Different mechanistic requirements for prokaryotic and eukaryotic chaperonins: a lattice study

MOTIVATION: The folding of many proteins in vivo and in vitro is assisted by molecular chaperones. A well-characterized molecular chaperone system is the chaperonin GroEL/GroES from Escherichia coli which has a homolog found in the eukaryotic cytosol called CCT. All chaperonins have a ring structure with a cavity in which the substrate protein folds. An interesting difference between prokaryotic and eukaryotic chaperonins is in the nature of the ATP-mediated conformational changes that their ring structures undergo during their reaction cycle. Prokaryotic chaperonins are known to exhibit a highly cooperative concerted change of their cavity surface while in eukaryotic chaperonins the change is sequential. Approximately 70% of proteins in eukaryotic cells are multi-domain whereas in prokaryotes single-domain proteins are more common. Thus, it was suggested that the different modes of action of prokaryotic and eukaryotic chaperonins can be explained by the need of eukaryotic chaperonins to facilitate folding of multi-domain proteins. RESULTS: Using a 2D square lattice model, we generated two large populations of single-domain and double-domain substrate proteins. Chaperonins were modeled as static structures with a cavity wall with which the substrate protein interacts. We simulated both concerted and sequential changes of the cavity surfaces and demonstrated that folding of single-domain proteins benefits from concerted but not sequential changes whereas double-domain proteins benefit also from sequential changes. Thus, our results support the suggestion that the different modes of allosteric switching of prokaryotic and eukaryotic chaperonin rings have functional implications as it enables eukaryotic chaperonins to better assist multi-domain protein folding.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.     

Stability of helix-rich proteins at high concentrations

A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 degrees C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (T(m)s) in concentrated rather than in dilute solutions (deltaT(m) approximately 2-10 degrees C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower T(m) than in dilute solutions (deltaT(m) approximately 2-20 degrees C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions.     

Snail基因及其蛋白、E-cadherin蛋白在胃癌中的表达及其临床意义

目的观察Snail mRNA及其蛋白、E-cadherin蛋白在胃癌组织中的表达及其与胃癌临床病理特征的关系,并探讨它们在胃癌发生、发展中的作用及其临床应用价值。方法收集96例手术切除胃癌标本,同时取80例癌旁组织作为对照。应用免疫组织化学S-P法检测胃癌组织、癌旁组织中snail蛋白、E-cadherin蛋白的表达;运用原位分子杂交技术检测胃癌组织、癌旁组织中Snail mRNA的表达。结果（1）Snail蛋白在胃癌组织阳性率（83.3%）显著高于癌旁组织（41.25%）（P〈0.05）;高、中分化组Snail蛋白阳性表达率显著低于低分化组（P〈0.05）;Snail蛋白的阳性表达率在乳头状腺癌、管状腺癌及低分化腺癌与黏液癌之间差异有显著性（P〈0.05）;Snail蛋白的表达与胃癌浸润深度、淋巴结转移及远处转移有关（P〈0.05）,与性别、年龄、肿瘤大小、肿瘤部位及临床分期无关（P〉0.05）;（2）胃癌组织中snail mR-NA的阳性率（76%）显著高于癌旁组织（30%）（P〈0.05）;高、中分化组Snail mRNA阳性表达率显著低于低分化组（P〈0.05）;Snail mRNA的阳性表达率在乳头状腺癌、管状腺癌及低分化腺癌与黏液癌之间差异有显著性（P〈0.05）;Snail mRNA的表达与浸润深度及淋巴结转移有关（P〈0.05）,与性别、年龄、肿瘤大小、肿瘤部位、临床分期及远处转移无关（P〉0.05）;（3）E-cadherin蛋白在胃癌组织阳性率（37.5%）显著低于癌旁组织（100%）（P〈0.05）;高、中分化组E-cadherin蛋白阳性率显著高于低分化组（P〈0.05）;E-cadherin蛋白阳性率在乳头状腺癌、管状腺癌及低分化腺癌与黏液癌之间差异有显著性（P〈0.05）;E-cadherin蛋白的表达与胃癌浸润深度、淋巴结转移、临床分期及远处转移有关（P〈0.05）,与性别、年龄、肿瘤大小、肿瘤部位均无关（P〉0.05）;（4）胃癌组织中snail mRNA和snail蛋白的表达呈正相关（r=0.594,P〈0.05）;Snail蛋白和E-cadherin蛋白的表达呈负相关（r=-0.234,P〈0.05）。结论（1）E-cadher-in蛋白低表达与Snail蛋白高表达可能是胃黏膜恶性转变以及胃癌发生浸润转移的重要生物学标志;联合检测E-cadherin蛋白与Snail蛋白对预测胃癌浸润转移有重要意义。（2）Snail蛋白可能在转录水平上调控E-cadherin蛋白的表达。     

