The Final Frontier of pH and the Undiscovered Country Beyond

The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, K _w = [H⁺]×[OH⁻].We used K _w [in a reversed way] to calculate the number of undissociated H₂O molecules required by this equilibrium constant to yield at least one of its daughter ions, H⁺ or OH⁻ at a given pH. In this way we obtained a formula that relates pH to the minimal volume V_pH required to provide a physical meaning to K _w, (where N _A is Avogadro’s number). For example, at pH 7 (neutral at 25°C) V_pH = 16.6 aL. Any deviation from neutral pH results in a larger V_pH value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H⁺ ions at equilibrium, thus the definition of pH based on K _w is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H⁺ ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H⁺ ions as a general source for biochemical reactions. Consequences of this finding are discussed.     

Mimicking Insect Communication: Release and Detection of Pheromone,Biosynthesized by an Alcohol Acetyl Transferase Immobilized in a Microreactor

Infochemical production, release and detection of (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone of the moth Spodoptera littoralis, is achieved in a novel microfluidic system designed to mimic the final step of the pheromone biosynthesis by immobilized recombinant alcohol acetyl transferase. The microfluidic system is part of an “artificial gland”, i.e., a chemoemitter that comprises a microreactor connected to a microevaporator and is able to produce and release a pre-defined amount of the major component of the pheromone from the corresponding (Z,E)-9,11-tetradecadienol. Performance of the entire chemoemitter has been assessed in electrophysiological and behavioral experiments. Electroantennographic depolarizations of the pheromone produced by the chemoemitter were ca. 40% relative to that evoked by the synthetic pheromone. In a wind tunnel, the pheromone released from the evaporator elicited on males a similar attraction behavior as 3 virgin females in most of the parameters considered.     

Loop-mediated isothermal amplification for the detection of goose circovirus

ABSTRACT: BACKGROUND: Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. Theinfection's clinical symptoms include growth retardation or feathering disorders but theinfection process may remain non-symptomatic what makes the infected birds moresusceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection ismade by histopathological examination, dot blot hybridization, polymerase chain reaction(PCR) and real-time PCR. However these techniques require application of thermocyclersand qualified staff which may be cost-consuming for some diagnostic units. The aim of thisstudy was to develop loop-mediated isothermal amplification assay (LAMP) as a simplemethod of GCV detection. RESULTS: The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus(GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate thereaction. The optimum reaction temperature and the time were 62°C for 30 min, respectively.The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eightisolates of GCV collected from geese flocks in Poland were examined. For comparison, realtimepolymerase chain reaction with F3 and B3 primers and SYBR Green dye wasconducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specificassay and alternative for PCR-based methods. CONCLUSIONS: The developed technique due to its simplicity may be applied by any veterinary laboratory oreven mobile diagnostics units for the routine detection of GCV.     

Interactions between CD44 protein and hyaluronan: insights from the computational study

CD44 is a protein, being a major cell surface receptor for hyaluronan (HA). Molecular modeling investigation was carried out on the murine CD44 in complex with a HA heptasaccharide in order to: (i) elucidate the nature and dynamics of interactions between the HA chain and CD44; (ii) find out if the existence of two conformational forms of CD44 discovered in the XRD (X-Ray Diffraction) study can be responsible for its switching between low and high affinity for HA. The results indicate that the contact of CD44 with HA is dominated by hydrogen bonding with small contribution of hydrophobic interactions and salt bridges. In addition, the two ('A' and 'B') conformational forms of the HA-CD44 complex reported experimentally by Banerji et al. cannot be observed during simulations when considering the distance between HA and the sidechain of the R45 residue. There exists, however, a free energy barrier associated with the change of the φ dihedral angle value at Y46. Additionally, some thermodynamic parameters (e.g. the Gibbs free energy change) accompanying the HA binding by CD44 were estimated.     

Polymorphism of the <Emphasis Type="Italic">CD36</Emphasis> Gene and Cardiovascular Risk Factors in Patients with Coronary Artery Disease Manifested at a Young Age

This study investigates potential associations between CD36 gene variants and the presence of risk factors in Caucasians with coronary artery disease (CAD) manifested at a young age. The study group consisted of 90 patients; the men were ≤ 50 years old and the women were ≤ 55 years old. Amplicons of exons 4 and 5 including fragments of introns were analyzed by DHPLC. Two polymorphisms were found: IVS3-6 T/C (rs3173798) and IVS4-10 G/A (rs3211892). The C allele of the IVS3-6 T/C polymorphism was associated with higher prevalence of obesity and diabetes, higher hsCRP, lower Lp(a) serum concentrations, and younger age at myocardial infarction. The A allele of the IVS4-10 G/A polymorphism was associated with older age of myocardial infarction and higher white blood cell count. The functional role of CD36 polymorphisms in CAD development needs further research.     

