A Chemical Relaxation Study of Human Prostatic Acid Phosphatase

Chemical relaxation methods and a dilution technique were applied to the study of the hydrolysis of p-nitrophenyl phosphate by human prostatic acid phosphatase. Although the reaction mechanism was not elucidated, rate constants and equilibrium constants were obtained for the reaction of enzyme and p-nitrophenol to form a complex. A slow, 2-sec relaxation effect which showed no concentration dependence was observed in various reaction mixtures, including some lacking the substrate and products of the hydrolytic reaction. The conclusion drawn is that there are two forms of the prostatic enzyme, which are normally in equilibrium with each other, but which undergo a relatively slow interconversion when this equilibrium is perturbed. A preliminary calculation indicates that these forms are present in the equilibrium ratio of 2:1.     

The chemical potential in solutions and the free energy of reaction

The expression for the chemical potential of a species in a multi-component solution is usually derived by a thermodynamic argument based on ideal gas theory. A direct statistical mechanical derivation is given below, using the methods of the preceding paper. The analysis is formulated in such a way that there is no need to introduce the notion of standard states. It is made clear why certain species (water, H⁺ in buffered systems) may be ignored in calculating the affinity (negative free energy change) of a chemical reaction. The equilibrium constant is expressed in terms of generalized phase integrals or partition functions.     

Measurements of Serotonin and Related Indoles Using Capillary Electrophoresis with Multiphoton-Induced Hyperluminescence

We report the use of multiphoton-excited photochemistry to generate highly fluorescent products from hydroxyindoles fractionated in submicron capillary electrophoresis channels. In this approach, the near-infrared (750 nm) output from a modelocked titanium:sapphire laser is focused at the outlet of a 0.6-μm i.d. capillary, producing pulse intensities of ∼10¹²W cm⁻²within a femtoliter focal volume. Hydroxyindole molecules migrating through the outlet aperture of the capillary intersect the beam focus, where absorption of three to four photons (∼1.65 eV photon⁻¹) initiates a photobleaching reaction. The resultant hydroxyindole photoproducts produce broadband visible emission (λ_max≈ 500 nm) when excited with two additional near-IR photons and appear substantially more resistant to photobleaching than the parent hydroxyindoles. This multiphoton-induced conversion of analytes to hyperluminescent derivatives thus offers a more sensitive approach than UV fluorescence for detecting extremely small quantities of material. Mixtures of the hydroxyindoles serotonin (5-hydroxytryptamine), 5-hydroxytryptophan, and 5-hydroxyindole acetic acid are reliably characterized (relative error ≈10%) in 100 s, with detection limits as low as ∼70 zmol (∼42,000 molecules). The sensitivity of this measurement strategy improves on the best previously reported results for capillary separations of indoles by more than one order of magnitude.     

Relationship between atorvastatin dose and the harm caused by torcetrapib

Development of the cholesteryl ester transfer protein (CETP) inhibitor, torcetrapib, was halted after the ILLUMINATE trial revealed an increase in both all-cause mortality (ACM) and major cardiovascular events (MCVEs) associated with its use. We now report that the harm caused by torcetrapib was confined to those in the 10 mg atorvastatin subgroup for both ACM [hazard ratio (HR) = 2.68, 95% CI (1.58, 4.54), P < 0.0001] and MCVEs [HR = 1.41, 95% CI (1.14, 1.74), P = 0.002], with no evidence of harm when torcetrapib was coadministered with higher doses of atorvastatin. In the atorvastatin 10 mg subgroup, age, prior heart failure and stroke were significantly associated with ACM, independent of torcetrapib treatment, whereas low apoA-I, smoking, hypertension, heart failure, myocardial infarction, and stroke were independently associated with MCVEs. After adjusting for these factors, the HR associated with torcetrapib treatment in the 10 mg atorvastatin subgroup remained elevated for both ACM [HR = 2.67, 95% CI (1.57, 4.54), P < 0.001] and MCVE [HR = 1.36, 95% CI (1.10, 1.69), P = 0.005]. Thus, the harm caused by torcetrapib was confined to individuals taking atorvastatin 10 mg. The harm could not be explained by torcetrapib-induced changes in lipid levels, blood pressure, or electrolytes. It is conceivable that higher doses of atorvastatin protected against the harm caused by torcetrapib.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

Dynamic rearrangement of the filamentous actin network occurs during zoosporogenesis and encystment in the oomycete phytophthora cinnamomi

The organization of filamentous actin (F-actin) in living cells of the oomycete Phytophthora cinnamomi was determined during zoosporogenesis and zoospore encystment by microinjecting sporangia with fluorescently labeled phalloidin and observing resultant fluorescence by confocal microscopy. In multinucleate sporangia prior to the induction of cleavage, phalloidin labeling took the form of plaques which occurred mainly in the periphery of the sporangia. After induction of cleavage, phalloidin labeling showed that the plaques disappeared and that F-actin began to accumulate along the developing cleavage planes and around nuclei and water expulsion vacuoles. F-actin labeling was also observed near the plasma membrane in zoospores and young cysts but reverted to the plaque form in older cysts. Localization of F-actin close to the developing cleavage planes is consistent with the idea that actin microfilaments function in the positioning and expansion of the cleavage membranes. Observations of plaques of actin in living sporangia provide evidence that plaques are not aldehyde-induced fixation artifacts. Copyright 1998 Academic Press.     

Serglycin expression during monocytic differentiation of U937-1 cells

Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.      

A microbead array chemical sensor using capillary-based sample introduction: toward the development of an "electronic tongue"

The development of a micromachined fluidic structure for the introduction of liquid samples into a chip-based sensor array composed of individually addressable polymeric microbeads is presented. The micromachined structure consists of micromachined storage cavities combined with a covering glass layer that confines the microbeads and fluidic channels. In our sensor array transduction occurs via optical (colorimetric and fluorescence) changes to receptors and indicator molecules that are covalently attached to termination sites on the polymeric microbeads. Spectral data are acquired for each of the individual microbeads using a charged-coupled device (CCD) allowing for the near-real-time analysis of liquid sample. Hence the micromachined fluidic structure must allow for both optical access to the microbeads and fluid flow through the micromachined cavities that serve as the microreactors/analysis chambers. One of the key parts of the structure is a passive fluid introduction system driven only by capillary force. This simple means of fluid introduction realizes a compact device. The capillary flow on the inlet channel has been studied, and the responses of the microbeads (alizarin complexone) to a liquid sample have been characterized. The test results show that this system is useful in a micro-total-analysis-system (mu-TAS) and biomedical applications.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.