Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases that are pivotal in cellular regulation. Since its discovery in 1977, PKCs have been known as cytosolic and peripheral membrane proteins. However, there are reports that PKC can insert into phospholipids vesicles in vitro. Given the intimate relationship between the plasma membrane and the activation of PKC, it is important to determine whether such “membrane-inserted” form of PKC exists in mammalian cells or tissues. Here, we report the identification of an integral plasma membrane pool for all the 10 PKC isozymes in vivo by their ability to partition into the detergent-rich phase in Triton X-114 phase partitioning, and by their resistance to extractions with 0.2 M sodium carbonate (pH 11.5), 2 M urea and 2 M sodium chloride. The endogenous integral membrane pool of PKC in mouse fibroblasts is found to be acutely regulated by phorbol ester or diacylglycerol, suggesting that this pool of PKC may participate in cellular processes known to be regulated by PKC. At least for PKCα, the C2–V3 region at the regulatory domain of the kinase is responsible for membrane integration. Further exploration of the function of this novel integral plasma membrane pool of PKC will not only shed new light on molecular mechanisms underlying its cellular functions but also provide new strategies for pharmaceutical modulation of this important group of kinases.

Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)

The MNK (Menkes disease protein; ATP7A) is a major copper- transporting P-type ATPase involved in the delivery of copper to cuproenzymes in the secretory pathway and the efflux of excess copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in Menkes disease, a fatal neurodegenerative copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic detergent DDM (n-dodecyl b-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 µM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6 µM; h=5.0). The purified MNK demonstrated active ATP-dependent vectorial 64Cu transport when reconstituted into soya-bean asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian copper-transporting P-type ATPase associated with a human disease.

Effects of laundering on conductivity of polypyrrole-coated textiles

This study examines the resistance change in conductive polypyrrole-coated PET fabrics under the AS 2001.4.15 – 1994 laundering test conditions. The effects of individual detergent components of a standard detergent, including auxiliary chemicals, at four different temperatures were studied. When the coated fabric was washed under the AS 2001.4.15-1994 conditions, the ECE reference detergent and pure soap flakes (sodium sterate) both decreased the conductivity of the coating at a rate exponentially proportional to the laundering temperature. Detergent types had an influence over the rate of degradation; pH conditions had a large influence on the rate of polymer deterioration with the acidic nonionic detergent giving rise to significantly improved laundering conditions. The auxiliary chemicals, sodium carbonate and sodium perborate were seen to cause large degradation of polymers during laundering. Ethylene diamine tetra acetic acid was seen to have only a slight influence on the reduction of conductivity of polymers.

Morphological and biochemical alterations of abalone testicular germ cells and spawned sperm and their fertilizing ability

In this study, we aimed to detect morphological and biochemical changes in developing germ cells (Gc), testicular sperm (Tsp), and spawned sperm (Ssp) using capacitation-associated characteristics. Gradual changes in the profiles of two membrane proteins, namely NaCl- and detergent-extractable proteins, were observed as compared Gc with Tsp and Tsp with Ssp. These membrane modifications were accomplished mostly through the introduction of new protein sets, both peripheral and integral, into Tsp and Ssp membranes. Activation of serine proteases, particularly in Ssp detergent-extracted proteins with the molecular masses of 38–130 kDa was evident and marked a major difference between Ssp and Tsp. An increase in the level of tyrosine phosphorylation of the proteins ranging from 15 to 20 kDa was noted in Tsp and remained constant in Ssp. Specifically, these three capacitation-associated characteristics could be detected in Ssp, possessing full fertilizing capacity. The lack of an activated proteolytic activity in Tsp resulted in a delayed fertilization, but not affected fertilizing ability. We believe that these characteristics should be advantageous in predicting abalone sperm fertilizing capability, particularly in cases when isolated germ cells or purified Tsp are used in place of spawned sperm in abalone aquaculture.

Study into the ultrasonic cleaning of wool

This research studied the use of ultrasonic irradiation during wool cleaning with an aim to improve the cleaning process and reduce its impact on the environment. The research found that ultrasonic cleaning reduced energy, detergent and chemicals consumption, reduced fibre entanglement and had no significant effect on fibre properties.

Thermal and mechanical properties of ultrasonically treated wool

Wool fabrics, ultrasonically treated in various chemical conditions and for different time durations, were analysed for thermal properties by thermo-gravimetric analysis and differential scanning calorimeter, in comparison with the untreated fabric. Fabric mechanical properties, such as bending and tensile performance, and changes in fibre morphology were also evaluated before and after ultrasonic treatment.It is found that wool treated with ultrasonics at the appropriate time, has less mass loss and a higher thermal degradation temperature than that without ultrasonic treatment or with prolonged ultrasonic treatment. Resistance to thermal degradation is reduced when wool is ultrasonically treated in the presence of alkali. Differential scanning calorimeter analysis shows that while ultrasonic treatment has little effect on fibre crystallinity, an appropriate treatment can provide wool with increased water absorption. Ultrasonic treatment stiffens wool fabric to some extent when the treatment time is prolonged. The addition of detergent alone to the ultrasonic bath has little effect on fabric tensile behaviour, whereas a treatment with both detergent and alkali produces severe fibre damage and significant loss of fabric tensile strength.

