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The Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a FADD-like interferon converting enzyme or caspase 8 (FLICE) inhibitory protein (vFLIP) that prevents death receptor-mediated apoptosis by inhibiting the recruitment and activation of FLICE. Since vFLIP physically interacts with tumor necrosis factor receptor associated factor 2 (TRAF2) and TRAF2 mediates activation of the jun NH(2)-terminal kinase (JNK)/activation protein 1 (AP1) pathway, we hypothesized that vFLIP might also activate this pathway. To evaluate this hypothesis, we transiently and stably transfected a vFLIP expression construct and performed several complementary assays to document that vFLIP activates the JNK/AP1 pathway and does so in a TRAF-dependent fashion. As vFLIP also activates the nuclear factor kappaB (NF-kappaB) signaling pathway and the NF-kappaB and JNK/AP1 pathways both modulate cellular interleukin-6 (cIL-6) expression, we postulated that vFLIP induces expression of this cytokine. We show that vFLIP induces cIL-6 expression and activates the cIL-6 promoter, and maximal activation of the cIL-6 promoter by vFLIP requires NF-kappaB and AP1 activation. In addition, vFLIP and latency-associated nuclear antigen (LANA), another KSHV-encoded latent protein, potentiate each other's ability to activate the cIL-6 promoter. Gene silencing experiments by RNA interference demonstrate that vFLIP in BCBL-1 endogenously infected primary effusion lymphoma (PEL) cells mediates JNK/AP1 activation and cIL-6 expression. Thus, we conclude that vFLIP, in addition to its known effects on NF-kappaB activation, also modulates the JNK/AP1 pathway and induces gene expression from the cIL-6 promoter in a JNK/AP1-dependent fashion.

Background: Isolated monomorphic premature ventricular contractions (PVCs) are not uncommon in the pediatric population. The degree of cardiac dysfunction caused by PVCs in children without structural heart disease is unknown. Purpose: To investigate the influence of PVCs on echocardiographic left ventricular (LV) systolic function in children without structural heart disease. Methods: Forty asymptomatic children with isolated monomorphic PVCs without structural heart disease were selected. The median age was 6 years, range of 3–12 years. The following subgroups were compared: frequent vs. infrequent PVCs (> or ≤10/min); short vs. long coupling interval (RR′/RR ratio ≤ or >0.6); and short vs. long QT interval (QT ≤ or >400 ms). Using echocardiography the left ventricular ejection fraction (LVEF in percentage) and cardiac index (CI in L/min/m2) were measured for both normal sinus beats (SB-LVEF and CI), PVCs (PVC-LVEF and CI) and the average LVEF and CI were calculated. All values were expressed as means ± SD. Results: In all children LV dimensions and the SB-LVEF and CI were within normal limits. The PVC-LVEF (48 ± 5) and PVC-CI (1.57 ± 0.19) were significantly decreased and the average CI was 2.41 ± 0.29. In 27 pts with >10 PVCs/min the average LVEF and CI decreased to 53 ± 5 and 2.08 ± 0.24 respectively. In 16 pts with PVCs and a short coupling interval (RR′/RR ≤ 0.6) the PVC-LVEF and PVC-CI was 43 ± 0.03 and 1.50 ± 0.14, which was significantly lower than in 24 pts with a long coupling interval (58 ± 4, 1.88 ± 0.11). In 11 pts with a prolonged QT interval (>400 ms) the PVC-LVEF and PVC-CI was significantly lower than in the 29 children with a shorter QT interval, 41 ± 5 vs. 55 ± 4 and 1.46 ± 0.13 vs. 1.86 ± 0.15 respectively (all p 10/min), have a short coupling interval or a prolonged QT interval.

A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension-induced atrophy or hindlimb suspension-induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting-induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting-induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteorne occur during disuse-induced soleus muscle atrophy and reweighting hypertrophy.

Kinetic interactions of β-lactam antibiotics such as penicillin-G and cefotaxime with normal, penicillin-susceptible PBP2x from Streptococcus pneumoniae and a penicillin-resistant PBP2x (PBP2xR) from a resistant clinical isolate (CS109) of the bacterium have been extensively characterized using electrospray mass spectrometry coupled with a fast reaction (quench flow) technique. Kinetic evidence for a two-step acylation of PBP2x by penicillin-G has been demonstrated, and the dissociation constant, K d of 0.9 mm, and the acylation rate constant, k 2 of 180 s−1, have been determined for the first time. The millimolar range K d implies that the β-lactam fits to the active site pocket of the penicillin-sensitive PBP rather poorly, whereas the extremely fast k 2 value indicates that this step contributes most of the binding affinity of the β-lactam. The values of K d (4 mm) and k 2 (0.56 s−1) were also determined for PBP2xR. The combined value ofk 2/K d, known as overall binding efficiency, for PBP2xR (137m −1 s−1) was over 1000-fold slower than that for PBP2x (200,000 m −1s−1), indicating that a major part is played by the acylation steps in penicillin resistance. Most of the decreased binding efficiency of PBP2xR comes from the decreased (∼300-fold)k 2. Kinetic studies of cefotaxime acylation of the two PBP2x proteins confirmed all of the above findings. Deacylation rate constants (k 3) for the third step of the interactions were determined to be 8 × 10−6s−1 for penicilloyl-PBP2x and 5.7 × 10−4 s−1 for penicilloyl-PBP2xR, corresponding to over 70-fold increase of the deacylation rate for the resistant PBP2xR. Similarly, over 80-fold enhancement of the deacylation rate was found for cefotaxime-PBP2xRcomplex (k 3 = 3 × 10−4s−1) as compared with that of cefotaxime-PBP2x complex (3.5 × 10−6 s−1). This is the first time that such a significant increase ofk 3 values was found for a β-lactam-resistant penicillin-binding protein. These data indicate that the deacylation step also plays a role, which is much more important than previously thought, in PBP2xR resistance to β-lactams.

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.

A proteomic analysis was performed comparing normal rat soleus muscle to denervated soleus muscle at 0.5, 1, 2, 4, 6, 8 and 10 days post denervation. Muscle mass measurements demonstrated that the times of major mass changes occurred between 2 and 4 days post denervation. Proteomic analysis of the denervated soleus muscle during the atrophy process demonstrated statistically significant (at the p < 0.01 level) changes in 73 soleus proteins, including coordinated changes in select groups of proteins. Sequence analysis of ten differentially regulated proteins identified metabolic proteins, chaperone and contractile apparatus proteins. Together these data indicate that coordinated temporally regulated changes in the proteome occur during denervation-induced soleus muscle atrophy, including changes in muscle metabolism and contractile apparatus proteins.

