Effect of concomitant statin treatment in postmenopausal patients with hormone receptor-positive early-stage breast cancer receiving adjuvant denosumab or placebo: a post hoc analysis of ABCSG-18.

BACKGROUND: Statins are cholesterol-lowering drugs prescribed for the prevention and treatment of cardiovascular disease. Moreover, statins may possess anticancer properties and interact with receptor activator of nuclear factor κB ligand expression. We aimed at evaluating a hypothetical synergistic effect of statins with denosumab in early-stage breast cancer (BC) patients from the Austrian Breast and Colorectal Cancer Study Group (ABCSG) trial 18. PATIENTS AND METHODS: ABCSG-18 (NCT00556374) is a prospective, randomized, double-blind, phase III study; postmenopausal patients with hormone receptor-positive BC receiving a nonsteroidal aromatase inhibitor were randomly assigned to denosumab or placebo. In this post hoc analysis, we investigated the effects of concomitant statin therapy on recurrence risk (RR) of BC, fracture risk and bone mineral density (BMD). RESULTS: In the study population (n = 3420), statin therapy (n = 824) was associated with worse disease-free survival (DFS) [hazard ratio (HR) 1.35, 95% confidence interval (CI) 1.04-1.75; P = 0.023]. While no significant effect of lipophilic statins (n = 710) on RR was observed (HR 1.30, 95% CI 0.99-1.72; P = 0.062), patients on hydrophilic statins (n = 87) had worse DFS compared with patients not receiving any statins (HR 2.00, 95% CI 1.09-3.66; P = 0.026). This finding was mainly driven by the effect of hydrophilic statins on DFS in the denosumab arm (HR 2.63, 95% CI 1.21-5.68; P = 0.014). However, this effect subsided after correction for confounders in the sensitivity analysis. No association between statin use and fracture risk or osteoporosis was observed. CONCLUSION: According to this analysis, hydrophilic statins showed a detrimental effect on DFS in the main model, which was attenuated after correction for confounders. Our data need to be interpreted with caution due to their retrospective nature and the low number of patients receiving hydrophilic statins.

Acute Gouty Knee Arthritis: Ultrasound Findings Compared With Dual-Energy CT Findings.

OBJECTIVE: The purpose of this study was to compare findings of ultrasound (US) with dual-energy CT (DECT) findings in patients presenting with suspected gouty knee arthritis. SUBJECTS AND METHODS: This prospective study included 65 patients (52 men and 13 women; median age, 61.7 years [range, 38-87 years]) with an initial clinical diagnosis of acute gouty knee arthritis who underwent DECT performed using a 128-MDCT scanner and US performed using a 5-18-MHz transducer. Both intra- and extraarticular findings obtained using each modality were tabulated. RESULTS: DECT identified gout as the final diagnosis for 52 of 65 patients (80.0%). An alternative diagnosis was confirmed for the remaining 13 patients. US detected gout in 31 of 52 patients (sensitivity, 59.6%) and produced findings negative for gout in seven of 13 patients (specificity, 53.8%). The double contour sign on US was positive for gout in 23 of 52 patients (44.2%) and negative in 12 of 13 patients (92.3%). Extraarticular urate deposition was identified by DECT in 44 of 52 patients, compared with identification by US in 11 of 52 patients (p < 0.001). CONCLUSION: The sensitivity of US for the diagnosis of gouty knee arthritis is limited, particularly with respect to extraarticular urate deposition. The double contour sign is the single most valuable sign for the assessment of gouty knee arthritis by US.

Immune reactivity after adenoviral-mediated aquaporin-1 cDNA transfer to human parotid glands.

