Mode of binding of [3H]dibenzocycloalkenimine (MK-801) to the N-methyl-D-aspartate (NMDA) receptor and its therapeutic implication

Binding of the labeled anticonvulsant drug [³H]dibenzocycloalkenimine (³H]MK-801) to the N-methyl-D-aspartate (NMDA) receptor and its dissociation from the receptor at 25°C are slow processes, both of which follow first order kinetics (t_1/270 and 180 min, respectively). Both reactions are markedly accelerated by glutamate and glycine (t_1/22-8 and 4 min, respectively), which allow bimolecular association kinetics of the labeled drug with the receptors whereas equilibrium binding of [³H]MK-801 (K_d 2–4 nM) is hardly affected by glutamate and glycine. The data suggest that MK-801 acts as a steric blocker of the NMDA receptor channel. The competitive antagonist D-(−)-2-amino-5-phosphovaleric acid (AP-5) freezes the receptor in a state which precludes either binding of [³H]MK-801 to the receptor channel or its dissociation from it. These findings have therapeutic implications.     

The B cell surface molecule B1 is functionally linked with B cell activation and differentiation

The B1 molecule is a 32,000 m.w. phosphorylated cell surface protein expressed exclusively by B cells from the mid pre-B until the plasma cell stage of differentiation. Two monoclonal antibodies (gamma 2a and mu) reactive with this molecule were used to assess the role of B1 in B cell activation, proliferation, and differentiation. The anti-B1 antibodies at concentrations ranging from 0.1 to 100 micrograms/ml significantly inhibited B cell proliferation induced by anti-mu antibodies, Staphylococcus aureus Cowan strain 1, activated T cells, and Epstein Barr virus. Although capable of inhibiting proliferation, anti-B1 antibody in soluble form or coupled to beads did not activate B cells or induce proliferation. Antibodies of comparable isotypes or against other B cell-restricted antigens, including B2, B4, B5, and HB-5, did not inhibit activation. Pretreatment of B cells with anti-B1 antibody did not inhibit activation, indicating that B cells had to be cultured with anti-B1 antibody for anti-B1-mediated inhibition to occur. Maximum inhibition was obtained when anti-B1 antibody was added at the initiation of culture. In agreement with this, growth factor-dependent proliferation of preactivated B cells was not inhibited by anti-B1 antibodies. Comparable inhibition of B cell activation was noted with antibodies reactive with class II antigens of the major histocompatibility complex with the exception that anti-B1 antibody inhibited immunoglobulin secretion in pokeweed mitogen assays, whereas anti-DR antibody did not. These results suggest that the B1 molecule may serve a central role in the regulation of B cell activation and differentiation.     

A model for the lateral diffusion of “stiff” chains in a lipid bilayer

We present random walk models for the diffusive motion of lipid probe molecules in a lipid bilayer and calculate the diffusion constants for probes spanning the entire bilayer and for probes extending through one lipid layer only. The stiffness of such molecules can explain the observed value of 2/3 for the ratio of these diffusion constants.     

Shuffling of amino acid sequence: an important control in synthetic peptide studies of nucleic acid-binding domains. Binding properties of fragments of a conserved eukaryotic RNA binding motif.

We have used synthetic peptides to study a conserved RNA binding motif in yeast poly(A)-binding protein. Two peptides, 45 and 44 amino acids in length, corresponding to amino and carboxyl halves of a 90-amino acid RNA-binding domain in the protein were synthesized. While the amino-terminal peptide had no significant affinity for nucleic acids, the carboxyl-terminal peptide-bound nucleic acids with similar characteristics to that for the entire 577 residue yeast poly(A)-binding protein. In 100 mM NaCl, the latter peptide retained over 50% of the intrinsic binding free energy of the protein, as well as, similar RNA versus DNA binding specificity. However, shuffling of the sequence of this 44 residue peptide had surprisingly little effect on its nucleic acid binding properties suggesting the overriding importance of amino acid composition as opposed to primary sequence. Deletion studies on the 44 residue peptide with the "correct" sequence succeeded in identifying amino acids important for conferring RNA specificity and for increasing our understanding of the molecular basis for nucleic acid binding by synthetic peptides. The shuffled peptide study, however, clearly indicates that considerable caution must be exercised before extrapolating results of structure/function studies on synthetic peptide analogues to the parent protein.     

Growth arrest of human B lymphocytes is accompanied by induction of the low molecular weight mammalian heat shock protein (Hsp28).

A large number of protein and molecular markers have been identified that delineate the early stages of human B cell activation and proliferation. In contrast, few if any molecules are transiently expressed precisely as activated B cells stop proliferating and undergo growth arrest. We demonstrate that the low molecular weight heat shock protein (hsp28) exhibits unique induction kinetics that specifically demarcates this interval. After mitogenic activation of unstimulated splenic B cells, hsp28 protein and phosphorylation transiently increase coinciding precisely with the peak of cellular proliferation and the onset of growth arrest. Although most neoplastic B cells constitutively express hsp28, three cell lines were identified that were hsp28-. No differences in phenotype or growth kinetics were detected between hsp28+ and hsp28- neoplastic B cells demonstrating that hsp28 expression is not essential for cell growth. However, when treated with phorbol ester or heat shock, these hsp28- cell lines synthesize hsp28 followed by the onset growth arrest. The consistency with which hsp28 induction transiently delineates the interval from peak proliferation to the onset of growth arrest suggests hsp28 itself is likely to be involved in regulating this process.     

