Theeraporn Puntheeranurak

Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4B Bacillus thuringiensis toxin and its alpha1-alpha5 fragment

Trypsin activation of Cry4B, a 130-kDa Bacillus thuringiensis (Bt) protein, produces a 65-kDa toxin active against mosquito larvae. The active toxin is made of two protease-resistant products of ca. 45 kDa and ca. 20 kDa. The cloned 21-kDa fragment consisting of the N-terminal region of the toxin was previously shown to be capable of permeabilizing liposomes. The present study was designed to test the following hypotheses: (1) Cry4B, like several other Bt toxins, is a channel-forming toxin in planar lipid bilayers; and (2) the 21-kDa N-terminal region, which maps for the first five helices (α1–α5) of domain 1 in other Cry toxins, and which putatively shares a similar tri-dimensional structure, is sufficient to account for the ion channel activity of the whole toxin. Using circular dichroism spectroscopy and planar lipid bilayers, we showed that the 21-kDa polypeptide existed as an α-helical structure and that both Cry4B and its α1–α5 fragment formed ion channels of 248±44 pS and 207±23 pS, respectively. The channels were cation-selective with a potassium-to-chloride permeability ratio of 6.7 for Cry4B and 4.5 for its fragment. However, contrary to the full-length toxin, the α1–α5 region formed channels at low dose; they tended to remain locked in their open state and displayed flickering activity bouts. Thus, like the full-length toxin, the α1–α5 region is a functional channel former. A pH-dependent, yet undefined region of the toxin may be involved in regulating the channel properties.

Single-molecule recognition force spectroscopy of transmembrane transporters on living cells

Atomic force microscopy (AFM) has proven to be a powerful tool in biological sciences. Its particular advantage over other high-resolution methods commonly used is that biomolecules can be investigated not only under physiological conditions but also while they perform their biological functions. Single-molecule force spectroscopy with AFM tip-modification techniques can provide insight into intermolecular forces between individual ligand-receptor pairs of biological systems. Here we present protocols for force spectroscopy of living cells, including cell sample preparation, tip chemistry, step-by-step AFM imaging, force spectroscopy and data analysis. We also delineate critical steps and describe limitations that we have experienced. The entire protocol can be completed in 12 h. The model studies discussed here demonstrate the power of AFM for studying transmembrane transporters at the single-molecule level.

Single Molecule Force Microscopy on Cells and Biological Membranes

Atomic force microscopy (AFM) enables high resolution topographic imaging of biological samples under near-physiological conditions. Therefore, the AFM is optimally suited for investigation of biological membranes and cell surfaces, as exemplified by studies on bacterial S-layers, purple membranes and cultured living cells. Topographic imaging allows visualizing single proteins and protein assemblies in native membranes, as well as substructures of live cells, such as cytoskeletal architecture. In addition to high-resolution imaging, the measurement of mechanical forces yields detailed insight into structure-function relationships of molecular processes in their native environment. In molecular recognition force microscopy, interaction forces between tip-bound ligands and membrane-embedded receptors can be studied under well-controlled buffer conditions and effectors concentrations. In case of low lateral density and inhomogeneous distribution of the target molecules in a cell membrane, fluorescence microscopy can help to guide the AFM tip to the membrane proteins of interest, which can subsequently be investigated by molecular recognition force microscopy.

Sensor pillow and bed sheet system: Unconstrained monitoring of respiration rate and posture movements during sleep

In this paper, we have developed a low-cost sleep monitoring system for patient based on polysomnography which will be useful for patient communication with healthcare personals and/or relatives. In particular, we have presented the sensor pillow and bed sheet system that employs wireless networks based on low-cost ZigBee technology and a sensor array of force sensitive resistors (FSR) based on polymer thick film (PTF) device, for classifying and specifically verifying the respiration rate during sleep. This paper also proposes a simple motion model that explains the change of head and body pressure distribution. In addition, we can detect some physiological parameters during the sleep stages and wakefulness as well as record respiration rate as related to different physiological factors. The integration of this sensor system and wireless technology with computer software could make this healthcare monitoring system a commercial product valuable for point-of-care applications.

