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This work will present the passive waveguide devices developed at CNR-IEIIT aimed at multi-beam scientific instruments operating in the Q and W bands. Layouts of high-performance OMTs, polarizers and marker injectors suitable for medium scale production will be presented. The presentation will focus on the analysis, design, manufacturing and measurements of these components.

Mechanical Measurements of the Q-band OMTs for the LSPE-STRIP instrument

RF measurements of the 49 Q-Band OMTs for the LSPE-STRIP instrument

This work will present the corrugated-horns, polarizers and ortho-mode transducers developed for the 49-element coherent polarimeter array working in [39, 48] GHz to be embarked in the Large Scale Polarization Explorer. The latter is an ASI-funded balloon experiment designed to measure the CMB B-modes on large angular scales. The presentation will focus on the analysis, design, manufacturing and measurements of these components.

One of the most challenging aspects of the new-generation Low-Frequency Aperture Array (LFAA) radio telescopes is instrument calibration. The operational LOw-Frequency ARray (LOFAR) instrument and the future LFAA element of the Square Kilometre Array (SKA) require advanced calibration techniques to reach the expected outstanding performance. In this framework, a small array, called Medicina Array Demonstrator (MAD), has been designed and installed in Italy to provide a test bench for antenna characterization and calibration techniques based on a flying artificial test source. A radio-frequency tone is transmitted through a dipole antenna mounted on a micro Unmanned Aerial Vehicle (UAV) (hexacopter) and received by each element of the array. A modern digital FPGA-based back-end is responsible for both data-acquisition and data-reduction. A simple amplitude and phase equalization algorithm is exploited for array calibration owing to the high stability and accuracy of the developed artificial test source. Both the measured embedded element patterns and calibrated array patterns are found to be in good agreement with the simulated data. The successful measurement campaign has demonstrated that a UAV-mounted test source provides a means to accurately validate and calibrate the full-polarized response of an antenna/array in operating conditions, including consequently effects like mutual coupling between the array elements and contribution of the environment to the antenna patterns. A similar system can therefore find a future application in the SKA-LFAA context.

RF measurements of the Q-Band OMTs and polarizers for the LSPE-STRIP instrument

We discuss the design and expected performance of STRIP (STRatospheric Italian Polarimeter), an array of coherent receivers designed to fly on board the LSPE (Large Scale Polarization Explorer) balloon experiment. The STRIP focal plane array comprises 49 elements in Q band and 7 elements in W-band using cryogenic HEMT low noise amplifiers and high performance waveguide components. In operation, the array will be cooled to 20 K and placed in the focal plane of a $\sim 0.6$ meter telescope providing an angular resolution of $\sim1.5$ degrees. The LSPE experiment aims at large scale, high sensitivity measurements of CMB polarization, with multi-frequency deep measurements to optimize component separation. The STRIP Q-band channel is crucial to accurately measure and remove the synchrotron polarized component, while the W-band channel, together with a bolometric channel at the same frequency, provides a crucial cross-check for systematic effects., In press on the Proceedings of the SPIE Conference Astronomical Telescopes + instrumentation 2012, Amsterdam, paper 8446-279

