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At least 217 records · Page 12Linked to original sources
PubMed1999

Essential components of the Ti plasmid trb system, a type IV macromolecular transporter.

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58DeltaaccR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.

Agrobacterium tumefaciens↗
PubMed2005

Adaptation to extreme environments: macromolecular dynamics in complex systems.

What we previously thought of as insurmountable physical and chemical barriers to life, we now see as yet another niche harbouring 'extremophiles'. Extremophiles and their macromolecules had to develop molecular mechanisms of adaptation to extreme physico-chemical conditions. Using neutron spectroscopy, we have demonstrated that molecular dynamics represents one of these molecular mechanisms of adaptation. To which extent do hyper-saline conditions and extreme temperatures influence molecular dynamics? First, molecular dynamics were analysed for halophilic malate dehydrogenase from Haloarcula marismortui (Hm MalDH) under different molar solvent salt concentration conditions influencing its stability. Secondly, mean macromolecular motions were measured in-vivo in psychrophile (Aquaspirillum arcticum), mesophile (Escherichia coli and Proteus mirabilis), thermophile (Thermus thermophilus), and hyperthermophile (Aquifex pyrofilus) bacteria. The mean constant force of Hm MalDH increases progessively with increasing stability. The results show that the molecular adaptation of Hm MalDH to hyper-saline conditions is achieved through an increasing resilience of its structure dominated by enthalpic mechanisms. The study of bacteria has provided tools to quantify the macromolecular adaptation to extreme temperatures in the naturally crowded environment of the cell. The macromolecular resilience of bacteria increases with adaptation to high temperatures.

Adaptation, Biological↗
PubMed2000

The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework.

The number of solved structures of macromolecules that have the same fold and thus exhibit some degree of conformational variability is rapidly increasing. It is consequently advantageous to develop a standardized terminology for describing this variability and automated systems for processing protein structures in different conformations. We have developed such a system as a 'front-end' server to our database of macromolecular motions. Our system attempts to describe a protein motion as a rigid-body rotation of a small 'core' relative to a larger one, using a set of hinges. The motion is placed in a standardized coordinate system so that all statistics between any two motions are directly comparable. We find that while this model can accommodate most protein motions, it cannot accommodate all; the degree to which a motion can be accommodated provides an aid in classifying it. Furthermore, we perform an adiabatic mapping (a restrained interpolation) between every two conformations. This gives some indication of the extent of the energetic barriers that need to be surmounted in the motion, and as a by-product results in a 'morph movie'. We make these movies available over the Web to aid in visualization. Many instances of conformational variability occur between proteins with somewhat different sequences. We can accommodate these differences in a rough fashion, generating an 'evolutionary morph'. Users have already submitted hundreds of examples of protein motions to our server, producing a comprehensive set of statistics. So far the statistics show that the median submitted motion has a rotation of approximately 10 degrees and a maximum Calpha displacement of 17 A. Almost all involve at least one large torsion angle change of >140 degrees. The server is accessible at http://bioinfo.mbb.yale. edu/MolMovDB

Computer Graphics↗
PubMed2003

Systemic concentrations of antioxidants and biomarkers of macromolecular oxidative damage in horses with grass sickness.

REASONS FOR PERFORMING STUDY: The aetiopathogenesis of equine grass sickness (EGS) is unknown. The role of free radical-mediated neuronal damage has not previously been investigated in this condition. OBJECTIVES: To investigate the potential contribution of oxidative damage and antioxidant status to neurodegeneration in EGS. METHODS: Systemic levels of surrogate biomarkers were determined in 10 horses with acute EGS and in 2 control populations; 10 healthy horses co-grazing with the 10 EGS horses at the onset of clinical disease, and 10 healthy mares grazing where EGS has not been reported. RESULTS: EGS horses had alterations in levels of several antioxidants, consistent with oxidative stress, the acute phase response and/or the secondary metabolic complications of EGS. EGS horses had elevated plasma dihydroxyphenylalanine (DOPA) levels. CONCLUSIONS: The elevated DOPA levels probably reflected a generalised disturbance of catecholamine metabolism rather than increased DOPA production via free radical-mediated oxidation of tyrosine. However, there was no evidence of systemic macromolecular oxidative damage. POTENTIAL CLINICAL RELEVANCE: Further work is required to determine whether macromolecular oxidative damage occurring at the neuronal level contributes to EGS.