OBJECTIVES: The purpose of this study was to examine the humoral and cellular immune reactivity to adenoviral vector (AdhAQP1) administration in the human parotid gland over the first 42 days of a clinical gene therapy trial. METHODS: Of eleven treated subjects, five were considered as positive responders (Baum et al, 2012). Herein, we measured serum neutralizing antibody titers, circulating cytotoxic lymphocytes, and lymphocyte proliferation in peripheral blood mononuclear cells. Additionally, after adenoviral vector stimulation of lymphocyte proliferation, we quantified secreted cytokine levels. RESULTS: Responders showed little to modest immune reactivity during the first 42 days following gene transfer. Additionally, baseline serum neutralizing antibody titers to serotype 5-adenovirus generally were not predictive of a subject's response to parotid gland administration of AdhAQP1. Cytokine profiling from activated peripheral blood mononuclear cells could not distinguish responders and non-responders. CONCLUSIONS: The data are the first to describe immune responses after adenoviral vector administration in a human parotid gland. Importantly, we found that modest (2-3 fold) changes in systemic cell-mediated immune reactivity did not preclude positive subject responses to gene transfer. However, changes beyond that level likely impeded the efficacy of gene transfer.

Decorin is a part of the ovarian extracellular matrix in primates and may act as a signaling molecule.

STUDY QUESTION: Is decorin (DCN), a putative modulator of growth factor (GF) signaling, expressed in the primate ovary and does it play a role in ovarian biology? SUMMARY ANSWER: DCN expression in the theca, the corpus luteum (CL), its presence in the follicular fluid (FF) and its actions revealed in human IVF-derived granulosa cells (GCs), suggest that it plays multiple roles in the ovary including folliculogenesis, ovulation and survival of the CL. WHAT IS KNOWN ALREADY: DCN is a secreted proteoglycan, which has a structural role in the extracellular matrix (ECM) and also interferes with the signaling of multiple GF/GF receptors (GFRs). However, DCN expression and action in the primate ovary has yet to be determined. STUDY DESIGN, SIZE, DURATION: Archival human and monkey ovarian samples were analyzed. Studies were conducted using FF and GC samples collected from IVF patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemistry, western blotting, RT-PCR, quantitative RT-PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) studies were complemented by cellular studies, including the measurements of intracellular Ca²âº, reactive oxygen species (ROS), epidermal GF receptor (EGFR) phosphorylation by DCN and caspase activity. MAIN RESULTS AND THE ROLE OF CHANCE: Immunohistochemistry revealed strong DCN staining in the connective tissue and follicular thecal compartments, but not in GCs of pre-antral and antral follicles. Pre-ovulatory follicles could not be studied, but DCN was associated with connective tissue of CL samples and the cytoplasm of luteal cells. DCN expression in monkey CL doubled (P < 0.05) towards the end of the luteal lifespan. DCN was found in human FF obtained from IVF patients (mean: 12.9 ng/ml; n = 20) as determined by ELISA. DCN mRNA and/or protein were detected in freshly isolated and cultured, luteinized human GCs. In the latter, exogenous human recombinant DCN increased intracellular Ca²âº levels and induced the production of ROS in a concentration-dependent manner. DCN, like epidermal GF, phosphorylated EGFR significantly (P < 0.05) and reduced the activity of caspase 3/7 in cultured GCs. The data indicate the expression of DCN in the theca of growing follicles, in FF of ovulatory follicles and in the CL. Therefore, DCN may exert paracrine actions via GF/GFR systems in multiple ovarian compartments. LIMITATIONS, REASONS FOR CAUTION: Functional studies were performed in cultures of human luteinized GCs, which are an apt model but may not fully mirror the pre-ovulatory GC compartment or the CL. Other human ovarian cells, including the thecal cells, were not available. WIDER IMPLICATIONS OF THE FINDINGS: In accordance with its evolving roles in other organs, ovarian DCN is an ECM-associated component, which acts as a multifunctional regulator of GF signaling in the primate ovary. DCN may thus be involved in folliculogenesis, ovulation and the regulation of the CL survival in primates. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Deutsche Forschungsgemeinschaft (DFG) MA1080/17-3 and in part DFG MA1080/21-1 (to AM), NIH grants HD24870 (S.R.O. and R.L.S.), the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement HD18185 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research (S.R.O.) and 8P51OD011092-53 for the operation of the Oregon National Primate Research Center (G.A.D., J.D.H., S.R.O. and R.L.S).

