Transthyretin and complex protein pattern in aqueous humor of patients with primary open-angle glaucoma.

PURPOSE: To analyze protein patterns in the aqueous humor of glaucoma patients in comparison to control subject using two different methods. METHODS: Aqueous humor was collected from 52 patients with primary open-angle glaucoma (POAG) and from 55 control subjects (CO). Twenty-two POAG samples and 24 CO samples were used for protein profiling through surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) ProteinChip arrays. The data were analyzed by multivariate statistical methods and artificial neural networks. One highly significant biomarker was identified through matrix assisted laser desorption/ionisation time of flight-mass spectrometry (MALDI-TOF). Thirty samples from patients with POAG and 31 control samples were analyzed through two-dimensional electrophoresis. Subsequently, the protein spots of all gels were detected, and the two groups were compared. One spot group exhibiting clear differential abundance was identified by mass spectrometry (electrospray ionization mass spectrometry). RESULTS: In the samples analyzed by SELDI-TOF-MS, about 250 protein peaks could be consistently clustered in both groups. The analyses revealed eight biomarkers, which discriminated glaucoma from non-glaucoma controls with a sensitivity of 90% and a specificity of 87%. These biomarkers were purified further, and one marker, which was upregulated in glaucoma patients (p=0.006), was identified as transthyretin. The upregulation of transthyretin in POAG patients was also confirmed by enzyme linked immunosorbent assay (ELISA; p=0.03). In all samples analyzed by two-dimensional electrophoresis, complex protein patterns were detected in a total of 177 spot groups. The aqueous humor of all glaucoma patients revealed some regions that were clearly different from the controls. Several spots were significantly increased in the aqueous humor of glaucoma patients. One of the proteins that is highly abundant in the aqueous of glaucoma patients was identified as transthyretin. CONCLUSIONS: The aqueous humor of glaucoma patients revealed characteristic differences in protein/peptide profiles from control patients using two different analytical methods, SELDI-TOF-MS and two-dimensional electrophoresis. Interestingly, we could detect elevated transthyretin concentrations in glaucoma samples. Transthyretin might play a role in the onset of glaucoma since it has been shown to form amyloid deposits. These particles could cause outflow obstructions thereby increasing intraocular pressure as a possible onset mechanism.

Number of nitrate groups determines reactivity and potency of organic nitrates: a proof of concept study in ALDH-2-/- mice.

BACKGROUND AND PURPOSE: Mitochondrial aldehyde dehydrogenase (ALDH-2) has been shown to provide a pathway for bioactivation of organic nitrates and to be prone to desensitization in response to highly potent, but not to less potent, nitrates. We therefore sought to support the hypothesis that bioactivation by ALDH-2 critically depends on the number of nitrate groups within the nitrovasodilator. EXPERIMENTAL APPROACH: Nitrates with one (PEMN), two (PEDN; GDN), three (PETriN; glyceryl trinitrate, GTN) and four (pentaerithrityl tetranitrate, PETN) nitrate groups were investigated. Vasodilatory potency was measured in isometric tension studies using isolated aortic segments of wild type (WT) and ALDH-2-/- mice. Activity of the cGMP-dependent kinase-I (reflected by levels of phosphorylated VAsodilator Stimulated Phosphoprotein, P-VASP) was quantified by Western blot analysis, mitochondrial dehydrogenase activity by HPLC. Following incubation of isolated mitochondria with PETN, PETriN-chromophore and PEDN, metabolites were quantified using chemiluminescence nitrogen detection and mass spectrometry. KEY RESULTS: Compared to WT, vasorelaxation in response to PETN, PETriN and GTN was attenuated about 10fold in ALDH-2-/- mice, identical to WT vessels preincubated with inhibitors of ALDH-2. Reduced vasodilator potency correlated with reduced P-VASP formation and diminished biotransformation of the tetranitrate- and trinitrate-compounds. None of these findings were observed for PEDN, GDN and PEMN. CONCLUSIONS AND IMPLICATIONS: Our results support the crucial role of ALDH-2 in bioactivating highly reactive nitrates like GTN, PETN and PETriN. ALDH-2-mediated relaxation by organic nitrates therefore depends mainly on the number of nitrate groups. Less potent nitrates like PEDN, GDN and PEMN are apparently biotransformed by other pathways.

