise to one daughter CSC and one daughter committed cell. By this mechanism of growth kinetics, the pool of primitive CSCs is preserved, and a myocyte progeny is generated together with endothelial and smooth muscle cells. Thus, CSCs regulate myocyte turnover that is heterogeneous across the heart, faster at the apex and atria, and slower at the base-midregion of the ventricle.

Animals↗
PubMed2002

Residual HIV-1 disease in seminal cells of HIV-1-infected men on suppressive HAART: latency without on-going cellular infections.

BACKGROUND: HIV-1-infected men on suppressive highly active antiretroviral therapy (HAART) have a reduction of viral replication in vivo, but HIV-1 RNA is still detectable by certain ultrasensitive reverse transcriptase-PCR assays in blood plasma. Replication-competent virus can also be isolated from both peripheral blood mononuclear cells (PBMC) and seminal cells of these patients. Despite HAART, on-going in vivo infection of HIV-1-seropositive patients' PBMC was demonstrated by the detection of episomal HIV-1 moieties, known as HIV-1 two-long terminal repeat (2-LTR) DNA circles. METHODS: The present study analyzes whether new cellular infections occur in vivo in seminal cells of HIV-1-infected men on suppressive HAART. PBMC and seminal cells were isolated from a cohort of HIV-1-seropositive men taking suppressive HAART (< 50 copies HIV RNA/ml blood plasma). Viral growth assays were performed in vitro, as well as semi-quantitative PCR to detect HIV-1 2-LTR circular DNA in PBMC and seminal mononuclear cells. RESULTS: Viral growth in vitro was demonstrated in 16 out of 28 (57%) patients' PBMC, and in five patients' seminal cells (18%). Although 18 patients' PBMC were positive for HIV-1 2-LTR DNA circles, importantly, 2-LTR circular DNA was not detected in any semen sample, even when replication-competent HIV-1 virus had been recovered from a patient's seminal cells by viral co-culture assays. CONCLUSIONS: The current study suggests that in HIV-1-infected men treated with suppressive HAART, new cellular infections occur in PBMC, but that new infections do not take place in seminal cells in vivo. Thus, these findings suggest that mainly latent HIV-1 occurs in seminal cells of men on suppressive HAART, which may be a compartment-specific mechanism of residual HIV-1 disease.

Antiretroviral Therapy, Highly Active↗
PubMed1995

Identification of the mouse cytomegalovirus genomic region affecting major histocompatibility complex class I molecule transport.

Mouse cytomegalovirus (MCMV) functions expressed at the beginning of the early phase of the viral replication cycle interfere with the major histocompatibility complex (MHC) class I-restricted pathway of antigen presentation (M. J. Reddehase, M. R. Fibi, G. M. Keil, and U. H. Koszinowski, J. Virol. 60:1125-1129, 1986; M. Del Val, K. Münch, M. J. Reddehase, and U. H. Koszinowski, Cell 58:305-315, 1989). Nascent MHC class I heavy chains associate with beta 2-microglobulin and peptide, but the assembled trimolecular complex is retained in the endoplasmatic reticulum/cis-Golgi compartment (M. Del Val, H. Hengel, H. Häcker, U. Hartlaub, T. Ruppert, P. Lucin, and U. H. Koszinowski, J. Exp. Med. 176:729-738, 1992). To locate the responsible genomic region, the cytoplasmic retention of MHC class I molecules after injection of MCMV DNA was tested. The function was mapped to the HindIII E fragment. A recombinant MCMV deletion mutant delta MS94.5 lacking 15.8 kb in HindIII-E was constructed. Restoration of MHC class I molecule maturation and recognition of antigenic peptides by cytolytic T lymphocytes during the first hours of the early phase in mutant virus-infected cells proved the correct location to a 6.8-kb region in the HindIII E fragment. At later stages of the early phase, membrane-resident MHC class I molecules and cytolytic T lymphocyte recognition disappeared in delta MS94.5 mutant virus-infected cells. These results demonstrate that more than one early-gene function of MCMV affects the MHC class I pathway of antigen presentation. The redundant MHC class I-reactive functions target the transport of MHC class I molecules at different steps.