SLC5 and SLC2 Transporters in Epithelia—Cellular Role and Molecular Mechanisms

Members of the SLC5 and SLC2 family are prominently involved in epithelial sugar transport. SGLT1 (sodium-glucose transporter) and SGLT2, as representatives of the former, mediate sodium-dependent uptake of sugars into intestinal and renal cells. GLUT2 (glucose transporter), as representative of the latter, facilitates the sodium-independent exit of sugars from cells. SGLT has played a major role in the formulation and experimental proof for the existence of sodium cotransport systems. Based on the sequence data and biochemical and biophysical analyses, the role of extramembranous loops in sugar and inhibitor binding can be delineated. Crystal structures and homology modeling of SGLT reveal that the sugar translocation involves operation of two hydrophobic gates and intermediate exofacial and endofacial occluded states of the carrier in an alternating access model. The same basic model is proposed for GLUT1. Studies on GLUT1 have pioneered the isolation of eukaryotic transporters by biochemical methods and the development of transport kinetics and transporter models. For GLUT1, results from extensive mutagenesis, cysteine substitution and accessibility studies can be incorporated into a homology model with a barrel-like structure in which accessibility to the extracellular and intracellular medium is altered by pinching movements of some of the helices. For SGLT1 and GLUT1, the extensive hydrophilic and hydrophobic interactions between sugars and binding sites of the various intramembrane helices occur and lead to different substrate specificities and inhibitor affinities of the two transporters. A complex network of regulatory steps adapts the transport activity to the needs of the body.

Identification of people from armpit odor region using networked electronic nose

Homeland security basically needs devices that can detect, track, and identify a terrorist from a distance. Body odor recognition offers an opportunity to confirm a persons identity based on ones unique odor pattern. In this paper, we have reported how to invent a networked electronic nose (E-nose), which can detect and recognize human odors from the armpit region. An array of metal oxide sensors was used to detect human odor. Principal component analysis (PCA) was employed to perform pattern recognition and discrimination. The method for correction of the sensor drift has been proposed. The results show that the networked E-nose has a capability to detect human body odor and can create the unique smell print of each person. Based on PCA with 95% confidence ellipse, this E-nose can identify a person from four persons by detecting odor from armpit region.

An artificial nose based on m-porphyrin (M = Mg, Zn) thin film and optical spectroscopy

Artificial nose has recently become an emerging instrument for quality assurance in the food industry. These paper presents the optical gas sensors based on Magnesium - 5,10,15,20 - tetra phenyl - porphyrin (MgTPP) and Zinc - 5,10,15,20 - tetra phenyl - porphyrin (ZnTPP) thin films and their application as an artificial nose. Based on the measurement of optical absorbance response using a general UV-Vis spectroscopy, this artificial noses was tested to discriminate various volatile organic compounds (VOCs) and Thai beverages. Atomic force microscopy (AFM) and X-rays diffraction were used to confirm the polycrystalline structure of the sensing materials. Density functional theory (DFT) calculations reveal that MgTPP interacts more strongly with the VOCs than ZnTPP, especially with water and methanol. The classification results of VOCs and Thai beverage vapors using the principle component analysis indicate that both MgTPP and ZnTPP-based artificial noses can be an efficient tool for quality assurance of alcoholic beverages.

Investigation of thermal and methanol-vapor treatments for MgTPP as an optical gas sensor

In this paper, we have investigated the sensing properties of magnesium - 5,10,15,20 - tetraphenyl - porphyrin (MgTPP) to various volatile organic compounds (VOCs). The spin-coated MgTPP thin films were subjected to thermal annealing and methanol-vapor exposure to study the effects of pre-treatment on the sensing properties. Atomic force microscopy (AFM) has shown that both pre-treatment techniques have induced re-crystallization of the film, thereby improving the sensitivity over the as-deposited film. The thermally annealed films were found more effective than the methanol-vapor treated ones. The in-house optical sensor setup was applied to discriminate various VOCs and alcoholic beverages. Principal component analysis (PCA) confirms that the thermally annealed MgTPP thin film can distinguish several kinds of VOCs. Computational density functional theory (DFT) indicates that the interaction energy between analyte and sensing molecules can be used to explain comparative sensitivity.

The role of transporter ectodomains in drug recognition and binding: phlorizin and the sodium–glucose cotransporter

This article reviews the role of segments of SLCs located outside the plasma membrane bilayer (ectodomains) using the inhibition of SGLTs (SLC5 family) by the aromatic glucoside phlorizin as a model system. Phlorizin has been the lead substance for the development of SGLT2 (SLC5A2) inhibitors that have been introduced recently for the treatment of type 2 diabetes. Using mainly biophysical methods, it is shown that three ectodomains form well-defined substructures, such as short helices, that are arranged in a vestibule and undergo significant conformational changes during binding of phlorizin. From these data, tentative structures of inhibitor/ectodomain complexes are derived by molecular modeling. The ectodomains provide an additional binding site for aglucones, which cooperates with the binding site for the sugar moiety of phlorizin in the sugar translocation pathway, buried inside the membrane. They play a significant role in determining the specificity, selectivity, and affinity of sugar transport inhibitors and might even explain the difference in sensitivity of various members of the SGLT family, located in different tissues and organs of the human body. Similar binding modes are suggested for several other SLCs, in particular the monoamine transporter (SLC6 family), that belong to the sodium/neurotransmitter cotransporter family.