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PubMed1995

Modification of a xylanase cDNA isolated from an anaerobic fungus Neocallimastix patriciarum for high-level expression in Escherichia coli.

A Neocallimastix patriciarum xylanase cDNA with the core coding sequence essentially identical to xynA was isolated and modified for high-level expression in Escherichia coli. The xylanase cDNA was truncated into individual catalytic domains, which were modified at the N-terminus. These modified xylanases were synthesised as non-fusion proteins under the control of the tac promoter. High-level expression was obtained with the modified domain II construct, accounting for approx. 25% of total cellular protein. However, with the same vector and expression cassette, expression levels of constructs containing domain I or domains I and II fused in tandem were very low. RNA analysis revealed that the striking difference in expression levels of these three constructs was not due to transcription efficiency, but was mainly related to transcript stability. Further analysis of the domain II construct revealed that the high-level expression of the domain II xylanase was largely attributed to the presence of a favourable N-terminal coding sequence, as mutation at the N-terminus of the domain II dramatically reduced the expression level. The modified domain II xylanase produced in E. coli had a specific activity of 1229 U mg-1 protein at pH 7 and 50 degrees C without purification. The availability of a recombinant fungal xylanase with high specific activity and in high yield offers a potentially attractive source of xylanase for industrial applications.

Animals↗
PubMed1995

Galvanic vestibular stimulation for analysis of postural adaptation and stability.

Human postural dynamics was investigated in 12 normal subjects by means of a force platform recording body sway, induced by bipolar transmastoid galvanic stimulation of the vestibular nerve and labyrinth. The model adopted was that of an inverted segmented pendulum, the dynamics of postural control being assumed to be reflected in the stabilizing forces actuated by the feet as a result of complex muscular activity subject to state feedback of body sway and position. Time-series analysis demonstrates that a transfer function from stimulus to sway-force response with specific parameters can be identified. In addition, adaptation to the vestibular stimulus is demonstrated to exist, and we describe this phenomenon using quantification in terms of a postural adaptation time constant in the range of 40-50 s. The results suggest means to evaluate adaptive behavior and postural control in the erect human being which may be useful in the rehabilitation of individuals striving to regain upright stance.

Adaptation, Physiological↗
PubMed2002

Hydroxynonenal inactivates cathepsin B by forming Michael adducts with active site residues.

Oxidation of plasma low-density lipoprotein (oxLDL) generates the lipid peroxidation product 4-hydroxy-2 nonenal (HNE) and also reduces proteolytic degradation of oxLDL and other proteins internalized by mouse peritoneal macrophages in culture. This leads to accumulation of undegraded material in lysosomes and formation of ceroid, a component of foam cells in atherosclerotic lesions. To explore the possibility that HNE contributes directly to the inactivation of proteases, structure-function studies of the lysosomal protease cathepsin B have been pursued. We found that treatment of mouse macrophages with HNE reduces degradation of internalized maleyl bovine serine albumin and cathepsin B activity. Purified bovine cathepsin B treated briefly with 15 microM HNE lost approximately 76% of its protease activity and also developed immunoreactivity with antibodies to HNE adducts in Western blot analysis. After stabilization of the potential Michael adducts by sodium borohydride reduction, modified amino acids were localized within the bovine cathepsin B protein structure by mass spectrometric analysis of tryptic peptides. Michael adducts were identified by tandem mass spectrometry at cathepsin B active site residues Cys 29 (mature A chain) and His 150 (mature B chain). Thus, covalent interaction between HNE and critical active site residues inactivates cathepsin B. These results support the hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE.

Aldehydes↗
PubMed1995

Mutational analysis of human U6 RNA: stabilizing the intramolecular helix blocks the spliceosomal assembly pathway.

U6 RNA undergoes several conformational transitions during the spliceosome cycle: after the interaction with U4, the singular form of U6 is converted into the U4-U6 base-paired form, and within the spliceosome, the U4-U6 duplex isomerizes into the active U6-U2 conformation. The secondary structure of the singular form contains an extended 3' stem-loop, the upper part of which (intramolecular helix) most likely reforms in the spliceosome. We have previously shown in the mammalian splicing complementation system that the loop and the three adjacent, highly conserved base pairs of the intramolecular helix function during both the U4-U6 interaction and the first step of splicing. Here we demonstrate that the balanced stability of the lower, less conserved part of the 3' stem-loop is also critical for U4-U6 interaction; however, no specific splicing function could be detected in this region. The analysis of the heterologous interaction between mammalian U4 snRNP and yeast U6 RNA derivatives suggests that there are--in addition to the 3' loop and the stability of the intramolecular helix--specific sequence determinants in the 3' terminal domain of U6 that are important for efficient U4/U6 snRNP assembly.