We report the application of nanoelectrospray ionization tandem mass spectrometry (nES-MS/MS) and capillary LC/microelectrospray MS/MS (cLC/µES-MS/MS) for sequencing sulfonic acid derivatized tryptic peptides. These derivatives were specifically prepared to facilitate low-energy charge-site-initiated fragmentation of C-terminal arginine-containing peptides, and to enhance the selective detection of a single series of y-type fragment ions. Both singly and doubly protonated peptides were analyzed by MS/MS and the results were compared with those from their derivatized counterparts. Model peptides and peptides from tryptic digests of gel-isolated proteins were analyzed. Derivatized singly protonated peptides fragment in the same way by nES-MS/MS as they do by post-source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD-MALDI-MS). They produce fragment ion spectra dominated by y-ions, and the simplified spectra are readily interpreted de novo. Doubly protonated peptides fragment in much the same way as their non-derivatized doubly protonated counterparts. The fragmentation of doubly protonated derivatives is especially useful for sequencing peptides that possess a proline residue near the N-terminus of the molecule. The singly protonated forms of these proline-containing derivatives often show enhanced fragmentation on the N-terminal side of the proline and considerably reduced fragmentation on the C-terminal side. In addition, sulfonic acid derivatization increases the in-source fragmentation of arginine-containing peptides. This could be useful for sequence verification and sequence tagging for use in single stage mass spectrometry. Copyright © 2000 John Wiley & Sons, Ltd.

We analyzed protein expression in the cytosolic fraction prepared from whole kidneys in male Swiss-Webster mice exposed 1 h/day for five days to aerosolized JP-8 jet fuel at a concentration of 1000 mg/m3, simulating military occupational exposure. Kidney cytosol samples were solubilized and separated via large-scale, high-resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant changes in soluble kidney proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass finger-printing and related to ultrastructural abnormalities, altered protein processing, metabolic effects, and paradoxical stress protein/detoxification system responses. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression in the kidney and provide novel molecular evidence of JP-8 nephrotoxicity. Human risk is suggested by these data but conclusive assessment awaits a noninvasive search for biomarkers in JP-8 exposed humans.

We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss-Webster mice exposed 1 h/day for seven days to aerosolized JP-8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid-base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.

Penicillin-binding protein 2a (PBP2a) is the primary beta-lactam resistance determinant of methicillin-resistant Staphylococcus aureus (MRSA). MecA, the gene coding for PBP2a, was cloned with the membrane-anchoring region at the N-terminus deleted. The truncated protein (PBP2a) was overexpressed in Escherichia coli mostly in the soluble form accounting for approximately 25% of soluble cell protein and was purified to homogeneity. The purified protein was shown to covalently bind beta-lactams in an 1:1 ratio as determined by electrospray mass spectrometry. A novel method based on HPLC-elctrospray mass spectrometry has been developed to quantitatively determine the formation of the covalent adducts or acyl-PBP2a complexes. By using this method, combined with kinetic techniques including quench flow, we have extensively characterized the interactions between PBP2a and three beta-lactams and determined related kinetic parameters for the first time. The apparent first-order rate constants (ka) of PBP2a acylation by benzylpenicillin showed a hyperbolic dependence on the concentration of benzylpenicillin. This is consistent with the mechanism that the binding of the penicillin to PBP2a consists of reversible formation of a Michaelis complex followed by formation of the penicilloyl-PBP2a adduct, and allowed the determination of the individual kinetic parameters for these two steps, the dissociation constant Kd of 13.3 mM and the first-order rate constant k2 of 0.22 s-1. From these values, the second-order rate constant k2/Kd, the value reflecting the overall binding efficiency of a beta-lactam, of 16.5 M-1 s-1 was obtained. The fairly high Kd value indicates that benzylpenicillin fits rather poorly into the protein active site. Similar studies on the interaction between PBP2a and methicillin revealed k2 of 0.0083 s-1 and Kd of 16.9 mM, resulting in an even smaller k2/Kd value of 0.49 M-1 s-1. The rate constants k3 for deacylation of the acyl-PBP2a complexes, the third step in the interactions, were measured to be

Enzymatic digests of proteins isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were separated by capillary high-performance liquid chromatography (HPLC). The column eluate was split to an electrospray mass spectrometer on one side and to both a UV detector and a microblotter on the other side. Using the microblotter, the peptides eluted from the column were collected directly onto a polyvinylidene difluoride (PVDF) membrane for Edman sequencing. Thus, a peptide mass map from the mass spectrometric analysis and a prepared PVDF membrane for subsequent Edman sequencing were generated in a single experiment. The addition of molecular mass information to the blotted LC eluate is useful for determining the most important peaks to undergo Edman sequencing. Coupling the capillary HPLC with a microblotter to electrospray mass spectrometry provides an integrated system for separation, collection, and structural analysis of protein digests. It provides high levels of sensitivity, recovery, and convenience for protein characterization. Proteins loaded onto SDS-PAGE at low picomole levels can be analyzed by the new integrated system.

Penicillin-binding protein 2a (PBP2a), a high molecular mass PBP, is the primary enzyme responsible for the β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by β-lactams is due to covalent modification of an active site serine residue. Based on the sequence alignment with well studied β-lactamases, DD-carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S403XXK in PBP2a was tentatively identified as the penicillin-binding site. However, direct evidence for the involvement of serine403 has not been reported. In this study, a method which combines liquid chromatography/electrospray mass spectrometry (LC/MS) and nano-electrospray MS for the identification of the active site serine in PBP2a is described. The covalent binding of the β-lactams was carried out in vitro with the recombinant PBP2a. Peptide mapping of the cyanogen bromide fragments from penicilloyl-PBP2a, using microbore LC/MS, provided a rapid identification of the modified peptide with a 334 Da mass increase. The acylated peptide was isolated and further digested with trypsin. Nano-electrospray MS/MS sequencing of the acylated peptide in the tryptic digest showed that the penicillin was indeed attached to serine403. © 1998 John Wiley & Sons, Ltd.

It has been reported that social isolation stress could be a key factor that leads to cognitive deficit for both humans and rodent models. However, detailed mechanisms are not yet clear. ADAR1 (Adenosine deaminase acting on RNA) is an enzyme involved in RNA editing that has a close relation to cognitive function. We have hypothesized that social isolation stress may impact the expression of ADAR1 in the brain of mice with cognitive deficit. To test our hypothesis, we evaluated the cognition ability of mice isolated for different durations (2, 4, and 8 weeks) using object recognition and object location tests; we also measured ADAR1 expression in hippocampus and cortex using immunohistochemistry and western blot. Our study showed that social isolation stress induced spatial and non-spatial cognition deficits of the tested mice. In addition, social isolation significantly increased both the immunoreactivity and protein expression of ADAR1 (p110) in the hippocampus and frontal cortex. Furthermore, re-socialization could not only recover the cognition deficits, but also bring ADAR1 (p110) immunoreactivity of hippocampus and frontal cortex, as well as ADAR1 (p110) protein expression of hippocampus back to the normal level for the isolated mice in adolescence. In conclusion, social isolation stress significantly increases ADAR1 (p110) expression in the hippocampus and frontal cortex of the mice with cognitive deficit. This finding may open a window to better understand the reasons (e.g., epigenetic change) that are responsible for social isolation-induced cognitive deficit and help the development of novel therapies for the resulted diseases.

