Systematic Investigation of Insulin Fibrillation on a Chip

A microfluidic protein aggregation device (microPAD) that allows the user to perform a series of protein incubations with various concentrations of two reagents is demonstrated. The microfluidic device consists of 64 incubation chambers to perform individual incubations of the protein at 64 specific conditions. Parallel processes of metering reagents, stepwise concentration gradient generation, and mixing are achieved simultaneously by pneumatic valves. Fibrillation of bovine insulin was selected to test the device. The effect of insulin and sodium chloride (NaCl) concentration on the formation of fibrillar structures was studied by observing the growth rate of partially folded protein, using the fluorescent marker Thioflavin-T. Moreover, dual gradients of different NaCl and hydrochloric acid (HCl) concentrations were formed, to investigate their interactive roles in the formation of insulin fibrils and spherulites. The chip-system provides a bird’s eye view on protein aggregation, including an overview of the factors that affect the process and their interactions. This microfluidic platform is potentially useful for rapid analysis of the fibrillation of proteins associated with many misfolding-based diseases, such as quantitative and qualitative studies on amyloid growth.

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Isolation and characterization of recombinant Drosophila Copia aspartic proteinase

Biochemical Journal ◽

10.1042/bj20060800 ◽

2006 ◽

Vol 399 (3) ◽

pp. 535-542 ◽

Cited By ~ 5

Author(s):

Senarath B. P. Athauda ◽

Katsuji Yoshioka ◽

Tadayoshi Shiba ◽

Kenji Takahashi

Keyword(s):

Active Site ◽

Cleavage Site ◽

Bovine Insulin ◽

Precursor Protein ◽

Coding Region ◽

Aspartic Proteinases ◽

E Coli ◽

Isolation And Characterization ◽

Nacl Concentration ◽

Molecular Masses

The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe–Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 °C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15−–Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.

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ESI-IMS–MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules

Methods ◽

10.1016/j.ymeth.2015.05.017 ◽

2016 ◽

Vol 95 ◽

pp. 62-69 ◽

Cited By ~ 35

Author(s):

Lydia M. Young ◽

Janet C. Saunders ◽

Rachel A. Mahood ◽

Charlotte H. Revill ◽

Richard J. Foster ◽

...

Keyword(s):

Protein Aggregation ◽

Small Molecules ◽

Rapid Analysis

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Determination of the Thermodynamics of B-lactoglobulin Aggregation Using Ultra Violet Light Scattering Spectroscopy

10.1101/246298 ◽

2018 ◽

Cited By ~ 1

Author(s):

James I Austerberry ◽

Daniel J Belton

Keyword(s):

Light Scattering ◽

Protein Aggregation ◽

Ultra Violet ◽

Ultra Violet Light ◽

Violet Light ◽

Light Scattering Spectroscopy ◽

Nacl Concentration ◽

Model Protein ◽

Β Lactoglobulin

AbstractThe problem of protein aggregation is widely studied across a number of disciplines, where understanding the behaviour of the protein monomer, and its behaviour with co-solutes is imperative in order to devise solutions to the problem. Here we present a method for measuring the kinetics of protein aggregation based on ultra violet light scattering spectroscopy (UVLSS) across a range of NaCl conditions. Through measurement of wavelength dependant scattering and using the model protein β-lactoglobulin, it is possible to isolate the thermodynamic contributions to thermal unfolding. We show that increasing NaCl concentration decreases the free energy of unfolding which is dominated by the decrease of the enthalpy contribution. This contribution is significantly larger than the decrease in change in entropy observed at higher salt concentrations between the folded and unfolded states.

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Grain Refinement in Titanium-Erbium Alloys

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100052626 ◽

1974 ◽

Vol 32 ◽

pp. 522-523

Author(s):

B. B. Rath ◽

J. E. O'Neal ◽

R. J. Lederich

Keyword(s):

Electron Microscopy ◽

Size Distribution ◽

Grain Refinement ◽

Grain Growth ◽

Volume Fraction ◽

Pure Titanium ◽

Systematic Investigation ◽

Second Phase ◽

Titanium Matrix ◽

Average Size

Addition of small amounts of erbium has a profound effect on recrystallization and grain growth in titanium. Erbium, because of its negligible solubility in titanium, precipitates in the titanium matrix as a finely dispersed second phase. The presence of this phase, depending on its average size, distribution, and volume fraction in titanium, strongly inhibits the migration of grain boundaries during recrystallization and grain growth, and thus produces ultimate grains of sub-micrometer dimensions. A systematic investigation has been conducted to study the isothermal grain growth in electrolytically pure titanium and titanium-erbium alloys (Er concentration ranging from 0-0.3 at.%) over the temperature range of 450 to 850°C by electron microscopy.

