[A new apparatus for separate and simultaneous functional analysis of both lungs].
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Humans↗
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[Model studies for the estimation of error in the "radiocardiographic function analysis" using Cr 51].
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Chromium Isotopes↗
Impact of mycophenolate mofetil dose posttransplantation on 12-month renal function: analysis of the MOST database.
INTRODUCTION: Mycophenolate mofetil (MMF) has greatly reduced the risk of acute rejection episodes (ARE) after renal transplantation, but dose reductions/withdrawals could jeopardize long-term results. METHODS: The MOST database of "de novo" patients treated with MMF at month 1 and functioning grafts at month 12 were divided into 2 groups: groups 1, 2 g MMF at month 1 and month 12; and group 2, 2 g MMF at month 1 but MMF <2 g at month 12 to evaluate renal function glonerular filtration rate (GFR). RESULTS: In this study, 1136 patients were receiving 2 g MMF at month 1. On month 12, 645 were on 2 g (56.8%, group 1) and 431 were on <2 g (43.2%, group 2). Group 1 included younger recipients of younger donors with fewer patients with delayed graft function (DGF). Group 1 showed more ARE during month 1 and more patients who received induction. Mean Neoral daily doses at month 1/month 12 were 5.3/3.0 and 5.3/3.1 mg/kg in group 1 and group 2, respectively (P = .05 at month 12). GFR in group 1 and group 2 were 59.06 (CI 57.10-60.60) and 53.81 (CI 52-55.7) at month 1 (P < .001); 63.7 (CI 62.1-65.30) and 55.9 (CI 54.1-57.7) mL/min*1.73 m(2) at month 12 (P < .001). The mean increases in GFR between month 1 and month 12 were 4.64 and 1.94 mL/min*1.73 m(2), respectively (P < .05). A multivariate analysis also included 795 patients from the "maintenance" patient database with retrospective detailed information. The following parameters were highly predictive for good renal function at month 12: donor age younger than 60 years, recipient age younger than 60 years, immediate graft function, 12-month MMF dose = 2 g, absence of CMV infection, and 12-month Neoral dose <3 mg/kg/d. CONCLUSIONS: Maintenance of MMF dose at 2 g/d during the first year appears to facilitate the attainment of optimal renal function at 12-months after kidney transplantation.
Analysis of Variance↗
Ensemble averaging and multiple statistical testing of EMG activities of cyclically repeated body motions. A tool for muscle function analysis in experimental and clinical orthopaedics.
Coupled with suitable computerized signal recording and processing methods surface electromyography can be a powerful tool for the analysis of muscle activity in specific body movements. It can be used for this purpose in experimental and in clinical diagnostic orthopaedics as well as in physiotherapy. We describe in this paper a motion analysis system comprising this feature. It has been employed for the diagnosis of the basic angular kinematics and muscle function in human gait and other cyclically repeatable movements of the human locomotive system. Changes in the temporal characteristics of the movements and the muscle activity due to changed physical or experimental conditions can be systematically investigated this way. Such changes can be the result of surgical and/or conservative orthopaedic therapy, a long term physiotherapeutic program, or modified walking conditions as in experimental orthopaedics. They are displayed and validated by signal ensemble averaging and subsequent multiple statistical testing (e.g. by a suitably adapted Bonferroni criterion). The efficiency of the system is demonstrated by an exemplary gait analysis of selected kinematic and muscular effects caused by an experimental simulation of a leg length inequality.
Algorithms↗
Distribution and functional analysis of memory antiviral CD8 T cell responses in HIV-1 and cytomegalovirus infections.
In the present study, we have investigated the anatomic distribution and the function of different populations of HIV-1- and cytomegalovirus (CMV)-specific memory CD8 T cells. The different populations of virus-specific memory CD8 T cells were distinguished on the basis of the expression of CD45RA and CCR7, and the composition of HIV-1- and CMV-specific memory CD8 T cell pools were compared in subjects with chronic HIV-1 and CMV co-infection. The distribution of HIV-1-specific CD8 T cells was similar between blood and lymph node. However, CMV-specific CD8 T cells were accumulated predominantly in the blood away from the lymphoid tissue. The majority (>70%) of HIV-1- and CMV-specific CD8 T cells in both blood and lymph node had a phenotype, e.g. CCR7-, typical of effector T cells. HIV-1-specific memory CD8 T cells were mostly (>80%) pre-terminally differentiated cells, e.g. CD45RA-CCR7-, in both blood and lymph node while 30-50% of CMV-specific CD8 T cells were terminally differentiated, e.g. CD45RA+CCR7-. Therefore, consistently with studies in mice, antigen-specific effector memory CD8 T cells accumulate predominantly in the target organ of the pathogen in humans, and the differences in the composition of HIV-1- and CMV-specific CD8 T cell pools were also present in the lymphoid tissue. A substantial proportion (30-40%) of virus-specific CD8+CCR7+ T cells produced IFN-gamma. Thus, indicating that the expression of CCR7 does not provide a clear-cut separation of memory CD8 T cells with distinct functional capacities. Taken together, these results provide further advances in the characterization of human memory CD8 T cells.