Crystallins, the structural proteins of the lens, have been isolated from human lenses using a combination of gel filtration and reversed phase high performance liquid chromatography (HPLC). The molecular weights of the isolated crystallins have been determined by electrospray ionization mass spectrometry. The isolated crystallins have also been proteolytically digested into peptides, the peptides fractionated by HPLC, and the masses of the peptides determined by fast atom bombardment mass spectrometry. With these techniques, it is possible to confirm and/or correct known protein sequences and identify and locate post translational modifications. I NTRODU CTl ON Cataract, which can be defined as an opacity of the eye lens, is the leading cause of blindness worldwide. In countries where surgery to remove a cataractous lens and replace it with a synthetic lens is readily available, cataract may not be a serious impairment; however, in much of the world this surgery is not available, and the development of cataract leads to blindness. The two types of cataract, cortical and nuclear, both involve changes in the lens proteins, called the crystallins. For the lens to be transparent, the crystallins must be tightly and uniformly packed producing a lens with a uniform refractive index. In cortical cataract, the density of the crystallins is altered, causing a non-uniform density with variations in the lens refractive index and consequent light scattering (ref. 1). Nuclear cataract is associated with the formation of insoluble protein aggregates. In both cases, there are modifications to the lens crystallins that either prevent their normal close packing in the cortex or cause their aggregation in the nucleus. The focus of our research is to determine which proteins are modified, where the modifications are located, and the identity of the modifications. Cataract is most commonly associated with old age. Most people over age 80 have at least early indications of cataract. Cataract occurs earlier in people with diabetes, renal failure, chronic diarrhea or who have experienced prolonged exposure to W radiation. The mechanisms that have been proposed for the development of cataract vary, depending on the disease with which it is associated. Cataract associated with old age, diabetes, renal failure and chronic diarrhea are all proposed to occur because of modifications to the lysyl residues of the crystallins. In old age, the concentration of glutathione in the lens is lower. This permits an increase in the concentration of dehydroascorbic acid, which is normally reduced by glutathione in younger lenses. Dehydroascorbic acid can react with the amino groups of the lysyl residues, forming a Schiff base which can then rearrange or possibly form cross-links (ref. 2). In diabetes, the elevated concentrations of glucose may lead to formation of a similar Schiff base between the lysyl residues and glucose (ref. 3). Both renal failure and chronic diarrhea are associated with elevated urea, which forms an equilibrium with isocyanate. Isocyanate can also react with lysyl residues forming a carbamylated protein (refs. 4, 5). A different mechanism, oxidation of the tryptophan residues to form kynurenine, has been proposed

The primary structure and posttranslational modifications of rabbit lens alpha-crystallins were examined using electrospray ionization mass spectrometry to determine the molecular weights of the intact proteins and fast atom bombardment mass spectrometry to analyze proteolytic digests of the alpha A- and alpha B-crystallins. The previously determined primary structure of alpha A-crystallin was confirmed. Posttranslational modifications detected included one phosphorylation site and the presence of a truncated form minus the five C-terminal residues. The previously undetermined amino acid sequence of rabbit alpha B-crystallin was determined to be the same as the bovine alpha B-crystallin sequence except at three residues: Thr 40, Thr 132, and Pro 153. Rabbit alpha B-crystallin showed evidence of phosphorylation at the same three sites as bovine alpha B-crystallin. The molecular weights of the intact proteins indicated that any one molecule had a maximum of two phosphorylations. Also, there was a truncated form which did not include the five C-terminal residues.

Background One of the beneficial effects of exercise training in chronic heart failure (CHF) is an improvement in baroreflex sensitivity (BRS), a prognostic index in CHF. In our hypothesis-generating study we propose that at least part of this effect is mediated by neural afferent information, and more specifically, by exercise-induced somatosensory nerve traffic. Objective To compare the effects of periodic electrical somatosensory stimulation on BRS in patients with CHF with the effects of exercise training and with usual care. Methods We compared in stable CHF patients the effect of transcutaneous electrical nerve stimulation (TENS, N =23, LVEF 30±9%) with the effects of bicycle exercise training (EXTR, N =20, LVEF 32±7%). To mimic exercise-associated somatosensory ergoreceptor stimulation, we applied periodic (2/s, marching pace) burst TENS to both feet. TENS and EXTR sessions were held during two successive days. Results BRS, measured prior to the first intervention session and one day after the second intervention session, increased by 28% from 3.07±2.06 to 4.24±2.61ms/mmHg in the TENS group, but did not change in the EXTR group (baseline: 3.37±2.53ms/mmHg; effect: 3.26±2.54ms/mmHg) ( P (TENS vs EXTR)=0.02). Heart rate and systolic blood pressure did not change in either group. Conclusions We demonstrated that periodic somatosensory input alone is sufficient and efficient in increasing BRS in CHF patients. This concept constitutes a basis for studies towards more effective exercise training regimens in the diseased/impaired, in whom training aimed at BRS improvement should possibly focus more on the somatosensory aspect.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1- 14 C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH 3 . This rate of incorporation was 6–7-fold more rapid than that obtained with [[ 14 C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the cyrstallin subunits. [1- 14 ]Ascorbic acid-label led α-crystallin was separated into into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N ϵ -(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.

Although the precise mechanisms of transient hypotension after intravenous infusion of hypertonic saline (HTS) are not yet clarified, a rapid infusion of HTS is widely used as the initial therapy of hypovolemia. We investigated the effect of the intravenous infusion of a small dose of 0.97-9.7% NaCl solutions in anesthetized rats. Intravenous infusion of HTS at a rate of 0.3 ml/kg/min for 1 min produced the transient hypotension lasting for several minutes. The depressor response to HTS was not abolished by bilateral cervical vagotomy. The HTS infusion into the femoral vein evoked the depressor response with a larger magnitude and a shorter latency than that into the aortic arch. While the arterial baroreceptor pressure was kept constant at the baseline level of systemic arterial pressure, HTS-induced hypotension was significantly augmented. The gain factor of the arterial baroreflex was reduced by intravenous HTS. Pretreatment with bretylium tosylate completely abolished the depressor response without affecting the baseline level of arterial pressure. These results suggest that the depressor response to the very small dose of intravenous HTS is the sympathosympathetic neural reflex with cardiopulmonary afferents and vasomotor efferents.

