An electron-capture gas–liquid-chromatographic method for the determination of prostaglandin F2α in biological fluids

A sensitive electron-capture gas-liquid-chromatographic method for the determination of sub-nanogram quantities of prostaglandin F(2alpha) was developed. The method is based on the sub-microgram scale conversion of the prostaglandin into the electron-capturing pentafluorobenzyl ester, and analysis of the latter as the tris-trimethylsilyl ether. The lower limit of detection was 12.5pg of the ester injected ;on-column' as the silylated product. The method was successfully applied to the determination of prostaglandin F(2alpha) in monkey plasma. The specificity of the analytical procedure was increased by incorporating a thin-layer chromatographic fractionation before gas-liquid chromatography. The utility of the analytical methodology developed was demonstrated by its application to the determination of plasma concentrations of intact prostaglandin F(2alpha) in a Rhesus monkey, after subcutaneous administration of a single dose of prostaglandin F(2alpha). The electron-capture gas-liquid-chromatographic assay is compared with the radioimmunoassay and the gas-liquid-chromatographic-mass-spectrometry assay for the determination of prostaglandin F(2alpha).     

Mechanism for increased leaf growth in elevated CO2

The effect of exposure to elevated CO₂ on the processes of leafcell production and leaf cell expansion was studied using primaryleaves of Phaseolus vulgaris L. Cell division and expansionwere separated temporally by exposing seedlings to dim red lightfor 10 d (when leaf cell division was completed) followed byexposure to bright white light for 14 d (when leaf growth wasentirely dependent on cell expansion). When plants were exposedto elevated CO₂ during the phase of cell expansion, epidermalcell size and leaf area development were stimulated. Three piecesof evidence suggest that this occurred as a result of increasedcell wall loosening and extensibility, (i) cell wall extensibility(WEx, measured as tensiometric extension using an Instron) wassignificantly increased, (ii) cell wall yield turgor (V, MPa)was reduced and (iii) xyloglucan endotransglycosylase (XET)enzyme activity was significantly increased. When plants wereexposed to elevated CO₂ during the phase of cell division, thenumber of epidermal cells was increased whilst final cell sizewas significantly reduced and this was associated with reducedfinal leaf area, WEx and XET activity. When plants were exposedto elevated CO₂ during both phases of cell division and expansion,leaf area development was not affected. For this treatment,however, the number of epidermal cells was increased, but cellexpansion was inhibited, despite exposure to elevated CO₂ duringthe expansion phase. Assessments were also made of the spatialpatterns of WEx across the expanding leaf lamina and the datasuggest that exposure to elevated CO₂ during the phase of leafexpansion may lead to enhanced extensibility particularly atbasal leaf margins which may result in altered leaf shape. The data show that both cell production and expansion were stimulatedby elevated CO₂, but that leaf growth was only enhanced by exposureto elevated CO₂ in the cell expansion phase of leaf development.Increased leaf cell expansion is, therefore, an important mechanismfor enhanced leaf growth in elevated CO₂, whilst the importanceof increased leaf cell production in elevated CO₂ remains tobe elucidated. Key words: Phaseolus vulgaris L., dwarf beans, elevated CO², biophysics of cell expansion, xyloglucan endotransglycosylase, XET, water relations     

A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.     

Juvenile hormone catabolism and oviposition in the codling moth, Cydia pomonella, as functions of age, mating status, and hormone treatment.

In vitro catabolism of juvenile hormone (JH) in haemolymph of adult female Cydia pomonella was ascribed mainly to juvenile hormone esterase (JHE) activity. No significant differences were noted between virgin and mated females 0-96 h post-emergence. Changes in JHE activity did not appear dependent upon fluctuations in JH titre; conversely, changes in JHE activity could not explain the changes in JH titres. Maximal JHE activity was recorded at 24 h (331.47 +/- 47.25 pmol/h/microl; 355.93 +/- 36.68 pmol/h/microl, virgin; mated insects, respectively) and preceded the peak in JH titres at 48 h. Topical application of JH II (10 ng-10 microg) or fenoxycarb (50 ng) enhanced JHE activity up to 640 and 56%, respectively. Treatment upon emergence with 10 microg JH II induced enzymic activity for less than 24 h, and when 10 microg JH II or 50 ng fenoxycarb were applied, circulating JH titres returned to control levels within 24 h. Oviposition was highly sensitive to exogenous JH and declined significantly with dosages >100 pg. To allow a degree of oocyte maturation before JH treatment, the hormone was administered at 6, 12, 24, or 48 h post-emergence and/or females were mated. Neither measure "protected" the system; oviposition declined immediately after JH application.     

