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At least 1,567 records · Page 87Linked to original sources
PubMed2003

Molecular cloning and functional analysis of zebrafish high-density lipoprotein-binding protein.

High-density lipoprotein-binding protein (HBP) plays a pivotal role in the endocrine regulation of both lipids and cholesterol. This first study of the zebrafish (Danio rerio) HBP gene in a piscine provides information on the complex molecular events that regulates lipid and cholesterol functions in fish, and allows a comparison with starvation and hormonal regulation. One identical zebrafish HBP cDNA clone was obtained from a 24-h-old zebrafish cDNA library. Zebrafish HBP is composed of 1273 amino acids as residues. The 1273-aa of HBP has 87.8% and 87.0% similarities to human and chicken HBP, respectively. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis showed that HBP is highly expressed in the 36 h of the developmental stage after fertilization as compared to other stages. As to tissue-specific expression, the HBP is highly expressed in the fin, liver and ovary. In the starvation experiment, results show significant differences between the control group and the group after 3-week starvation. After injecting GH, IGF-I, IGF-II or insulin, no significant differences were shown between the control and the experimental groups. These results suggest that in vivo HBP expression is not regulated by the insulin family or by growth hormone, but other factors present during the starvation may down- or up-regulate the HBP. Although the exact function of the HBP is unknown, its high expression in the liver and ovary suggests a role for this molecule in the cumulative efficiency of fish intake of food or lipid transfer; these results can possibly be applied to aquaculture in the near future.

Animals↗
PubMed2006

Genomic and functional analysis of the IncP-9 naphthalene-catabolic plasmid NAH7 and its transposon Tn4655 suggests catabolic gene spread by a tyrosine recombinase.

The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.

Bacterial Proteins↗
PubMed2001

Predicting mastery level on a large-scale standardized patient test: a comparison of case and instrument score-based models using discriminant function analysis.

Clinical skills assessments have traditionally been scored via experts' ratings of examinee performance. However, this approach to scoring may be impractical in a large-scale context due to logistical and cost considerations as well as the increased probability of rater error. The purpose of this investigation was therefore to identify, using discriminant analysis, weighted score-based models that maximize the accuracy with which mastery level can be estimated for examinees taking a nationally administered standardized patient test. Additionally, the accuracy with which the resulting classification functions can be applied to predict mastery level for a cross-validation sample of examinees was also examined. Results suggest that it might be feasible to implement an automated scoring procedure in a cost-effective manner while still retaining the important facets of the decision-making process of expert raters. Cost-benefit, test development and psychometric implications of these results are important and discussed in the full paper.

Clinical Competence↗
PubMed1986

Functional analysis of the murine IgH enhancer: evidence for negative control of cell-type specificity.

We have carried out a mutational analysis of the mouse IgH enhancer. Consistent with previous reports, deletions extending from either the 5' side or the 3' side of the enhancer fail to reveal distinct boundaries which define enhancer function in lymphoid cells. Interestingly, internal point mutations and deletions within the "enhancer core" regions fail to identify any necessary functional role for these conserved elements. When tested in CV1 cells, which do not normally respond to the IgH enhancer, certain deletions exhibit significant enhancer activity. We take these findings to indicate that the functional domains of the IgH enhancer are complex and that cell type specificity is defined in part by negative factors present in non-lymphoid cells.

Animals↗
PubMed2002

Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice.

Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.

Animals↗
PubMed2001

Functional analysis of the influenza A virus cRNA promoter and construction of an ambisense transcription system.

While influenza A viral RNA is known to act as a template for the synthesis of both viral mRNA and complementary cRNA, the latter has been observed so far only to function as an intermediate in replication and give rise to progeny vRNA molecules. Here it is shown that the cRNA promoter is also capable of initiating viral mRNA synthesis, similar to vRNA-promoted transcription adhering to the cap-snatching mode of primer recruitment. Detection of cRNA promoted transcription required an inversion of the reporter gene coding sequence plus relocation of the viral polyadenylation signal. Construction of cRNA promoter variants through RNA polymerase I reverse genetics allowed us to determine the RNA polymerase-associated, base-paired conformation in a reporter gene read-out system. It again turned out to adhere to the "corkscrew" model, similar, but slightly different in its binding interactions from the corresponding vRNA conformation. The observation of two transcription reactions, initiated in either direction from influenza vRNA and cRNA template molecules, allowed us to construct bicistronic, ambisense RNA molecules for simultaneous expression of two proteins from a single segment of viral RNA.

Cell Line↗
PubMed2000

Therapeutic potential of chimeric anti-(ganglioside GD3) antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis.

KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore, we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the nude mouse model.

Animals↗
PubMed2004

Functional analysis in serum from atypical Hemolytic Uremic Syndrome patients reveals impaired protection of host cells associated with mutations in factor H.