This paper described a facile method for preparation of gold nanoparticles with high efficient SERS activity within short reaction time in the presence of an environmentally benign and low-cost reagent named inositol hexaphosphate (IP(6)). IP(6) acted as a tunable cross-linker to obtain a suitable separation (about 2nm) between the neighboring particles for certain surface plasmons by adjusting the dosages of IP(6). When the molar ratio of Au to IP(6) was 10:1, gold particles presented in nano-pearl-necklace pattern, demonstrating the high enhancement factor (>10(7)) for 2-mercaptopyridine (2-Mpy) Raman scattering and high stability (>2months).

The main objective of this work was to use positive-ion fast atom bombardment mass spectrometry (FAB-MS) B/E linked scan spectra to investigate the possibility of differentiating positional isomers of various authentic glycine- and taurine-conjugated bile acids. Sodium salts of 14 conjugated bile acids were individually ionized by FAB-MS and characterized by scanning simultaneously the magnetic field B and the electric sector field E such that B/E remained constant throughout the scan. The dominant fragment ions could be related to cleavage of the aliphatic side chain with charge retention on the conjugated end of the bile acids. However, fragment ions arising from ring cleavages were also observed and could be used to distinguish the positions of substituent hydroxyl groups. For example, ring cleavage of conjugated dihydroxy bile acids at C-7/C-8 and C-9/C-10 permitted the differentiation of chenodeoxycholyltaurine (3 alpha,7 alpha-substitution pattern) from deoxycholyltaurine (3 alpha,12 alpha-substitution pattern) based on the presence of fragment ions at m/z 388 or m/z 404, which were indicative of hydroxyl group substitutions at either the 7- or 12-positions, respectively. It was concluded that B/E linked scans can be used to discriminate positional isomers of conjugated bile acids.

Prior studies suggest Staphylococcus aureus exotoxins are not produced when the organism is cultured in human blood. Human blood was fractionated into plasma and water-lysed red blood cells, and it was demonstrated that mixtures of alpha and beta globins of hemoglobin (as low as 1 mug/mL) inhibited S. aureus exotoxin production while increasing production of protein A and not affecting bacterial growth. Pepsin but not trypsin digestion destroyed the ability of alpha and beta globin to inhibit exotoxin production. Exotoxin production by both methicillin-resistant and methicillin-susceptible organisms was inhibited. Production of streptococcal pyrogenic exotoxin A by Streptococcus pyogenes was unaffected by alpha and beta globin chains but was inhibited when produced in S. aureus. Use of isogenic S. aureus strains suggested the targets of alpha and beta globin chains, leading to inhibition of staphylococcal exotoxins, included the two-component system SrrA-SrrB. delta hemolysin production was also inhibited, suggesting the two-component (and quorum sensing) system AgrA-AgrC was targeted. The alpha and beta globin chains represent promising molecules to interfere with the pathogenesis of serious staphylococcal diseases.

Fungi in the genus Malassezia are ubiquitous skin residents of humans and other warm-blooded animals. Malassezia are involved in disorders including dandruff and seborrheic dermatitis, which together affect >50% of humans. Despite the importance of Malassezia in common skin diseases, remarkably little is known at the molecular level. We describe the genome, secretory proteome, and expression of selected genes of Malassezia globosa . Further, we report a comparative survey of the genome and secretory proteome of Malassezia restricta , a close relative implicated in similar skin disorders. Adaptation to the skin environment and associated pathogenicity may be due to unique metabolic limitations and capabilities. For example, the lipid dependence of M. globosa can be explained by the apparent absence of a fatty acid synthase gene. The inability to synthesize fatty acids may be complemented by the presence of multiple secreted lipases to aid in harvesting host lipids. In addition, an abundance of genes encoding secreted hydrolases (e.g., lipases, phospholipases, aspartyl proteases, and acid sphingomyelinases) was found in the M. globosa genome. In contrast, the phylogenetically closely related plant pathogen Ustilago maydis encodes a different arsenal of extracellular hydrolases with more copies of glycosyl hydrolase genes. M. globosa shares a similar arsenal of extracellular hydrolases with the phylogenetically distant human pathogen, Candida albicans , which occupies a similar niche, indicating the importance of host-specific adaptation. The M. globosa genome sequence also revealed the presence of mating-type genes, providing an indication that Malassezia may be capable of sex.

s / Biol Blood Marrow Transplant 19 (2013) S178eS193 S186 may be warranted to discern if DM pts may benefit from different methods of mobilization or if long term transplant outcomes are impacted.

Advanced renal cell carcinoma (RCC) is resistant to cytotoxic chemotherapy, and immunotherapy has modest activity. Proteasome inhibitors represent a novel class of anticancer agents that have activity across a wide spectrum of tumor types. We investigated the efficacy of the proteasome inhibitor bortezomib (VELCADE, formerly known as PS-341) in RCC and found that bortezomib potently induces apoptosis of RCC cell lines. Blockade of the nuclear factor-kappaB (NF-kappaB) pathway is considered a crucial effect in bortezomib-induced apoptosis, but the dependence on NF-kappaB inhibition for bortezomib-mediated death has not been formally demonstrated. Thus, we also studied the contribution of NF-kappaB inhibition as a mechanism of bortezomib-induced apoptosis in RCC cells, which display constitutive NF-kappaB activation. Ectopic expression of the NF-kappaB family members, p65 (Rel A) and p50 (NF-kappaB1), markedly reduced bortezomib-induced apoptosis. However, when we used selective genetic and chemical inhibitors of NF-kappaB, we found that NF-kappaB blockade was not sufficient to induce apoptosis of RCC cells. Thus, we conclude that maximal bortezomib-induced apoptosis is dependent on its NF-kappaB inhibitory effect, but NF-kappaB-independent effects also play a critical role in the induction of apoptosis by bortezomib. This represents the first report to formally demonstrate that bortezomib-induced NF-kappaB blockade is required to achieve the maximum degree of apoptosis by this drug.

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.

