Biomedical subjects

T Rose

Publications and source records attributed to T Rose.

At least 37 records · Page 2Linked to original sources

PubMed2001

Characterization of an IL-2 mimetic with therapeutic potential.

Human interleukin-2 (IL-2) interacts with two types of functional receptors (IL-2R alpha betagamma and IL-2R betagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. IL-2 is also used in different clinical trials aimed at improving the treatment of some cancers and the recovery of CD4 lymphocytes by HIV patients. The therapeutic index of IL-2 is limited by various side effects dominated by the vascular leak syndrome. We have shown that a chemically synthesised fragment of the IL-2 sequence can fold into a helical tetramer likely mimicking the quatemary structure of an hemopoietin. Indeed, peptide p1-30 (containing amino acids 1 to 30, including the sequence corresponding to the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T-cell lines expressing human IL-2R beta, whereas shorter versions of the peptide lack helical structure and are inactive. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8 low lymphocytes and natural killer cells, which constitutively express IL-2R beta. A significant IFN-gamma production is also detected following p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys) which is likely unable to induce vascular leak syndrome remains capable to generate LAK cells like the original p1-30 peptide. Altogether our data suggest that p1-30 has therapeutic potential.

CD8-Positive T-Lymphocytes↗

PubMed2000

Complementation between dimeric mutants as a probe of dimer-dimer interactions in tetrameric dihydrofolate reductase encoded by R67 plasmid of E. coli.

The effect of mutations on the interactions between dimers in R67 dihydrofolate reductase (R67 DHFR), a tetrameric enzyme conferring resistance to trimethoprim, was investigated by site-directed mutagenesis combined with phenotypic, enzymatic, and biochemical analysis. Some 14 mutants at two positions involved in a hydrogen bond between dimers were constructed. All were shown to be dimers. However, complementation between pairs of dimeric mutated proteins resulted in the restoration of the enzymatic activity and heterotetramer formation. A combinatorial approach was set up to create efficiently such heterotetramers and identify the complementing pairs of mutations. A dozen of such pairs were found. An accurate method was set up to measure the association of the complementing dimers in a "quasi-isologous" heterotetramer and used to study the effects of mutations and pH on the association. Thus, the pair of proteins bearing respectively the S59A and H62L mutations was shown to form heterotetramers with catalytic properties close to those of the wild-type protein. Its association was as strong as that of the wild-type protein at cytoplasmic pH (6. 5), and was more stable at lower pH values.A double-mutant protein bearing simultaneously the S59A and H62L mutations was produced and analyzed. Its association was weakened by 1.2 kcal/mol as compared to the wild-type enzyme at pH 6.5 but was insensitive to pH. Comparing the energy of association between dimers in the wild-type protein, the heterotetramer and the double mutant allowed us to dissect the effects of the pH and of the molecular context on a subset of interactions between the R67 DHFR subunits.

Amino Acid Substitution↗

PubMed2000

Identification of a binding site for quaternary amines in factor Xa.

In the process of characterizing the Na(+)-binding properties of factor Xa, a specific inhibition of this enzyme by quaternary amines was identified, consistent with previous observations. The binding occurs with K(i) in the low millimolar range, with trimethylphenylammonium (TMPA) showing the highest specificity. Binding of TMPA inhibits substrate hydrolysis in a competitive manner, does not inhibit the binding of p-aminobenzamidine to the S1 pocket, and is positively linked to Na(+) binding. Inhibition by TMPA is also seen in thrombin and tissue plasminogen activator (tPA), though to a lesser extent compared to factor Xa. Computer modeling using the crystal structure of factor Xa suggests that TMPA binds to the S2/S3 specificity sites, with its hydrophobic moiety making van der Waals interactions with the side chains of Y99, F174, and W215, and the charged amine coupling electrostatically with the carboxylates of E97. Site-directed mutagenesis of factor Xa, thrombin, and tPA confirms the predictions drawn by docking calculations and reveal a dominant role for residue Y99. Binding of TMPA to factor Xa is drastically (25-fold) reduced by the Y99T replacement. Likewise, the Y99L substitution compromises binding of TMPA to tPA. On the other hand, the affinity of TMPA is enhanced 4-fold in thrombin with the substitution L99Y. The identification of a binding site for quaternary amines in factor Xa has a bearing on the rational design of selective inhibitors of this clotting enzyme.