Base Composition↗
PubMed2001

Modification of the Fc region of a primatized IgG antibody to human CD4 retains its ability to modulate CD4 receptors but does not deplete CD4(+) T cells in chimpanzees.

Keliximab, a Primatized IgG1 CD4 mAb, was reconfigured to an IgG4 antibody. The gamma4 constant region was further modified by substituting glutamic acid for serine at position 235 in the CH2 domain (IgG4-E), to remove residual binding to Fcgamma receptors, and substitution of serine with proline at position 228 in the hinge region (IgG4-PE) for greater stability. Pharmacokinetic analysis in rats gave a t(1/2) of approximately 4 days for IgG4-E and 9 days for IgG4-PE, consistent with a greater stability of the IgG4-PE molecule. The effects on T cell subsets were assessed in chimpanzees given escalating doses of IgG4-PE: 0.05 mg/kg on Day 16, 1.5 mg/kg dose on Day 43, and 15 mg/kg on Day 85. Receptor modulation was observed at the two highest doses, but no depletion of T cells at any dose. The in vitro and in vivo results demonstrate the potential of this IgG4-PE mAb for use in human trials.

Amino Acid Substitution↗
PubMed1992

Life events, personality, and physical risk factors in recent-onset duodenal ulcer. A preliminary study.

To clarify the interactions between stressful life events and other risk factors in the development of duodenal ulcer disease, we studied 33 patients with active ulcer, symptomatic for less than or equal to 6 months and untreated during the previous year, using the Paykel Interview for stressful life events, the Minnesota Multiphasic Personality Inventory (MMPI), and Zung's Anxiety and Depression scales. MMPIs were abnormal in 64% of the patients, and 61% showed some degree of depression. The 16 patients whose symptoms had been preceded by severe stress and more pathological MMPIs, especially on paranoia and dependency scales; were more depressed; and had used less nonsteroidal anti-inflammatory drugs than those without (p less than 0.05). They were somewhat more likely to be single, to be of low social class, and to have recently increased use of cigarettes, alcohol, or coffee (though their absolute level of alcohol consumption was low). Anxiety levels did not differ between stress and nonstress groups. Correspondence analysis yielded four clusters of patients, characterized by (a) alcohol/cigarette use, personality disorder, chronicity; (b) early onset, neurosis; (c) depression, life events; (d) late onset, psychosocial stability. The analysis changed little according to whether life events were or were not considered in cluster formation. We conclude that ulcer patients who become ill under stress from a distinct subgroup, that depressed mood and stress-related increases in use of alcohol and cigarettes may mediate between stress and ulcer formation, and that life events are a quantitatively minor factor in ulcerogenesis.

Adult↗
PubMed1992

Complexation of steroid hormones with cyclodextrin derivatives: substituent effects of the guest molecule on solubility and stability in aqueous solution.

The inclusion complexation of homologous derivatives of steroid hormones with cyclodextrins and 2-hydroxypropyl-beta-cyclodextrin (2-HP-beta-CD) was investigated with regard to underlying structure-interaction relationship. The interaction was studied by phase solubility analysis and stabilization effects of complex formation with 2-HP-beta-CD. The solubilizing and stabilizing abilities of 2-HP-beta-CD were generally more effective for testosterone derivatives than for estradiol esters. Within a homologous series of steroid hormones, the steepest linear solubility isotherms were found for 17-methyl and 3-methyl derivatives. The solubilization of steroid esters by 2-HP-beta-CD depended on the structure and length of the ester side chain. The interaction of 2-HP-beta-CD with the steroids was hindered by long-chain fatty acid ester groups. With increasing length of the side chain, a decline of the isotherms occurred and the phase solubility behavior changed from linear to exponential. Contrary to expectations, benzoylation of steroids considerably decreased the guest-host interaction. The observed rates of degradation of the steroid esters were significantly reduced by 2-HP-beta-CD, depending on the chain length, and correlated well with the order found in phase solubility analysis. The degradation showed no deviations from pseudo-first-order kinetics, and the degradation mechanism was not changed because of complexation. The results suggest that interaction of 2-HP-beta-CD with steroid esters involves the ester functions of the prodrugs and is more suitable for unsubstituted guest molecules.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed2004

Impact of spatial heterogeneity on a predator-prey system dynamics.