Isolated monomorphic premature ventricular contractions (PVCs) are not uncommon in the pediatric population. The degree of cardiac dysfunction caused by PVCs in children without structural heart disease is unknown.To investigate the influence of PVCs on echocardiographic left ventricular (LV) systolic function in children without structural heart disease.Forty asymptomatic children with isolated monomorphic PVCs without structural heart disease were selected. The median age was 6 years, range of 3-12 years. The following subgroups were compared: frequent vs. infrequent PVCs (oror = 10/min); short vs. long coupling interval (RR'/RR ratioor = or0.6); and short vs. long QT interval (QTor = or400 ms). Using echocardiography the left ventricular ejection fraction (LVEF in percentage) and cardiac index (CI in L/min/m2) were measured for both normal sinus beats (SB-LVEF and CI), PVCs (PVC-LVEF and CI) and the average LVEF and CI were calculated. All values were expressed as means +/- SD.In all children LV dimensions and the SB-LVEF and CI were within normal limits. The PVC-LVEF (48 +/- 5) and PVC-CI (1.57 +/- 0.19) were significantly decreased and the average CI was 2.41 +/- 0.29. In 27 pts with10 PVCs/min the average LVEF and CI decreased to 53 +/- 5 and 2.08 +/- 0.24 respectively. In 16 pts with PVCs and a short coupling interval (RR'/RRor = 0.6) the PVC-LVEF and PVC-CI was 43 +/- 0.03 and 1.50 +/- 0.14, which was significantly lower than in 24 pts with a long coupling interval (58 +/- 4, 1.88 +/- 0.11). In 11 pts with a prolonged QT interval (400 ms) the PVC-LVEF and PVC-CI was significantly lower than in the 29 children with a shorter QT interval, 41 +/- 5 vs. 55 +/- 4 and 1.46 +/- 0.13 vs. 1.86 +/- 0.15 respectively (all p0.01).In asymptomatic children with isolated monomorphic PVCs the average ejection fraction and cardiac output is markedly reduced if PVCs are frequent (10/min), have a short coupling interval or a prolonged QT interval.

The Kaposi sarcoma–associated herpesvirus (KSHV)–encoded latency-associated nuclear antigen (LANA) modulates viral and cellular gene expression, including interleukin 6 (IL-6), a growth factor for KSHV-associated diseases. LANA-driven IL-6 expression is dependent on the activator protein 1 (AP1) response element (RE) within the IL-6 promoter. We show that LANA activates the AP1 RE in a Jun-dependent fashion and that LANA enhances the transcriptional activity of a GAL4-Jun fusion protein. Coimmunoprecipitation studies documented a physical interaction between LANA and c-Jun in transiently transfected 293 cells as well as the KSHV-infected BCBL-1 primary effusion lymphoma (PEL) cell line. Taken together, these data indicate that LANA is a transcriptional coactivator of c-Jun. In addition, electrophoretic mobility shift assays demonstrated that LANA induces binding of a c-Jun-Fos heterodimer to the AP1 RE, but does not itself bind to the AP1 RE. RNA interference experiments confirmed that LANA activates the AP1 RE, stimulates binding of a c-Jun-Fos heterodimer to the AP1 RE, and induces expression of IL-6. These data indicate that LANA is a transcriptional coactivator of c-Jun, a function that may have implications for the pathogenesis of KSHV-associated diseases.

Cytotoxic T-lymphocytes are critical in the clearance of chronic viral infections such as HTLV-1. Peptide-based vaccines may have potential application in invoking antiviral CTL responses. In the development of vaccination strategies, it is becoming increasingly important to elicit a broad immune response against several epitopes simultaneously that may provide large population coverage. In the present study, we addressed this issue, namely the processing and presentation of multiple CTL epitopes simultaneously for the generation of multispecific CTL responses. We designed a novel multivalent peptide consisting of three HLA-A(*)0201 restricted CTL epitopes, with intervening double arginine residues in tandem. These epitopes were derived from the HTLV-1 regulatory protein Tax, which is an attractive target for vaccine development against HTLV-1. Arginine residues were included to provide cleavage sites for proteasomes, to generate the intended MHC Class I ligands. Proteasomal digestion studies and mass spectrometry analysis showed cleavage of the multivalent construct to generate the individual epitopes. Immunization of HLA-A(*)0201 transgenic mice with this construct efficiently elicited cellular responses to each intended epitope in vivo, further validating the applicability of this approach. These data may have potential in the development of immunotherapeutic strategies for the treatment of HTLV-1 disease and in the future design of multivalent subunit peptide vaccines.

The phase function of an SAR signal in the two dimensional wavenumber domain is usually derived by using the principal of stationary phase. From this phase function, the phase error of the conventional range-Doppler algorithm when used for high resolution large squint angle SAR imaging is obvious [Bamler 1992]. The secondary range compression and other approximate wavenumber domain algorithm can be derived based on the phase function. But the physical meaning of the secondary range compression can not be directly perceived. In this paper we derive the phase function from SAR imaging geometry by using the Doppler-azimuth coordinate relationship in different RF frequency. From this point of view, it is easy to understand the phase error originated from the range-Doppler algorithm.

The radar cross section (RCS) of sea surface measured by a radar altimeter is dominated by specular reflection. There are two factors affecting the normalized RCS of this type of target: one is the mean surface slope; the other is the radar look angle. For nadir looking altimeter, a long wave length wave has the effect of changing the local incident angle of the coming electromagnetic waves. The authors investigate the possibility of using synthetic aperture radar altimeter for estimation of directional wave spectrum. Synthetic aperture processing inherently can give a high spatial resolution in the along track direction by combining of several pulses. Then by appropriate sampling within each pulse they can sample the sea surface at high resolution in the across-track direction. They study this novel process by using computer simulation. They simulate the image got by synthetic aperture radar altimeter. The resolution achieved by SAR altimeter in the simulation is about 25 m/spl times/25 m. The results show they can estimate the directional wave spectrum but with an ambiguity in wave direction.

A proteomic analysis was performed comparing normal slow twitch type fiber rat soleus muscle and normal fast twitch type fiber tibialis anterior muscle to immobilized soleus and tibialis anterior muscles at 0.5, 1, 2, 4, 6, 8 and 10 days post immobilization. Muscle mass measurements demonstrate mass changes throughout the period of immobilization. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 17 proteins. Proteomic analysis of normal and atrophied tibialis anterior muscle demonstrated statistically significant changes in the relative levels of 45 proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both soleus and tibialis anterior muscles. Four differentially regulated soleus proteins and six differentially regulated tibialis anterior proteins were identified. The identified proteins can be grouped according to function as metabolic proteins, chaperone proteins, and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the proteome occur during immobilization-induced atrophy in both slow twitch and fast twitch fiber type skeletal muscle.

A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension-induced atrophy or hindlimb suspension-induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting-induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting-induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteome occur during disuse-induced soleus muscle atrophy and reweighting hypertrophy.