Abstract Petroleum engineers rely on periodic acquisition of bottom-hole data to monitor changes in well and reservoir inflow characteristics, and to quantify well and reservoir parameters using Pressure Transient Analysis (PTA). However, the acquisition of this data, especially in an offshore high pressure and sour gas environment, presents many logistical challenges and operational risks, and can negatively affect expenditure and downtime performance. On the other hand, surface data is routinely available, sometimes with sufficient frequency and precision to carry out PTA. Surface data is affected by wellbore transients that must be taken into consideration for converting the surface data to bottom hole conditions with the accuracy required for proper analysis. This paper describes a novel technique to properly account for wellbore transient effects and convert surface data to downhole conditions to enable conventional PTA without the need for well intervention. The technique combines the transient wellbore modelling with a novel approach of defining coefficients in a modified bottom hole pressure equation which is described in the paper. Coefficients are calibrated using concurrent surface and downhole data sets and then utilised to convert future surface data to downhole conditions. The paper describes the findings and conclusions from a five-well pilot, which involved wells with a range of reservoir characteristics, single and multi-layer production, and with/without cross-flow. The pilot work compared PTA results utilising this technique and surface data with results generated using downhole data. Introduction RasGas and SPT Group (Schlumberger) have conducted a joint evaluation of transient modelling capabilities to accurately predict bottom hole pressure using wellhead pressure data as an input for offshore gas producers. The primary objective of the trial was to enable conventional transient well test analysis using the surface data converted to downhole conditions. This would reduce the need for recording downhole pressure buildup data to obtain wellbore skin and kh during routine production logging operations and hence reduce the operational risk associated with well intervention. The technique will also enable more frequent acquisition of pressure buildup data whenever the well is shut-in for any reason since the wellhead operating parameters are continuously recorded by the Real Time Information System (RTIS) on all RasGas wells.

Abstract Current technologies for assessing corrosion damage in downhole tubing and casing strings have several limitations. Under certain conditions, mechanical, electromagnetic and ultrasonic tools can be run inside a downhole tubing string to quantify corrosion and wall loss in that string. But these tools, at best, may only be able to qualitatively assess the condition of tubing strings which are in contact or close proximity to the tool. In wells that develop communication between the production/injection tubing and casing and allow ingress of potentially corrosive fluids into the annulus, the ability to effectively assess the condition of the production casing is important. This knowledge can drive critical decisions around well operating limits, surveillance programmes, workovers, or abandonment operations. This paper describes the results of corrosion modelling and testing conducted on carbon steel to understand the extent of internal corrosion damage expected on a production casing string when sour gas enters the tubing-casing annulus through a leak source. A wide range of conditions including various hydrogen sulfide (H2S) and carbon dioxide (CO2) concentrations were modelled using proprietary corrosion modelling software. Laboratory tests on corrosion coupons were also performed and compared to the model results. Key findings around the expected corrosion potential of production casing exposed to sour gas include:Corrosion rates are generally low over a wide range of H2S concentrations. The presence of H2S reduces the general corrosion rate by forming a protective iron sulfide (FeS) scale.Corrosion rates are sensitive to the chemical composition of the water in the annulus. Higher bicarbonates levels significantly reduce corrosion rates.General corrosion rates in a sweet gas environment with CO2 can be very high because of the discontinuous nature of iron carbonate scale formed at test conditions. This case study demonstrates how corrosion modelling can be used with laboratory testing to provide reliable insight about the condition of tubulars which cannot be directly measured. Introduction Corrosion assessment of downhole components in general is recognised as a major challenge faced by the oil and gas industry. Understanding corrosion of carbon steel in sour downhole environments is critical for ensuring asset integrity. Sour corrosion, or CO2/H2S corrosion is a complex process that is affected by many factors, including temperature, pressure, sour gas composition, flow regime, flow shear, and water chemistry. Over time the industry has studied, discussed and documented different types of corrosion mechanisms and corrosion management solutions.1–3 The limits in inspecting and monitoring the onset of corrosion have also been explored and recognised. One of the notable technology limits that the industry struggles with today is the ability to monitor the condition of concentric casing strings in a well. Existing multi-finger caliper tools can give direct quantitative mechanical measurement of the inner tubing walls. The caliper readings then can be translated into wall thickness readings, with assumptions around the external condition of the tubing, but these assumptions often entail a high degree of uncertainty.