Molecular scanner experiment with human plasma: improving protein identification by using intensity distributions of matching peptide masses

The development of high throughput utilities to identify proteins is a major challenge in present research in the field of proteomics. One such utility, the molecular scanner, uses proteins separated by two-dimensional polyacrylamide gel electrophoresis that are digested in the gel and during transfer onto a collecting membrane. After adding a matrix, the membrane is inserted into a matrix-assisted laser desorption/ionization-time of flight mass spectrometer and a peptide mass fingerprint (PMF) is measured for every scanned site. Since the spacing between scanned sites is much smaller than the size of the most abundant protein spots, there is a certain redundancy in the data that was used in an earlier experiment with Escherichia coli [1] to improve mass calibration and PMF identification results. It was observed that the signal intensity of a peptide mass as a function of the position on the membrane showed similar patterns if peptides stemmed from the same protein. Taking account of these similarities a clustering algorithm was used to find lists of experimental masses with similar intensity distributions, which provided clearer identification of the corresponding proteins. Here, these methods are applied to a human plasma scan, where proteins were highly modified and less separated. The presence of very abundant proteins like albumin and immunoglobulins added another difficulty. The calibration of the initial PMFs was not satisfactory and masses had to be recalibrated. After discarding chemical noise, the membrane was partitioned into regions and for each region protein identification was carried out separately. A new scoring method was used, where the PMF score was multiplied by a factor that measures the similarity of matching peptides. This method proved to be more robust than the method developed in [1] if the region where a protein was found had an extended, nonspherical shape and strong overlap with regions of other proteins. Many proteins annotated on the SWISS-2D PAGE human plasma master gel could be clearly identified and many interesting properties were observed.     

Activity-Dependent Modulation of Odorant Receptor Gene Expression in the Mouse Olfactory Epithelium

Activity plays critical roles in development and maintenance of the olfactory system, which undergoes considerable neurogenesis throughout life. In the mouse olfactory epithelium, each olfactory sensory neuron (OSN) stably expresses a single odorant receptor (OR) type out of a repertoire of ∼1200 and the OSNs with the same OR identity are distributed within one of the few broadly-defined zones. However, it remains elusive whether and how activity modulates such OR expression patterns. Here we addressed this question by investigating OR gene expression via in situ hybridization when sensory experience or neuronal excitability is manipulated. We first examined the expression patterns of fifteen OR genes in mice which underwent neonatal, unilateral naris closure. After four-week occlusion, the cell density in the closed (sensory-deprived) side was significantly lower (for four ORs), similar (for three ORs), or significantly higher (for eight ORs) as compared to that in the open (over-stimulated) side, suggesting that sensory inputs have differential effects on OSNs expressing different OR genes. We next examined the expression patterns of seven OR genes in transgenic mice in which mature OSNs had reduced neuronal excitability. Neuronal silencing led to a significant reduction in the cell density for most OR genes tested and thinner olfactory epithelium with an increased density of apoptotic cells. These results suggest that sensory experience plays important roles in shaping OR gene expression patterns and the neuronal activity is critical for survival of OSNs.     