Nurses’ advice regarding sterile or clean urinary drainage bags for individuals with a long-term Indwelling urinary catheter

PURPOSE: The purpose of this study was to identify, compare, and explore advice nurses give to community-dwelling long-term indwelling catheter users on the use of sterile or clean urinary drainage bags, and to obtain information that would inform the design of a larger-scale international survey.


SUBJECTS AND SETTINGS: A survey was targeted to nurse members of the International Continence Society (n = 130). Respondents (n = 28; 21.5%) included nurses from Australia, Canada, Belgium, Switzerland, the United Kingdom, and the United States, who specialized in managing incontinence.


METHODS: The project was conducted as a descriptive, exploratory pilot study. Respondents completed an online anonymous survey that was distributed by the International Continence Society. The survey instrument was designed by the investigators and comprised 14 questions with both fixed and open-ended response options.


RESULTS: Most respondents in this survey advised indwelling catheter users to reuse their catheter bags (n = 15; 68%). Factors that influenced advice included concerns about the cost of catheter bags, an evaluation of the individual's infection risk, local and national policies, evidence-based guidelines, users' living arrangements, and their ability to clean the bags. Advice on decontamination methods varied; however, the most commonly recommended cleaning agent was water and vinegar, followed by a sterilizing or bleach solution or dishwashing detergent.


CONCLUSION: Nurses play a key role in educating and supporting indwelling catheter users. Results of this study highlight variability in the advice nurses give to community-dwelling long-term indwelling catheter users about sterile or clean urinary drainage bags. This variability requires further investigation and affirms the need for a larger-scale study that draws on a broader sample of nurses.

The signature pedagogy in chemistry education

Laboratories are the signature pedagogy in chemistry education. The chemical sciences are based in investigations that are reproducible, and objectively testable. Some investigations might involve testing a hypothesis – does a carbonate produce carbon dioxide gas when reacted with acid? Other activities may not have an obvious hypothesis – how much salt is in this detergent package? Nevertheless, laboratory work is a distinctive part of science generally, and of chemistry in particular.

Laboratory work is a significant part of working in the chemistry profession. The best way for students to learn what scientists do, is to do what scientists do. The only way to conduct a laboratory investigation is to get into a laboratory and to do it!

Learning and doing chemistry in a laboratory is an important and irreplaceable part of a chemistry education.

Effects of forage type and extruded linseed supplementation on methane production and milk fatty acid composition of lactating dairy cows

Replacing dietary grass silage (GS) with maize silage (MS) and dietary fat supplements may reduce milk concentration of specific saturated fatty acids (SFA) and can reduce methane production by dairy cows. The present study investigated the effect of feeding an extruded linseed supplement on milk fatty acid (FA) composition and methane production of lactating dairy cows, and whether basal forage type, in diets formulated for similar neutral detergent fiber and starch, altered the response to the extruded linseed supplement. Four mid-lactation Holstein-Friesian cows were fed diets as total mixed rations, containing either high proportions of MS or GS, both with or without extruded linseed supplement, in a 4×4 Latin square design experiment with 28-d periods. Diets contained 500 g of forage/kg of dry matter (DM) containing MS and GS in proportions (DM basis) of either 75:25 or 25:75 for high MS or high GS diets, respectively. Extruded linseed supplement (275 g/kg ether extract, DM basis) was included in treatment diets at 50 g/kg of DM. Milk yields, DM intake, milk composition, and methane production were measured at the end of each experimental period when cows were housed in respiration chambers. Whereas DM intake was higher for the MS-based diet, forage type and extruded linseed had no significant effect on milk yield, milk fat, protein, or lactose concentration, methane production, or methane per kilogram of DM intake or milk yield. Total milk fat SFA concentrations were lower with MS compared with GS-based diets (65.4 vs. 68.4 g/100 g of FA, respectively) and with extruded linseed compared with no extruded linseed (65.2 vs. 68.6 g/100 g of FA, respectively), and these effects were additive. Concentrations of total trans FA were higher with MS compared with GS-based diets (7.0 vs. 5.4 g/100 g of FA, respectively) and when extruded linseed was fed (6.8 vs. 5. 6g/100 g of FA, respectively). Total n-3 FA were higher when extruded linseed was fed compared with no extruded linseed (1.2 vs. 0.8 g/100 g of FA, respectively), whereas total n-6 polyunsaturated FA were higher when feeding MS compared with GS (2.5 vs. 2.1 g/100 g of FA, respectively). Feeding extruded linseed and MS both provided potentially beneficial decreases in SFA concentration of milk, and no significant interactions were found between extruded linseed supplementation and forage type. However, both MS and extruded linseed increased trans FA concentration in milk fat. Neither MS nor extruded linseed had significant effects on methane production or yield, but the amounts of supplemental lipid provided by extruded linseed were relatively small.