Studies on Aspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grown Aspergillus niger was increased 3-5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH4 + , and further, the enzyme is repressed by increasing concentrations of NH4 +. In contrast to other micro-organisms, the Aspergillus niger enzyme was neither specifically inactivated by NH4+ or L-glutamine nor regulated by covalent modification.Glutamine synthetase from Aspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn2+ or Mg2+-dependent synthetase and Mn2+-dependent transferase activity.Aspergillus niger glutamine synthetase was completely inactivated by two mol of phenylglyoxal and one mol of N-ethylmaleimide with second order rate constants of 3·8 M–1 min–1 and 760 M–1 min–1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH4+, Mn2+ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.

Use of semi-active dampers in seismic mitigation of building structures

This research provides information for providing the required seismic mitigation in building structures through the use of semi active and passive dampers. The Magneto-Rheological (MR) semi-active damper model was developed using control algorithms and integrated into seismically excited structures as a time domain function. Linear and nonlinear structure models are evaluated in real time scenarios. Research information can be used for the design and construction of earthquake safe buildings with optimally employed MR dampers and MR-passive damper combinations.

Web-based active learning and frequent feedback: Engaging first-year university students

Web-based technology is particularly well-suited to promoting active student involvement in the processes of learning. All students enrolled in a first-year educational psychology unit were required to complete ten weekly online quizzes, ten weekly student-generated questions and ten weekly student answers to those questions. Results of an online survey of participating students strongly support the viability and perceived benefits of such an instructional approach. Although students reported that the 30 assessments were useful and reasonable, the most common theme to emerge from the professional reflections of participating lecturers was that the marking of questions and answers was unmanageable.

GDP-L-fucose: synthesis and role in inflammation

GDP-L-fucose: synthesis and role in inflammation The migration of leukocytes from intravascular locations to extravascular sites is essential to the immune responses. The initial attachment of leukocytes to the endothelium and the rolling step of the leukocyte extravasation cascade are mediated by selectins, a family of cell adhesion molecules on cell surfaces. Selectins are able to recognize glycoproteins and glycolipids containing the tetrasaccharide sialyl Lewis x (sLex, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc). Several glycosyltransferases are involved in the biosynthesis of sLex, fucosyltransferase VII (Fuc-TVII) being the last enzyme to modify the sLex structure. Fuc-TVII transfers L-fucose from GDP-L-fucose to sialylated N-acetyllactosamine. GDP-L-fucose is synthesized in the cytosol via two different metabolic pathways. The major, constitutively active de novo pathway involves conversion of GDP-α-D-mannose to GDP-β-L-fucose. In the alternative salvage pathway, L-fucokinase synthesizes from free fucose L-fucose-1-phosphate, which is further converted to GDP-L-fucose by GDP-L-fucose pyrophosphorylase. GDP-L-fucose is translocated from the cytosol to Golgi for fucosylation via the GDP-fucose transporter. This thesis involved the study of the synthesis of GDP-L-fucose via the salvage pathway: cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. The gene expression levels of these enzymes were found to be relatively high in various tissues; the mRNA levels were highest in brain, ovary and testis. This study also describes molecular cloning of rat fucosyltransferase VII (FUT7) and its expression as a functional enzyme. Gene expression levels of GDP-L-fucose synthesizing enzymes, GDP-fucose transporter and FUT7 were determined in inflamed tissues as well as cancer cells. Our results revealed a clear upregulation of the enzymes involved in the synthesis of GDP-L-fucose via de novo pathway, GDP-fucose transporter and FUT7 in inflamed tissues and in cancer cells. On the contrary, the GDP-L-fucose salvage pathway was found to be irrelevant in inflammation and in tumorigenesis. Furthermore, our results indicated the transcriptional coregulation of Golgi transporters involved in the synthesis of sulfo sLex, i.e. CMP-sialic acid, GDP-fucose and 3 phosphoadenosine 5 -phosphosulfate transporters, in inflammation.