Abstract This paper discusses an approach used to assess liquid film erosion/corrosion effects in the tubing strings of sour, high-rate, wet gas producers. This was done as an alternative to API RP 14E, which utilises an empirical erosional velocity factor "C" to estimate maximum velocity limits to minimise the potential for tubing metal loss from erosional effects. Many RasGas wells are completed with a full L-80 carbon steel or a combination L-80/Corrosion Resistant Alloy (CRA) production string. Once on production, a thin iron sulfide scale develops on the tubing wall significantly retarding the rate of metal loss due to internal corrosion. However, shear stresses generated from the condensate/water film flowing along the tubing wall could potentially remove this protective iron sulfide coating and expose fresh metal to much higher corrosion rates. This paper describes the approach adopted to assess the magnitude of shear stress created across a range of flow conditions including well production rates, fluid properties, and completion sizes using transient 1D flow simulation and more detailed 3D computational fluid dynamics modelling. The results will be used to design future laboratory experiments to assess the effect of these stresses on the integrity and effectiveness of the iron sulfide scale in reducing corrosion rates. Introduction RasGas Company Limited initiated a study to investigate the potential effect of high flow velocity on the stability of iron sulfide scales formed in carbon steel tubing during late field life when the wells will be required to maintain high rates at low wellhead pressures. Higher flow velocities will generate higher shear stresses that might be capable of stripping iron sulfide scale leading to high general corrosion rate and/or severe localised corrosion. The former will be expected in the case of uniform scale erosion while the latter is expected in the case of non-uniform scale erosion. In order to meet the study objective, the first step is to understand the flow behaviour (wall shear stress, flow regime and temperature distribution) in the tubing string. Four different tubing strings were investigated: a 7-inch monobore-vertical (MB-V) and deviated (MB-D), a 9-5/8 inch x 7 inch bigbore-vertical (BB-V) and deviated (BB-D). The completion schemes for MB and BB wells are illustrated in Figure 1. 1-D flow simulations using a Transient Flow Simulation Software (TFSS) were conducted to determine the flow regime, wall shear stress, and temperature distribution and the portion of liquid flowing as wall film in the tubing strings. 3-D Computational Fluid Dynamics (CFD) simulations were also conducted to determine the wall shear stress at specific locations (X-overs) where there is a change in internal diameter of the tubing string that is greater than 10%. The objective of these simulations was to obtain shear stress predictions that would be utilised in a customised test loop to establish the stability of the iron sulfide film. If this film is not stable at the predicted shear stresses, then the wellbore integrity management programme would have to be focused on corrosion protection since historical well corrosion monitoring data has established high corrosion rate in tubing strings where the iron sulfide film has not been formed.

The Unmanned Aerial Vehicle (UAV) technology has been recently used to develop a pattern measurement system for low-frequency arrays in their real installation conditions. This paper presents the most important results that have been obtained on the Italian Medicina Array Demonstrator (MAD).

Abstract Well and reservoir surveillance data gathering activities can present a number of logistical challenges and operational risks associated with well intervention work, especially in offshore sour gas fields. This paper describes an approach adopted by RasGas Company Limited (RasGas) to reduce these risks by implementing a workflow that facilitates full integration of the acquired data to maximise its value and improve the understanding of well/reservoir performance. Hence, the workflow is used to arrive at a need-based strategy to plan, design and execute yearly well/reservoir surveillance programmes. The need for early well and reservoir performance data when a new gas field is placed on production often necessitates a calendar-based and broadly applied surveillance programme to develop reservoir deliverability and well productivity trends across the field. These baseline data are used to proactively identify and quantify potential threats to well productivity and production sustainability from issues such as skin build up from scale or condensate banking, unexpected formation water production, corrosion damage, etc. Once the sufficient baseline data set has been acquired, there is a need to develop an optimum approach towards well and reservoir surveillance programmes. This ensures that surveillance programme is focused on delivering truly needed data while reducing the risks inherent to well intervention activities. RasGas achieved this optimisation through development of a workflow called "DIET" (Data Integration and Evaluation Technique). Implementation of DIET has significantly reduced the reliance on a set-frequency logging strategy, thus reducing overall well intervention job counts, costs and risks. In addition to integrating all relevant data for single well evaluation, the DIET workflow is also being expanded to enable field-wide data integration.

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by arterial and/or venous thrombosis, recurrent fetal losses or other pregnancy complications, and the presence of antiphospholipid antibodies (aPL). Ocular manifestations occur in 8—88% of patients with APS and are typically due to vaso-occlusive disease involving retinal and choroidal vessels. We report an unusual case of neuroretinitis as a first presentation of lupus-like illness with APS. Lupus (2010) 19, 1662—1664.