Animals↗
PubMed1996

Use of macromolecular assemblies as expression systems for peptides and synthetic vaccines.

The past decade has witnessed the development of numerous systems for the presentation of antigens on the surface of self-assembling macromolecules. Although the sites for insertion were initially chosen empirically, the determination of the three-dimensional structures of a number of carrier macromolecules has enabled structure-based insertional mutagenesis to be used increasingly. Furthermore, it is now feasible to determine the structure of an inserted sequence as presented in a heterologous environment, making it possible to correlate the detailed structure of a peptide with its immunological properties.

Animals↗
PubMed1997

Pharmacokinetics of methotrexate-albumin conjugates in tumor-bearing rats.

Linking chemotherapeutic drugs to a macromolecular carrier system may enhance tumor targeting, reduce toxicity and overcome drug resistance mechanisms. As an elementary model to evaluate the pharmacological properties of macromolecular drug carrier systems we chose rat serum albumin (RSA) for carrier and methotrexate (MTX) as antineoplastic drug. The conjugation procedure yielded conjugates with an approximate 1:1 molar loading rate (MTX(1)-RSA). In the first part of the study a residualizing [111In]DTPA protein label was used for mapping in vivo the catabolic sites of the native carrier protein and of the MTX(1)-RSA drug conjugate in Walker 256 carcinosarcoma bearing rats. The tumor accumulation was about 14% of the injected dose for the RSA and MTX(1)-RSA tracers after 24 h. Tracer entrapment by organs with an active mononuclear phagocyte system was low (liver below 7% and spleen below 1.5% of the injected dose after 24 h). The 1:1 conjugation of MTX to RSA did not decisively alter the pharmacokinetic properties nor the tumor or tissue distribution of the native carrier protein RSA. In the second part of the study the different properties of the MTX(1)-RSA conjugate were compared with MTX in vivo. About 2 mg MTX/kg body weight either of the drug conjugate or of the original drug were injected after being additionally spiked with radiolabeled tracers. Plasma concentrations were simultaneously determined by immunological and radioactive means. After 24 h about 12% MTX(1)-RSA was found in circulation compared to 0.03% MTX. Favorable tumor accumulation rates of about 14% were achieved for MTX(1)-RSA versus 0.04% for MTX. About 45-fold more of the injected dose of [3H]MTX accumulated in the liver as compared to the tumor (1.5 versus 0.03%) after 24 h. Conjugation of MTX to RSA reversed this ratio in favor of the tumor to 1:1.4 (13.6 versus 9.6%). In conclusion, the potential therapeutic benefit of the MTX(1)-RSA conjugate lies in its very long tumor exposure time and its improved tumor accumulation rate compared to conventional MTX. In addition the conjugation to albumin might enhance the therapeutic effects over those achieved by long-term continuous infusion of MTX, as MTX(1)-RSA enters the cells by a different uptake mechanism. This might also help to circumvent MTX resistance mechanisms, such as a reduction in folate receptor numbers or impaired MTX polyglutamylation.

Animals↗
PubMed1998

Crystallography & NMR system: A new software suite for macromolecular structure determination.

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.

Computer Simulation↗
PubMed2003

Conceptual design of novel IP-conveyor-belt Weissenberg-mode data-collection system with multi-readers for macromolecular crystallography. A comparison between Galaxy and Super Galaxy.

