Structure and folding of a designed knotted protein

A very small number of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophysical characterization and X-ray crystal structure determination show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approximately 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic experiments suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy--connecting two monomers of an intertwined homodimer into a single protein chain--supports a model for evolution of knotted structures via gene duplication.     

Application of neural networks for the analysis of intravascular ultrasound and histological aortic wall appearance-an in vitro tissue characterization study

The role of tissue characterization by intravascular ultrasound (IVUS) imaging of the aortic wall has not been well established. The artificial neural networks (ANNs) are a promising tool for image classification. The aim of the study was to assess the texture correlation between matching IVUS and histologic images of the aortic wall. The computer-based discrimination of pathology within the data sets was also evaluated. In vitro IVUS images and histologic sections from 36 aortic segments were compared using texture parameters that produced the best correlation or the highest discriminative value. The images were classified as normal or abnormal with variable degrees of pathology. Tissue characterization was performed by a nearest neighbor classifier, linear discriminant analysis (LDA) and the ANN-based approach. Good agreement was observed between IVUS and the histologic reference with a correlation coefficient of r = 0.89, r = 0.76 and r = 0.71 for the three most successful texture parameters. The ANN-based approach was the most effective in discriminant analysis, with a correct classification rate of 87.5% for histologic images and 79.2% for IVUS data. The study shows that ANNs are a potentially effective tool for assessment of IVUS aortic images.     

Nonalcoholic fatty liver disease: a review of the spectrum of disease, diagnosis, and therapy

Worldwide, there is an epidemic of obesity and overweight, with two-thirds of Americans affected. A strong association exists between excessive body weight and nonalcoholic fatty liver disease (NAFLD), the most common etiology of abnormal liver function tests. Nonalcoholic fatty liver disease is a spectrum of liver disease, from a "bland" fatty infiltration to chronic hepatitis (nonalcoholic steatohepatitis or NASH), that can result in cirrhosis and organ failure. With the increasing prevalence of obesity in the world, the proportion of people affected by NAFLD is only expected to be parallel. Although primarily noted in obese individuals, NAFLD has also been associated with a number of surgical procedures, metabolic conditions, and medications. NASH is commonly underdiagnosed as most affected patients are symptom free, and routine screening is not performed. Noninvasive diagnostic testing is not sensitive in diagnosis or staging the severity of disease. Fatty infiltration and oxidative injury to the hepatocytes are believed to be the major factors behind the progression of disease from simple fatty infiltration of the liver to chronic hepatitis. Understanding the inflammatory pathways involved in NASH is a subject of extensive research. Currently, few proven treatment options exist, and controlled weight reduction is the only safe modality recommended for treatment of NASH.     

Synthesis and biological evaluation of new amino acid and dipeptide derivatives of neocryptolepine as anticancer agents

The syntheses of neocryptolepine derivatives containing an amino acid or a dipeptide at the C-9 position and their evaluation for antitumor activity in vitro and in vivo are reported. To establish the influence of an amino acid or a peptide on the physicochemical properties of 5H-indolo[2,3-b]quinoline (DiMIQ), lipophilic and hemolytic properties were investigated. Most of the compounds displayed a high antiproliferative activity in vitro and strongly inhibited growth of tumor in mice compared to cyclophosphamide. The attachment of the hydrophilic amino acid or the peptide to the hydrophobic DiMIQ increased its hydrophilic properties and decreased its hemolytic activity. The glycylglycine conjugate (7a) was the most promising derivative. It strongly inhibited the growth of the tumor in mice (at dose 50 mg kg(-1) day(-1) it inhibited the tumor growth by 46-63% on days 11-16 and by 29-43% on days 18-23) and significantly decreased hemolytic activity and lowered the in vivo toxicity compared to DiMIQ.     

Injections of botulinum A toxin for the treatment of anal fissures.