Thymosin β4 promotes the migration of endothelial cells without intracellular Ca2+ elevation

Numerous studies have demonstrated the effects of Tβ4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which Tβ4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with Tβ4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that Tβ4 interacts with Ku80, which may operate as a novel receptor for Tβ4 and mediates its intracellular activity. In this paper, we provide evidence that Tβ4 induces cellular processes without changes in the intracellular Ca(2+) concentration. External treatment of HUVECs with Tβ4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (Tβ4(AcSDKPT/4A)) or the actin-binding sequence KLKKTET (Tβ4(KLKKTET/7A)) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by Tβ4 was not associated with the intracellular Ca(2+) elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added Tβ4 induces HUVEC migration via the surface membrane receptors known to generate Ca(2+) influx. Our data confirm the concept that externally added Tβ4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.     

Association of C-terminal region of phosphoglycerate mutase with glycolytic complex regulates energy production in cancer cells

Cancer cells prefer anaerobic ATP synthesis, regardless of the availability of oxygen. It has been hypothesized that in these cells, glycolytic enzymes associate into a large complex, which results in an increased efficiency of glycolytic flux. However, there is no convincing in vivo evidence supporting this hypothesis. Here, we show that all the enzymes of triose phosphate metabolism, from aldolase to pyruvate kinase consecutively, form a macromolecular complex in vivo and that disruption of such complex significantly inhibits lactate release and ATP synthesis in the glycolytic pathway. Composition of the complex and the effectiveness of the glycolytic flux depends on lactate and glucose concentration. High concentrations of exogenous lactate reduces association of the C-terminal region phosphoglycerate mutase (PGAM) with the complex which results in its disruption and inhibition of ATP synthesis. Additionally, high lactate affects nuclear localization of PGAM and ceases cell proliferation. Our findings might provide new prospects for cancer treatment using low-molecular weight competitors to destabilize the glycolytic complex and reduce proliferative potential of cancer cells.     

A Thermodynamic Model of Microtubule Assembly and Disassembly

Microtubules are self-assembling polymers whose dynamics are essential for the normal function of cellular processes including chromosome separation and cytokinesis. Therefore understanding what factors effect microtubule growth is fundamental to our understanding of the control of microtubule based processes. An important factor that determines the status of a microtubule, whether it is growing or shrinking, is the length of the GTP tubulin microtubule cap. Here, we derive a Monte Carlo model of the assembly and disassembly of microtubules. We use thermodynamic laws to reduce the number of parameters of our model and, in particular, we take into account the contribution of water to the entropy of the system. We fit all parameters of the model from published experimental data using the GTP tubulin dimer attachment rate and the lateral and longitudinal binding energies of GTP and GDP tubulin dimers at both ends. Also we calculate and incorporate the GTP hydrolysis rate. We have applied our model and can mimic published experimental data, which formerly suggested a single layer GTP tubulin dimer microtubule cap, to show that these data demonstrate that the GTP cap can fluctuate and can be several microns long.     

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein: Is PAM involved in the capacitative calcium entry?

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca²⁺ signalling and maintenance of Ca²⁺ homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca²⁺-ATPase, Na⁺, K⁺-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca²⁺ ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca²⁺ entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca²⁺ entry, and their formation and rebuilding have an important regulatory role in cellular Ca²⁺ homeostasis.     

Biomarkers of HIV-1 associated dementia: proteomic investigation of sera

Background

Acute lymphoblastic leukemia (ALL) is a common form of cancer in children. Currently, bone marrow biopsy is used for diagnosis. Noninvasive biomarkers for the early diagnosis of pediatric ALL are urgently needed. The aim of this study was to discover potential protein biomarkers for pediatric ALL.

Methods

Ninety-four pediatric ALL patients and 84 controls were randomly divided into a "training" set (45 ALL patients, 34 healthy controls) and a test set (49 ALL patients, 30 healthy controls and 30 pediatric acute myeloid leukemia (AML) patients). Serum proteomic profiles were measured using surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy (SELDI-TOF-MS). A classification model was established by Biomarker Pattern Software (BPS). Candidate protein biomarkers were purified by HPLC, identified by LC-MS/MS and validated using ProteinChip immunoassays.

Results

A total of 7 protein peaks (9290 m/z, 7769 m/z, 15110 m/z, 7564 m/z, 4469 m/z, 8937 m/z, 8137 m/z) were found with differential expression levels in the sera of pediatric ALL patients and controls using SELDI-TOF-MS and then analyzed by BPS to construct a classification model in the "training" set. The sensitivity and specificity of the model were found to be 91.8%, and 90.0%, respectively, in the test set. Two candidate protein peaks (7769 and 9290 m/z) were found to be down-regulated in ALL patients, where these were identified as platelet factor 4 (PF4) and pro-platelet basic protein precursor (PBP). Two other candidate protein peaks (8137 and 8937 m/z) were found up-regulated in the sera of ALL patients, and these were identified as fragments of the complement component 3a (C3a).

Conclusion

Platelet factor (PF4), connective tissue activating peptide III (CTAP-III) and two fragments of C3a may be potential protein biomarkers of pediatric ALL and used to distinguish pediatric ALL patients from healthy controls and pediatric AML patients. Further studies with additional populations or using pre-diagnostic sera are needed to confirm the importance of these findings as diagnostic markers of pediatric ALL.