Galaxy is a Weissenberg-type high-speed high-resolution and highly accurate fully automatic data-collection system using two cylindrical IP-cassettes each with a radius of 400 mm and a width of 450 mm. It was originally developed for static three-dimensional analysis using X-ray diffraction and was installed on bending-magnet beamline BL6C at the Photon Factory. It was found, however, that Galaxy was also very useful for time-resolved protein crystallography on a time scale of minutes. This has prompted us to design a new IP-conveyor-belt Weissenberg-mode data-collection system called Super Galaxy for time-resolved crystallography with improved time and crystallographic resolution over that achievable with Galaxy. Super Galaxy was designed with a half-cylinder-shaped cassette with a radius of 420 mm and a width of 690 mm. Using 1.0 A incident X-rays, these dimensions correspond to a maximum resolutions of 0.71 A in the vertical direction and 1.58 A in the horizontal. Upper and lower screens can be used to set the frame size of the recorded image. This function is useful not only to reduce the frame exchange time but also to save disk space on the data server. The use of an IP-conveyor-belt and many IP-readers make Super Galaxy well suited for time-resolved, monochromatic X-ray crystallography at a very intense third-generation SR beamline. Here, Galaxy and a conceptual design for Super Galaxy are described, and their suitability for use as data-collection systems for macromolecular time-resolved monochromatic X-ray crystallography are compared.

Crystallography, X-Ray↗
PubMed1978

Selective translation of mengovirus RNA over Host mRNA in homologous, fractionated, cell-free translational systems from Ehrlich-ascites-tumor cells.

The selective translation of viral RNA in mengovirus-infected Ehrlich ascites tumor cells was investigated using fractionated translational systems whose macromolecular components were derived entirely from uninfected or virus-infected cells. Both systems translate host mRNA from uninfected cells, host mRNA from virus-infected cells, and mengovirus RNA. In competition experiments, where viral RNA and host mRNA were translated together in systems from uninfected cells, the relative amounts of virus-specific and host-specific proteins synthesized were proportional to the relative concentrations of the RNA templates. In systems whose components were obtained from virus-infected cells, mengovirus RNA was preferentially translated. 70% of the selectivity found in the translational systems derived from infected cells was due to the initiation factor fraction, the remaining 30% to components of the pH 5 enzyme fraction. In addition, host mRNA isolated after virus infection is translated in vitro to a lower extent in the presence of mengovirus RNA than is host mRNA from uninfected cells.

Animals↗
PubMed2004

The macromolecular polymers for the preparation of hydrodynamically balanced systems--methods of evaluation.

Evaluation of macromolecular polymers used as excipients for the preparation of hydrodynamically balanced systems (HBS) was carried out. Hard gelatine capsules were filled with polymeric substances belonging to various chemical groups (chitosan, sodium alginate, hydroxypropylmethycellulose--HPMC). The following properties of the HBS were investigated: density, hydration, erosion and floating force. The solvent penetration process into the HBS was visualized using magnetic resonance imaging (MRI) technique. Densities of the HBS in hydrochloric acid (0.1 M) ranged from 0.37 g/cm3 to 0.71 g/cm3. Each polymer demonstrated different hydration/erosion abilities and floating properties. The maximum floating force (F(float max)) for capsules size 0, ranged from 26.7 mN (sodium alginate) to 64.7 mN (chitosan). HBS formulations also varied in time to reach maximum floating force (T(float max)). HPMC and sodium alginate formulation reached F(float max) within half an hour after immersion, while in the case of chitosan formulations (deacetylation degree (d.d.) 66% and d.d. 93%), the time was 184 minutes and 218 minutes respectively. The floating properties of the dosage forms were reliant on type of the polymer and the medium-fasted state simulated gastric fluid (FaSSGF) or fed state simulated gastric fluid (FeSSGF). The size of the HBS influenced the floating force value. The mechanisms of erosion and swelling of the polymeric matrices play a dominant role in flotation of the dosage forms.

Alginates↗
PubMedDate not supplied

Release characteristics of chitosan treated alginate beads: I. Sustained release of a macromolecular drug from chitosan treated alginate beads.