Treatment of perianal sepsis and long-term outcome of recurrence and continence.

AIM: The study investigated the fate of patients with perianal sepsis of cryptoglandular origin. METHOD: All patients treated for perianal sepsis between January 1994 and December 2000 were retrospectively analysed regarding recurrence and faecal incontinence. Data collection was conducted by chart review and by telephone questionnaire using the Vaizey incontinence score. RESULTS: One hundred seventy-three (58%) of 300 patients were available for follow-up at a median period of 121 (77-171) months. Fistula-in-ano was diagnosed in 156 (90%) patients. After a single surgical procedure, 55 (32%) patients had no recurrence of perianal sepsis. In 118 (68%), recurrence required multiple procedures (median 3, range 2-19). If only a single incision and drainage was performed (n = 10, 6%), no faecal incontinence occurred. Drainage with fistulotomy (n = 45, 26%) induced mild incontinence in 9% and severe incontinence in 4%. After multiple procedures that were required in 118 (68%) patients, mild and severe faecal incontinence was found in 16% and 4% of them, respectively. CONCLUSION: Treatment of anal sepsis is associated with a high recurrence rate and a substantial risk of faecal incontinence.

Structural analysis of free and enzyme-bound amaranth alpha-amylase inhibitor: classification within the knottin fold superfamily and analysis of its functional flexibility.

The three-dimensional structure of the amaranth alpha-amylase inhibitor (AAI) adopts a knottin fold of abcabc topology. Upon binding to alpha-amylase, it adopts a more compact conformation characterized by an increased number of intramolecular hydrogen bonds, a decreased volume and in addition a trans to cis isomerization of Pro20. A systematic analysis of the 3-D structural databanks revealed that similar proteins and domains share with AAI the characteristic presence of proline residues, many of which are in a cis backbone conformation. As these proteins fulfil a variety of functional roles and are expressed in very different organisms, we conclude that the structure of the knottin fold, including the propensity of the cis bond, are the result of convergent evolution.

Structural basis for possible calcium-induced activation mechanisms of calpains.

The calpains form a growing family of structurally related intracellular multidomainal cysteine proteinases, which exhibit a catalytic domain distantly related to papain. In contrast to papain, however, their activity in most cases depends on calcium. The calpains are believed to play important roles in cytoskeletal remodeling processes, cell differentiation, apoptosis and signal transduction, but have also been implicated in muscular dystrophy, ischemia, traumatic brain injury, neurodegenerative diseases, rheumatoid arthritis and cataract formation. The best characterized calpains are the ubiquitously expressed mu- and m-calpains, consisting of a common 30 kDa small S-subunit (domains V and VI) and slightly differing 80 kDa large L-subunits (domains I to IV). We have recently determined the 2.3 A structure of recombinant full-length human m-calpain in the absence of calcium, which reveals that the catalytic domain and the two calmodulin-like domains, previously believed to represent the unique calcium switch, are not positioned adjacent to each other, but are separated by the beta-sandwich domain III, which distantly resembles C2 domains. Although the catalytic domain of apocalpain is strongly disrupted compared to papain (which explains its inactivity in the absence of calcium), the crystal structure reveals several sites where calcium could bind, thereby causing a subdomain fusion to form a papain-like catalytic center. All current evidence points to the cooperative interaction of several calcium binding sites. Sites identified include the three EF-hand binding sites in each calmodulin-like domain, the negatively charged segments arranged around the active-site cleft (provided by both catalytic subdomains), as well as an exposed acidic loop of domain III, whose charge compensation could allow the adjacent barrel-like subdomain IIb to move toward the helical subdomain IIa. The Gly-rich S-chain N-terminus and the calcium-loaded acidic loop could target the conventional calpains to cellular/nuclear membranes, thereby explaining their strongly reduced calcium requirement in vivo and in vitro in the presence of acidic phospholipids.