Proopiomelanocortin (POMC) is a precursor polypeptide for various bioactive peptides, including adrenocorticotropic hormone, alpha-, beta-, and gamma-melanotropin, beta-endorphin, and beta-lipotropin. Although the classical source of POMC is the pituitary, various studies indicate the expression of POMC in several nonpituitary tissues. In this study, in situ hybridization with anti-sense cRNA riboprobe was used to show expression of POMC mRNA in human epidermis and cultured human epidermal cells (melanocytes and keratinocytes). POMC mRNA was amplified by reverse transcriptase-polymerase chain reaction using anti-sense and sense primers designed from Exons 2 and 3 of POMC gene. A approximately 300 bp product was present in normal human skin, grafted human skin, and cultured normal human melanocytes and keratinocytes. By Southern analysis this product was hybridized specifically to the POMC cDNA. Sequence analysis of the reverse transcriptase polymerase chain reaction product from tissues or cells showed 85% homology to POMC cDNA from human, bovine, pig, and monkey sources. This suggests the existence of a putative isoform or variant of POMC mRNA in human epidermis.

The proposed ‘Gravitational-Wave Antenna Detector InteRferometer (GWADIR)’ will be a 3 km × 3 km Fabry-Perot type laser interferometer. The laser oscillator to be used will be a DL excited Nd-YAG laser with a minimum output of 100 Watts. The optical system will consist of 25 cm diameter Recycler, Beam Splitter, and Near/End Mirrors for each of two Ducts. The mirror system will be suspended by fine wires to isolate the system from the seismic noises of the earth. The output light emerging from the interferometer will be split into 8 beams and sent to 8 different interference detectors. These detectors will measure the interference intensities for the recombined light from the separate legs of the interferometer. The effective amplification due to the interferometer cavities will allow measurements of displacements to better than 10 times the wavelength of the laser light. A high vacuum of 10-8 Torrs will be maintained throughout the system, using turbo-molecular and ion pumps. The detector is designed to reach strain sensitivities from gravitational sources of h

This work will present the research activities ongoing at the CNR-IEIIT institute concerning the development of scientific instrumentation for astrophysical surveys. The presentation will focus both on the analysis, design, manufacturing and measurement techniques and on the layouts conceived for a large class of passive waveguide components.

This contribution presents a manufacturing-oriented Ortho-mode Transducer configuration for polarimeter clusters at millimeter-waves. It is based on a turnstile junction with a proper recombination network. All the discontinuities were properly conceived so that the overall geometry can be manufactured in a multi-layer (platelet) fashion, using commercially-available metal layers. All the metal layers can be machined using the spark-erosion technique, which provides high accuracy and high repeatability for a large number of devices. A W-band implementation with standard WR10 output waveguides has been designed in the operative bandwidth 80-108 GHz. The simulated return loss is better than 25 dB in the overall bandwidth for both polarizations. The predicted insertion loss is 0.35 dB at 94 GHz (metal resistivity 10 ??cm). The isolation between the two polarizations is better than 60 dB owing to the symmetry of the device.

Previously, split-thickness human skin grafted onto athymic mice has been shown to become markedly hyperpigmented, but the factor(s) responsible for this hyperpigmentation had not been isolated. The present study describes the isolation and characterization of a potent melanogenic inhibitor from grafted human skin. Extracts from grafted skin inhibited, in a concentration-dependent manner, tyrosinase activity of normal human melanocytes and of Cloudman S91 murine melanoma in culture. Sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis of extracts from pre- and post-grafted skin demonstrated the presence of a protein doublet of approximately 14 kD exclusively in the post-grafted skin. This protein inhibited both tyrosinase activity and cellular proliferation in a concentration-dependent manner. The inhibition of tyrosinase activity in normal human melanocytes was 53% at 0.5 microgram/ml concentration, whereas this inhibition was almost complete in murine melanoma cultures at 1.0 microgram/ml. The protein did not inhibit either cellular proliferation or protein synthesis in normal human fibroblast cultures, and therefore may act specifically on melanocytes. Injections of the inhibitor corresponded with a delay and reduction in the quantity of pigment in human skin 2 weeks after grafting. Multiple injections of the inhibitor into the hyperpigmented xenografts (20 weeks after grafting) reversed the hyperpigmentation with no observable inflammatory or toxic responses. The results indicate that hyperpigmented human skin xenografts contain a potent inhibitor of melanogenesis and melanocyte proliferation.