Alginate and chitosan treated alginate beads were prepared and compared as an oral controlled release system for macromolecular drugs. Dextran (M.W. 70,000) was used as a model substance. The beads were prepared by the ionotropic gelation method and the effect of various factors (alginate, chitosan, drug and calcium chloride concentrations, the volume of external and internal phases and drying methods) on bead properties were investigated. The addition of chitosan increased the drug loading capacity of the beads, and larger beads were obtained in the presence of chitosan. On the other hand, addition of chitosan in the gel structure reduced the drug release from beads. The erosion of the beads was suppressed by chitosan treatment. The drying method was important to the properties of the chitosan-alginate beads. It is proposed that chitosan treated alginate beads may be used as a potential controlled release system of such macromolecules.

Alginates↗
PubMed2003

Comparison of dendrimer-based macromolecular contrast agents for dynamic micro-magnetic resonance lymphangiography.

Few methods are currently available to visualize the entire lymphatic system. A method known as micro-magnetic resonance lymphangiography (MRL), which employs a dendrimer-based MRI contrast agent (PAMAM-G8) and a clinical-grade 1.5T MRI instrument, was recently developed for use in mice. In the present study, three dendrimer-based MRI contrast agents (PAMAM-G8, DAB-G5, and PAMAM-G4) with different pharmacokinetic characteristics were compared to determine the best reagent to visualize the lymphatic system under physiological or pathological conditions. In addition, two established MRI contrast agents (Gadomer-17 and Gd-[DTPA]-dimeglumine (Magnevist)) were used as control agents. In experiments with mice, most of the deep lymphatic system was visualized by micro-MRL with all agents except Gd-[DTPA]-dimeglumine. PAMAM-G8 was best for visualizing lymphatic vessels, whereas DAB-G5 was better for visualizing lymph nodes. PAMAM-G4 was intermediate in character between PAMAM-G8 and DAB-G5, except in exhibiting a low background signal (especially in the liver). The lymphatic system was not clearly visualized with Gd-[DTPA]-dimeglumine; however, the lymph nodes were visualized with Gadomer-17, although not as well as with dendrimer-based agents. In conclusion, DAB-G5 and PAMAM-G4 can be used to identify lymph nodes and lymphatic vessels, respectively. Their rapid excretion makes these compounds potentially attractive for human use.

Animals↗
PubMed2005

Peptidoglycan degradation by specialized lytic transglycosylases associated with type III and type IV secretion systems.

Specialized lytic transglycosylases are muramidases capable of locally degrading the peptidoglycan meshwork of Gram-negative bacteria. Specialized lytic transglycosylase genes are present in clusters encoding diverse macromolecular transport systems. This paper reports the analysis of selected members of the specialized lytic transglycosylase family from type III and type IV secretion systems. These proteins were analysed in vivo by assaying their ability to complement the DNA transfer defect of the conjugative F-like plasmid R1-16 lacking a functional P19 protein, the specialized lytic transglycosylase of this type IV secretion system. Heterologous complementation was accomplished using IpgF from the plasmid-encoded type III secretion system of Shigella sonnei and TrbN from the type IV secretion system of the conjugative plasmid RP4. In contrast, neither VirB1 proteins (Agrobacterium tumefaciens, Brucella suis) nor IagB (Salmonella enterica) could functionally replace P19. In vitro, IpgF, IagB, both VirB1 proteins, HP0523 (Helicobacter pylori) and P19 displayed peptidoglycanase activity in zymogram analyses. Using an established test system and a newly developed assay it was shown that IpgF degraded peptidoglycan in solution. IpgF was active only after removal of the chaperonin GroEL, which co-purified with IpgF and inhibited its enzymic activity. A mutant IpgF protein in which the predicted catalytic amino acid, Glu42, was replaced by Gln, was completely inactive. IpgF-catalysed peptidoglycan degradation was optimal at pH 6 and was inhibited by the lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A.

Bacterial Proteins↗