Implications of high level pseudogene transcription in Mycobacterium leprae

Background: The Mycobacterium leprae genome has less than 50% coding capacity and 1,133 pseudogenes. Preliminary evidence suggests that some pseudogenes are expressed. Therefore, defining pseudogene transcriptional and translational potentials of this genome should increase our understanding of their impact on M. leprae physiology. Results: Gene expression analysis identified transcripts from 49% of all M. leprae genes including 57% of all ORFs and 43% of all pseudogenes in the genome. Transcribed pseudogenes were randomly distributed throughout the chromosome. Factors resulting in pseudogene transcription included: 1) co-orientation of transcribed pseudogenes with transcribed ORFs within or exclusive of operon-like structures; 2) the paucity of intrinsic stem-loop transcriptional terminators between transcribed ORFs and downstream pseudogenes; and 3) predicted pseudogene promoters. Mechanisms for translational ``silencing'' of pseudogene transcripts included the lack of both translational start codons and strong Shine-Dalgarno (SD) sequences. Transcribed pseudogenes also contained multiple ``in-frame'' stop codons and high Ka/Ks ratios, compared to that of homologs in M. tuberculosis and ORFs in M. leprae. A pseudogene transcript containing an active promoter, strong SD site, a start codon, but containing two in frame stop codons yielded a protein product when expressed in E. coli. Conclusion: Approximately half of M. leprae's transcriptome consists of inactive gene products consuming energy and resources without potential benefit to M. leprae. Presently it is unclear what additional detrimental affect(s) this large number of inactive mRNAs has on the functional capability of this organism. Translation of these pseudogenes may play an important role in overall energy consumption and resultant pathophysiological characteristics of M. leprae. However, this study also demonstrated that multiple translational ``silencing'' mechanisms are present, reducing additional energy and resource expenditure required for protein production from the vast majority of these transcripts.

Saga land: Richard Fidler and Kári Gíslason in Iceland

This is a four-part radio documentary produced for the Australian Broadcasting Corporation's Radio National. Richard Fidler and Kári Gíslason travel to Iceland to tell the stories of the ancient Vikings, and to settle a longstanding family mystery.

Effect of amplifier imperfections on active networks

The deviation in the performance of active networks due to practical operational amplifiers (OA) is mainly because of the finite gain bandwidth productBand nonzero output resistanceR_0. The effect ofBandR_0on two OA impedances and single and multi-OA filters are discussed. In filters, the effect ofR_0is to add zeros to the transfer function often making it nonminimum phase. A simple method of analysis has been suggested for 3-OA biquad and coupled biquad circuits. A general method of noise minimization of the generalized impedance converter (GIC), while operating OA's within the prescribed voltage and current limits, is also discussed. The 3-OA biquadratic sections analyzed also exhibit noise behavior and signal handling capacity similar to the GIC. The GIC based structures are found to be better than other configurations both in biquadratic sections and direct realizations of higher order transfer functions.

Structure-Based Design of DevR Inhibitor Active against Nonreplicating Mycobacterium tuberculosis

Antitubercular treatment is directed against actively replicating organisms. There is an urgent need to develop drugs targeting persistent subpopulations of Mycobacterium tuberculosis. The DevR response regulator is believed to play a key role in bacterial dormancy adaptation during hypoxia. We developed a homology-based model of DevR and used it for the rational design of inhibitors. A phenylcoumarin derivative (compound 10) identified by in silico pharmacophore-based screening of 2.5 million compounds employing protocols with some novel features including a water-based pharmacophore query, was characterized further. Compound 10 inhibited DevR binding to target DNA, down-regulated dormancy genes transcription, and drastically reduced survival of hypoxic but not nutrient-starved dormant bacteria or actively growing organ ` isms. Our findings suggest that compound 10 ``locks'' DevR in an inactive conformation that is unable to bind cognate DNA and induce the dormancy regulon. These results provide proof-of-concept for DevR as a novel target to develop molecules with sterilizing activity against tubercle bacilli.

Buckling, stiffening, and negative dissipation in the dynamics of a biopolymer in an active medium

We present a generic theory for the dynamics of a stiff filament under tension, in an active medium with orientational correlations, such as a microtubule in contractile actin. In sharp contrast to the case of a passive medium, we find the filament can stiffen, and possibly oscillate or buckle, depending on both the contractile or tensile nature of the activity and the filament-medium anchoring interaction. We also demonstrate a strong violation of the fluctuation-dissipation (FD) relation in the effective dynamics of the filament, including a negative FD ratio. Our approach is also of relevance to the dynamics of axons, and our model equations bear a remarkable formal similarity to those in recent work [Martin P, Hudspeth AJ, Juelicher F (2001) Proc Natl Acad Sci USA 98: 14380-14385] on auditory hair cells. Detailed tests of our predictions can be made by using a single filament in actomyosin extracts or bacterial suspensions.

Semi-active vibration isolation using a near-zero-spring-rate device:steady state analysis of a single-degree-of-freedom model

A vibration isolator is described which incorporates a near-zero-spring-rate device within its operating range. The device is an assembly of a vertical spring in parallel with two inclined springs. A low spring rate is achieved by combining the equivalent stiffness in the vertical direction of the inclined springs with the stiffness of the vertical central spring. It is shown that there is a relation between the geometry and the stiffness of the individual springs that results in a low spring rate. Computer simulation studies of a single-degree-of-freedom model for harmonic base input show that the performance of the proposed scheme is superior to that of the passive schemes with linear springs and skyhook damping configuration. The response curves show that, for small to large amplitudes of base disturbance, the system goes into resonance at low frequencies of excitation. Thus, it is possible to achieve very good isolation over a wide low-frequency band. Also, the damper force requirements for the proposed scheme are much lower than for the damper force of a skyhook configuration or a conventional linear spring with a semi-active damper.