Amines↗

PubMed2000

De novo 46,XX,t(6;7)(q27;q11;23) associated with severe cardiovascular manifestations characteristic of supravalvular aortic stenosis and Williams syndrome.

Supravalvular aortic stenosis may present as an isolated finding or as part of Williams syndrome. Williams syndrome is a contiguous gene syndrome associated with neurodevelopmental and multisystemic manifestations caused by hemizygous deletion at 7q11.23. We report on the prenatal and histopathological findings in a patient with a chromosome translocation involving the Williams syndrome critical region. The initial abnormality on fetal ultrasound was hydrops fetalis detected at 30 weeks and echocardiography showed narrowing of the aorta and the pulmonary arteries. The baby died shortly after delivery and an autopsy revealed diffuse tubular thickening with luminal narrowing of the aorta, aortic branches, and the pulmonary arteries. Histopathology showed dysplasia of the media with reduced elastic content and "cartwheel" arrangement of collagen, elastic, and muscle fascicles. The karyotype was 46,XX,t(6;7)(q27;q11.23). Three signals were detected using the Oncor fluorescent in situ hybridization probe for elastin-Williams syndrome (WSCR) suggesting that the break in chromosome 7 is within the elastin-Williams gene. This patient is of special interest because of the prenatal presentation and the chromosomal translocation involving the elastin-Williams syndrome locus.

Aortic Valve Stenosis↗

PubMed2000

The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1-30 (containing amino acids 1-30, covering the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1-30 activates Tyk2, thus suggesting that IL-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma production is also detected after p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1-30 peptide. Altogether, our data suggest that p1-30 has therapeutic potential.

Amino Acid Sequence↗

PubMed2000

Structure and dynamics of the pore of inwardly rectifying K(ATP) channels.

Inwardly rectifying K(+) currents are generated by a complex of four Kir (Kir1-6) subunits. Pore properties are conferred by the second transmembrane domain (M2) of each subunit. Using cadmium ions as a cysteine-interacting probe, we examined the accessibility of substituted cysteines in M2 of the Kir6.2 subunit of inwardly rectifying K(ATP) channels. The ability of Cd(2+) ions to inhibit channels was used as the estimate of accessibility. The distribution of Cd(2+) accessibility is consistent with an alpha-helical structure of M2. The apparent surface of reactivity is broad, and the most reactive residues correspond to the solvent-accessible residues in the bacterial KcsA channel crystal structure. In several mutants, single channel measurements indicated that inhibition occurred by a single transition from the open state to a zero-conductance state. Analysis of currents expressed from mixtures of control and L164C mutant subunits indicated that at least three cysteines are required for coordination of the Cd(2+) ion. Application of phosphatidylinositol 4,5-diphosphate to inside-out membrane patches stabilized the open state of all mutants and also reduced cadmium sensitivity. Moreover, the Cd(2+) sensitivity of several mutants was greatly reduced in the presence of inhibitory ATP concentrations. Taken together, these results are consistent with state-dependent accessibility of single Cd(2+) ions to coordination sites within a relatively narrow inner vestibule.

Adenosine Triphosphate↗

PubMed2000

[Retropatellar autologous cartilage-bone transplantation. A treatment option in severe cartilage damage].

In a 32 years old professional handball layer, the micro-traumata typical of this game led to retropatellar cartilage lesion, which became sympthomatic following an distortion-impact trauma. Since the patient remains symptomatic after initial conservative treatment as well as arthroscopic chondroplastic, osteo-chondral transplantation was performed. There was a good functional result 6 months after surgery.

Adult↗

PubMed2000

Sequence determinants of function and evolution in serine proteases.

Animals↗

PubMed2000

Two distinct lineages of macaque gamma herpesviruses related to the Kaposi's sarcoma associated herpesvirus.

