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PubMed1995

Multivariate discriminant function analysis of neurologic, pain, and psychiatric patients with the MMPI.

The ability of the MMPI to classify five well-defined patient groups was investigated (N = 394; control, neurologic, psychiatric, chronic pain, and random). Clinical inspection and discriminant function analyses of basic clinical and research scales could not classify groups correctly, but discriminant function analyses of 37 variables loaded with CNS items (Cripe Neurologic Symptom items) correctly classified the groups with 78% overall accuracy (70% of neurologic, 62% of psychiatric, 81% of pain, 84% of controls, and 100% random). Results indicate that differential diagnosis is not possible with clinical inspection of scales, but complex statistical analysis of the MMPI is potentially useful in diagnosis and decision making. A method for applying the discriminant function analysis to individual cases is provided.

Activities of Daily Living↗
PubMed1990

Structure-function analysis of human interleukin-6. Evidence for the involvement of the carboxy-terminus in function.

C-terminally deleted analogs of human interleukin-6 (IL-6) have been constructed at the cDNA level, and after cell-free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL-6 resulted in 58% helix, 14% beta-structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL-6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% beta-structure, and 18% turn and coil.

Animals↗
PubMed1984

Construction and functional analysis of a series of synthetic RNA polymerase III promoters.

RNA polymerase III promoters were constructed by cloning chemically synthesized double stranded analogues of the box A and box B consensus sequences into suitable vectors. In contrast to approaches adopted previously for the analysis of RNA polymerase III promoters, this method has no limitation on the structure and number of variants generated, and allows critical sequences in various permutations to be studied. Furthermore, the series of synthetic polymerase III promoters created constitute a collection of point mutation variants and hence provide a powerful tool for the analysis of nucleotides essential for promoter function. The results demonstrate that these two boxes, when separated by approximately 50 base pairs, are sufficient to direct efficient transcription, and that substitution of certain nucleotides causes reduced template activity.

Base Sequence↗
PubMed1995

Well ordered crystals of a short-chain alcohol dehydrogenase from Drosophila lebanonensis: re-evaluation of the crystallographic data and rotation-function analysis.

Alcohol dehydrogenase prepared from Drosophila lebanonensis yields well ordered plate-like crystals which diffract to better than 2.3 A resolution. The crystals belong to space group P2(1) of the monoclinic system; the unit-cell dimensions are a = 65.25, b = 55.77, c = 70.02 A, alpha = 90, beta = 107.08, gamma = 90 degrees. The asymmetric unit of the crystal cell is most probably occupied by a dimer, corresponding to a packing density of 2.15 A(3) Da-L. The orientation of the non-crystallographic twofold symmetry axes is determined by analysis of a self-rotation function calculated with native intensity data.

Journal Article↗
PubMed1989

Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin.

Pseudomonas exotoxin is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation. The substitution of the cell recognition domain (domain Ia) with a growth factor such as transforming growth factor alpha (TGF alpha), creates a cell-specific cytotoxic agent, TGF alpha-PE40, which kills cells bearing epidermal growth factor (EGF) receptors. We have used TGF alpha-PE40 to define the role of sequences in domains II, Ib, and III. Various mutations were made in these domains and mutant forms of TGF alpha-PE40 expressed in Escherichia coli. Mutant proteins were then tested for their ADP-ribosylation, EGF receptor-binding, and cell-killing activities. Additionally, the amino boundary of domain III, which contains the ADP-ribosylation activity, was determined by deletion analysis. Data indicate that (i) the functional amino terminus of domain III is near amino acid 400; (ii) deletion of various regions in domain II or conversion of cysteines 265 and 268 to serines results in a loss of cytotoxicity which ranged from 10-fold to more than 150-fold, indicating that domain II is essential for full expression of cytotoxicity; (iii) deletion of the amino terminus of domain Ib results in a molecule with somewhat increased cytotoxic activity, indicating that domain Ib is not essential for the cytotoxic effect of TGF alpha-PE40; and (iv) TGF alpha-PE40, produced by denaturing and refolding of insoluble material from inclusion bodies, binds better to EGF receptors and is about 10-fold more cytotoxic to cells bearing EGF receptors than is the secreted form of soluble TGF alpha-PE40.