This paper deals with the study of a predator-prey model in a patchy environment. Prey individuals moves on two patches, one is a refuge and the second one contains predator individuals. The movements are assumed to be faster than growth and predator-prey interaction processes. Each patch is assumed to be homogeneous. The spatial heterogeneity is obtained by assuming that the demographic parameters (growth rates, predation rates and mortality rates) depend on the patches. On the predation patch, we use a Lotka-Volterra model. Since the movements are faster that the other processes, we may assume that the frequency of prey and predators become constant and we would get a global predator-prey model, which is shown to be a Lotka-Volterra one. However, this simplified model at the population level does not match the dynamics obtained with the complete initial model. We explain this phenomenom and we continue the analysis in order to give a two-dimensional predator-prey model that gives the same dynamics as that provided by the complete initial one. We use this simplified model to study the impact of spatial heterogeneity and movements on the system stability. This analysis shows that there is a globally asymptotically stable equilibrium in the positive quadrant, i.e. the spatial heterogeneity stabilizes the equilibrium.

Animals↗
PubMed1984

Stability of large systems.

We use the May-Wigner Stability Theorem (Geman (1984) preprint, Brown University; Hastings (1984) preprint, Hofstra University), to study the Lyapunov and structural stability of "real" large systems. Here are our new main results. For large systems which satisfy certain natural scaling relations (Harrison, Am. Natur., 113 (1979) 659; May (1979) Blackwell Scientific, Oxford), Lyapunov stability tends to increase with increasing complexity. However, at least one aspect of structural stability decreases: both competitive and cooperative effects can rapidly destabilize such a system. Finally, we observe that random matrices which satisfy the hypotheses and stability criterion of the May-Wigner theorem are asymptotically of the form 'rotation followed by multiplication by lambda,lambda less than 1'. This allows an easy analysis of the effects of noise in these systems. We conclude by briefly discussing applications to analysis of stability of systems such as the world economy, power networks, and the immune system.

Cybernetics↗
PubMed2003

Overview of stability study designs.

Stability requirements for the worldwide registration of pharmaceutical products have changed dramatically in the past few years. A series of guidelines on the design, conduct, and data analysis of stability studies of pharmaceuticals have been published by the International Conference on Harmonization (ICH); however, the statistical discussion on study design is limited. In this paper, stability designs including full, bracketing, and matrixing designs will be exemplified. The statistical aspects of these stability designs will be discussed in relation to the recent ICH guidelines. The statistical and regulatory considerations on the selection of stability design will also be presented. The stability study should be well designed so the shelf life of the drug product can be estimated with a high degree of accuracy and precision. Several commonly used criteria for design comparison will be presented. Finally, a case study is presented to illustrate the potential problems of an overreduced design from the statistical and regulatory perspectives.

Chemistry, Pharmaceutical↗
PubMed2002

Structure-function analysis of the glucose-6-phosphate transporter deficient in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), a 10 transmembrane domain endoplasmic reticulum protein. To date, 69 G6PT mutations, including 28 missenses and 2 codon deletions, have been identified in GSD-Ib patients. We previously characterized 15 of the missense and one codon deletion mutations using a pSVL-based expression assay. A lack of sensitivity in this assay limited the discrimination between mutations that lead to loss of function and mutations that leave a low residual activity. We now report an improved G6PT assay, based on an adenoviral vector-mediated expression system and its use in the functional characterization of all 30 codon mutations found in GSD-Ib patients. Twenty of the naturally occurring mutations completely abolish microsomal G6P uptake activity while the other 10 mutations, including 5 previously characterized ones, partially inactivate the transporter. This information should greatly facilitate genotype-phenotype correlation. We also report a structure-function analysis of G6PT. In addition to the 3 destabilizing mutations reported previously, we now show that the G50R, C176R, V235del, G339C and G339D mutations also compromise the G6PT stability. Mutation analysis of the amino-terminal domain of G6PT shows that it is required for optimal G6P uptake activity. Finally, we show that degradation of both wild-type and mutant G6PT is inhibited by a potent proteasome inhibitor, lactacystin, demonstrating that G6PT is a substrate for proteasome-mediated degradation.

Acetylcysteine↗
PubMed1984

Stability of union after tibial shaft fracture. Analysis by a non-invasive technique.