Crystallization and preliminary X-ray analysis of recombinant full-length human m-calpain.

m-Calpain constitutes the prototype of the superfamily of neutral calcium-activated cysteine proteinases. It is a heterodimer consisting of an 80 and a 30 kDa subunit. Recombinant full-length human m-calpain has been crystallized using macro-seeding techniques and vapour-diffusion methods. Two different monoclinic crystal forms (space group P2(1)) were obtained from a solution containing polyethylene glycol (M(W) = 10 000) as a precipitating agent. Complete data sets have been collected to 2.3 and 3.0 A resolution using cryo-cooling conditions and synchrotron radiation. The unit-cell parameters are a = 64.86, b = 133.97, c = 78.00 A, beta = 102.43 degrees and a = 51.80, b = 171.36, c = 64.66 A, beta = 94.78 degrees, respectively. The V(m) values indicate that there is one heterodimer in each asymmetric unit.

The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium.

Calpains (calcium-dependent cytoplasmic cysteine proteinases) are implicated in processes such as cytoskeleton remodeling and signal transduction. The 2.3-A crystal structure of full-length heterodimeric [80-kDa (dI-dIV) + 30-kDa (dV+dVI)] human m-calpain crystallized in the absence of calcium reveals an oval disc-like shape, with the papain-like catalytic domain dII and the two calmodulin-like domains dIV+dVI occupying opposite poles, and the tumor necrosis factor alpha-like beta-sandwich domain dIII and the N-terminal segments dI+dV located between. Compared with papain, the two subdomains dIIa+dIIb of the catalytic unit are rotated against one another by 50 degrees, disrupting the active site and the substrate binding site, explaining the inactivity of calpains in the absence of calcium. Calcium binding to an extremely negatively charged loop of domain dIII (an electrostatic switch) could release the adjacent barrel-like subdomain dIIb to move toward the helical subdomain dIIa, allowing formation of a functional catalytic center. This switch loop could also mediate membrane binding, thereby explaining calpains' strongly reduced calcium requirements in vivo. The activity status at the catalytic center might be further modulated by calcium binding to the calmodulin domains via the N-terminal linkers.

Cycling of human dendritic cell effector phenotypes in response to TNF-alpha: modification of the current 'maturation' paradigm and implications for in vivo immunoregulation.

Dendritic cells (DCs) are potent antigen presenting cells reported to undergo irreversible functional 'maturation' in response to inflammatory signals such as TNF-alpha. The current paradigm holds that this DC maturation event is required for full functional capacity and represents terminal differentiation of this cell type, culminating in apoptotic cell death. This provides a possible mechanism for avoiding dysregulated immunostimulatory activity, but imposes constraints on the capacity of DCs to influence subsequent immune responses and to participate in immunological memory. We report that the cell surface and functional effects induced by TNF-alpha are reversible and reinducible. These effects are accompanied by a concordant modulation of cytokine mRNA expression that includes the induction of proinflammatory factors (IL-15, IL-12, LT-alpha, LT-beta, TNF-alpha, RANTES) which is coincident with the down-regulation of counter-regulatory cytokines (IL-10, TGF-beta1, TGF-beta2, IL-1 RA, MCP-1). The resultant net effect is a dendritic cell activation state characterized by a transient proinflammatory posture. These results demonstrate that 1) human DCs do not undergo terminal 'maturation' in response to TNF-alpha, 2) DC phenotypes are more pleiotropic than previously thought, and 3) DCs are potential immunoregulatory effector cells with implications for control of immune responses in both in vivo and in vitro systems.

Specific inhibition of insect alpha-amylases: yellow meal worm alpha-amylase in complex with the amaranth alpha-amylase inhibitor at 2.0 A resolution.