In the human epidermis, melanocytes are distributed at a distance from each other. In contrast, melanocytes in nevi, which are considered benign neoplasms of melanocytes, are grouped in nests. Although still not well defined, environmental factors are thought to play an important role in the development of nevi. We found that chronic growth stimulation by leukotriene C4, a compound found in increased amounts in inflamed skin, induced pleiotropic modifications in the normal melanocyte phenotype. These changes include loss of contact inhibition and formation of structures resembling tumor spheroids. In parallel with these changes, there was a constitutive expression of Fos protein. Switching these cultures to medium supplemented with phorbol ester sustained growth with reversion of the altered phenotype. In contrast, a cAMP stimulator, cholera toxin, induced features of terminal differentiation. Our findings suggest a role for inflammatory mediators in human epidermal melanocytes. This observation provides insight into melanocyte growth alterations which may have relevance in early stages of melanocyte oncogenesis.

Vessel buckling is integrated into oscillometry to determine the continuous mean arterial pressure. A microprocessor based feedback control is utilized to maintain the arterial pressure at the buckling point. The derivative of arterial volume is employed to indicate when the vessel is buckling. Implementation of an automated control loop improves the time it takes to track mean arterial pressure and allows the determination of frequency response parameters. Therefore, tracking of mean arterial pressure (MAP) will allow for the instantaneous determination of arterial pressure by later introducing a second simultaneous control loop.

Vessel buckling is integrated into oscillometry to determine the continuous mean arterial pressure. A microprocessor based feedback control is utilized to maintain the arterial pressure at the buckling point. Implementation of an automated control loop improves the time it takes to track mean arterial pressure and allows the determination of frequency response parameters. Therefore, tracking of mean arterial pressure (MAP) will allow for the instantaneous determination of arterial pressure by later introducing a second simultaneous control loop.

We investigated the presence of bullous pemphigoid antibodies in bullous pemphigoid sera that are negative by standard indirect immunofluorescence. We incubated each of four indirect immunofluorescence-positive bullous pemphigoid sera, seven indirect immunofluorescence-negative bullous pemphigoid sera, one indirect immunofluorescence-negative herpes gestationis serum, three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera, five indirect immunofluorescence-negative epidermolysis bullosa acquisita sera, and two normal human sera with fresh human skin explants in medium 199 at 4 degrees C for 48 h. All bullous pemphigoid sera, herpes gestations serum, and the three indirect immunofluorescence-positive epidermolysis bullosa acquisita sera had IgG that bound the basement membrane zone of skin explants with moderate to marked intensity as demonstrated by immunofluorescence. Normal sera and indirect immunofluorescence-negative epidermolysis bullosa acquisita sera failed to bind the explant basement membrane zone. Immunoblotting of bullous pemphigoid sera showed five of seven indirect immunofluorescence-negative bullous pemphigoid sera to bind high-molecular weight and/or low-molecular weight bullous pemphigoid antigens from epidermal extracts. We conclude that the skin explant culture system is a very sensitive method for the detection of bullous pemphigoid antibodies in all bullous pemphigoid sera.