To study the effect of dehydroepiandrosterone sulfate (DHEAS) treatment of osteoporosis in men with T(BMD)or = 2.5SD.Eighty-six patients were randomly divided into two groups: treatment group (n = 44) and control group (n = 42). DHEAS (100 mg q.d.) was given to the treatment group for 6 months. Bone mineral density, (BMD), biochemical markers of bone absorption and formation and other serum biochemical markers were measured before and after DHEAS treatment. Drug side effects were also evaluated.After oral administration of DHEAS (100 mg q.d.) for 6 months, the serum concentrations of DHEAS and IGF-I in the treatment group were 93.75% +/- 16.1% and 17.71% +/- 4.2% higher respectively than those in the control group (P0.01). The BMD of L2, L3, L4, L2 - 4 and Neck sections increased in the treatment group by 2.65% +/- 0.65%, 2.70% +/- 0.48%, 3.10% +/- 0.41%, 2.82% +/- 0.37% and 2.32% +/- 0.31%, respectively, as compared with that the control group (P0.05 or 0.01). No significant changes were observed in serum FT, E(2) and PSA concentrations in the treatment group as compared with the control group.The treatment of osteoporosis in men with DHEAS is safe and effective.

A data-aided method of joint frequency offset and chromatic dispersion estimation based on the Chu training sequences is shown in 112 Gb/s polarization-multiplexed (PM) quadrature phase-shift keying and 224 Gb/s PM-16-quadrature amplitude modulation with different pulse-shaping filters, respectively. The proposed method achieves a good accuracy and is verified to be robust to polarization mode dispersion.

Single phase samples (Bi, Pb)2Sr2Ca2Cu3Oy have been prepared under different heat treatments. The midpoint transition temperature (Tc) is determined by both AC susceptibility and resistance measurement. The resistance-temperature coefficient is found changed from positive to negative when the sample is fast cooled. The Hall coefficient measurement shows that Tc increases monotonically with the effective carrier concentration (nH) and that nH increases with temperature linearly. The authors draw the conclusion that the reduction of the carrier concentration is caused not by the reduction of the oxygen content, but by the weak localization of the carriers which is unfavorable to High Tc.

Publisher Summary Nano-electrospray (nES) is a new technique for characterizing biomolecules in small volumes (0.5–2 μl) at low picomole levels. In nES, signals from a single sample loading typically last more than 30 minutes, which permits optimization of instrument parameters and MS/MS sequencing with high sensitivity. Both nES/MS and nES/MS/MS data can be obtained from a single sample loading. These features make nES an attractive off-line technique for sequencing peptides collected from capillary electrophoresis (CE). In this chapter, an off-line approach that combines nES/MS analysis and Edman sequencing of peptide/protein fractions collected from CE is presented. Automatic peak collection is accomplished using a computer-controlled Beckman P/ACE 5000 instrument. An off-line approach that is simple and useful for peptide/protein sequencing using 5–10 picomoles of material has been demonstrated. Several different samples of material, including a peptide mixture (angiotensin-I, methionine enkephalin, and substance-P), a tryptic digest of cytochrome-C and proteins like myoglobin, insulin and lysozyme, are used to demonstrate this method. For this purpose, peptide and protein samples are first separated by capillary electrophoresis. Selected peaks are fraction collected and analyzed by both nano-electrospray mass spectrometry and Edman sequencing.

P.R.ChinaABSTRACTIn this paper we show the relationship among the Lagrange invariant, the interference invariant and thespace bandwidth product. they are important in geometrical optics, physical optics and information optics. Andwe gie a method of caIcuating the space bandvidth product of opticai system.I . INTRODUCTIONThe Lagrange invariant is important in geometrical optics such as in aberration calculations and in thephotometry of optical instruments. in this paper we will ihtroduce a new invariant — interference invariant — inphysical optics. it shows the conditions for forming clear interference fringes and for space coherence of lightfield. And it is well known that the space bandwidth product is significant in holography and optical informationprocessing. It can be used for describing information quantity of optical imaging system and capability of opticalprocessing system and storage capability of hologram etc. Furthermore, we Will

Optical image differentiation operations by means of the wavelet transform (WT) are given in this paper. We detail the advantages of Haar's function and its various types as differentiation wavelets over other functions. We give the appropriate type and parameters of the functions they can demonstrate the best effects of image differentiation. In this paper two types of power spectrum nonlinear processing of joint Fourier transform correlator in hybrid system are also reported. Both the processing of fractional power of Fourier spectrum and the filtering of the zero order spectrum have some special properties, respectively. Combined the Haar's wavelet transform with the power spectrum nonlinear processing, we demonstrate the differentiation of the upper right or down left of optical iamge.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

alpha-Crystallins from the water-soluble and the water-insoluble, guanidine-soluble portions of lenses from four renal failure patients and two normal donors of similar age were isolated and enzymatically digested into peptides. Molecular weights of the peptides, determined by fast atom bombardment mass spectrometry, indicated modifications specifically associated with renal failure. The only modifications observed in the alpha-crystallins from renal failure patients, but not in the normal old lenses, were glutathione adducts to Cys 131 and Cys 142. These adducts were present in the lenses of all four renal failure patients, but not in the two normal old lenses. The four lenses from the renal failure patients were searched for evidence of carbamylation at lysyl or cysteinyl residues: carbamylation was not detected. Because the same mass spectrometric methods had previously demonstrated sufficient sensitivity and specificity to detect as little as 5% modification in the examination of in vitro carbamylated bovine lenses, these results indicated that carbamylation is not a major modification of the lens alpha-crystallins of renal failure patients.