Low-energy X-radiation for prevention of restenosis results in localized inhibition of V79 fibroblast cell proliferation

Local delivery of high-energy ionizing radiation by using 3 or 3 emitters to injured vessels demonstrated inhibition of cell proliferation (CP) and neointima formation. Low-energy ('soft') X-radiation (LEXR) offers logistic and safety advantages over the use of disposable radioisotopes. This study evaluated the efficacy of LEXR in penetration and inhibition of CP at doses similar to those prescribed for the use of radioisotopes for prevention of restenosis. Serial measurements in an ion chamber detected the attenuation of LEXR using potentials of 17 and 40 kV at a distance of 17 cm of air through 0-10 mm depths of serum-containing tissue culture medium. The effect of inhibition on CP was determined by exposing V79 fibroblasts to a potential of 17 kV in order to deliver a prescribed dose of 13 Gy at a dose rate of 2.17 Gy/min to the surface of the cells. Complete inhibition of CP at a height of 0.00 mm occurred with 13 Gy; however, a 50% attenuation of the dose was measured at a medium depth of 1.22 mm and was associated with a reduction of 60% of the CP. LEXR demonstrated an ability to inhibit CP at doses equivalent to those used in techniques involving 3 and 3 irradiation. Under such conditions, the dose gradient is too high, especially for large vessels. However, a catheter-based LEXR that could be inserted into the artery with the capability of varying effective energy would be ideal for intravascular applications.     

Combining bulk segregation analysis and microarrays for mapping of the pH trait in melon

The availability of sequence information for many plants has opened the way to advanced genetic analysis in many non-model plants. Nevertheless, exploration of genetic variation on a large scale and its use as a tool for the identification of traits of interest are still rare. In this study, we combined a bulk segregation approach with our own-designed microarrays to map the pH locus that influences fruit pH in melon. Using these technologies, we identified a set of markers that are genetically linked to the pH trait. Further analysis using a set of melon cultivars demonstrated that some of these markers are tightly linked to the pH trait throughout our germplasm collection. These results validate the utility of combining microarray technology with a bulk segregation approach in mapping traits of interest in non-model plants.     

Leaf biomechanics, morphology, and anatomy of the deciduous mesophyte Prunus serrulata (Rosaceae) and the evergreen sclerophyllous shrub Heteromeles arbutifolia (Rosaceae)

Leaf tensile properties were compared between the mesic deciduous tree Prunus serrulata (var. "Kwanzan") and the xeric and sclerophyllous chaparral evergreen shrub Heteromeles arbutifolia (M. Roem). All values for biomechanical parameters for H. arbutifolia were significantly greater than those of P. serrulata. The fracture planes also differed between the two species with P. serrulata fracturing along the secondary veins, while H. arbutifolia most often fractured across the leaf irrespective of the vein or mesophyll position, thus yielding qualitative differences in the stress-strain curves of the two species. Anatomically, P. serrulata exhibits features typical for a deciduous mesophytic leaf such as a thin cuticle, a single layer of palisade mesophyll, isodiametric spongy mesophyll, and extensive reticulation of the laminar veins. Heteromeles arbutifolia leaves, however, are typically two- to three-fold thicker with a 35% higher dry mass/fresh mass ratio. The vascular tissue is restricted to the interface of the palisade and spongy mesophyll near the center of the leaf. Both epidermal layers have a thick cuticle. The palisade mesophyll is tightly packed and two to three layers thick. The spongy mesophyll cells are ameboid in shape and tightly interlinked both to other spongy cells as well as to the overlying palisade layer. We conclude that the qualitative and quantitative biomechanical differences between the leaves of these two species are likely due to a complex interaction of internal architectural arrangement and the physical/chemical differences in the properties of their respective cell walls. These studies illustrate the importance that morphological and anatomical correlates play with mechanical behavior in plant material and ultimately reflect adaptations present in the leaves of chaparral shrubs that are conducive to surviving in arid environments.