BACKGROUND: alpha-Amylases constitute a family of enzymes that catalyze the hydrolysis of alpha-D-(1,4)-glucan linkages in starch and related polysaccharides. The Amaranth alpha-amylase inhibitor (AAI) specifically inhibits alpha-amylases from insects, but not from mammalian sources. AAI is the smallest proteinaceous alpha-amylase inhibitor described so far and has no known homologs in the sequence databases. Its mode of inhibition of alpha-amylases was unknown until now. RESULTS: The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with AAI was determined at 2.0 A resolution. The overall fold of AAI, its three-stranded twisted beta sheet and the topology of its disulfide bonds identify it as a knottin-like protein. The inhibitor binds into the active-site groove of TMA, blocking the central four sugar-binding subsites. Residues from two AAI segments target the active-site residues of TMA. A comparison of the TMA-AAI complex with a modeled complex between porcine pancreatic alpha-amylase (PPA) and AAI identified six hydrogen bonds that can be formed only in the TMA-AAI complex. CONCLUSIONS: The binding of AAI to TMA presents a new inhibition mode for alpha-amylases. Due to its unique specificity towards insect alpha-amylases, AAI might represent a valuable tool for protecting crop plants from predatory insects. The close structural homology between AAI and 'knottins' opens new perspectives for the engineering of various novel activities onto the small scaffold of this group of proteins.

The 2.2 A crystal structure of human chymase in complex with succinyl-Ala-Ala-Pro-Phe-chloromethylketone: structural explanation for its dipeptidyl carboxypeptidase specificity.

Human chymase (HC) is a chymotrypsin-like serine proteinase expressed by mast cells. The 2.2 A crystal structure of HC complexed to the peptidyl inhibitor, succinyl-Ala-Ala-Pro-Phe-chloromethylketone (CMK), was solved and refined to a crystallographic R-factor of 18.4 %. The HC structure exhibits the typical folding pattern of a chymotrypsin-like serine proteinase, and shows particularly similarity to rat chymase 2 (rat mast cell proteinase II) and human cathepsin G. The peptidyl-CMK inhibitor is covalently bound to the active-site residues Ser195 and His57; the peptidyl moiety juxtaposes the S1 entrance frame segment 214-217 by forming a short antiparallel beta-sheet. HC is a highly efficient angiotensin-converting enzyme. Modeling of the chymase-angiotensin I interaction guided by the geometry of the bound chloromethylketone inhibitor indicates that the extended substrate binding site contains features that may generate the dipeptidyl carboxypeptidase-like activity needed for efficient cleavage and activation of the hormone. The C-terminal carboxylate group of angiotensin I docked into the active-site cleft, with the last two residues extending beyond the active site, is perfectly localized to make a favorable hydrogen bond and salt bridge with the amide nitrogen of the Lys40-Phe41 peptide bond and with the epsilon-ammonium group of the Lys40 side-chain. This amide positioning is unique to the chymase-related proteinases, and only chymases from primates possess a Lys residue at position 40. Thus, the structure conveniently explains the preferred conversion of angiotensin I to angiotensin II by human chymase.

Phase I trial of anti-CD3-stimulated CD4+ T cells, infusional interleukin-2, and cyclophosphamide in patients with advanced cancer.