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation‐derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC‐MS³‐based targeted detection of low‐abundant human leukocyte antigen (HLA) class‐I‐presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen‐derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA‐peptide complexes, while keeping high isolation yields of low‐abundant target peptides. The subsequent targeted LC‐MS³ detection allowed for increased sensitivity, which resulted in successful detection of the known HLA‐A2‐restricted epitope E7_11–19 and ten additional E7‐derived peptides on the surface of HPV16‐transformed cells. T‐cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low‐abundant candidate epitopes to be true immunotherapy targets.     

Plant cell culture technology in the cosmetics and food industries: current state and future trends

The production of drugs, cosmetics, and food which are derived from plant cell and tissue cultures has a long tradition. The emerging trend of manufacturing cosmetics and food products in a natural and sustainable manner has brought a new wave in plant cell culture technology over the past 10 years. More than 50 products based on extracts from plant cell cultures have made their way into the cosmetics industry during this time, whereby the majority is produced with plant cell suspension cultures. In addition, the first plant cell culture-based food supplement ingredients, such as Echigena Plus and Teoside 10, are now produced at production scale. In this mini review, we discuss the reasons for and the characteristics as well as the challenges of plant cell culture-based productions for the cosmetics and food industries. It focuses on the current state of the art in this field. In addition, two examples of the latest developments in plant cell culture-based food production are presented, that is, superfood which boosts health and food that can be produced in the lab or at home.

     

Release of Glutamate and Aspartate from CA1 Synaptosomes: Selective Modulation of Aspartate Release by Ionotropic Glutamate Receptor Ligands

Abstract: Synaptosomes prepared from area CA1 of the rat hippocampus were used to determine (a) whether Schaffer collateral-commissural-ipsilateral associational terminals release both aspartate and glutamate in a Ca²⁺-dependent manner when reuptake of released glutamate is minimal and (b) whether autoreceptor mechanisms described in CA1 or hippocampal slices could reflect direct actions of glutamate receptor ligands on the synaptic terminal. When challenged for 1 min with either 25 m M K⁺ or 300 µ M 4-aminopyridine, CA1 synaptosomes released both glutamate and aspartate in a Ca²⁺-dependent manner. The glutamate/aspartate ratio was ∼5:1 in each case. K⁺-evoked glutamate release was unaffected by ligands active at NMDA or ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Unlike glutamate release, the release of aspartate was enhanced by NMDA, and this effect was blocked by d -2-amino-5-phosphonovalerate ( d -AP5). Kainate selectively depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) selectively increased the K⁺-evoked release of aspartate. AMPA enhanced aspartate release, like the antagonist CNQX. When applied in the presence of diazoxide, which blocks the desensitization of AMPA receptors, AMPA and kainate both depressed aspartate release. These findings support the view that Schaffer collateral-commissural-ipsilateral associational terminals release aspartate as well as glutamate and that these two release processes are regulated by different autoreceptor mechanisms.     

Use of Complementary and Alternative Medicine (CAM) as Part of the Oncological Treatment: Survey about Patients’ Attitude towards CAM in a University-Based Oncology Center in Germany

IntroductionTo understand if and which patients would be open-minded to Complementary and Alternative Medicine (CAM) use parallel to their oncological treatment. Moreover, we sought to determine which methods are most accepted and which are the primary motivators to use CAM.MethodsWe developed and anonymously conducted a questionnaire for patients in the oncology center (TU Munich). Questions focus on different CAM methods, previous experiences, and willingness to apply or use CAM when offered in a university-based setting.ResultsA total of 171 of 376 patients (37.4% women, 62.0% men, 0.6% unknown) participated. This corresponds to a return rate of 45%. Median age was 64 years (17–87 years). Of all participants, 15.2% used CAM during their oncological therapy; 32.7% have used it in the past. The majority (81.9%) was not using CAM during therapy; 55.5% have not used CAM in the past respectively. The analysis revealed a significant correlation between education and CAM use during therapy (r = 0.18; p = 0.02), and CAM use in the past (r = 0.17; p = 0.04). Of all patients using CAM during therapy, favored methods were food supplements (42.3%), vitamins/minerals (42.3%), massage (34.6%). Motivations are especially the reduction of side effect and stress, the positive effect of certain CAM-treatments on the immune system and tumor therapy. Results showed no difference between women and men. Most patients not having had any experience with CAM complain about the deficiency of information by their treating oncologist (31.4%) as well as missing treatment possibilities (54.3%).ConclusionSince many patients believe in study results demonstrating the efficacy of CAM, it stresses our task to develop innovative study protocols to investigate the outcomes of certain CAM on symptom reduction or other endpoints. Thus, prospective trials and innovative evidence-based treatment concepts to include CAM into high-end oncology is what patients demand and what a modern oncology center should offer.