Abstract 3080 Background: Salvage therapeutic options are limited for multiple myeloma (MM) patients who relapse after autologous hematopoietic stem cell transplantation (ASCT). A second ASCT using a different conditioning regimen may provide long-term disease control. We report efficacy and safety of daily intravenous busulfan (IV Bu) conditioning given with bortezomib for second ASCT. Materials and Methods: In this prospective, multicenter, Phase IIa study, thirty MM patients who relapsed ≥ 1 year after initial ASCT and were candidates for second ASCT were enrolled at eleven centers in the US and Canada. Patients received a test dose of IV Bu (0.8 mg/kg) over 2 hours between Days -12 and -9 prior to ASCT. Pharmacokinetic (PK) analysis from test dose determined Bu exposure as area under the concentration-time curve (AUC). This analysis was used to determine individualized Bu PK-directed dosing for the conditioning regimen in order to achieve a total regimen AUC of 20,000 mM*min. IV Bu was administered over 3 hours once daily from Day -5 to Day -2. Confirmatory PK analysis was conducted in all patients on Day -5. Bu doses were adjusted on Days -3 and -2, if needed. Bortezomib (1.3 mg/m2 QD) was administered as an IV bolus injection on Day -1. Disease response was evaluated prior to the ASCT and at 3 and 6 months post-transplant, based on the International Myeloma Working Group uniform response criteria in 2006. Results: Patient Demographics: Median age at second ASCT was 59 years (range: 48–73). All patients had previously been treated with bortezomib (86.7%), thalidomide (46.7%), and/or lenalidomide (66.7%). All subjects underwent first ASCT with high-dose melphalan. Median time from first ASCT to second ASCT was 28.0 months (range: 12–119). The disease status at second ASCT was seven very good partial response (VGPR; 23.3%), twelve partial response (PR; 40.0%), two stable disease (SD; 6.7%); and nine progressive disease (PD; 30.0%). Safety: The most common grade 3 or 4 adverse event (CTCAE v3.0) was febrile neutropenia in 15 patients (50.0%), followed by stomatitis in 13 patients (43.3%), nausea in four (13.3%) and hypokalemia in three (10.0%). One transplant-related death due to pulmonary complications was reported for a patient with Parkinsonism on post-transplant Day 20. There was no instance of seizure, worsening neuropathy, or hepatic veno-occlusive disease (VOD) meeting the Baltimore criteria. Efficacy: 28 patients had evaluable disease response at least at one time point after second ASCT. Disease response at 3 months were two complete responses (CR; 6.7%), five VGPR (16.7%), four PR (13.3%), eight SD (26.7%), nine PD (30.0%), and two cases without evaluable assessment (6.7%). Two patients who achieved CR at 3 months had PD and VGPR prior to ASCT, respectively. Disease response at 6 months were one stringent CR (sCR; 3.3%), one CR (3.3%), four VGPR (13.3%), seven SD (23.3%), fourteen PD (46.7%), and three cases without evaluable assessment (10.0%). Median progression-free survival was 191 days, while median overall survival has not been reached yet. PK: 40.0% (n=12/30) of patients had AUC outside the expected range from pre-transplant test dose, 0.8 mg/kg of IV Bu: eleven cases with AUC 1,500 μM*min. If only weight was used (e.g. 3.2 mg/kg daily) to determine the dose without considering difference in individual busulfan metabolism, this 40% would have been dosed outside the total target AUC range. Based on test PK, IV Bu dosing for conditioning was individualized ranging between 1.99 and 4.73 mg/kg, which resulted in 93.3% of patients (n=28/30) falling between 16,000 and 24,000 μM*min as a total target AUC without any further dose alteration during conditioning. Only 2 patients (6.7%) needed dose reduction on Days -3 and -2. Mean Bu clearance for test dose and on Day -5 were comparable, 3.00 and 2.92 ml/min/kg, respectively. Conclusions: Disclosures: Stadtmauer: Millenium: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Off Label Use: IV busulfan and bortezomib-based conditioning regimen prior to transplant for myeloma. Freytes:Otsuka Pharmaceuticals: Research Funding. Shaughnessy:Otsuka: Honoraria, Speakers Bureau. White:Otsuka: Honoraria, Research Funding. Rodriguez:Otsuka: Consultancy, Research Funding, Speakers Bureau; Millennium: Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; SOBI: Consultancy, Speakers Bureau. Sun:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Armstrong:Otsuka: Employment. Smith:Otsuka Pharmaceutical Development & Commercialization, Inc: Consultancy. Elekes:Otsuka Pharmaceutical Development & Commercialisation., Inc.: Employment. Kato:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Reece:Otsuka: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Millinneum Pharmaceuticals: Research Funding; Merck: Consultancy, Honoraria, Research Funding.

Abstract 813 Background: Published reports indicate that oral or intravenous (IV) busulfan in combination with cyclophosphamide and VP-16 (BuCyVP-16) is an effective conditioning regimen with acceptable safety profile for lymphoma patients prior to autologous hematopoietic stem cell transplantation (ASCT). Since the therapeutic window of Bu is narrow, it is important to standardize the systemic exposure during conditioning. Although the IV formulation of Bu eliminates the problem of variable drug absorption, unpredictable systemic exposure can still occur due to interpatient differences in Bu clearance. Therefore, tighter control of systemic Bu exposure using pharmacokinetics (PK) may lead to improved efficacy and further decrease in toxicity. The aim of this multi-center, single-arm, Phase II study was to prospectively evaluate the safety and efficacy of IV BuCyVP-16 regimen in lymphoma patients undergoing ASCT, after optimizing Bu exposure using PK-directed dosing. Methods: Patients with chemosensitive, relapsed or primary-refractory Hodgkin and B-cell non-Hodgkin lymphoma undergoing the first ASCT received a test dose of IV Bu (0.8 mg/kg) given as a 2-hour infusion 11 to 14 days before transplant. Bu exposure was determined as area under the concentration-time curve (AUC) using six serial blood samples after the test dose administration. Doses for the conditioning regimen were then calculated to result in total AUC (conditioning + test) of 20,000 mM*min. One-fourth of the resulting calculated Bu dose was given as a 3-hour infusion on Day -8, followed by a second, confirmatory PK analysis. The same daily Bu dose was administered on the next 3 days, unless the confirmatory PK analysis showed that this would result in total AUC outside the target range (>24,000 mM*min or Results: A total of 202 subjects with Hodgkin (n=65) and non-Hodgkin lymphoma (n=137) were enrolled from 32 centers in the US and Canada. 196 subjects had both test PK and confirmatory PK results. Test PK demonstrated that 36.2% (n=71) of the patients had exposure outside of expected range (1,250 μM*min ± 20%): higher AUC (>1,500 μM*min) in five patients (2.6 %) and lower AUC ( An early subset-analysis by age in June 2011 revealed that 4 of 18 subjects older than 65 years suffered TRM. Consequently, the protocol was amended to lower the inclusion age limit to 65. The final TRM at day 100 was 4/18 (22.2% [95%CI; 6.4–47.6%]) for patients older than 65 years and 5/184 (2.7% [95%CI; 0.9–6.2%]) for those 65 years old or younger. PK results indicated no extraordinary Bu exposure in TRM cases. The most frequently observed grade ≥ 3 adverse events (CTCAE ver. 3.0) were febrile neutropenia 55.1 % (Grade 3: 52.2%; Grade 4: 2.9%; no Grade 5), stomatitis 37.7% (all Grade 3 events) and nausea 9.7% (all Grade 3 events). No case of hepatic veno-occlusive disease (HVOD) meeting Baltimore criteria was reported. Conclusions: A pre-conditioning test dose estimated individual PK parameters and accurately predicted Day-8 PK in 95% of the subjects. This strategy personalized the dosing for optimal Bu exposure preventing the Bu overexposure or underexposure that would have occurred in over a third of patients. The toxicity of PK-guided IV BuCyVP-16 preparative regimen was low for patients younger than 66 years of age. This approach resulted in no incidence of HVOD. Disclosures: Costa: Otsuka: Research Funding. Off Label Use: Off label use of busulfan in non hodgkin and hodgkin lymphoma. Waller:Outsuka: Research Funding. Freytes:Otsuka Pharmaceuticals: Research Funding. Shea:Otsuka : Research Funding. Rodriguez:Otsuka: Consultancy, Research Funding, Speakers Bureau; Millennium: Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; SOBI: Consultancy, Speakers Bureau. Sun:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Armstrong:Otsuka: Employment. Smith:Otsuka America Pharmaceuticals Inc: Consultancy. Elekes:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Kato:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Vaughan:Pierre Fabre: Honoraria.