A subgroup of patients with the most severe form of the Hemolytic Uremic Syndrome (HUS) presents mutations in the complement regulatory protein factor H. The functional analyses of the factor H mutant proteins purified from some of these patients have shown a specific defect in the capacity to control complement activation on cellular surfaces. Here, we show that these factor H-related complement regulatory defects can be detected in the patients' serum with a simple hemolytic assay. Data obtained from HUS patients and control individuals indicate that this assay is a useful tool for the molecular diagnosis of factor H-related HUS.

Animals↗
PubMed2001

Functional analysis of BRCA1 C-terminal missense mutations identified in breast and ovarian cancer families.

Germline mutations in the breast and ovarian cancer susceptibility gene BRCA1 are responsible for the majority of cases involving hereditary breast and ovarian cancer. Whereas all truncating mutations are considered as functionally deleterious, most of the missense variants identified to date cannot be readily distinguished as either disease-associated mutations or benign polymorphisms. The C-terminal domain of BRCA1 displays an intrinsic transactivation activity, and mutations linked to disease predisposition have been shown to confer loss of such activity in yeast and mammalian cells. In an attempt to clarify the functional importance of the BRCA1 C-terminus as a transcription activator in cancer predisposition, we have characterized the effect of C-terminal germline variants identified in Scandinavian breast and ovarian cancer families. Missense variants A1669S, C1697R, R1699W, R1699Q, A1708E, S1715R and G1738E and a truncating mutation, W1837X, were characterized using yeast- and mammalian-based transcription assays. In addition, four additional missense variants (V1665M, D1692N, S1715N and D1733G) and one in-frame deletion (V1688del) were included in the study. Our findings demonstrate that transactivation activity may reflect a tumor-suppressing function of BRCA1 and further support the role of BRCA1 missense mutations in disease predisposition. We also report a discrepancy between results from yeast- and mammalian-based assays, indicating that it may not be possible to unambiguously characterize variants with the yeast assay alone. We show that transcription-based assays can aid in the characterization of deleterious mutations in the C-terminal part of BRCA1 and may form the basis of a functional assay.

Adult↗
PubMed2000

Structure-function analysis of hepatitis C virus envelope-CD81 binding.

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease. We have recently found that the large extracellular loop (LEL) of human CD81 binds HCV. This finding prompted us to assess the structure-function features of HCV-CD81 interaction by using recombinant E2 protein and a recombinant soluble form of CD81 LEL. We have found that HCV-E2 binds CD81 LEL with a K(d) of 1.8 nM; CD81 can mediate attachment of E2 on hepatocytes; engagement of CD81 mediates internalization of only 30% of CD81 molecules even after 12 h; and the four cysteines of CD81 LEL form two disulfide bridges, the integrity of which is necessary for CD81-HCV interaction. Altogether our data suggest that neutralizing antibodies aimed at interfering with HCV binding to human cells should have an affinity higher than 10(-9) M, that HCV binding to hepatocytes may not entirely depend on CD81, that CD81 is an attachment receptor with poor capacity to mediate virus entry, and that reducing environments do not favor CD81-HCV interaction. These studies provide a better understanding of the CD81-HCV interaction and should thus help to elucidate the viral life cycle and to develop new strategies aimed at interfering with HCV binding to human cells.

Amino Acid Sequence↗
PubMed1996

Molecular studies on bromovirus capsid protein. II. Functional analysis of the amino-terminal arginine-rich motif and its role in encapsidation, movement, and pathology.

The N-terminal region of the brome mosaic bromovirus (BMV) coat protein (CP) contains an arginine-rich motif that is conserved among plant and nonplant viruses and implicated in binding the RNA during encapsidation. To elucidate the functional significance of this conserved motif in the BMV CP, a series of deletions encompassing the arginine-rich motif was introduced into a biologically active clone of BMV RNA3, and their effect on replication, encapsidation, and infection in plants was examined. Analysis of infection phenotypes elicited on Chenopodium quinoa revealed the importance of the first 19 N-proximal amino acids of BMV CP in encapsidation and pathogenicity. Inoculation of C. quinoa with three viable variants of BMV RNA3 lacking the first 11, 14, and 18 N-terminal amino acids of the CP resulted in the development of necrotic local lesions and restricted the spread of infection to inoculated leaves. Progeny analysis from symptomatic leaves revealed that, in each case, virus accumulation was severely affected by the introduced mutations and each truncated CP differed in its ability to package genomic RNA. In contrast to these observations in C. quinoa, none of the CP variants was able to establish either local or systemic infections in barley plants. The intrinsic role played by the N-terminal arginine-rich motif of BMV CP in packaging viral RNAs and the interactions between the host and the truncated CPs in modulating symptom expression and movement are discussed.

Amino Acid Sequence↗
PubMed1996

Transfer function analysis of central arc of aortic baroreceptor reflex in rabbits.