BACKGROUND: KSHV, Kaposi's sarcoma-associated herpesvirus, is a necessary cofactor for the development of Kaposi's sarcoma (KS). We have previously reported KSHV-related DNA sequences in retroperitoneal fibromatosis (RF) tissue from two species of macaque. The putative herpesvirus was called RFHV for RF-associated herpesvirus. These data suggested that KSHV is a human representative of a larger family of primate herpesviruses. OBJECTIVE: To identify and characterize other members of a putative family of KSHV-related herpesviruses in macaques in order to obtain information on the evolutionary history of KSHV infection in humans. STUDY DESIGN: Lymphoid tissue cells and blood leukocytes from rhesus-, cynomolgus- and pigtailed-macaques were tested for the presence of unknown herpesviruses using degenerate primer-driven PCR amplification. The sequences obtained were compared against known herpesvirus sequences. RESULTS: We have identified new herpesvirus DNA sequences in each of the three macaque species. Sequence comparisons indicate that these new viruses are most related to each other and form a separate phylogenetic lineage within the gamma herpesviruses. Screening of PBMC from Indonesian-origin quarantine animals suggests that these viruses (MGV, macaque gamma virus) are species-specific, and highly prevalent in the wild. They are readily cultured in vivo, and share a common tissue tropism with the previously identified RFHV. CONCLUSIONS: MGV and RFHV represent two independent introductions of an ancestral gamma herpesvirus into macaque precursors.

Amino Acid Sequence↗

PubMed2000

A randomized, placebo-controlled study to evaluate the efficacy of 12-month famciclovir treatment in patients with chronic hepatitis B e antigen-positive hepatitis B.

We conducted a randomized, placebo-controlled clinical study evaluating famciclovir (500 mg 3 times daily and 1.5 g once daily) for 1 year (6 months post-treatment follow-up) in patients with chronic hepatitis B e antigen (HBeAg)-positive hepatitis B virus (HBV) infection. The study was conducted in 80 centers in North America, Europe, and Australia/New Zealand. A total of 417 patients with histologically documented chronic hepatitis B (histologic activity index [HAI] 9.5-11.0) received famciclovir (500 mg 3 times daily or 1.5 g once daily) or placebo. Famciclovir 500 mg 3 times daily significantly reduced HBV DNA and median HAI scores versus placebo. By week 8, median HBV DNA decreased from 1,645 to 283 MEq/mL (famciclovir 500 mg 3 times daily) and from 1,147 to 304 MEq/mL (famciclovir 1.5 g once daily), while increasing for placebo (1,617 to 1,685 MEq/mL). Median change in HBV DNA at the end of therapy was -76% (famciclovir 500 mg 3 times daily; P <.01) and -60% (famciclovir 1.5 g once daily; P =.25) versus -37% for placebo. Median change in HAI was -1.5 points (famciclovir 500 mg 3 times daily; P =.02) and -1.0 point (famciclovir 1.5 g once daily; P =.35) and zero for placebo. Fifty percent of patients receiving famciclovir 500 mg 3 times daily (P =.07) and 43% receiving 1.5 g once daily (P =.41) experienced >/=2 points improvement in HAI versus 37% for placebo. Nine percent of patients treated with famciclovir 500 mg 3 times daily underwent anti-HBeAg seroconversion with undetectable HBV DNA at end of follow-up versus 3% in the placebo group (P =.05). Famciclovir was well tolerated; the incidence of post-treatment alanine transaminase (ALT) elevations was comparable with placebo. In conclusion, famciclovir 500 mg 3 times daily gave modest suppression of viral replication, but translated into significant histologic improvement in median HAI score at 1 year.

2-Aminopurine↗

PubMed2000

In vitro identification of the cytochrome P450 isoform responsible for the metabolism of alpha-dihydroergocryptine.