Adenosine Diphosphate Ribose↗
PubMed1985

Relationship between immune system and gram-negative bacteria. III. Functional analysis of human peripheral blood lymphocyte subpopulations separated by cytoadherence with Salmonella minnesota Rb.

Spontaneous adherence of bacteria to human peripheral blood mononuclear cells (PBMC) represents a useful tool for analysis of lymphocyte subsets with different functions. We have recently shown that PBMC can be divided into 2 populations based on their ability to bind Salmonella minnesota R345 (Rb) bacteria. By using these procedures, here, we provide evidence that Rb-bound and Rb-unbound PBMC populations give similar proliferative responses to phytohemagglutinin (PHA) and concanavalin A (Con A), while the pokeweed mitogen (PWM)-induced proliferative and differentiative responses are higher in the Rb-unbound than in the Rb-bound PBMC fraction. Moreover, enhanced PWM-induced responses are obtained in Rb-unbound cell cultures enriched for T4+ cells. When B (non-E rosetting) cells are cultured with purified T lymphocytes from the Rb-bound (T-Rb+) and Rb-unbound (T-Rb-) fractions, comparable PWM-induced mitogenic responses are observed. The T-Rb- population contains a higher percentage of cells expressing T4+ phenotype, and when added to B cell cultures a more elevated PWM-induced IgA, IgG and IgM synthesis is observed than in B cell cultures containing T-Rb+ cells. These results suggest that the T-Rb- fraction is enriched for T cells which help IgA, IgG and IgM responses.

B-Lymphocytes↗
PubMed1984

Construction and functional analysis of a series of synthetic RNA polymerase III promoters.

RNA polymerase III promoters were constructed by cloning chemically synthesized double-stranded analogues of the box A and box B consensus sequences into suitable vectors. In contrast to approaches adopted previously for the analysis of RNA polymerase III promoters, this method has no limitation on the structure and number of variants generated and allows critical sequences in various permutations to be studied. Furthermore, the series of synthetic polymerase III promoters created constitute a collection of point mutation variants and hence provide a powerful tool for the analysis of nucleotides essential for promoter function. The results demonstrate that these two boxes, when separated by 51 base pairs, are sufficient to direct efficient transcription and that substitution of certain nucleotides causes reduced template activity.

Base Sequence↗
PubMed1998

[The 13C-methacetin breath test for quantitative noninvasive liver function analysis with an isotope-specific nondispersive infrared spectrometer in liver cirrhosis].

BACKGROUND AND OBJECTIVE: Cytochrome-P450-dependent liver function can be measured with the 13C-methacetin breath test (MBT). This has heretofore been done with the use of a mass-spectrometer. This study was undertaken to evaluate the MBT (NDIRS) done with the isotope-selective nondispersive infrared spectrometer. PATIENTS AND METHODS: 20 healthy volunteers (ten women, ten men, aged 22-76 years) and 16 patients (ten women, six men, aged 48-71 years) with histologically confirmed liver cirrhosis (Child-Pugh stage A [n = 7], B [n = 5] or C [n = 4]) were given 13C-methacetin in 100 ml of tea after a 12-hour fasting period. Breath tests were performed before the test drink and 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 120, 150 and 180 min thereafter. The ratio of 13C to 12C was determined, as delta (/1000), and from it the maximal percentage rate (PDRmax) calculated, as well as the cumulative rate (cPDRmax) after 30, 60, 120 and 180 min (Median and 5th and 95th percentiles). RESULTS: For patients with liver cirrhosis there were significantly lower values for cPDRmax and cPDR after 30, 60, 120 and 180 min than in the healthy subjects (P < 0.002): PDR [%]/h: 3.9 (0.7-15.9) vs. 36.5 (23.1-50.0); cDPR 30 min [%]: 1.1 (-0.2-6.0) vs. 12.4 (7.6-17.1); cDPR 3 h [%]: 9.8 (-2.3-27.5) vs. 36.0 (29.9-45.1). There were significant differences among the patients, depending on their Child-Pugh staging. CONCLUSION: The MBT with the cost-effective NDIRS can reliably and noninvasively distinguish between healthy subjects and patients with liver cirrhosis. The test is therefore suitable for the quantitative analysis of liver functions.