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Optical Properties of HVEM Accelerators

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100114529 ◽

1975 ◽

Vol 33 ◽

pp. 180-181

Author(s):

A. Strojnik ◽

J.W. Scholl ◽

V. Bevc

Keyword(s):

Optical Properties ◽

Electron Accelerator ◽

Systematic Investigation ◽

Electron Source ◽

High Brightness ◽

The Past ◽

Wide Range ◽

Optical Behavior

The electron accelerator, as inserted between the electron source (injector) and the imaging column of the HVEM, is usually a strong lens and should be optimized in order to ensure high brightness over a wide range of accelerating voltages and illuminating conditions. This is especially true in the case of the STEM where the brightness directly determines the highest resolution attainable. In the past, the optical behavior of accelerators was usually determined for a particular configuration. During the development of the accelerator for the Arizona 1 MEV STEM, systematic investigation was made of the major optical properties for a variety of electrode configurations, number of stages N, accelerating voltages, 1 and 10 MEV, and a range of injection voltages ϕ0 = 1, 3, 10, 30, 100, 300 kV).

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An Ultra-Structural Study of Human Melanoma in Vivo and Vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100091275 ◽

1976 ◽

Vol 34 ◽

pp. 258-259

Author(s):

James R. Gaylor ◽

Fredda Schafer ◽

Robert E. Nordquist

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Protein Aggregation ◽

Structural Study ◽

Human Melanoma ◽

Protein Matrix ◽

Early Form ◽

Endoplasmic Reticulum Protein ◽

Mitochondrial Origin

Several theories on the origin of the melanosome exist. These include the Golgi origin theory, in which a tyrosinase-rich protein is "packaged" by the Golgi apparatus, thus forming the early form of the melanosome. A second theory postulates a mitochondrial origin of melanosomes. Its author contends that the melanosome is a modified mitochondria which acquires melanin during its development. A third theory states that a pre-melanosome is formed in the smooth or rough endoplasmic reticulum. Protein aggregation is suggested by one author as a possible source of the melanosome. This fourth theory postulates that the melanosome originates when the protein products of several genetic loci aggregate in the cytoplasm of the melanocyte. It is this protein matrix on which the melanin is deposited. It was with these theories in mind that this project was undertaken.

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Magnitude Estimation and Cross-Modal Matching of Lingual Vibrotactile and Auditory Sensation

Journal of Speech Language and Hearing Research ◽

10.1044/jshr.3203.698 ◽

1989 ◽

Vol 32 (3) ◽

pp. 698-702 ◽

Cited By ~ 2

Author(s):

Daniel Harris ◽

Donald Fucci ◽

Linda Petrosino

Keyword(s):

Auditory Stimulus ◽

Magnitude Estimation ◽

Sensory Processing ◽

Test Site ◽

Systematic Investigation ◽

Thenar Eminence ◽

Stimulus Range ◽

Psychophysical Scaling ◽

Scaling Methods ◽

Response Biases

The present experiment was a preliminary attempt to use the psychophysical scaling methods of magnitude estimation and cross-modal matching to investigate suprathreshold judgments of lingual vibrotactile and auditory sensation magnitudes for 20 normal young adult subjects. A 250-Hz lingual vibrotactile stimulus and a 1000-Hz binaural auditory stimulus were employed. To obtain judgments for nonoral vibrotactile sensory magnitudes, the thenar eminence of the hand was also employed as a test site for 5 additional subjects. Eight stimulus intensities were presented during all experimental tasks. The results showed that the slopes of the log-log vibrotactile magnitude estimation functions decreased at higher stimulus intensity levels for both test sites. Auditory magnitude estimation functions were relatively constant throughout the stimulus range. Cross-modal matching functions for the two stimuli generally agreed with functions predicted from the magnitude estimation data, except when subjects adjusted vibration on the tongue to match auditory stimulus intensities. The results suggested that the methods of magnitude estimation and cross-modal matching may be useful for studying sensory processing in the speech production system. However, systematic investigation of response biases associated with vibrotactile-auditory psychophysical scaling tasks appears to be a prerequisite.

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