Manganese administration induces the increased production of dopamine sulfate and depletion of dopamine in Sprague-Dawley rats

Sprague-Dawley rats were used as an experimental model for investigating the effects of manganese poisoning on the serum levels of unsulfated and sulfated forms of dopamine and its biosynthetic precursors, L-Dopa and L-p-tyrosine. Groups of rats were treated daily with Mn(2+) (20 mg or 40 mg; in the form of MnSO(4)) or Na(+) (20 mg; in the form of Na(2)SO(4)). High performance liquid chromatography (HPLC) analysis of the serum samples taken after a 50-day experimental period revealed that the serum level of dopamine sulfate increased by more than 10 times compared with untreated control rats or rats treated with sodium sulfate. In contrast, there was a dramatic decrease (by as much as 4.8 times) in the serum level of unsulfated dopamine in manganese-treated rats. The serum levels of L-Dopa sulfate and L-p-tyrosine sulfate were also markedly elevated, although not as much as those of dopamine sulfate. Meanwhile, the serum levels of unsulfated L-Dopa and L-p-tyrosine showed no dramatic changes. Atomic absorption spectrophotometric analysis revealed in general an accumulation of manganese in the four organ samples taken from manganese-treated rats. Compared with liver, heart, and kidney, the highest degree of manganese accumulation in manganese-treated rats appeared to be in brain. These results together suggested a role for manganese in stimulating the dopamine-sulfating sulfotransferases in brain, thereby leading to the depletion of dopamine in vivo.     

Chemical Characterization of Gallstones: An Approach to Explore the Aetiopathogenesis of Gallstone Disease in Sri Lanka

Introduction

Records on gallstones and associated ailments in Sri Lankan community are scarce, despite frequent detection of gallstone disease. Identification of the chemical composition of gallstones in the local setting is important in defining aetiopathogenic factors which in turn are useful in implementing therapeutic and preventive strategies. This study aimed to describe the chemical composition of gallstones and the socio-demographic factors of a cohort of Sri Lankan patients with gallstone disease.

Materials and Methods

Data on clinical and socio-demographic factors, and gallstones removed at surgery were collected from patients with cholelithiasis admitted to Teaching Hospital, Peradeniya, Sri Lanka from May 2011 to December 2012. External and cross sectional morphological features of gallstones were recorded by naked eye observation. Compositional analysis was carried out by Fourier Transform Infrared Spectroscopy, X - ray Powder Diffraction, and Atomic Absorption Spectrophotometry. Scanning Electron Microscopy was used to identify the microstructure of gallstones.

Results

Data of 102 patients were analyzed. Of them majority (n = 77, 76%) were females with a female: male ratio of 3:1. Mean age of the study group was 46.1±11.6 years. All the patients had primary gallbladder stones. According to the physical and chemical analysis, majority (n = 54, 53%) were pigment gallstones followed by mixed cholesterol gallstones (n = 38, 37%). Only 10 (9%) had pure cholesterol gallstones. Calcium bilirubinate, calcium carbonate and calcium phosphate were the commonest calcium salts identified in pigment gallstones and core of mixed cholesterol gallstones.

Conclusion

Presence of a pigment nidus in gallstones is a common feature in majority of Sri Lankan patients denoting the possible role of elevated unconjugated bilirubin in bile on the pathogenesis of GS. Hence it is imperative to explore this further to understand the aetiopathogenesis of GS among Sri Lankans.     

Silent ovulation, based on walking activity and milk progesterone concentrations, in Holstein cows housed in a free-stall barn

The objectives of the current study were to determine the incidence of silent ovulation (based on walking activity and milk progesterone profiles), identify risk factors for silent ovulation, and investigate its impact on reproductive performance in high-yielding dairy cows in free-stall housing. Overall, 277 lactations in 161 Holstein Friesian cows from a commercial dairy herd in northern Japan were studied. Walking activity (measured with pedometers) >80% above the mean for the preceding 2 d was defined as estrus, whereas day of ovulation was estimated using milk progesterone concentrations. Ovulation not preceded by increased walking activity was considered silent ovulation; the incidence was 55.2%, 23.8%, 21.3%, and 10.5% at the first, second, third, and fourth ovulations postpartum, respectively. Moderate and high milk yield significantly increased the risk of silent ovulation at second (odds ratio [OR] = 2.7 and 1.2; P = 0.04) and third and/or fourth ovulations (OR = 6.7 and 12.9; P = 0.03). Based on survival analysis, silent ovulations at the first, second, third, and/or fourth ovulations were associated with 28% (hazard ratio [HR] = 0.72), 55% (HR = 0.45), and 47% (HR = 0.53) reductions in pregnancy rate, respectively, and 41% (HR = 0.59), 66% (HR = 0.34), and 65% (HR = 0.35) reductions in artificial insemination (AI) submission rate. Cows with at least one silent ovulation (with the exception of the first ovulation) had a longer interval from calving to first AI (72 vs. 54 d, P < 0.001) and to achievement of pregnancy (133 vs. 80 d, P < 0.001). In conclusion, approximately one third of the ovulations (based on milk progesterone concentrations) in Holstein cows within 90 d postpartum were silent. Silent ovulations at the second to fourth ovulations were associated with high milk yields and at all ovulations were associated with impaired reproductive performance.     