Acetamides↗
PubMed1998

Functional analysis of a voltage-gated sodium channel and its splice variant from rat dorsal root ganglia.

Neurons of the dorsal root ganglia (DRG) express a diversity of voltage-gated sodium channels. From rat DRG we have cloned and functionally expressed a tetrodotoxin-sensitive sodium channel alpha subunit, NaCh6/Scn8a/rPN4, and a splice variant, rPN4a. Primary structure analysis shows NaCh6/Scn8a/rPN4 to be highly homologous (99%) to NaCh6 and most likely represents the same transcript. The splice variation in rPN4a is homologous in sequence and location to that of rat brain I. Tissue distribution analyzed by RT-PCR showed NaCh6/Scn8a/rPN4 to be expressed at its highest levels in rat brain, at moderate levels in spinal cord, and at lower levels in DRG, nodose ganglia, and superior cervical ganglia and to be absent from sciatic nerve, heart, and skeletal muscle. In contrast, rPN4a shows no expression in brain and low-level expression in spinal cord, whereas in DRG its expression is comparable to that of NaCh6/Scn8a/rPN4. Functional analysis of these channels expressed in Xenopus oocytes showed that NaCh6/Scn8a/rPN4 and rPN4a exhibited similar properties, with V(1/2) approximately -100 mV for steady-state inactivation and V(1/2) approximately -40 mV for activation. rPN4a recovered from inactivation significantly faster than NaCh6/Scn8a/rPN4. NaCh6/Scn8a/rPN4 was inhibited by tetrodotoxin with an IC50 approximately 1 nM. Coexpression of the beta1 subunit accelerated inactivation kinetics, but the beta2 subunit was without effect.

Alternative Splicing↗
PubMed1983

Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60src.

To study the function of pp60src, the transforming protein encoded for by Rous sarcoma virus, we have raised antibodies against synthetic oligopeptides corresponding to the primary structure of pp60src. All eight investigated peptides were immunogenic in rabbits, and four induced pp60src-specific antibodies. We screened tumor-bearing rabbit (TBR) sera for antibodies against the peptides; this revealed that five out of six of the peptides, chosen according to a high hydrophilicity plot, were related to epitopes of native pp60src, in contrast to two peptides of low hydrophilicity, which contained a cleavage site for protease. Antibodies against three of the peptides appeared to react with the kinase-active site of pp60src, as these antibodies were phosphorylated in their heavy chain upon immune precipitation. Antibodies against two of the peptides, in contrast to the others, did not precipitate pp60src when this molecule was complexed with two cellular proteins, pp50 and pp90. This observation allows speculation about the location of the pp60src site involved in the formation of this complex.

Amino Acid Sequence↗
PubMed2006

Using Caenorhabditis elegans for functional analysis of genes of parasitic nematodes.

Information on the functional genomics of Caenorhabditis elegans has increased significantly in the last few years with the development of RNA interference. In parasitic nematodes, RNA interference has shown some success in gene knockdown but optimisation of this technique will be required before it can be adopted as a reliable functional genomics tool. Comparative studies in C. elegans remain an appropriate alternative for studying the function and regulation of some parasite genes and will be extremely useful for fully exploiting the increasing parasite genome sequence data becoming available.

Animals↗
PubMed1977

Heteroantisera prepared against B lymphocytes at different stages of differentiation. II. Functional analysis of cytotoxicity to different B-cell populations.

Functional studies on heteroantisera raised against mouse B cells in rabbits suggest a correlation between thymus-independent B cells and LPS-responsive B cells, and differences in either from thymus-dependent B cells. Furthermore it seems possible to follow B-lymphocyte differentiation from antigen-uncommitted precursors to antigen committed immature B cells to mature B lymphocytes by the loss and acquisition of surface markers distinguished by these heteroantisera. Additional evidence is provided that there are distinctive antigenic differences between antibody forming cells raised from the same B-cell source after in vivo or in vitro immunization.

Animals↗
PubMed2006

Assessing normative cut points through differential item functioning analysis: an example from the adaptation of the Middlesex Elderly Assessment of Mental State (MEAMS) for use as a cognitive screening test in Turkey.