Membrane-assisted liquid-liquid extraction coupled with gas chromatography-mass spectrometry for determination of selected polycyclic musk compounds and drugs in water samples.

Selected polycyclic musk compounds and drugs were extracted from water samples by membrane-assisted micro liquid-liquid extraction. The two-phase extraction system consisted of polyethylene membrane bags filled with an organic solvent. Chloroform proved to be most suited as acceptor phase to extract caffeine, Galaxolide, Tonalide, phenazone and carbamazepine from aqueous samples. The compounds were enriched from 50 mL sample into a volume of 500 microL of chloroform. Gas chromatography-mass spectrometry (GC-MS) was applied for analysis. The extraction procedure was optimised in regard to membrane material, extraction time and temperature. The evaluation of the entire analysis protocol found limits of detection that ranged from 20 to 200 ng/L. The linear range of calibration covered one magnitude with standard deviations between 4 and 12%. Method comparison with standard analysis techniques such as solid-phase extraction (SPE) combined with GC-MS as well as LC-MS-MS confirmed this method as an easy and reliable protocol, even for the monitoring of matrix-loaded wastewater. The analysis of real samples established the feasibility of the technique.

Basic fibroblast growth factor induces 3T3 fibroblasts to synthesize and secrete a cyclophilin-like protein and beta 2-microglobulin.

When stimulated by fibroblast growth factor (FGF) BALB/c 3T3 cells synthesize and secrete elevated amounts of five proteins called the 'superinducible proteins', or SIPs. The expression of these proteins is greatly enhanced if the cells are treated with cycloheximide during induction. The 24 kDa protein (SIP24) has been purified and antiserum raised against it. This protein is N-glycosylated and probably structurally constrained by one or more intramolecular disulfide bonds. The amino acid sequences of three of four peptides show significant identity with cyclophilin, an abundant cytoplasmic protein believed to mediate the immunosuppressive effects of cyclosporin A. Several members of the cyclophilin family have been identified, and cDNA clones of two cyclophilin-like proteins with signal sequences have been reported. Here we show that at least one cyclophilin-like protein is secreted and that its expression is regulated by growth factors. The 12.5 kDa protein (SIP12.5) was found to be immunoprecipitated by an antiserum raised to human beta 2-microglobulin. This protein is strongly induced by interferon, which is a characteristic of the beta 2-microglobulin gene. Thus, FGF stimulates mouse embryo 3T3 cells to produce two proteins related to immune regulatory molecules. This may reflect an interaction between immune cells and nonimmune cells that occurs in vivo during processes such as wound healing when growth factors are released locally.

Effects of multipurpose contact lens solutions on the protein composition of the tear film.

OBJECTIVES: To analyze the influence of multipurpose contact lens cleaning solutions on tear proteins. Changes in tear film protein profiles of contact lens wearers who used several marketed brands of multipurpose contact lens care solutions, were assessed by ProteinChip analysis. METHODS: Three studies were conducted. Study I was a comparison of Complete and OptiFree multipurpose solutions. Study II was a study with Complete Moisture Plus solution, Study II was a comparison of Renu and Solocare contact lens solutions. Wearers of soft contact lenses were assigned to use the contact lens care solutions for 4 weeks. Non-contact lens wearing patients were used as controls. Tear samples of each participant were analyzed with the ProteinChip (SELDI-TOF) system. Multivariate statistical analysis and artificial neural networks were used to determine the tear protein profiles of each study group. RESULTS: Before starting the use of the solutions, the tear protein composition in all contact lens wearers deviated from the tear composition of the non-contact lens wearing controls. After 4 weeks of using the different care regimens, the tear protein composition of the patients using Complete or Complete Moisture Plus solutions tended to move toward that of the non-contact lens wearing controls. The tear protein composition of patients using the OptiFree, Renu or Solocare solutions did not undergo a measureable change in the protein level. CONCLUSIONS: The ProteinChip system can analyze protein profiles for large-scale applications as in clinical studies. Two multipurpose solutions, Complete and Complete Moisture Plus, demonstrated a beneficial effect on the tear proteins in contact lens wearers.