Adult↗
Functional analysis of Cdc42 in actin filament assembly, epithelial morphogenesis, and cell signaling during Drosophila development.
Cdc42, a member of the Rho family of GTP binding proteins, functions in the formation of polarized actin structures, in elongation of cell shape, and in cell signaling. Although genetic mutations previously have not been available in multicellular organisms, studies have attempted to discern Cdc42 functions in organisms, including Drosophila, using dominant active or interfering alleles. Here, for the first time, we examine the functions of Cdc42 in developing tissues using loss-of-function mutations in the Drosophila Cdc42 gene. We find that Cdc42(-) epithelial cells fail to elongate into a columnar cell shape and cannot maintain a monolayered epithelial structure. In contrast to previous studies, we find no requirement for Cdc42 in cell division or in activation of the Jun N-terminal kinase pathway. In addition, Cdc42 function is not required for cytoplasmic actin filament assembly in the nurse cells during oogenesis, although it may facilitate this process. Furthermore, our results indicate that Cdc42 plays a role in intercellular interactions between the germ line and the somatic follicle cells. These results confirm the role of Cdc42 in actin filament assembly and provide new insights into its functions in epithelial morphogenesis and regulating intercellular signaling events.
Actins↗
In silico structural and functional analysis of the human cytomegalovirus (HHV5) genome.
The open reading frames of human cytomegalovirus (human herpesvirus-5, HHV5) encode some 213 unique proteins with mostly unknown functions. Using the threading program, ProCeryon, we calculated possible matches between the amino acid sequences of these proteins and the Protein Data Bank library of three-dimensional structures. Thirty-six proteins were fully identified in terms of their structure and, often, function; 65 proteins were recognized as members of narrow structural/functional families (e.g. DNA-binding factors, cytokines, enzymes, signaling particles, cell surface receptors etc.); and 87 proteins were assigned to broad structural classes (e.g. all-beta, 3-layer-alphabetaalpha, multidomain, etc.). Genes encoding proteins with similar folds, or containing identical structural traits (extreme sequence length, runs of unstructured (Pro and/or Gly-rich) residues, transmembrane segments, etc.) often formed tandem clusters throughout the genome. In the course of this work, benchmarks on about 20 known folds were used to optimize adjustable parameters of threading calculations, i.e. gap penalty weights used in sequence/structure alignments; new scores obtained as simple combinations of existing scoring functions; and number of threading runs conducive to meaningful results. An introduction of summed, per-residue-normalized scores has been essential for discovery of subdomains (EGF-like, SH2, SH3) in longer protein sequences, such as the eight "open sandwich" cytokine domains, 60-70 amino acids long and having the 3beta1alpha fold with one or two disulfide bridges, present in otherwise unrelated proteins.
Amino Acid Sequence↗
Nuclease sensitivity and functional analysis of a maize histone H3 gene promoter.
A 1 kb region of a maize H3 histone gene promoter has been analysed at a structural and functional level. Micrococcal nuclease digestion of isolated nuclei showed that the promoter region is organized into nucleosomes but a zone extending over approximately one nucleosome (20 to 230 bp upstream of the TATA box) displays remarkable accessibility to digestion. Three DNase I-hypersensitive sites were found within this zone at the vicinity of consensus sequences, some of which are already known to act as cis elements. This promoter region is able to direct faithful expression of the GUS reporter gene in meristematic tissues of transgenic tobacco plants.
Base Sequence↗
In vitro morpho-functional analysis of pancreatic islets isolated from the domestic chicken.
The technique whereby islets were isolated from the pancreas of chicken and their in vitro histological and functional characterization are described in this paper. Our isolation procedure consisted of two steps: initially the pancreas removed from the chicken was perfused with a 2% solution of collagenase and enzymatic digestion was then carried out using the same solution. After this, density gradient separation was performed on the digested tissue, by means of differing Histopaque solutions: at the end of the separation, the islets were studied by light microscopy after treatment with diphenylthiocarbazone, which selectively stains beta-cells, and by scanning and transmission electron microscopy. The results reported here show the pattern closely resembled that encountered when islets were studied in situ. The beta-cells of the islets proved in vitro to preserve their functional capability of producing insulin even after stimulation with glucose or arginine.
Animals↗
Studies of rheumatoid synovial fluid lymphocytes. III. Phenotypic and functional analysis of natural killer-like cells.