BACKGROUND: The Middlesex Elderly Assessment of Mental State (MEAMS) was developed as a screening test to detect cognitive impairment in the elderly. It includes 12 subtests, each having a 'pass score'. A series of tasks were undertaken to adapt the measure for use in the adult population in Turkey and to determine the validity of existing cut points for passing subtests, given the wide range of educational level in the Turkish population. This study focuses on identifying and validating the scoring system of the MEAMS for Turkish adult population. METHODS: After the translation procedure, 350 normal subjects and 158 acquired brain injury patients were assessed by the Turkish version of MEAMS. Initially, appropriate pass scores for the normal population were determined through ANOVA post-hoc tests according to age, gender and education. Rasch analysis was then used to test the internal construct validity of the scale and the validity of the cut points for pass scores on the pooled data by using Differential Item Functioning (DIF) analysis within the framework of the Rasch model. RESULTS: Data with the initially modified pass scores were analyzed. DIF was found for certain subtests by age and education, but not for gender. Following this, pass scores were further adjusted and data re-fitted to the model. All subtests were found to fit the Rasch model (mean item fit 0.184, SD 0.319; person fit -0.224, SD 0.557) and DIF was then found to be absent. Thus the final pass scores for all subtests were determined. CONCLUSION: The MEAMS offers a valid assessment of cognitive state for the adult Turkish population, and the revised cut points accommodate for age and education. Further studies are required to ascertain the validity in different diagnostic groups.

Adolescent↗
PubMed2006

Alcohol and injury: a risk function analysis from the Emergency Room Collaborative Alcohol Analysis Project (ERCAAP).

While emergency room (ER) studies have documented a strong association of alcohol and injury, less is known about the level of risk at which various quantities of alcohol or particular patterns of drinking place the individual for injury. Comparative risk function analyses are carried out in ER samples in seven countries that cover sites in 14 studies included in the Emergency Room Collaborative Alcohol Analysis Project (ERCAAP). Risk of injury is analyzed for the mean number of drinks consumed per day and the number of occasions in which 5 or more drinks were consumed at one time (5+) during the last year. All countries showed similar increases in injury risk to an average volume of about 2 drinks per day, with a leveling off of risk at higher average daily volumes, with the exception of Italy. Risk of injury increased to 12 or more 5+ days for the USA, Canada and Mexico, but leveled off after only 3 5+ days for Argentina and Spain. Poland showed increased risk to 30 5+ days. Similar risk curves were found for both males and females, although females were at lower risk of injury in all countries expect Spain and Poland. In low detrimental drinking pattern societies, risk curves showed higher risk for any drinking and any frequency of 5+ but at higher levels of each, risk levels decreased nearly to levels found for abstainers. Risk functions were also consistent across gender and age groups in low detrimental drinking pattern societies, with higher risk for males and those <30. For those countries with high detrimental drinking patterns, injury risk increased with volume and 5+ drinking primarily among males. This ER-based risk function analysis suggests that risk of injury increases proportionally with increased alcohol consumption at lower consumption levels, but a threshold effect is achieved at relatively low levels of mean daily consumption and higher consumption times. Risk may be culturally specific, dependent, in part, on the manner in which alcohol is used in the culture.

Adolescent↗
PubMed2006

Functional analysis of gene duplications in Saccharomyces cerevisiae.

Gene duplication can occur on two scales: whole-genome duplications (WGD) and smaller-scale duplications (SSD) involving individual genes or genomic segments. Duplication may result in functionally redundant genes or diverge in function through neofunctionalization or subfunctionalization. The effect of duplication scale on functional evolution has not yet been explored, probably due to the lack of global knowledge of protein function and different times of duplication events. To address this question, we used integrated Bayesian analysis of diverse functional genomic data to accurately evaluate the extent of functional similarity and divergence between paralogs on a global scale. We found that paralogs resulting from the whole-genome duplication are more likely to share interaction partners and biological functions than smaller-scale duplicates, independent of sequence similarity. In addition, WGD paralogs show lower frequency of essential genes and higher synthetic lethality rate, but instead diverge more in expression pattern and upstream regulatory region. Thus, our analysis demonstrates that WGD paralogs generally have similar compensatory functions but diverging expression patterns, suggesting a potential of distinct evolutionary scenarios for paralogs that arose through different duplication mechanisms. Furthermore, by identifying these functional disparities between the two types of duplicates, we reconcile previous disputes on the relationship between sequence divergence and expression divergence or essentiality.