A flexible tool for integrated planning of active distribution networks

This paper presents a flexible and integrated planning tool for active distribution network to maximise the benefits of having high level s of renewables, customer engagement, and new technology implementations. The tool has two main processing parts: “optimisation” and “forecast”. The “optimization” part is an automated and integrated planning framework to optimize the net present value (NPV) of investment strategy for electric distribution network augmentation over large areas and long planning horizons (e.g. 5 to 20 years) based on a modified particle swarm optimization (MPSO). The “forecast” is a flexible agent-based framework to produce load duration curves (LDCs) of load forecasts for different levels of customer engagement, energy storage controls, and electric vehicles (EVs). In addition, “forecast” connects the existing databases of utility to the proposed tool as well as outputs the load profiles and network plan in Google Earth. This integrated tool enables different divisions within a utility to analyze their programs and options in a single platform using comprehensive information.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Canadian Stroke Best Practice Recommendations: Mood, Cognition and Fatigue Following Stroke practice guidelines, update 2015

Every year, approximately 62 000 people with stroke and transient ischemic attack are treated in Canadian hospitals, and the evidence suggests one-third or more will experience vascular-cognitive impairment, and/or intractable fatigue, either alone or in combination. The 2015 update of the Canadian Stroke Best Practice Recommendations: Mood, Cognition and Fatigue Module guideline is a comprehensive summary of current evidence-based recommendations for clinicians in a range of settings, who provide care to patients following stroke. The three consequences of stroke that are the focus of the this guideline (poststroke depression, vascular cognitive impairment, and fatigue) have high incidence rates and significant impact on the lives of people who have had a stroke, impede recovery, and result in worse long-term outcomes. Significant practice variations and gaps in the research evidence have been reported for initial screening and in-depth assessment of stroke patients for these conditions. Also of concern, an increased number of family members and informal caregivers may also experience depressive symptoms in the poststroke recovery phase which further impact patient recovery. These factors emphasize the need for a system of care that ensures screening occurs as a standard and consistent component of clinical practice across settings as stroke patients transition from acute care to active rehabilitation and reintegration into their community. Additionally, building system capacity to ensure access to appropriate specialists for treatment and ongoing management of stroke survivors with these conditions is another great challenge.

Isolation and visualization of active presynaptic filaments of recA protein and single-stranded DNA

In the presence of ATP, recA protein forms a presynaptic complex with single-stranded DNA that is an obligatory intermediate in homologous pairing. Presynaptic complexes of recA protein and circular single strands that are active in forming joint molecules can be isolated by gel filtration. These isolated active complexes are nucleoprotein filaments with the following characteristics: (i) a contour length that is at least 1.5 times that of the corresponding duplex DNA molecule, (ii) an ordered structure visualized by negative staining as a striated filament with a repeat distance of 9.0 nm and a width of 9.3 nm, (iii) approximately 8 molecules of recA protein and 20 nucleotide residues per striation. The widened spacing between bases in the nucleoprotein filament means that the initial matching of complementary sequences must involve intertwining of the filament and duplex DNA, unwinding of the latter, or some combination of both to equalize the spacing between nascent base pairs. These experiments support the concept that recA protein first forms a filament with single-stranded DNA, which in turn binds to duplex DNA to mediate both homologous pairing and subsequent strand exchange.

Active nucleoprotein filaments of single-stranded binding protein and recA protein on single-stranded DNA have a regular repeating structure

When E. coli single-stranded DNA binding protein (SSB) coats single-stranded DNA (ssDNA) in the presence of 1 mM MgCl2 it inhibits the subsequent binding of recA protein, whereas SSB binding to ssDNA in 12 mM MgCl2 promotes the binding of recA protein. These two conditions correspond respectively to those which produce 'smooth' and 'beaded' forms of ssDNA-SSB filaments. By gel filtration and immunoprecipitation we observed active nucleoprotein filaments of recA protein and SSB on ssDNA that contained on average 1 monomer of recA protein per 4 nucleotides and 1 monomer of SSB per 20-22 nucleotides. Filaments in such a mixture, when digested with micrococcal nuclease produced a regular repeating pattern, approximately every 70-80 nucleotides, that differed from the pattern observed when only recA protein was bound to the ssDNA. We conclude that the beaded ssDNA-SSB nucleoprotein filament readily binds recA protein and forms an intermediate that is active in the formation of joint molecules and can retain substantially all of the SSB that was originally bound.