Direct monitoring of protein-chemical reactions utilising nanoelectrospray mass spectrometry.

The feasibility of nanoelectrospray mass spectrometry (nanoESI) for the direct analysis of protein chemical reactions and structural changes of proteins has been evaluated. Taking advantage of the long spraying time and the capability of nanoESI for employing a wide range of solvent conditions such as buffers and detergents, applications of monitoring reaction pathways, and dynamics have been carried out with several peptides and proteins. The time course of proteolytic digestions with trypsin and pepsin was investigated for several model polypeptides, and nanoESI showed to provide an efficient tool for optimising digestion conditions for the mass spectrometric peptide mapping analysis. Examples of specific protein chemical modification reactions at arginine and tyrosine residues illustrate the feasibility of nanoESI to monitoring reaction yields and modification sites for more than 180 min. Furthermore, changes of the pattern of protonated molecules caused by temperature effects and by protein unfolding due to disulfide bond reduction have been studied with the model proteins cytochrome c and hen eggwhite lysozyme. The results indicate that nanoESI is an efficient technique for the direct, molecular characterisation of protein-chemical reactions in solution.

Regulation of the expression of mitogen-regulated protein (MRP; proliferin) and cathepsin L in cultured cells and in the murine placenta.

The genes encoding mitogen-regulated protein (MRP; also called proliferin; PLF) and procathepsin L (CL; also called major excreted protein; MEP) are expressed to high levels in the mouse placenta. Although they are both regulated by epidermal growth factor (EGF) and fibroblast growth factor (FGF) in 3T3 cells, expression of these genes is differently regulated with growth state. The expression patterns of MRP and CL as a function of murine development are also different. Basal and growth factor-stimulated levels of MRP expression are much higher in growing than in quiescent 3T3 cells, whereas CL levels are similar. These changes in gene expression in cultured quiescent cells parallel the changes in MRP and CL expression observed in the late-gestational quiescent placenta. These results suggest growth factors may regulate the expression of these genes, but other influences also regulate the expression of MRP and CL in vivo.

Thymectomy in multiple sclerosis. Two preliminary trials.

In two preliminary trials, thymectomy was performed on 35 multiple sclerosis (MS) patients, with (Group 1) or without (Group II) azathioprine therapy for 1 year. Formal studies of clinical neurologic status were conducted at yearly intervals after operation. Each group was compared with carefully matched control patients. Group I patients showed significant improvement in total functional groups (Kurtzke scale) and pyramidal functions 1 and 2 years following thymectomy. Other individual functions showed no significant difference. There was a statistical improvement in disability status (Kurtzke) for patients with relapsing-remitting MS 1 and 2 years following thymectomy. This subset also had a significant decrease in MS exacerbations. Group II patients showed none of the favorable trends seen in Group I. Functional groups, disability status scale, and exacerbations were similar to those of the control patients. Several studies indicate azathioprine therapy alone is of questionable benefit in MS. In myasthenia gravis (MG), the full immunosuppressive effect of thymectomy may not be realized for several years. Therefore, it is possible that in Group I patients a dual mechanism of immunosuppression--azathioprine and thymectomy--is yielding a favorable response not yet apparent in the Group II patients having thymectomy alone. If our data still appear favorable after a third follow-up year, a formal pilot study will be undertaken.

Isolation, primary structure characterization and identification of the glycosylation pattern of recombinant goldfish neurolin, a neuronal cell adhesion protein.