1. The in vitro metabolism of alpha-dihydroergocryptine (DHEC, Almirid), an ergot-derived dopamine agonist for the treatment of Parkinson's disease, has been studied in cultured cell lines following incubation with DHEC. Human hepatocytes as well as two sets of metabolically competent cell lines expressing one single human cytochrome P450 (1A1, 1A2, 1B1, 2A6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4) were used. 2. Mono- and dihydroxy metabolites of DHEC could only be detected in the culture media of the cell line expressing human cytochrome CYP3A4. The same metabolites were found in the media of cultured human hepatocytes derived from three different donors. After 24-h incubation with 1 microM DHEC, approximately 60% mono- and approximately 20% dihydroxy metabolites were detected, i.e. approximately 80% of DHEC was metabolized. Further, DHEC demonstrated an inhibitory effect on CYP3A4-mediated testosterone metabolism and additionally could induce CYP3A4 and CYP2E1 mRNA when added at 10 microM to cultured human hepatocytes. 3. The data suggest that DHEC metabolism in humans is primarily mediated by the CYP3A4 isoform. The results are in accordance with findings derived from other ergot alkaloids.

Aged↗

PubMed2000

Reducing nosocomial pressure ulcers in an acute care facility.

In 1996, a nursing committee at an acute care facility organized the first pressure ulcer point prevalence survey for that hospital. In 1996, hospital-acquired pressure ulcers were 90 percent of the predicted prevalence rate; in 1997, the rate dropped to 59 percent of the predicted prevalence and in 1998, to 53 percent of the predicted prevalence. The severity index decreased markedly from 291 (1996) to 98 (1997) then to 62 (1998). These improvements are attributed to the purposeful addition of multidimensional interventions, including best practices and research-based protocols, to prevent and treat nosocomial pressure ulcers.

Benchmarking↗

PubMed2000

CardioOp: an integrated approach to teleteaching in cardiac surgery.

INTRODUCTION/PURPOSE: The complexity of cardiac surgery requires continuous training, education and information addressing different individuals: physicians (cardiac surgeons, residents, anaesthesiologists, cardiologists), medical students, perfusionists and patients. Efficacy and efficiency of education and training will likely be improved by the use of multimedia information systems. Nevertheless, computer-based education is facing some serious disadvantages: 1) multimedia productions require tremendous financial and time resources; 2) the obtained multimedia data are only usable for one specific target user group in one specific instructional context; 3) computer based learning programs often show deficiencies in the support of individual learning styles and in providing individual information adjusted to the learner's individual needs. In this paper we describe a computer-system, providing multiple re-use of multimedia-data in different instructional sceneries and providing flexible composition of content to different target user groups. TOOLS AND METHODS: The ZYX document model has been developed, allowing the modelling and flexible on-the-fly composition of multimedia fragments. It has been implemented as a DataBlade module into the object-relational database system Informix Dynamic Server and allows for presentation-neutral storage of multimedia content from the application domain, delivery and presentation of multimedia material, content based retrieval, re-use and composition of multimedia material for different instructional settings. Multimedia data stored in the repository, that can be processed and authored in terms of our identified needs is created by using a next generation authoring environment called CardioOP-Wizard. High-quality intra-operative video is recorded using a video-robot. Difficult surgical procedures are visualized with generic and CT-based 3D-animations. RESULTS: An on-line architecture for multiple re-use and flexible composition of media data has been established. The system contains the following instructional applications (prototypically implemented): a multimedia textbook on operative techniques, an interactive module for problem based-training, a module for creation and presentation of lectures and a module for patient information. Principles of cognitive psychology and knowledge management have been employed in the program. These instructional applications provide information ranging from basic knowledge at the beginner's level, procedural knowledge for the advanced level to implicit knowledge for the professional level. For media-annotation with meta-data a metainformation system, the CardioOP-Clas has been developed. The prototype focuses on aortocoronary bypass grafting and heart transplantation. CONCLUSION: The demonstrated system reflects an integrated approach in terms of information technology and teaching by means of multiple re-use and composition of stored media-items to the individual user and the chosen educational setting on different instructional levels.

Computer Simulation↗

PubMed1999

The C-terminal sequence encodes function in serine proteases.