OBJECTIVE: To find out how injections of botulinum A toxin influence the healing of anal fissures. DESIGN: Retrospective study. SETTING: Medical University of Lodz, Poland. SUBJECTS: 13 patients (6 women, 7 men), mean age 49 (range 31-78), treated with injections of botulinum A toxin 50 units on either side of the anal fissure into the internal anal sphincter from May to December 1999. MAIN OUTCOME MEASURES: Complications and relapse. RESULTS: Seven fissures had healed by one month and four by two months. Two remained unhealed but asymptomatic. There was no incontinence of flatus or faeces after three months of treatment. Resting anal pressure was significantly lower in 10 of 13 patients compared with before treatment (p < 0.05). One fissure relapsed after 4 months and this patient had a successful anal stretch. CONCLUSION: Injection of botulinum A toxin gives good results in the treatment of anal fissures.     

Kindled rats are more sensitive than non-kindled rats to the behavioural effects of combined treatment with MK-801 and valproate.

The effects of combined treatment with low doses (0.025-0.05 mg/kg i.p.) of the non-competitive NMDA receptor antagonist, MK-801 (dizocilpine), and the antiepileptic drug, valproate, were studied in amygdala-kindled and non-kindled rats. MK-801, 0.05 mg/kg, did not exert anticonvulsant effects in fully kindled rats but increased the anticonvulsant potency of valproate, 100 mg/kg i.p. However, the increase in anticonvulsant activity was paralleled by a marked increase in adverse effects such as motor impairment and hyperactivity, resulting in a considerable reduction of the therapeutic index of the combined treatment compared to valproate alone. Furthermore, MK-801 potentiated the adverse effects but not the anticonvulsant activity of 50 mg/kg valproate. Combined treatment with MK-801 and valproate induced much less marked adverse effects in non-kindled rats than in kindled rats. The competitive NMDA receptor antagonist, CGP 37849 1 mg/kg i.p., did not alter the effects of valproate in kindled rats. The data on combined treatment with MK-801 and valproate substantiate the conclusion that kindling alters the susceptibility to manipulations of NMDA receptor-mediated events.     

Phylogenetic analysis of the genus Plasmodium based on the gene encoding adenylosuccinate lyase.

Phylogenetic studies of the genus Plasmodium have been performed using sequences of the nuclear, mitochondrial and plastid genes. Here we have analyzed the adenylosuccinate lyase (ASL) gene, which encodes an enzyme involved in the salvage of host purines needed by malaria parasites for DNA synthesis. The ASL gene is present in several eukaryotic as well as prokaryotic organisms and does not have repeat regions, which facilitates the accuracy of the alignment. Furthermore, it has been shown that ASL is not subject to positive natural selection. We have sequenced the ASL gene of several different Plasmodium species infecting humans, rodents, monkeys and birds and used the obtained sequences along with the previously known P. falciparum ASL sequence, for structural and phylogenetic analysis of the genus Plasmodium. The genetic divergence of ASL is comparable with that observed in other nuclear genes such as cysteine proteinase, although ASL cannot be considered conserved when compared to aldolase or superoxide dismutase, which exhibit a slower rate of evolution. Nevertheless, a protein like ASL has a rate of evolution that provides enough information for elucidating evolutionary relationships. We modeled 3D structures of the ASL protein based on sequences used in the phylogenetic analysis and obtained a consistent structure for four different species despite the divergence observed. Such models would facilitate alignment in further studies with a greater number of plasmodial species or other Apicomplexa.     

Structure of natural killer receptor 2B4 bound to CD48 reveals basis for heterophilic recognition in signaling lymphocyte activation molecule family

Natural killer (NK) cells eliminate virally infected and tumor cells. Among the receptors regulating NK cell function is 2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM) family that binds CD48. 2B4 is the only heterophilic receptor of the SLAM family, whose other members, e.g., NK-T-B-antigen (NTB-A), are self-ligands. We determined the structure of the complex between the N-terminal domains of mouse 2B4 and CD48, as well as the structures of unbound 2B4 and CD48. The complex displayed an association mode related to, yet distinct from, that of the NTB-A dimer. Binding was accompanied by the rigidification of flexible 2B4 regions containing most of the polymorphic residues across different species and receptor isoforms. We propose a model for 2B4-CD48 interactions that permits the intermixing of SLAM receptors with major histocompatibility complex-specific receptors in the NK cell immune synapse. This analysis revealed the basis for heterophilic recognition within the SLAM family.