Synovial fluid and peripheral blood lymphocytes from patients with rheumatoid arthritis have been compared in terms of phenotypic and functional expression of natural killer (NK) activity. Following pretreatment with the monoclonal antibodies anti-HNK-1 (anti-Leu-7) or anti-Leu-11b and complement, NK activity against K562 cells was assessed in 4-hr chromium release assays. Rheumatoid peripheral blood lymphocytes resembled those of normal individuals, in that complement-mediated lysis of Leu-11b+ cells virtually eliminated all NK activity. Complement-mediated lysis of Leu-7+ cells resulted in only a modest reduction in NK activity. Rheumatoid synovial fluid lymphocytes differed from autologous peripheral blood lymphocytes: the overall cytotoxicity was generally less and only a fraction of the NK-like activity appeared to be mediated by Leu-11b+ cells. This apparent lack of Leu-11b expression by rheumatoid synovial fluid NK-like cells could not be accounted for by blockage or alteration of the Leu-11b antigen by factors present in synovial fluid. These observations support and extend previous data showing a discordance of phenotype and function of lymphocytes in rheumatoid synovial fluid possibly representing activation of such cells within the rheumatoid joint.
Arthritis, Rheumatoid↗
Structure-function analysis of HKE4, a member of the new LIV-1 subfamily of zinc transporters.
The KE4 proteins are an emerging group of proteins with little known functional data. In the present study, we report the first characterization of the recombinant human KE4 protein in mammalian cells. The KE4 sequences are included in the subfamily of ZIP (Zrt-, Irt-like Proteins) zinc transporters, which we have termed LZT (LIV-1 subfamily of ZIP zinc Transporters). All these LZT sequences contain similarities to ZIP transporters, including the consensus sequence in transmembrane domain IV, which is essential for zinc transport. However, the new LZT subfamily can be separated from other ZIP transporters by the presence of a highly conserved potential metalloprotease motif (HEXPHEXGD) in transmembrane domain V. Here we report the location of HKE4 on intracellular membranes, including the endoplasmic reticulum, and its ability to increase the intracellular free zinc as measured with the zinc-specific fluorescent dye, Newport Green, in a time-, temperature- and concentration-dependent manner. This is in contrast with the zinc influx ability of another LZT protein, LIV-1, which was due to its plasma membrane location. Therefore we have added to the functionality of LZT proteins by reporting their ability to increase intracellular-free zinc, whether they are located on the plasma membrane or on intracellular membranes. This result, in combination with the crucial role that zinc plays in cell growth, emphasizes the importance of this new LZT subfamily, including the KE4 sequences, in the control of intracellular zinc homoeostasis, aberrations of which can lead to diseases such as cancer, immunological disorders and neurological dysfunction.
Amino Acid Sequence↗
[A renal function analysis using computed tomography. CT clearance versus 131I-hippuran clearance].
The CT clearance in a number of urological patients (39 patients, 25 with normal renal function) was studied by means of an "area under curve" (AUC) method, using a region of interest. 31 patients were given meglumine ioxithalamate and 8 patients Iopamidol as a contrast medium. Both for total and partial function there was highly significant correlation with isotope clearance with good reproducibility. The difference in the pharmacokinetics of the contrast media affected the absolute clearance values.
Contrast Media↗
Functional analysis of AeSCP-2 using gene expression knockdown in the yellow fever mosquito, Aedes aegypti.
The effect of gene expression knockdown was used to study the function of the sterol carrier protein-2 (AeSCP-2) in the yellow fever mosquito, Aedes aegypti. Injection of small double stranded AeSCP-2 RNAs into mosquito larvae resulted in the knockdown of gene products. The lack of AeSCP-2 in larvae coincided with a reduction in accumulated cholesterol in pupae, supporting the hypothesis that AeSCP-2 may be involved in cholesterol uptake in mosquito larvae. Knockdown of AeSCP-2 caused a high mortality rate in developing adult and reduced egg viability. Results from this study indicate that AeSCP-2 is important for adult development and for the viability of the eggs.
Aedes↗
Functional analysis of human MUC7 mucin gene 5'-flanking region in lung epithelial cells.
The human MUC7 gene encodes a low-molecular-mass mucin glycoprotein that functions in modulation of microbial flora in the oral cavity and respiratory tracts. MUC7 gene expression is tissue- and cell-specific, with dominant expression in salivary gland acinar cells. To begin to understand the molecular mechanisms responsible for controlling MUC7 gene expression, we analyzed the promoter activity of MUC7 5'-flanking region in a human lung epithelial cell line A549. We demonstrated that MUC7 gene is expressed constitutively in this cell line and is upregulated by TNF-alpha stimulation. The promoter activities of a 2,762-bp fragment of the human genomic DNA (-2,732/+30 bp) and its deletion series, subcloned into a luciferase reporter vector, were characterized at the basal level and under stimulation by TNF-alpha. The results indicated that the minimal functional MUC7 promoter is in the region of -138/+30 bp. This region also revealed the greatest increase in the promoter activity upon TNF-alpha stimulation. Two putative AP1-binding elements and one NF-kappaB-binding element were identified within the proximal promoter. Further analyses demonstrated that mutations of these elements dramatically reduced specific DNA-protein binding ability and reporter gene expression. AP1 elements played an essential role in the constitutive expression, while the NF-kappaB element was crucially important in the response to TNF-alpha stimulation, demonstrating that TNF-alpha activates MUC7 transcription via NF-kappaB signaling pathway.