Serine proteases of the chymotrypsin family have maintained a common fold over an evolutionary span of more than one billion years. Notwithstanding modest changes in sequence, this class of enzymes has developed a wide variety of substrate specificities and important biological functions. Remarkably, the C-terminal portion of the sequence in the protease domain accounts fully for this functional diversity. This portion is often encoded by a single exon and contains most of the residues forming the contact surface in the active site for the P1-P3 residues of the substrate, as well as domains responsible for the modulation of catalytic activity. The evolution of serine proteases was therefore driven by optimization of contacts made with the unprimed subsites of the substrate and targeted a relatively short portion of the sequence toward the C-terminal end. The dominant role of the C-terminal sequence should facilitate the identification of function in newly discovered genes belonging to this class of enzymes.

Animals↗

PubMed1999

Unexpected crucial role of residue 225 in serine proteases.

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

Amino Acid Sequence↗

PubMed1999

[Acute and chronic osteochondral lesions of the talus].

Osteochondral lesions of the talus are often not or delayed diagnosticated. They mostly are related to traumatic lesions of the upper ankle joint, where by traumatic distorsion are most common among. The degenerative change classified as osteochondrosis dissecans tali shows in the most cases also an originally traumatic generic. The classification by osteochondrosis dissecans tali in four stages by Berndt and Harty is nowadays accepted. These can be distinguished by means of several diagnostic methods. They are regarded as state-of-the-art of therapy and prognosis. Basically for diagnostic purposis the conventional X-ray in two planes with the right feet-allignement is sufficient. Despite the fact, that Szintigraphy and Computertomography for particular questions are the right tools, the MRI gives the highest amount of information. In the case of low levels of defects (Stage I and II) the conservative therapy is appropriate. In more serious cases (Stage III and IV) the surgical intervention has to be used. Beside the open surgical approach the arthroscopy has a growing importancy. In 60% of cases good long term results can be achieved. Beside some advantage there are some limits compared to the arthrotomy. The efficiency of new therapeutic methods like bone-cartilage-transplantation and chondrocyte-transplantation compared to the conventional wound toilet, microfractures and fragmentrefixation has to be proved by long term studies. The results depend on the stage and the localisation of the osteochondral lesion. In the developed stages III and IV surgical actions as wound toilet, removement of dissecate with microfragmentation respectivaly refixation are indicated, since conservative therapy methods lead undoubtedly to worse results. Generally mostly good and very good results connected with painless and weight bearing could be achieved.

Acute Disease↗

PubMed1997

Patient capacity and judicial decisionmaking.

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Data Collection↗

PubMed1996

Non-isotopic competitive reverse transcription polymerase chain reaction coupled with high performance liquid chromatography to measure beta 2-receptor messenger RNA in the human heart.

We describe an application of competitive reverse transcription-polymerase chain reaction (PCR) coupled with HPLC for quantification of beta 2-adrenergic receptor messenger RNA (mRNA) in human atrial tissues removed during cannulation for cardiopulmonary bypass operations. We constructed an internal standard which was reverse transcribed in different concentrations together with constant levels of cellular RNA and subsequently PCR amplified. The competitor RNA shows the same beta 2-adrenergic receptor primer sequences as the cellular mRNA but yields a different-sized product. This allows resolution of the amplified copy DNA (complementary DNA, cDNA) fragments with a specific HPLC column. The concentration of beta 2-adrenergic receptor mRNA is derived from the ratio between the peak intensities corresponding to the amplified competitor and target products. We assessed the imprecision, accuracy and sensitivity of the method. Concentrations of beta 2-adrenergic receptor mRNA of 22.7 +/- 15.2 x 10(6) molecules per micrograms total RNA in patients treated with beta 2-antagonists were not significantly different from control patients showing 16.8 +/- 9.9 x 10(6) beta 2-adrenergic receptor mRNA molecules per microgram total RNA (Mean +/- SD). Competitive reverse transcription PCR is a highly specific, non-radioactive procedure for quantification of beta 2-adrenergic receptor mRNA and simultaneously other gene expression levels of interest in atrial tissue specimens and may therefore be used to advance our understanding of heart muscle disease.

Adrenergic beta-Antagonists↗