Neurolin is a growth-associated cell surface glycoprotein from goldfish and zebra fish which has been shown to be involved in axonal path-finding in the goldfish retina and suggested to function as a receptor for axon guidance molecules. Being a member of the immunoglobulin superfamily of cell adhesion proteins, neurolin consists of five N-terminal extracellular immunoglobulin (Ig)-like domains, a transmembrane and a short cytoplasmatic domain. Repeated injections of polyclonal Fab fragments against neurolin and of monoclonal antibodies against either Ig domains cause path-finding errors and disturbance of axonal fasciculation. In order to obtain a complete structural characterization and a molecular basis for structure-function determination, recombinant neurolin with the complete extracellular part but lacking the transmembrane and cytoplasmatic domain was expressed in Chinese hamster ovary (CHO) cells (CHO-neurolin). The isolation of CHO-neurolin was carried out by Ni-affinity chromatography and subsequent high-performance liquid chromatography (HPLC). An exact molecular mass determination was obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and revealed 60.9 kDa, which suggested that approximately 10 kDa are due to glycosylation. The predicted molecular mass is 51.5 kDa, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) yielded an apparent molecular mass of 72 kDa. Gel shift assays using SDS-PAGE and Western blot analysis with anti-neurolin antibodies provided consistent molecular mass data. The complete primary structure and N-glycosylation patterns were identified using specific lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS in combination with specific glycosidase degradation. HPLC isolation of glycosylated peptide fragments and MS after selective deglycosylation revealed heterogeneous glycosylations at all five N-glycosylation consensus sites. All attached N-glycans are of the complex type and show a mainly biantennary structure; they are fucosylated with alpha(2,3)-terminal neuraminic acid. These data serve as a first detailed model to characterize the molecular recognition structures exhibited by the extracellular domains.

The relationships among body weight, body composition, and intramuscular fat content in steers.

Angus steers of known age (265 +/- 17 d) and parentage were used in a 2-yr study (yr 1, n = 40; yr 2, n = 45) to evaluate the relationship between percentage of i.m. fat content of the longissimus dorsi at the 12th rib and carcass characteristics during growth of nonimplanted steers. Steers were sorted by age and EPD of paternal grandsire for marbling into high- and low-marbling groups so that steers with varying degrees of genetic potential for marbling were evenly distributed across slaughter groups. All steers were fed a 90% concentrate corn-based diet. Steers were allotted to five slaughter groups targeted to achieve hot carcass weights (HCW) of 204, 250, 295, 340, and 386 kg over the course of the feeding period. Data were analyzed as a completely random design with a factorial arrangement of treatments (year, marbling group, and slaughter group). Marbling group did not affect backfat, LM area, yield grade (YG), or marbling score. Regression equations were developed to quantify the change in carcass characteristics and composition over slaughter groups. Hot carcass weight increased in a linear fashion and differed (P < 0.01) among the slaughter groups as anticipated by design. Yield grade followed a quadratic upward pattern (P < 0.01) as HCW increased. Slaughter group affected the degree of marbling linearly (P < 0.01). There were no slaughter group x marbling group interactions, indicating that no differences occurred in the pattern of marbling attributable to paternal grandsire EPD. Carcasses expressed small degrees of marbling at 266 kg of HCW and obtained a YG of 3.0 at 291 kg of HCW. Fractional growth rates decelerated with increasing HCW. Greater advances in marbling relative to total carcass fatness occurred at HCW less than 300 kg. Management practices early in growth may influence final quality grade if compensatory i.m. fat content development does not occur.

Circulating ghrelin and leptin concentrations and growth hormone secretagogue receptor abundance in liver, muscle, and adipose tissue of beef cattle exhibiting differences in composition of gain.