While electrically stimulating the aortic depressor nerve (ADN) pseudorandomly, we recorded renal sympathetic nerve activity (RSNA) and systemic arterial pressure (SAP) in 19 alpha-chloralose-anesthetized rabbits with sinoaortic denervation. From the recorded signals, we determined the transfer functions from ADN stimulation by a pseudorandom binary sequence to RSNA [HCMD.RSNA(f)] and to SAP [HCMD.SAP(f)]. The modulus of HCMD.RSNA(f) was flat over 0.0122-0.8 Hz, whereas the phase lag increased linearly with frequency. Thus the central transduction appeared not to modify the relative amplitude of the signals from the baroreflex afferents but to provide a fixed time delay (approximately 400 ms). In contrast, the modulus of HCMD.SAP(f) decreased precipitously toward high frequencies, and the degree of the phase lag was larger than that of HCMD.RSNA(f). We conclude that 1) the transfer property of the central are does not significantly modify the relative amplitude of the frequency components of the baroreflex afferents but provides a fixed time delay and 2) the frequency independence of the modulus of the transfer property is not preserved when the analysis is extended to SAP.

Animals↗
PubMed2026

Functional analysis of stem-loop structures within the SARS-CoV-2 5' untranslated region using a plasmid-based reporter system.

The 5' untranslated region (5'UTR) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains highly conserved stem-loop structures that regulate viral gene expression. This study investigated the functional contributions of selected 5'UTR stem-loop elements to reporter gene expression using a plasmid-based mammalian expression system. Five constructs were tested using a non-integrating plasmid: the wild-type (WT) 5'UTR fused to GFP under the CMV promoter, and four deletion variants (&#x394;B, &#x394;C, &#x394;D, and &#x394;E) corresponding to deletions of stem-loop 4 (SL4), SL4.5, SL5, and SL5a, respectively. Following transfection into HEK293 cells, GFP fluorescence was quantified using a fluorescence microplate reader, and relative GFP transcript abundance was assessed by RT-qPCR. Deletion of SL4 (&#x394;B) resulted in marked reduction in both fluorescence and relative transcript abundance compared to WT construct, indicating substantially reduced reporter gene expression. In contrast, deletion of SL4.5, SL5, or SL5a did not produce the pronounced reduction observed for &#x394;B, although descriptive RT-qPCR analysis indicated differences in relative transcript abundance among these variants. Statistical analysis of fluorescence data demonstrated significant differences among constructs (one-way ANOVA, p&#x2009;<&#x2009;0.05). Because the reporter assay was based on plasmid expression, the observed differences likely reflect combined contributions from transcription, transcript abundance, RNA stability, and translation rather than translation alone. These findings demonstrate that the SL4 region contributes substantially to reporter gene expression in this experimental system, whereas the remaining stem-loop regions examined exert comparatively modest effects. This study provides additional insight into the functional organization of the SARS-CoV-2 5'UTR and establishes a framework for future investigations aimed at distinguished the transcriptional, post-transcriptional, and translational contributions of individual RNA structural elements.

5' Untranslated Regions↗
PubMed2001

Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription.

Human annexin A5 is a ubiquitous protein implicated in diverse signal transduction processes associated with cell growth and differentiation, and its gene regulation is an important component of this function. Promoter transcriptional activity was determined for a wide 5' portion of the human annexin A5 gene, from bp -1275 to +79 relative to the most 5' of several discrete transcription start points. Transfection experiments carried out in HeLa cells identified the segment from bp -202 to +79 as the minimal promoter conferring optimal transcriptional activity. Two canonical Sp1 sites in the immediate 5' flanking region of a CpG island were required for significant transcription. Strong repressive activity in the distal promoter region between bp -717 to -1153 was attributed to the presence of an endogenous retroviral long terminal repeat, homologous with long terminal repeat 47B. The downstream sequence from bp position +31 to +79 in untranslated exon 1 was also essential for transcription, as its deletion from any of the plasmid constructs abolished activity in transfection assays. Electrophoretic mobility-shift assays, Southwestern-blot analysis and affinity chromatography were used to identify a protein doublet of relative molecular mass 35 kDa that bound an octanucleotide palindromic sequence in exon 1. The DNA cis-element resembled an E-box, but did not bind higher molecular mass transcription factors, such as upstream stimulatory factor or activator protein 4. The discovery of a downstream element crucial for annexin A5 gene transcription, and its interaction with a potentially novel transcription factor or complex, may provide a clue to understanding the initiation of transcription by TATA-less, multiple start site promoters.

Amino Acid Motifs↗
PubMed1995

Functional analysis of the phosphoprotein PII (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942.

The PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942 signals the cellular N status by being phosphorylated or dephosphorylated at a seryl residue. Here we show that the PII-modifying system responds to the activity of ammonium assimilation via the glutamine synthase-glutamate synthase pathway and to the state of CO2 fixation. To identify possible functions of PII in this microorganism, a PII-deficient mutant was created and its general phenotype was characterized. The analysis shows that the PII protein interferes with the regulation of enzymes required for nitrogen assimilation, although ammonium repression is still detectable in the PII-deficient mutant. We suggest that the phosphorylation and dephosphorylation of PII are part of a complex signal transduction network involved in global nitrogen control in cyanobacteria. In this regulatory process, PII might be involved in mediating the tight coordination between carbon and nitrogen assimilation.

Bacterial Proteins↗