Abstract 4509 BACKGROUND: Once multiple myeloma (MM) relapses after a first autologous hematopoietic stem cell transplantation (HSCT), long-term disease control is challenging because of limited therapeutic options. A second autologous HSCT is one of the options although the choice of conditioning regimens has not been thoroughly investigated. Past studies demonstrated that adding oral busulfan (Bu) to melphalan for conditioning regimen prior to a first HSCT resulted in better disease control of MM than melphalan single administration [Lahuerta et al, Haematologica. 2010;95:1913]. Nonetheless, the risk of hepatic veno-occlusive disease (VOD) hinders the use of oral Bu as a part of the conditioning regimen. Intravenous (IV) Bu eliminates the unpredictable bioavailability of the oral drug, whereas pharmacokinetic (PK)-directed dose optimization reduces inter-individual variability in the metabolism of Bu, resulting in decreased incidence of VOD and improvement in the control of hematologic malignancies [Andersson et al, Biol Blood Marrow Transplant 2002;8:477]. In addition, a proteasome inhibitor like bortezomib may have synergy with an alkylating agent for conditioning prior to HSCT in myeloma [Lonial et al, Clin Cancer Res 2010; 16: 5079], but the safety of a combination with Bu has not been investigated to date. Here we report results from a multicenter, prospective Phase 2 study to examine the pharmacokinetics of IV Bu and safety with bortezomib as a conditioning regimen for a second autologous HSCT. METHODS: 30 patients with relapsed MM who had a first autologous HSCT ≥1 year prior to the planned HSCT were enrolled from eleven centers in the US and Canada. Patients received a test dose of IV Bu (0.8 mg/kg) over 2 hours between days 12 and 9 prior to HSCT. Serial blood samples were drawn up to 360 min after the start of the infusion. A central laboratory measured Bu concentrations, determined Bu exposure as area under the concentration-time curve (AUC) using WinNonlin software, and recommended individualized PK-directed dosing in order to achieve a total regimen AUC of 20,000 μM·min. Using the patient-specific PK-directed dose, IV Bu was administered over 3 hours once daily from Day -5 to Day -2. In all patients, a second PK analysis was conducted on Day -5 to confirm the PK-directed dose. Bu doses were adjusted on Day -3 and Day -2, if needed. Bortezomib (1.3 mg/m2 QD) was administered as an IV bolus injection on Day -1. RESULTS: All 30 patients who enrolled in this study had been treated with high dose melphalan before a first HSCT. The median time from the first to second HSCT was 31.4 months (range 12.0 – 119.2 months). Disease status prior to the second HSCT was VGPR (n=6), PR (n=12), SD (n=2), and PD (n=10). Test PK revealed that 40.0 % (n=12/30) of the patients had doses outside of target range (1,250 μM·min +/− 20%), based on expected exposure using a fixed dose of 0.8 mg/kg. Specifically, infusion of the test dose resulted in 1,500 μM·min or higher AUC in 3.3% (n=1/30) and 1,000 μM·min or lower AUC in 36.7% (n=11/30) of patients. In order to achieve the target AUC, daily PK-directed dose of IV Bu when conditioning started on Day -5 ranged between 1.99 mg/kg and 4.73 mg/kg: lower than 3.2 mg/kg in 12 subjects and higher than 3.2 mg/kg in the remaining 18. Confirmatory PK on Day -5 demonstrated that mean Bu clearances were comparable to that from test PK: 3.03 ml/min/kg for test dose and 2.93 ml/min/kg for Day - 5. Consequently, using a test dose followed by PK-directed Bu dosing, 93.3% of patients (n=28/30) fell within the target range (AUC, 20,000 μM·min +/−20%). Only two patients (6.7%) needed subsequent daily dose adjustment on Day -3 and Day -2. No instances of VOD, seizure, or worsening neuropathy have been reported to date. One death was reported by Day 30 after transplant in a patient with Parkinsonism who died of pulmonary complications. CONCLUSION: This multicenter, prospective study reveals that a pre-transplant test dose PK allows accurate targeting of busulfan dosing. The conditioning regimen of bortezomib and IV busulfan with PK-directed dose optimization is well tolerated in patients with relapsed MM who undergo second autologous HSCT. Disclosures: Freytes: Otsuka Pharmaceuticals: Research Funding. Off Label Use: Busulfex and bortezomib as conditioning regimen for autologous transplant in patients with multiple myeloma. Yeh:Otsuka Pharmaceutical Development & Commercialization, Inc.: Research Funding. Shaughnessy:Otsuka: Consultancy, Honoraria, Speakers Bureau. White:Otsuka: Consultancy, Honoraria. Rodriguez:Millennium: Research Funding, Speakers Bureau; Otsuka: Research Funding. Yu:Otsuka Pharmaceutical Development & Commercialization, Inc.: Research Funding. Sun:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Armstrong:Otsuka Pharmaceutical Development and Commercialization, Inc.: Employment. Elekes:Otsuka Pharmaceutical: Employment. Kato:Otsuka Pharmaceutical Development & Commercialization, Inc.: Employment. Reece:Novartis: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Johnson & Johnson: Research Funding.

A combination of mass spectrometric techniques has been used to investigate the amino acid sequence and post-translational modifications of alpha B-crystallin isolated from bovine lenses by gel filtration chromatography and reversed-phase high performance liquid chromatography. Chromatographic fractions were analyzed by electrospray ionization mass spectrometry to determine the homogeneity and molecular weights of proteins in the fractions. The alpha B-crystallin primary gene product, its mono- and diphosphorylated forms, its N- and C-terminal truncated forms, as well as other lens proteins unrelated to the alpha B-crystallins were identified by their molecular weights. Detailed information about the sites of phosphorylation, as well as evidence supporting reassignment of Asn to Asp at position 80, was obtained by analyzing proteolytic digests of these proteins by fast atom bombardment mass spectrometry. Results of this investigation indicate that alpha B-crystallin is phosphorylated in vivo at Ser 45, Ser 59, and either Ser 19 or 21. From the specificity of phosphorylation of alpha-crystallins, it appears that there may be two different kinases responsible for their phosphorylation.