The present study was designed to examine the expression of proopiomelanocortin (POMC) and its related derivative peptide adrenocorticotropic hormone (ACTH) in murine derived Thy-1+ dendritic cells. Immunostaining using a polyclonal antibody specific to ACTH and parent POMC molecule indicated the presence of POMC and its derivative peptide, ACTH, in cultures of Thy-1+ dendritic cells. To explore whether the POMC peptide is present as a reservoir or synthesized de novo in Thy-1+ dendritic cells. Northern blot analysis using 30-mer oligonucleotide probe for alpha-MSH/ACTH precursor POMC was carried out in total RNA from these cells. Northern blot analysis revealed the presence of POMC like mRNA transcript. However, the observed size of transcript was smaller (approximately 0.9 kb) than that expressed by murine AtT20 cells (approximately 1.2 kb), an anterior pituitary tumor cell line used as a positive control. These observations suggest that the epidermal Thy-1+ lymphocytes, like thymic lymphocytes, might serve the epidermis as one source for the synthesis of POMC. The synthesis and presence of POMC in the epidermis may be related to some of the pigmentary anomalies observed in many mucocutaneous disorders.

Melanocytes are pigmented cells distributed in humans in several organs like the epidermis, the leptomeninges, the eye, and the inner ear. Epidermal melanocytes, whether derived from adult or neonatal skin, proliferate well in a medium supplemented with phorbol esters and other mitogens before they undergo senescence. Potent cAMP inducers like cholera toxin are also growth promoters for neonatal melanocytes but only transient growth stimulators for cells derived from adults. We used this cellular system to delineate biochemical pathways involved in proliferation and in terminal differentiation. Here we show that after a period of 4-8 wk of sustained proliferation in the presence of cholera toxin, the adult melanocytes became round, flat, and enlarged. These changes were associated with terminal growth and preceded by a five- to sixfold increase in cAMP levels and an 8- to 10-fold increase in melanin content. The simultaneous addition of phorbol esters and cholera toxin did not prevent cells from reaching terminal differentiation. Identified targets for phorbol esters are protein kinase C (PKC) and the mitogen-activated kinases (MAPKs), also called extracellular signal-regulated kinases (ERKs). PKC was found to be similarly regulated in proliferating and in terminally differentiated melanocytes. Proliferating melanocytes in early or late passage showed identical activation of the kinase ERK2. This kinase was rapidly phosphorylated upon phorbol 12-myristate 13-acetate (PMA) addition and specifically accumulated in the nucleus of the cells, whereas in unstimulated cells it had a perinuclear distribution. In contrast, senescent and terminally differentiated cells were unable to phosphorylate tyrosine residues of the ERK2 gene product in spite of presenting normal amounts of ERK2 protein. In addition, ERK2 did not show the nuclear accumulation observed in proliferating melanocytes after PMA activation and remained localized in the perinuclear area. These results demonstrate that senescent and terminally differentiated melanocytes share a common block in a critical pathway thought to integrate multiple intracellular signals transmitted by various second messengers and specifically prevent the continuation of the signal transduction cascade initiated by PMA activation of PKC.

The mechanisms for hyperpigmentation observed in human cutaneous xenografts placed on athymic nude mice was investigated. Histologic, biochemical, histochemical, and ultrastructural examinations were performed on human skin prior to grafting and at various times ranging from 2 weeks to 30 weeks post-grafting (PG). Hyperpigmentation was macroscopically visible on the graft as early as 4-6 weeks. The number of Dopa-positive melanocytes per unit area was increased at 2 weeks PG and remained elevated until 20 weeks PG. The surface area of the melanocytes, a measure of the activity of the cells, also increased significantly and remained above the pre-grafting size throughout the study. Western blot analysis using tyrosinase specific antibody (alpha Ty-SP) revealed the presence of tyrosinase exclusively in the grafted skin from 2 weeks to 12 weeks PG tested. Histological and ultrastructural observations revealed the presence of numerous dendritic melanocytes, indeterminant clear cells suggestive of Langerhans cells, and dermal melanophages. The results of this study suggest that the observed hyperpigmentation in grafted tissue is caused by an increase in the number of Dopa-positive melanocytes and probably from enhanced melanin production. Extracts of proteins from the xenografts exhibited prominent differences in low and high molecular proteins between pre- and post-grafted skin. Among them, the exclusive appearance of a protein doublet with apparent mw approximately 14 kDa was found in grafted skin, and subsequent studies indicate it has potent effects on melanocyte function.