The stability of union following the conservative treatment of tibial shaft fractures has been examined in 157 patients by a non-invasive method. With this technique it is possible to ascertain when the fragments are united and whether the strength of union is sufficient for full weight-bearing without protection. The mean time required for union was 14.0 +/- 9.2 weeks, with a range of 4 to 48 weeks. In 31 cases union was judged to be delayed; in 22 of these, intended operations were avoided because repeated stability determinations indicated progressive union. Of nine fracture variables examined, the only ones which significantly affected the time required to achieve union were the age and the weight of the patient. Irrelevant factors were the type and level of the fracture, the energy of trauma, soft-tissue injury and the presence of multiple injuries.

Adolescent↗
PubMedDate not supplied

Medical screening using periodic spirometry for detection of chronic lung disease.

Approximately half of a worker population may benefit from the addition of a longitudinal comparison of their spirometry results, over only using annual comparisons with a cross-sectional LLN. The ATS recommendation of 15% for year-to-year changes appears to be essentially equivalent to a longitudinal LLN method based on the SEE. Therefore, a practical method for longitudinal interpretations is to establish a baseline value for a worker's FEV1 through several initial spirometric examinations. The FEV1 longitudinal LLN is calculated by taking 85% of this baseline value minus the expected decline over the time period based on the individual's age (e.g., for individuals older than 35 years at their initial examination, one approach is to use 30 mL/year times the number of years of follow-up). However, before any final classification is rendered, the data should be reviewed for stability. This analysis demonstrates that longitudinal spirometry adds sensitivity to spirometry screening efforts. The spirometry examinations should probably be performed annually in order to detect survey biases and determine the stability of the FEV1 measurements. If spirometry results indicate that someone has crossed either the longitudinal or the cross-sectional LLN, intervention activities should be initiated for that individual. As new data and studies become available, these suggested procedures will need to be revised-particularly estimates for the expected annual decline in FEV1.

Bias↗
PubMed2005

Conversion analysis for mutation detection in MLH1 and MSH2 in patients with colorectal cancer.

CONTEXT: The accurate identification and interpretation of germline mutations in mismatch repair genes in colorectal cancer cases is critical for clinical management. Current data suggest that mismatch repair mutations are highly heterogeneous and that many mutations are not detected when conventional DNA sequencing alone is used. OBJECTIVE: To evaluate the potential of conversion analysis compared with DNA sequencing alone to detect heterogeneous germline mutations in MLH1, MSH2, and MSH6 in colorectal cancer patients. DESIGN, SETTING, AND PARTICIPANTS: Multicenter study with patients who participate in the Colon Cancer Family Registry. Mutation analyses were performed in participant samples determined to have a high probability of carrying mismatch repair germline mutations. Samples from a total of 64 hereditary nonpolyposis colorectal cancer cases, 8 hereditary nonpolyposis colorectal cancer-like cases, and 17 cases diagnosed prior to age 50 years were analyzed from June 2002 to June 2003. MAIN OUTCOME MEASURES: Classification of family members as carriers or noncarriers of germline mutations in MLH1, MSH2, or MSH6; mutation data from conversion analysis compared with genomic DNA sequencing. RESULTS: Genomic DNA sequencing identified 28 likely deleterious exon mutations, 4 in-frame deletion mutations, 16 missense changes, and 22 putative splice site mutations. Conversion analysis identified all mutations detected by genomic DNA sequencing--plus an additional exon mutation, 12 large genomic deletions, and 1 exon duplication mutation--yielding an increase of 33% (14/42) in diagnostic yield of deleterious mutations. Conversion analysis also showed that 4 of 16 missense changes resulted in exon skipping in transcripts and that 17 of 22 putative splice site mutations affected splicing or mRNA transcript stability. Conversion analysis provided an increase of 56% (35/63) in the diagnostic yield of genetic testing compared with genomic DNA sequencing alone. CONCLUSIONS: The data confirm the heterogeneity of mismatch repair mutations and reveal that many mutations in colorectal cancer cases would be missed using conventional genomic DNA sequencing alone. Conversion analysis substantially increases the diagnostic yield of genetic testing for mismatch repair mutations in patients diagnosed as having colorectal cancer.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Degradation of transcription factor IRF-1 by the ubiquitin-proteasome pathway. The C-terminal region governs the protein stability.