A comparison of methods for studying pressure and solute potentials in xylem and also in phloem laticifers of Hevea brasiliensis

In this study, several ways have been examined to measure latexpressure, including some novel methods, with the aim of findingbetter methods than are presently available. A novel method,similar to the pressure probe as used for single cells, hasbeen applied to laticifers, exerting a back-pressure to preventexudation. The type of capillary bubble manometer developedby Bourdeau and Schopmeyer for resin pressures (1958) and thenused for latex pressures by Buttery and Boatman (1964, 1966)has been improved and the construction and standardization ofbubble manometer gauges for routine field use has been greatlysimplified. It is concluded that the back-pressure method in its presentstate of development provides neither sufficient rapidity norprecision on account of the slow response time. This methoddoes not justify the two operators needed. The requirement forrelatively large quantities of escaping latex limits its applicationsand, in certain instances, pressurization might be incomplete.Nevertheless, it has provided some additional confidence inthe accuracy of the other methods because readings can oftenbe repeated many times on a single puncture, which is impossiblewith a bubble manometer. Also the gauge used is linear and thereforemore accurate at high pressures than a bubble manometer. The newly designed Buttery and Boatman gauge requires fewerparts and utilizes standard plastic pip-petor tips. These greatlyspeed and simplify construction of the gauges, which are neededin large numbers as they are essentially disposable. These gaugeshave proved to be remarkably reliable, even with minimal latexvolumes, and have always indicated the highest latex pressuresof all methods used at a particular site, suggesting that leakagerates are minimal at the junction between gauge and laticifers.The plastic tip holders facilitate the manual insertion of thesedelicate gauges into the tree bark. Xylem sap is contaminated by phloem sap if collected from petiolesusing a pressure bomb. However, this problem can be overcomein stems by bark ringing. Alternatively, the vacuum extractionmethod can be used which does not seem to suffer from this contaminationproblem. Several ways have been tested to maximize the reliability oflatex serum samples for analysis in a vapour osmometer. Pressureand solute components of water potential can be integrated togive total water potentials. Centrifugation provides the bestmethod to remove rubber particles in Hevea latex: valid osmoticpotentials of serum can be made providing appropriate precautionsare observed. Key words: Latex pressure potentials, laticifer solute potentials, pressure pump, manometer, xylem solute potentials     

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.     

Evaluating vaccinia virus cytokine co-expression in TLR GKO mice

Using Toll-like receptor (TLR) and MyD88 gene knock-out (GKO) mice the effect of TLRs and MyD88 on virus replication, interferon (IFN)-β production, natural killer (NK) cell and CD8T cell responses were assessed following ectromelia virus (ECTV) and recombinant vaccinia virus (rVV) infection. The capacity for rVVs encoding cytokines to restore immune function in MyD88(-/-) mice was clearly demonstrated. Results showed that TLR2(-/-), TLR4(-/-)and TLR7(-/-) mice survived ECTV infection whereas MyD88(-/-) and TLR9(-/-)mice, in contrast, were highly susceptible. Next, following infection with rVV, MyD88(-/-) mice elicited reduced serum IFN-β, NK cell and CD8T cell responses compared with wild-type mice, whereas TLR9(-/-) mice showed elevated CD8T cell responses. When MyD88(-/-)mice were infected with rVV co-expressing IFN-β these mice were able to restore IFN-β levels and CD8T cell responses but not NK cell activation. Interestingly, even though rVV co-expressing interleukin (IL)-2 enhanced NK cell activation in MyD88(-/-) mice, this was not associated with an antiviral effect, as observed in normal mice. Surprisingly, co-infection with rVV IL-2/rVV IL-12, but not rVV IL-2/rVV IFN-β, restored the attenuated phenotype of rVV IL-2 in MyD88(-/-) mice indicating that the IL-2/IL-12 combination promotes antiviral responses. Our results clearly show that the CD8T cell defect observed in MyD88(-/-) mice to vaccinia virus infection can be restored by rVV-encoding IFN-β demonstrating the critical role of this cytokine in T cell mediated immunity and illustrates that the model can provide an effective platform for the elucidation of cytokine immunobiology.