Gas chromatographic examination of medium composition during growth of mammalian cells in suspension culture

The trimethylsilyl derivatives of 18 amino acids, 3 sugars and 2 vitamins present in a complex tissue culture medium have been prepared and separated by temperature-programmed gas-liquid chromatography. Identification is based upon retention time and quantitation upon the area under the elution peak. As little as 0.02 μg of compound can be identified. During growth of L cells in suspension culture at controlled pH and redox potential, the concentration of most components rapidly declined. However, seven components initially increased in concentration before decreasing, and two components increased during the growth cycle.     

Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a null mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.     

Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly

The age-associated decrease in functionality of the human immune system is thought to have a negative impact on the capacity to provide protection against infection, in turn leading to increased incidence of mortality. In a previous longitudinal study of octogenarians, we identified an immune risk phenotype (IRP) in the very elderly defined by an inverted CD4/CD8 ratio, which was associated with increased mortality and persistent CMV infection. In this study, we analyzed the CD8 clonal composition of nonagenarians and middle-aged individuals. An increased number of CD8 T cell clones was observed in the nonagenarians, and was associated with CMV-seropositivity. Surprisingly, CMV-seropositive nonagenarians with the IRP had a significantly lower number of clones compared with non-IRP individuals. The decrease in clone numbers in IRP individuals was associated with shorter survival time. MHC/peptide multimer staining indicated that the frequency of CMV-specific T cells was higher in nonagenarians than in the middle-aged, but the ratio of functionally intact cells was significantly lower. The lowest ratio of functional CMV-specific T cells was found in an IRP individual. A thorough longitudinal analysis of the CMV-specific T cells in nonagenarians showed a stable pattern with respect to frequency, phenotype, and clonal composition. We hypothesize that the number of different CD8 T cell clonal expansions increases as the individual ages, possibly, as a compensatory mechanism to control latent infections, e.g., CMV, but eventually a point is reached where clonal exhaustion leads to shrinkage of the CD8 clonal repertoire, associated with decreased survival.     

Interleukin-21 Receptor Signalling Is Important for Innate Immune Protection against HSV-2 Infections

Interleukin (IL) -21 is produced by Natural Killer T (NKT) cells and CD4⁺ T cells and is produced in response to virus infections, where IL-21 has been shown to be essential in adaptive immune responses. Cells from the innate immune system such as Natural Killer (NK) cells and macrophages are also important in immune protection against virus. These cells express the IL-21 receptor (IL-21R) and respond to IL-21 with increased cytotoxicity and cytokine production. Currently, however it is not known whether IL-21 plays a significant role in innate immune responses to virus infections. The purpose of this study was to investigate the role of IL-21 and IL-21R in the innate immune response to a virus infection. We used C57BL/6 wild type (WT) and IL-21R knock out (KO) mice in a murine vaginal Herpes Simplex Virus type 2 (HSV-2) infection model to show that IL-21 – IL-21R signalling is indeed important in innate immune responses against HSV-2. We found that the IL-21R was expressed in the vaginal epithelium in uninfected (u.i) WT mice, and expression increased early after HSV-2 infection. IL-21R KO mice exhibited increased vaginal viral titers on day 2 and 3 post infection (p.i.) and subsequently developed significantly higher disease scores and a lower survival rate compared to WT mice. In addition, WT mice infected with HSV-2 receiving intra-vaginal pre-treatment with murine recombinant IL-21 (mIL-21) had decreased vaginal viral titers on day 2 p.i., significantly lower disease scores, and a higher survival rate compared to infected untreated WT controls. Collectively our data demonstrate the novel finding that the IL-21R plays a critical role in regulating innate immune responses against HSV-2 infection.     

Behaviour of butyrylcholinesterase in the intestinal epithelial cells of starved and refed rats.

1. The activity and the molecular characteristics of butyrylcholinesterase were studied in the epithelial cells of the following intestinal segments: duodenum, jejunum, ileum, caecum and colon of starved and refed rats. 2. After starvation, the specific activity of the enzyme is found to increase in the jejunum. The same level of activity was maintained after refeeding. No notable changes were observed in the other intestinal segments after either starvation or refeeding. 3. The behaviour of aminopeptidase, a well-characterized intestinal enzyme, is comparable to that of butyrylcholinesterase, except in the duodenum where the aminopeptidase activity is increased after refeeding. 4. In this cell type, BuChE is found only in its globular forms (G1, G2 and G4). Starvation resulted in a higher value of the sedimentation coefficient of the ileal G2 form, suggesting the existence of a complex between the enzyme and non-cholinesterase components. 5. After refeeding, the sedimentation profile was similar to that of control.     

Three high-resolution crystal structures of cadmium-substituted carboxypeptidase A provide insight into the enzymatic function

Three high-resolution crystal structures of Cd(II)-substituted carboxypeptidase A (CPA) have been determined by X-ray diffraction from crystals prepared in three different buffer systems to assess the effect of pH and ionic strength on the Cd(II) coordination geometry. All crystallize in the space group P2(1) with identical cell dimensions. Cd-CPA(7.5): Cd(II)-substituted CPA prepared at pH 7.5 with [Cl(-)]=2 mM determined to 1.70 A resolution ( R=17.4% and R(free)=19.8%); Cd-CPA(5.5): Cd(II)-substituted CPA prepared at pH 5.5 with [Cl(-)]=2 mM to 2.00 A resolution ( R=16.1% and R(free)=18.6%); Cd-CPA(7.5)-Cl: Cd(II)-substituted CPA prepared at pH 7.5 with [Cl(-)]=250 mM to 1.76 A resolution ( R=16.7% and R(free)=17.8%). No noticeable structural changes were observed between the three structures. Two water molecules coordinate to Cd(II), in contrast to the single water molecule coordinating to Zn(II) in the Zn-CPA structure. No binding sites for anions could be identified, even in the structure with a high concentration of chloride ions. It is suggested that the anion inhibition is due to weak outer-sphere association of Cl(-) ions at several binding sites, shielding the strong positive charge distribution at the surface of the protein near the active site. Based on structural data and a sequence alignment of 18 non-redundant carboxypeptidases, a more elaborate version of the earlier reaction model is proposed that also addresses the transport of water to and from the active site. Conserved residues whose function was not addressed previously delineate the proposed pathways used in the transport of water during catalysis.     

Lactococcus lactis dihydroorotate dehydrogenase A mutants reveal important facets of the enzymatic function

Dihydroorotate dehydrogenases (DHODs) are flavoenzymes catalyzing the oxidation of (S)-dihydroorotate to orotate in the biosynthesis of UMP, the precursor of all other pyrimidine nucleotides. On the basis of sequence, DHODs can be divided into two classes, class 1, further divided in subclasses 1A and 1B, and class 2. This division corresponds to differences in cellular location and the nature of the electron acceptor. Herein we report a study of Lactococcus lactis DHODA, a representative of the class 1A enzymes. Based on the DHODA structure we selected seven residues that are highly conserved between both main classes of DHODs as well as three residues representing surface charges close to the active site for site-directed mutagenesis. The availability of both kinetic and structural data on the mutant enzymes allowed us to define the roles individual structural segments play in catalysis. We have also structurally proven the presence of an open active site loop in DHODA and obtained information about the interactions that control movements of loops around the active site. Furthermore, in one mutant structure we observed differences between the two monomers of the dimer, confirming an apparent asymmetry between the two substrate binding sites that was indicated by the kinetic results.     

Curariform antagonists bind in different orientations to acetylcholine-binding protein

Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to approximately 170 degrees rotation and approximately 30 degrees degree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.