Bayes Theorem↗
PubMed2004

Novel UBA domain mutations of SQSTM1 in Paget's disease of bone: genotype phenotype correlation, functional analysis, and structural consequences.

UNLABELLED: Three novel missense mutations of SQSTM1 were identified in familial PDB, all affecting the UBA domain. Functional and structural analysis showed that disease severity was related to the type of mutation but was unrelated to the polyubiquitin-binding properties of the mutant UBA domain peptides. INTRODUCTION: Mutations affecting the ubiquitin-associated (UBA) domain of Sequestosome 1 (SQSTM1) gene have recently been identified as a common cause of familial Paget's disease of bone (PDB), but the mechanisms responsible are unclear. We identified three novel SQSTM1 mutations in PDB, conducted functional and structural analyses of all PDB-causing mutations, and studied the relationship between genotype and phenotype. MATERIALS AND METHODS: Mutation screening of the SQSTM1 gene was conducted in 70 kindreds with familial PDB. We characterized the effect of the mutations on structure of the UBA domain by protein NMR, studied the effects of the mutant UBA domains on ubiquitin binding, and looked at genotype-phenotype correlations. RESULTS AND CONCLUSIONS: Three novel missense mutations affecting the SQSTM1 UBA domain were identified, including a missense mutation at codon 411 (G411S), a missense mutation at codon 404 (M404V), and a missense mutation at codon 425 (G425R). We also identified a deletion leading to a premature stop codon at 394 (L394X). None of the mutations were found in controls. Structural analysis showed that M404V and G425R involved residues on the hydrophobic surface patch implicated in ubiquitin binding, and consistent with this, the G425R and M404V mutants abolished the ability of mutant UBA domains to bind polyubiquitin chains. In contrast, the G411S and P392L mutants bound polyubiquitin chains normally. Genotype-phenotype analysis showed that patients with truncating mutations had more extensive PDB than those with missense mutations (bones involved = 6.05 +/- 2.71 versus 3.45 +/- 2.46; p < 0.0001). This work confirms the importance of UBA domain mutations of SQSTM1 as a cause of PDB but shows that there is no correlation between the ubiquitin-binding properties of the different mutant UBA domains and disease occurrence or extent. This indicates that the mechanism of action most probably involves an interaction between SQSTM1 and a hitherto unidentified protein that modulates bone turnover.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Morphological and functional analysis of rat hepatocyte spheroids generated on poly(L-lactic acid) polymer in a pulsatile flow bioreactor.

Liver neo-tissue suitable for transplantation has not been established. Primary rat hepatocytes were cultured on three-dimensional biodegradable polymer matrices in a pulsatile flow bioreactor with the intention of inducing tissue formation and improving cell survival. Functional and structural analysis of the hepatocytes forming liver neo-tissue was performed. Biodegradable poly(L-lactic acid) (PLLA) polymer discs were seeded with 4 x 10(6) primary rat hepatocytes each, were exposed to a pulsatile medium flow of 24 mL/min for 1, 2, 4, or 6 days and were investigated for monoethylglycinexylidine (MEGX) formation, ammonia detoxification, Cytokeratin 18 (CK18) expression, and preserved glycogen storage. Fine structural details were obtained using scanning and transmission electron microscopy. Spheroids of viable hepatocytes were formed. MEGX-specific production was maintained and ammonia removal capacity remained high during the entire flow-culture period of 6 days. CK18 distribution was normal. Periodic-acid- Schiff reaction demonstrated homogenous glycogen storage. The hepatocytes reassembled to form intercellular junctions and bile canaliculi. Functional and morphological analysis of rat hepatocytes forming spheroids in a pulsatile flow bioreactor indicated preserved and intact hepatocyte morphology and specific function. Pulsatile flow culture on PLLA scaffolds is a promising new method of hepatic tissue engineering leading to liver neo-tissue formation.

Animals↗