Interferon regulatory factor-1(IRF-1) is a transcriptional activator of interferon genes and interferon-inducible genes. It has been shown that IRF-1 functions not only as a regulator of the interferon-responsive system but also as a regulator of cell growth and apoptosis. In addition, it is known that IRF-1 is a short-lived protein, but the mechanism that regulates its stability has not yet been clarified. Here, we show that IRF-1 is degraded via the ubiquitin-proteasome pathway. IRF-1 protein degradation in HeLa and NIH3T3 cells was inhibited by treatment with proteasome-specific inhibitors. Overexpression of IRF-1 protein and ubiquitin in COS7 cells revealed specific multiubiquitination of IRF-1. Although the full-length IRF-1 was unstable, IRF-1 mutants with C-terminal truncations larger than 39 amino acids were found to be almost stable, suggesting that the 39-residue C-terminal region controls the stability of IRF-1. Further analysis of the stability of a green fluorescent protein-fusion protein containing the 39-residue C-terminal region of IRF-1 showed that this C-terminal region confers instability on green fluorescent protein, a normally stable protein, suggesting that this region functions as a protein-degradation signal. Taking the results together, it can be concluded that the 39-residue C-terminal region is necessary and sufficient to control the stability of the IRF-1 protein.

3T3 Cells↗
PubMed2002

Pharmaceutical development of a parenteral lyophilized formulation of the antimetastatic ruthenium complex NAMI-A.

This paper describes the development of a stable pharmaceutical dosage form for NAMI-A, a novel antimetastatic ruthenium complex, for Phase I testing. NAMI-A drug substance was characterized using several spectrometric and chromatographic techniques. In preformulation studies, it was found that NAMI-A in aqueous solution was not stable enough to allow sterilization by moist heat. The effect of several excipients on the stability of the formulation solution was investigated. None of them provided sufficient stability to allow long-term storage of an aqueous solution of NAMI-A. Therefore, a lyophilized product was developed. Five different formulations were prepared and subjected to thermogravimetric (TG) analysis and stability studies at various conditions for 1 year. Minimal degradation during the production process is achieved with a formulation solution of pH 3-4. Of the acids tested, only hydrochloric acid (HCl 0.1 mM) both stabilized the formulation solution and was compatible with the lyophilized product. This product was stable for at least 1 year when stored at -20 degrees C, 25 degrees C/60% relative humidity (RH) and 40 degrees C/75% RH, and was also photostable.

Antineoplastic Agents↗
PubMed1995

Wide complex tachycardia.

Wide complex tachycardias present diagnostic challenges for emergency physicians. The history, physical examination, and ECG provide information required to arrive at the correct diagnosis. When a previous history of heart disease exists, VT should be suspected; however, no single clinical feature is sufficiently reliable for distinguishing VT from SVT. Patients with VT may tolerate their dysrhythmias for several hours and maintain hemodynamic stability. ECG analysis is the most useful process in differentiating SVT and VT. Characteristics suggestive of VT include evidence of AV dissociation, QRS duration of longer than 0.16 seconds, and QRS axis between -90 degrees +/- 180 degrees. Predictive QRS morphologic criteria also have been established for VT. A four-step approach to ECG analysis has been reported to accurately identify patients with VT, but prospective validation in an ED setting is lacking. The initial approach to treating patients with wide QRS tachycardias depends on hemodynamic stability. Until the identify of a dysrhythmia is certain, consider all patients to be suffering from VT. Unstable patients require immediate cardioversion. Acute treatment of stable patients includes lidocaine or procainamide. Adenosine is appropriate when wide QRS SVT is the diagnosis, and it also has been used as a diagnostic aid to identify dysrhythmias. Reports of complications with the use of adenosine as a diagnostic agent have not yet appeared but may occur after sufficient numbers of cases have accumulated. Magnesium sulfate may be useful in refractory cases of VT and torsades de pointes. Chronic treatment of patients prone to VT may include complex pharmacotherapy and AICDs. Development of new class III agents and enhancement of AICD technology may result in improved patient outcomes and the availability of more choices for emergent therapy of wide QRS tachycardias.

Decision Trees↗
PubMed2001

Chromosome analysis after ex vivo expansion of CD34(+) cells from human cord blood.

Ex vivo expansion of cord blood hematopoietic progenitors is an attractive way to prepare a sufficient number of transplantable cells for cord blood transplantation in adult patients. The expanded cells need to have genetic stability. Karyotypic analysis of the expanded cells from cord blood CD34(+) cells by 7-day culture with stem cell growth factor, interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor, and erythropoietin was performed. Several-fold increases in total cell number and CFU-GM were 186.7 +/- 62.1 and 27.1 +/- 9.4 (mean +/- standard deviation of data from 6 samples), respectively. Karyotypes were analyzed in 600 expanded cells in total, and all of them showed normal karyotypes. This observation suggests that multifactor supported expansion of cord blood cells may not induce karyotypic abnormality, although a limited number of ex vivo expanded cells were tested.

Antigens, CD34↗