Data from species other than cattle indicate that ghrelin and GH secretagogue receptor (GHS-R) could play a key role in fat deposition, energy homeostasis, or glucose metabolism by directly affecting liver and adipose tissue metabolism. Beef steers (n = 72) were used to test the hypothesis that plasma ghrelin and leptin concentrations and abundance of the GHS-R in liver, muscle, and adipose tissues differ in steers exhibiting differences in composition of gain. At trial initiation (d 0), 8 steers were slaughtered for initial carcass composition. The remaining 64 steers were stratified by BW, allotted to pen, and treatment was assigned randomly to pen. Steers were not implanted with anabolic steroids. Treatments were 1) a low-energy (LE) diet fed during the growing period (0 to 111 d) followed by a high-energy (HE) diet during the finishing period (112 to 209 d; LE-HE) or 2) the HE diet for the duration of the trial (1 to 209 d; HE-HE). Eight steers per treatment were slaughtered on d 88, 111, 160, and 209. Carcass ninth, tenth, and eleventh rib sections were dissected for chemical composition and regression equations were developed to predict compositional gain. Liver, muscle, and subcutaneous adipose tissues were frozen in liquid nitrogen for subsequent Western blotting for GHS-R. Replicate blood samples collected before each slaughter were assayed for ghrelin and leptin concentrations. When compared at a common compositional fat end-point, the rate of carcass fat accretion (g·kg of shrunk BW(-1)) was greater (P < 0.001) in HE-HE steers whereas the rate of carcass protein accretion (g·kg of shrunk BW(-1)) was less (P < 0.001) compared with LE-HE steers. When compared at a common compositional fat end-point, plasma leptin, ghrelin, and insulin concentrations were greater (P < 0.05) for HE-HE compared with LE-HE steers. Abundance of the GHS-R, to which ghrelin binds, increased over time in liver and adipose tissue but did not differ as a result of treatment. Plasma ghrelin concentrations were increased for cattle continuously fed the HE diet as they became increasingly fatter; however, abundance of the GHS-R in liver, muscle, and subcutaneous adipose tissue was not different between treatment groups. The role of ghrelin in cattle metabolism warrants further investigation as it could have a significant effect on composition of BW gain, feed efficiency, and metabolic disorders such as ketosis and fatty liver.

The effect of stage of growth and implant exposure on performance and carcass composition in steers.

Angus and Angus x Limousin cross steers (n = 182; initial BW = 309 +/- 27.8 kg) were used to evaluate the influence of an estradiol-trenbolone acetate implant (containing 24 mg of estradiol and 125 mg of trenbolone acetate) on production efficiency and carcass traits when administered at specific stages of growth. Treatments were 1) control, no implant (NI); 2) early implant (EI) on d 1 (BW = 309 kg); or 3) delayed implant (DI) on d 57 (BW = 385 kg). Comparisons were also made between the NI and implanted treatments (I; EI + DI). Steers were procured at weaning and were backgrounded (47 d) before the initiation of the experiment. Initial predicted carcass composition was 14.9% protein, 13.3% fat, 54.6% moisture, and 17.2% bone. Days on feed were constant across treatment. After 56 d, ADG and G:F were improved (P < 0.01) by implants, NI vs. EI (1.68 vs. 1.90 kg and 0.227 vs. 0.257). At d 57, predicted carcass composition did not differ among treatments. From 57 to 112 d, DI caused higher ADG than NI or EI (NI = 1.65, EI = 1.57, and DI = 1.78 kg; P < 0.05) and higher G:F (NI = 0.155, EI = 0.150, and DI = 0.173; P < 0.01). Cumulative ADG and G:F were improved by implants (1.65 vs. 1.73 kg; P < 0.05) and (0.175 vs. 0.186; P < 0.01) for NI vs. I, respectively, with no differences between treatments that involved implants. Cumulative DMI was similar for all treatments. Implanting increased dressing percentage (63.5 vs. 64.1%; P < 0.05) and increased (P < 0.01) hot carcass weight (341 vs. 353 kg) and LM area (76.5 vs. 81.4 cm(2)) for NI vs. I, respectively. Rib fat and kidney, pelvic, and heart fat were not affected by treatment, and treatment had no effect on the whole carcass proportions of fat, protein, or water. Implants advanced maturity scores (NI = A(51) vs. EI + DI = A(59); P < 0.01). Marbling scores were decreased (P < 0.05) by EI but not by DI (NI = Small(65), EI = Small(20), DI = Small(36)). The percentage of i.m. fat content of the LM was decreased (P < 0.10) by EI and was not affected by DI (NI = 5.1, EI = 4.0, DI = 4.8%). Treatment affected (P < 0.10) the proportion of carcasses with marbling scores greater than Modest(0) (NI = 23.6, EI = 7.8, DI = 22.6%). The results of this study suggest that growth of i.m. fat is sensitive to anabolic growth promotants administered during early periods of growth.