PURPOSE: We performed a phase I trial to determine whether in vivo expansion of activated CD4+ T cells was possible in cancer patients. 111Indium labeling was used to observe trafficking patterns of the infused stimulated CD4+ T cells. The influence of cyclophosphamide (CTX) dosing on immunologic outcome was also examined. PATIENTS AND METHODS: Patients with advanced solid tumors or non-Hodgkin's lymphoma received CTX at 300 or 1,000 mg/m2 intravenously (i.v.). Leukapheresis was performed to harvest peripheral-blood mononuclear cells (PBMCs) either just before the CTX dose, or when the patient was either entering or recovering from the leukocyte nadir induced by CTX. An enriched population of CD4+ T cells was obtained by negative selection. The CD4+ T cells were activated ex vivo with anti-CD3, cultured with interleukin-2 (IL-2) for 4 days, and adoptively transferred. After adoptive transfer, patients received IL-2 (9.0 x 10(6) IU/m2/d) by continuous infusion for 7 days. RESULTS: The absolute number of CD4+, CD4+/DR+, and CD4+/CD45RO+ T cells increased in a statistically significant fashion in all cohorts after the first course of therapy. The degree of CD4 expansion was much greater than CD8 expansion, which resulted in a CD4:CD8 ratio that increased in 26 of 31 patients. The greatest in vivo CD4 expansion occurred when cells were harvested as patients entered the CTX-induced nadir. One complete response (CR), two partial responses (PRs), and eight minor responses were observed. Trafficking of 111Indium-labeled CD4 cells to subcutaneous melanoma deposits was also documented. CONCLUSION: CD4+ T cells can be expanded in vivo in cancer patients, which results in increased CD4:CD8 ratios. The timing of pheresis in relation to CTX administration influences the degree of CD4 expansion. Tumor responses with this regimen were observed in a variety of tumors, including melanoma and non-Hodgkin's lymphoma; a high percentage of patients had at least some tumor regression from the regimen that produced the greatest CD4+ T-cell expansion.

A novel strategy for inhibition of alpha-amylases: yellow meal worm alpha-amylase in complex with the Ragi bifunctional inhibitor at 2.5 A resolution.

BACKGROUND: alpha-Amylases catalyze the hydrolysis of alpha-D-(1,4)-glucan linkages in starch and related compounds. There is a wide range of industrial and medical applications for these enzymes and their inhibitors. The Ragi bifunctional alpha-amylase/trypsin inhibitor (RBI) is the prototype of the cereal inhibitor superfamily and is the only member of this family that inhibits both trypsin and alpha-amylases. The mode of inhibition of alpha-amylases by these cereal inhibitors has so far been unknown. RESULTS: The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with RBI was determined at 2.5 A resolution. RBI almost completely fills the substrate-binding site of TMA. Specifically, the free N terminus and the first residue (Ser1) of RBI interact with all three acidic residues of the active site of TMA (Asp185, Glu222 and Asp287). The complex is further stabilized by extensive interactions between the enzyme and inhibitor. Although there is no significant structural reorientation in TMA upon inhibitor binding, the N-terminal segment of RBI, which is highly flexible in the free inhibitor, adopts a 3(10)-helical conformation in the complex. RBI's trypsin-binding loop is located opposite the alpha-amylase-binding site, allowing simultaneous binding of alpha-amylase and trypsin. CONCLUSIONS: The binding of RBI to TMA constitutes a new inhibition mechanism for alpha-amylases and should be general for all alpha-amylase inhibitors of the cereal inhibitor superfamily. Because RBI inhibits two important digestive enzymes of animals, it constitutes an efficient plant defense protein and may be used to protect crop plants from predatory insects.

Crystal structure of yellow meal worm alpha-amylase at 1.64 A resolution.

The three-dimensional structure of the alpha-amylase from Tenebrio molitor larvae (TMA) has been determined by molecular replacement techniques using diffraction data of a crystal of space group P212121 (a=51.24 A; b=93.46 A; c=96.95 A). The structure has been refined to a crystallographic R-factor of 17.7% for 58,219 independent reflections in the 7.0 to 1.64 A resolution range, with root-mean-square deviations of 0.008 A for bond lengths and 1.482 degrees for bond angles. The final model comprises all 471 residues of TMA, 261 water molecules, one calcium cation and one chloride anion. The electron density confirms that the N-terminal glutamine residue has undergone a post-transitional modification resulting in a stable 5-oxo-proline residue. The X-ray structure of TMA provides the first three-dimensional model of an insect alpha-amylase. The monomeric enzyme exhibits an elongated shape approximately 75 Ax46 Ax40 A and consists of three distinct domains, in line with models for alpha-amylases from microbial, plant and mammalian origin. However, the structure of TMA reflects in the substrate and inhibitor binding region a remarkable difference from mammalian alpha-amylases: the lack of a highly flexible, glycine-rich loop, which has been proposed to be involved in a "trap-release" mechanism of substrate hydrolysis by mammalian alpha-amylases. The structural differences between alpha-amylases of various origins might explain the specificity of inhibitors directed exclusively against insect alpha-amylases.