3T3 Cells↗
PubMed2005

A new equivalence based metric for predictive check to qualify mixed-effects models.

The main objective of any modeling exercise is to provide a rationale for effective decision making during drug development. The aim of the current simulation experiment was to evaluate the properties of predictive check as a covariate model qualification technique and, more importantly, to introduce and evaluate alternative criteria to qualify models.Original concentration-time profiles (yod) were simulated using a 1-compartment model for an intravenous drug administered to 25 men and 25 women. The typical clearance for male subjects (TVCLm) was assumed to be 5-fold higher than that for female subjects (TVCLf). Fifty such trials under the same design were generated randomly. Predictive check was used as the model qualification tool to study predictive performance of true (males not equal females) and false (males = females) models in the context of maximum likelihood estimation. For each yod, 200 replications were generated to study the properties of a discrepancy variable, a statistic that depends on the model, and a test statistic, a statistic that does not depend on the model. Several qualification criteria were evaluated in assessing predictive performance, such as, predictive p-value (Pp), probability of equivalence (peqv), and probability of rejecting the null hypothesis (data = model) using the Kolmogorov-Smirnov test (pks). The Pp value was calculated using sum of squared errors as a discrepancy variable. For both of the models, the Pp values uniformly ranged between 0 and 1. The pattern of Pp values suggests that qualification of the false model is unlikely. For both of the models, the range of peqv is about 0.95 to 1.0 for concentration at 0.5 hours. However, this is not the case for the concentration at 4 hours, which is primarily dependent on the clearance. The false model (0.35 to 0.50) has poor predictive performance compared with the true model (0.65 to 0.80) using peqv. The pks suggests no difference in the distributions of replicated and original concentrations at all of the time points for both of the models. Discrepancy variables cannot aid in rejecting false models, whereas the use of a test statistic can aid in rejecting false models. However, selection of an informative test statistic is challenging. As far as the qualification criteria are considered, the equivalence-based comparison of a test statistic is more informative than a significance-based comparison. No convincing evidence exists in the literature demonstrating the added advantages of predictive check as a routine model qualification tool over the existing tools, such as diagnostic plots or mechanistic reasoning. However, when a model is to be used for designing a trial, it should at least be able to regenerate the data used to build the model. In such cases, predictive check might offer insights into potential inconsistencies.

Humans↗
PubMed2003

Peptides inhibitory for the transcriptional regulatory function of human papillomavirus E2.

PURPOSE: Human papillomavirus (HPV) infections are associated with cervical neoplasia. Cellular and viral proteins are known to interact with the papillomavirus E2 protein to initiate transcription and DNA replication in the HPV life cycle. Our aim was to identify peptides that bind to the HPV16 E2 protein and thereby inhibit its ability to alter the transcriptional activity of other genes. EXPERIMENTAL DESIGN: The HPV16 E2 protein was expressed and purified to near homogeneity in bacteria. We screened a phage display library of random peptides for ones that bound to HPV16 E2 protein. Among the isolated phage clones, we found that tryptophan-rich peptide sequences appeared repetitively in successive cycles of phage library panning. Replacement of the tryptophan amino acids in these dodecapeptides reduced the degree to which these peptides bound to the E2 protein. These E2-binding peptides were tested for their ability to inhibit the transcriptional regulatory function of E2 in a test cell line, which contained an E2 gene and a luciferase reporter gene driven by an E2-dependent transcriptional promoter. RESULTS: Delivery of four of the E2 binding peptides into the intracellular compartment of the test cell line resulted in suppression of the E2-dependent luciferase expression. Deletion of the tryptophan residues from these peptides reduced their E2 binding and their ability to suppress E2-dependent luciferase expression in the test cell line. CONCLUSIONS: These results suggest a strategy for the development of chemical inhibitors of E2-dependent transcription of viral genes in HPV-infected cells as an approach to the therapy of chronic HPV infections.

Cells, Cultured↗