Postoperative care and review of complications.

This chapter is an overview of the management of a patient who has undergone a surgical procedure in an outpatient setting. It addresses the complications that are most common in this setting and their management. Without attempting a thorough discussion of every conceivable complication, this discussion seeks nonetheless to be reasonably comprehensive. Perhaps the most straightforward means of presenting this information is to follow chronologically the patient's experience from the moment the procedure has been concluded, through his recovery room care, and finally look at the patient's needs and experiences after leaving the ambulatory care facility.

Partial purification and characterization of an acetylcarnitine hydrolase from bovine epididymal spermatozoa.

We previously reported that intact epididymal spermatozoa from bulls and hamsters oxidize [1-14C]acetyl-L-carnitine to 14CO2 at about the same rate as they oxidize [1-14C]acetate. In addition, we showed that acetylcarnitine is hydrolyzed by a hydrolase present in the plasma membrane and that the carnitine moiety does not enter the cell. Here we report the partial purification of the acetylcarnitine hydrolase from bovine spermatozoa and describe some of its properties. The detergent-extracted enzyme was purified by FPLC using an anion-exchange Mono-Q column. The hydrolase activity eluted from the column with the application of 0.22 to 0.30 M NaCl and was separated from acetylcholinesterase activity, which eluted with 0.35 to 0.40 M NaCl. Specific inhibitors of acetylcholinesterase had little effect on acetylcarnitine hydrolase but p-hydroxymercuriphenylsulfonate was a potent inhibitor of the hydrolase. Kinetic studies of the hydrolase yielded a K'm of 6-10 mM for acetylcarnitine and a V'max of 0.16 nmol min-1 mg protein-1. Similar studies with the acetylcholinesterase yielded a K'm for acetylcholine of about 300 microM and a V'max of 165 nmol min-1 mg protein-1. Acetylcarnitine was a poor substrate for the acetylcholinesterase. Several acyl-L-carnitines were tested as substrates for the hydrolase and the preferred substrate was acetylcarnitine. The role of acetylcarnitine hydrolase in the metabolism of acetylcarnitine by epididymal spermatozoa is discussed.

The metabolism of acetylcarnitine and acetate by bovine and hamster epididymal spermatozoa.

Since acetylcarnitine has been identified in the epididymal plasma of many mammalian species, we investigated whether acetylcarnitine could serve as an energy substrate for epididymal bull and hamster spermatozoa. Intact caudal cells from both species oxidized [I-14C]acetyl-l-carnitine to 14CO2, in vitro, and the amount oxidized was dependent on time, substrate concentration, and cell number. Within each species, the rate of oxidation was the same as the rate at which free [1-14C]acetate was oxidized. Spermatozoa incubated with [3H]acetyl-L-carnitine hydrolyzed the compound and [3H]acetate accumulated in the medium. Unlabeled acetate added to the incubation medium competed with cellular uptake of [3H]acetate and resulted in further increase in [3H]acetate accumulation in the medium. Furthermore, the acetyl group of acetylcarnitine was oxidized by spermatozoa without concomitant uptake of the carnitine group. Purified plasma membrane vesicles contained an acetylcarnitine hydrolase activity that was solubilized from whole cells by detergents and that could be distinguished from acetylcholinesterase also present in the cells. The solubilized acetylcarnitine hydrolase activity was inhibited by p-hydroxymercuriphenylsulfonate, but not by the specific acetylcholinesterase inhibitors, eserine or BW63C47. The sulfhydryl blocker also inhibited the production of 14CO2 from [1-14C]acetylcarnitine by intact cells; acetylcholinesterase inhibitors did not. From estimates of sperm energy requirements, our results indicate that extracellular acetylcarnitine serves as a physiologically important energy substrate for maturing sperm cells.

Analytical development of electrospray and nanoelectrospray mass spectrometry in combination with liquid chromatography for the characterization of proteins.