Two forms of cytochrome c-specific methyltransferases from Euglena gracilis were purified approximately 100- and 50-fold, respectively, using DEAE-cellulose and gel-filtration chromatography. The methylation product of enzyme I was identified as S-methylmethionine and that of enzyme II as NG-monomethylarginine. Both enzymes were located in the cytosol and exhibit maximum activity at pH 7.0. Among the various proteins tested as substrates, the enzymes were highly specific toward cytochrome c. Various types of histones, in particular, were not modified by either enzyme. The molecular weights of enzyme I and II were 28,000 and 36,000, respectively. Various S-adenosyl-L-homocysteine analogs were tested for their inhibitory activity toward the enzymes. Only the D- and L-isomers of S-adenosylhomocysteine and sinefungin were significantly inhibitory. The Ki values for S-adenosyl-L-homocysteine were 8.13 X 10(-6) and 1.17 X 10(-5) M for enzyme I and II, respectively. Two-dimensional peptide mapping revealed the methylation site of enzyme I to be the methionine residue at position 65 while that of enzyme II to be the arginine residue at position 38. The methylation of either methionine or arginine residues by enzyme I and II, respectively, lowers the isoelectric point (pI) of cytochrome c: 9.23, 9.33, and 10.06 for S-methylmethionine-, NG-monomethylarginine-modified, and unmodified cytochrome c, respectively.

Two forms of a histone H1-specific S-adenosylmethionine:protein-lysine N-methyltransferase (protein methylase III) have been purified from Euglena gracilis 48- and 214-fold, respectively, with yields of 3.4 and 4.6%. The enzymes were purified on DEAE-cellulose and histone-Sepharose affinity chromatography and found to be highly specific toward histone H1 as a substrate. However, one of the enzymes also methylates other histone subfractions to a limited extent. Of the proteins other than histones, only myosin showed measurable methyl-accepting capability. Both enzymes were found to be inhibited by S-adenosylhomocysteine (D and L forms), S-adenosyl-L-ethionine, and sinefungin. While the Ki values for S-adenosyl-L-ethionine were similar for both enzymes, the values for S-adenosyl-L-homocysteine and sinefungin were 10-fold lower for the second form. The Km values for histone H1 and S-adenosyl-L-methionine were found to be 3.1 X 10(-7) and 2.7 X 10(-5) M, respectively, for the first enzyme, and 4.4 X 10(-7) and 3.45 X 10(-5) M for the second. Peptide analysis of methyl-14C-labeled H1 revealed that the two enzymes methylate different sites within the histone H1 molecule. The two enzymes were found to have molecular weights of 55,000 and 34,000, respectively. Both enzymes have an optimum pH of 9.0, which is identical to that of other protein-lysine N-methyltransferases thus far identified.

A monoclonal antibody, B10, generated against pure human lecithin-cholesterol acyltransferase (EC 2.3.1.43) caused the inhibition of the esterolytic and cholesterol esterifying activities of the enzyme. This antibody also reacted with a number of pancreatic and snake venom phospholipases A2 species but not phospholipase A1. A concentration-dependent inhibition of phospholipase A2 was also seen in the presence of B10. Treatment of lecithin-cholesterol acyltransferase or B10-reacting phospholipases with phenacyl bromide, a reagent known to interact with the active site of phospholipase A2, inhibited both their esterolytic activity and their capacity to bind to b10. A dimeric phospholipase A2 species with a known occluded active site did not cross-react with B10. Thus, lecithin-cholesterol acyltransferase and some enzymes of the phospholipase A2 family share a common antigenic determinant which is probably located near or at their esterolytic active site.