The alpha-amylase from the yellow meal worm: complete primary structure, crystallization and preliminary X-ray analysis.

The alpha-amylase from Tenebrio molitor larvae (TMA) was purified from a crude larval extract. After removal of the N-terminal pyroglutamate residue and identification of the following 17 residues by Edman sequencing, the cDNA of mature TMA was cloned from larval mRNA. The encoded enzyme consists of 471 amino acid residues and has 57-79% sequence identity to other insect alpha-amylases and also shows high homology to the mammalian enzymes. TMA was crystallized in form of well-ordered orthorhombic crystals of space group P2(1)2(1)2(1) diffracting beyond 1.6 A resolution with unit cell dimensions of a = 51.24 A, b = 93.46 A, c = 96.95 A. TMA may serve as model system for the future analysis of interactions between insect alpha-amylase and proteinaceous plant inhibitors on the molecular level.

Alterations in T cell receptor and signal transduction molecules in melanoma patients.

We have recently described molecular changes in T cells from tumor-bearing patients that are associated with depressed immune function. The present work investigates changes in T-cell signal transduction proteins including the T-cell receptor-zeta (TCR-zeta) chain and receptor-associated tyrosine kinases in patients with metastatic malignant melanoma. A marked decrease in the expression of the TCR-zeta chain was observed in the peripheral blood T cells of 19 (43%) of 44 patients. Decreases in several tyrosine kinases were found in 12 (57%) of 21 patients tested. T cells from patients with diminished TCR-zeta chain expression also showed statistically significant differences in cytokine production pattern, with lower interleukin 2 and IFN-zeta production compared with normal subjects and melanoma patients with normal TCR-zeta chain status. The overall survival of melanoma patients with low TCR-zeta chain expression was significantly shorter than that of patients with normal TCR-zeta chain expression (P = 0.0013). TCR-zeta-deficient patients showed a trend toward having faster growing tumors. There was no correlation between the pretreatment TCR-zeta chain status and albumin or performance status. These findings suggest that alterations in T-cell function occur commonly in melanoma patients and may be independent predictors of clinical outcome.

Determination of the three-dimensional structure of the bifunctional alpha-amylase/trypsin inhibitor from ragi seeds by NMR spectroscopy.

The three-dimensional structure of the bifunctional alpha-amylase/trypsin inhibitor (RBI) from seeds of ragi (Eleusine coracana Gaertneri) has been determined in solution using multidimensional 1H and 15N NMR spectroscopy. The inhibitor consists of 122 amino acids, with 5 disulfide bridges, and belongs to the plant alpha-amylase/trypsin inhibitor family for which no three-dimensional structures have yet been available. The structure of the inhibitor was determined on the basis of 1131 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 52 phi angles, supplemented by 9 psi and 51 chi 1 angles. RBI consists of a globular four-helix motif with a simple "up-and-down" topology. The helices are between residues 18-29, 37-51, 58-65, and 87-94. A fragment from Val 67 to Ser 69 and Gln 73 to Glu 75 forms an antiparallel beta-sheet. The fold of RBI represents a new motif among the serine proteinase inhibitors. The trypsin binding loop of RBI adopts the "canonical", substrate-like conformation which is highly conserved among serine proteinase inhibitors. The binding loop is stabilized by the two adjacent alpha-helices 1 and 2. This motif is also novel and not found in known structures of serine proteinase inhibitors. The three-dimensional structure of RBI together with biochemical data suggests the location of the alpha-amylase binding site on the face of the molecule opposite to the site of the trypsin binding loop. The RBI fold should be general for all members of the RBI family because conserved residues among the members of the family from the core of the structure.