5' Flanking Region↗
Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum).
Polyketides are a class of secondary metabolites that exhibit a vast diversity of form and function. In fungi, these compounds are produced by large, multidomain enzymes classified as type I polyketide synthases (PKSs). In this study we identified and functionally disrupted 15 PKS genes from the genome of the filamentous fungus Gibberella zeae. Five of these genes are responsible for producing the mycotoxins zearalenone, aurofusarin, and fusarin C and the black perithecial pigment. A comprehensive expression analysis of the 15 genes revealed diverse expression patterns during grain colonization, plant colonization, sexual development, and mycelial growth. Expression of one of the PKS genes was not detected under any of 18 conditions tested. This is the first study to genetically characterize a complete set of PKS genes from a single organism.
Amino Acid Sequence↗
Multislice MR perfusion imaging and regional myocardial function analysis: complimentary findings in chronic myocardial ischemia.
PURPOSE: The purpose of this study is to assess the reliability of multislice MR perfusion imaging in comparison to regional wall function and nuclear medicine and to test different qualitative and quantitative parameters for perfusion assessment. MATERIAL AND METHODS: 15 patients with chronic myocardial ischemia underwent CINE and first-pass perfusion MR imaging. Functional myocardial imaging was performed using a segmented CINE FLASH sequence and systolic myocardial wall thickening was assessed after semiautomated segmentation. MR first-pass perfusion studies were performed using a multislice saturation recovery TurboFLASH sequence. Different parameters were calculated for assessment of hypoperfused segments and results of MR imaging compared to 99mTc-SestaMIBI SPECT. RESULTS: MR perfusion imaging showed a sensitivity of 72% and a specificity of 98%. In combination with MR CINE imaging and wall thickening analysis we calculated a sensitivity of 100% and a specificity of 93%. Qualitative and quantitative perfusion parameter analysis showed significant differences between normal and hypoperfused segments for the signal intensity increase (p < 0.001), the signal intensity upslope (p < 0.001) as well as for the myocardial mean transit time (p < 0.001). CONCLUSION: The combination of systolic wall thickening analysis and myocardial perfusion can markedly improve the sensitivity of MRI in depiction of LV myocardial perfusion abnormalities. For assessment of hypoperfusion, different quantitative and qualitative parameters can be calculated showing significant differences between normal state and hypoperfusion.
Aged↗
Expression and functional analysis of SURF1 in Leigh syndrome patients with cytochrome c oxidase deficiency.
Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an autosomal recessive neurodegenerative disorder caused by mutations in SURF1. Although SURF1 is ubiquitously expressed, its expression is lower in brain than in other highly aerobic tissues. All reported SURF1 mutations are loss of function, predicting a truncated protein (hSurf1) product. Western blot analysis with anti-hSurf1 antibodies demonstrated a specific 30 kDa protein in control fibroblasts, but no protein in LS patient cells. Steady-state levels of both nuclear- and mitochondrial-encoded COX subunits were also markedly reduced in patient cells, consistent with a failure to assemble or maintain a normal amount of the enzyme complex. An epitope (FLAG)-tagged hSurf1 was targeted to mitochondria in COS7 cells and a mitochondrial import assay showed that the hSurf1 precursor protein (35 kDa) was imported and processed to its mature form (30 kDa) in a membrane potential-dependent fashion. The protein was resistant to alkaline carbonate extraction and susceptible to proteinase K digestion in mitoplasts. Mutant proteins in which the N-terminal transmembrane domain or central loop were deleted, or the C-terminal transmembrane domain disrupted, did not accumulate and could not rescue COX activity in patient cells. Co-expression of the N- and C-terminal transmembrane domains as independent entities also failed to rescue the enzyme deficiency. These data demonstrate that hSurf1 is an integral inner membrane protein with an essential role in the assembly or maintenance of the COX complex and that insertion of both transmembrane domains in the intact protein is necessary for function.
Animals↗
Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis.
Cytosine methylation is important for transposon silencing and epigenetic regulation of endogenous genes, although the extent to which this DNA modification functions to regulate the genome is still unknown. Here we report the first comprehensive DNA methylation map of an entire genome, at 35 base pair resolution, using the flowering plant Arabidopsis thaliana as a model. We find that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only approximately 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation.
Arabidopsis↗