Purified human lecithin:cholesterol acyltransferase (LCAT) was covalently labeled by [3H]diisopropylflourophosphate with concomitant loss of enzymatic activity (M. Jauhiainen and P.J. Dolphin (1986) J. Biol. Chem. 261, 7023-7043). Some 60% of the enzyme was labeled in 1 h. Cyanogen bromide (CNBr) cleavage of the labeled, reduced, and carboxymethylated protein, followed by gel permeation chromatography yielded a 5- to 6-kDa peptide (LCAT CNBr-III) containing at least 60-70% of the incorporated label. Comparison of the amino acid composition of LCAT CNBr-III with that of the CNBr peptides predicted from the LCAT sequence (J. McLean et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339) indicates that LCAT CNBr-III is peptide 168-220. In 22 cycles of automated Edman degradation of CNBr-III a radioactive derivative was only observed at cycle 14, and of the predicted CNBr fragments only peptide 168-220 contains a serine at position 14 from the amino terminus. Tryptic peptides predicted from the sequence should contain Ser181 at positions 22 and 23 from the N-terminus of fragments 160-199 and 159-199, respectively. On the other hand, Ser216 should be in position 15 from the N-terminus in fragment 202-238. Radiolabel sequencing of the tryptic digest of [3H]diisopropylphosphate-LCAT resulted in recovery of radioactivity in cycles 22 and 23, whereas cycle 15 yielded negligible radioactivity. These results establish that Ser181 is the major active site serine in human LCAT.

The existence of metabolically distinct pools of S-adenosyl-L-methionine in Saccharomyces cerevisiae and isolated rat hepatocytes was investigated. Utilizing a relatively long labeling period with [methyl-14C]methionine, a metabolically 'stable' pool was labeled. A subsequent short labeling with [methyl-3H]methionine selectively labeled a putative metabolically 'labile' pool. The existence of these distinguishable pools was ascertained by following the 3H and 14C label disappearance in S-adenosyl-L-methionine during the chase-period in label-free media containing cycloleucine to prevent further synthesis of S-adenosyl-L-methionine. In both yeast and hepatocytes, the 3H/14C ratio in S-adenosyl-L-methionine decreased sharply. The individual 3H and 14C decrease in S-adenosyl-L-methionine showed t1/2 values of 3 and 8 min for yeast and 4 and 18 min for hepatocytes. The results strongly indicate that at least two metabolically distinct S-adenosyl-L-methionine pools actually do exist in both systems. Subcellular fractionation revealed that the 'labile' pool exist in the cytosol for both yeast and hepatocytes while the 'stable' pool exists in the vacuolar and the mitochondrial fraction for the yeast and hepatocytes respectively. The S-adenosyl-L-methionine pools were also studied in normal yeast under anaerobic chase condition and petite mutant yeast. Sharply contrasting with aerobically chased normal yeast, both showed closely parallel 3H and 14C decreases in S-adenosyl-L-methionine.

Two forms of a histone H1-specific S-adenosylmethionine:protein-lysine N-methyltransferase (protein methylase III) have been purified from Euglena gracilis 48- and 214-fold, respectively, with yields of 3.4 and 4.6%. The enzymes were purified on DEAE-cellulose and histone-Sepharose affinity chromatography and found to be highly specific toward histone H1 as a substrate. However, one of the enzymes also methylates other histone subfractions to a limited extent. Of the proteins other than histones, only myosin showed measurable methyl-accepting capability. Both enzymes were found to be inhibited by S-adenosylhomocysteine (D and L forms), S-adenosyl-L-ethionine, and sinefungin. While the Ki values for S-adenosyl-L-ethionine were similar for both enzymes, the values for S-adenosyl-L-homocysteine and sinefungin were 10-fold lower for the second form. The Km values for histone H1 and S-adenosyl-L-methionine were found to be 3.1 X 10(-7) and 2.7 X 10(-5) M, respectively, for the first enzyme, and 4.4 X 10(-7) and 3.45 X 10(-5) M for the second. Peptide analysis of methyl-14C-labeled H1 revealed that the two enzymes methylate different sites within the histone H1 molecule. The two enzymes were found to have molecular weights of 55,000 and 34,000, respectively. Both enzymes have an optimum pH of 9.0, which is identical to that of other protein-lysine N-methyltransferases thus far identified.