Mass spectrometry has significantly extended its applicability in the area of characterization of protein structures. Electrospray ionization enables on-line coupling with liquid chromatography which has become a powerful tool for the characterization of peptide and protein mixtures. The most recent development of a nanoelectrospray source, using capillary forces for a particularly mild analyte transport and ionization into the mass spectrometer, opens a wide field for applications to protein structure analysis. In this paper, the analytical development of liquid chromatography-electrospray ionization mass spectrometry and nano-electrospray ionization mass spectrometry, adapted to an electrospray ionization quadrupole mass spectrometer and its application to the characterization of noncovalent protein complexes are described.

Isolation and identification of the plasma membrane-associated intracellular protein reggie-2 from goldfish brain by chromatography and Fourier-transform ion cyclotron resonance mass spectrometry.

The neuronal protein reggie-2 is localized at the cytoplasmic face of the plasma membrane and participates, together with reggie-1, in the formation of plasma membrane microdomains. Reggie-2 exhibits several potential phosphorylation sites but whether the relevant sites are modified accordingly is not yet known. In order to obtain a detailed, molecular characterization of the primary structure of the native protein, an effective procedure for the isolation of the different reggie proteins from animal tissue is required. The specific properties of the proteins, particularly their membrane association and low abundance, make approaches for isolation such as affinity chromatography and 2D gel electrophoresis unfeasible. This study describes a rapid and efficient procedure for the isolation of reggie-2 by use of two consecutive HPLC steps and subsequent SDS-PAGE. The protein fractions were characterized by SDS-PAGE and Western blot analysis as well as by mass spectrometry. In the primary structure analysis by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS), the efficiency of high-resolution Fourier-transform ion cyclotron resonance-MALDI-MS was demonstrated, enabling the direct, unequivocal, and sensitive characterization of posttranslationally and/or chemically modified proteins.

Requirement of specific intrahelical interactions for stabilizing the inactive conformation of glycoprotein hormone receptors.

Systematic analysis of structural changes induced by activating mutations has been frequently utilized to study activation mechanisms of G-protein-coupled receptors (GPCRs). In the thyrotropin receptor and the lutropin receptor (LHR), a large number of naturally occurring mutations leading to constitutive receptor activation were identified. Saturating mutagenesis studies of a highly conserved Asp in the junction of the third intracellular loop and transmembrane domain 6 suggested a participation of this anionic residue in a salt bridge stabilizing the inactive receptor conformation. However, substitution of all conserved cationic residues at the cytoplasmic receptor surface did not support this hypothesis. Asp/Glu residues are a common motif at the N-terminal ends of alpha-helices terminating and stabilizing the helical structure (helix capping). Since Asp/Glu residues in the third intracellular loop/transmembrane domain 6 junction are not only preserved in glycoprotein hormone receptors but also in other GPCRs we speculated that this residue probably participates in an N-terminal helix-capping structure. Poly-Ala stretches are known to form and stabilize alpha-helices. Herein, we show that the function of the highly conserved Asp can be mimicked by poly-Ala substitutions in the LHR and thyrotropin receptor. CD and NMR studies of peptides derived from the juxtamembrane portion of the LHR confirmed the helix extension by the poly-Ala substitution and provided further evidence for an involvement of Asp in a helix-capping structure. Our data implicate that in addition to well established interhelical interactions the inactive conformation of GPCRs is also stabilized by specific intrahelical structures.

A simple technique for the purification of plasma membranes from ejaculated boar spermatozoa.

Spermatozoa were initially separated from fresh boar ejaculates using a 1.0 M sucrose density gradient. Spermatozoa (1 x 10(8) cells/ml) were subjected to gas cavitation (650 psi, 10 minutes), followed by a 4-step centrifugation technique to yield the final plasma membrane preparation. Purity of the plasma membrane isolate was determined using microscopic techniques (i.e. differential interference contrast and transmission electron microscopy) and marker enzymes for biochemical characterization. Plasma membranes were found to be removed primarily from the periacrosomal region of the sperm. Acrosomes appeared to remain intact on the cavitated spermatozoa. Transmission electron microscopy yielded a homogenous population of 100-200 microns unilamellar vesicles. Enzyme markers specific for plasma, acrosome and mitochondrial membranes substantial the purity observed under visual examination.