Validity of transfer-function representation of input-output relation in allosteric models

A transfer-function representation of reaction velocity is devised to describe analytically and approximately an input-output response of allosteric enzyme around a steady state. The transfer function is derived on assuming an exponential change in reaction velocity for the indicial response to substrate influx rate. The validity of the representation with variation in the kinetic parameters and flow rates is examined for the response of Koshland-Nemethy-Filmer (KNF) and Monod-Wyman-Changeux (MWC) dimeric models by comparing with the exact response obtained from the computer simulation, that is, by numerical integration of the rate equation. The representation has a wider valid region with a decrease in influx rate than with an increase. For the KNF model the representation is valid for negative cooperativity, but invalid for positive cooperativity. For the MWC model the validity decreases with stronger cooperativity. With the transfer functions valid for the Michaelis-Menten and allosteric reactions, we may derive the transfer-function representation for many metabolic pathways.     

Immune deficiency in the X-linked lymphoproliferative syndrome. I. Epstein-Barr virus-specific defects

Eleven males with XLP were evaluated for EBV-specific antibodies during periods of 2 to 7 yr. Variable responses to EBV-specific antigens were found. All 11 patients had subnormal anti-EBNA titers, which probably reflected a T cell deficiency. The patients showed four different patterns in their anti-VCA response: 1) two boys who had experienced malignant lymphoma mounted no antibodies at all; 2) two patients showed intermittent anti-VCA titers; 3) four males had persistently elevated anti-VCA titers; and 4) three patients showed normal anti-VCA titers. ADCC against EBV-infected cells was abnormally low in six patients and was elevated in two patients given gamma-globulin. ADCC titers did not correlate with anti-VCA titers. However, most patients with XLP failed to effect regression of autologous EBV-infected lymphoblastoid cell lines, indicating a deficiency in long-lived T cell-mediated immunity to EBV.     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

Application of avidin-ferritin and peroxidase as contrasting electron-dense markers for simultaneous electron microscopic immunocytochemical labelling of glutamic acid decarboxylase and tyrosine hydroxylase in the rat arcuate nucleus

Summary A pre-embedding immunostaining procedure was developed using ferritin and peroxidase to enable simultaneous electron microscopic localization of two antigens in the same tissue section. This method was used to study the anatomic relationship between glutamic acid decarboxylase (GAD) immunoreactive axons and tyrosine hydroxylase (TH) — containing neurons of the rat arcuate nucleus. The findings provide ultrastructural evidence that GAD-immunoreactive terminals establish symmetric (Gray II) synapses on TH-reactive neurons.     

Two distinct HLA-A * 0101-specific submotifs illustrate alternative peptide binding modes

 Previous studies have defined two different peptide binding motifs specific for HLA-A ^* 0101. These motifs are characterized by the presence of tyrosine (Y) at the C-termini of 9-mer and 10-mer peptides, and either a small polar or hydrophobic (S, T, M) residue in position 2, or a negatively charged (D or E) residue in position 3. In this study, the structural requirements for peptide binding to A ^* 0101 have been further analyzed by examining the binding capacity of large sets of peptides corresponding to naturally occurring sequences which bore one or the other of these two A ^* 0101-specific motifs. By correlating the presence of specific residue types at each position along the peptide sequence with increased (or decreased) binding affinity, the prominent influence of secondary anchor residues was revealed. In most cases, the two anchors in positions 2 and 3 appear to act synergistically. With the exception of the DE₃ submotif in 9-mer peptides, a positive role for aromatic residues in position 1 and the center of the peptide (positions 4 or 5 of 9- or 10-mer peptides, respectively), and proline at C-3, were also consistently detected. However, secondary anchor residues also appear to differ significantly between the two different submotifs, demonstrating that A ^* 0101 can utilize alternative modes in binding its peptide ligands. According to these analyses, specific refined submotifs were also established, and their merit verified by independent sets of potential A ^* 0101 binding peptides. Besides providing useful insight into the nature of the interaction of the A ^* 0101 allele with its peptide ligands, such refined motifs should also facilitate accurate prediction of potential A ^* 0101-restricted peptide epitopes. Received: 16 July 1996 / Revised: 18 September 1996     

Simple Elastic Network Models for Exhaustive Analysis of Long Double-Stranded DNA Dynamics with Sequence Geometry Dependence

Simple elastic network models of DNA were developed to reveal the structure-dynamics relationships for several nucleotide sequences. First, we propose a simple all-atom elastic network model of DNA that can explain the profiles of temperature factors for several crystal structures of DNA. Second, we propose a coarse-grained elastic network model of DNA, where each nucleotide is described only by one node. This model could effectively reproduce the detailed dynamics obtained with the all-atom elastic network model according to the sequence-dependent geometry. Through normal-mode analysis for the coarse-grained elastic network model, we exhaustively analyzed the dynamic features of a large number of long DNA sequences, approximately ∼150 bp in length. These analyses revealed positive correlations between the nucleosome-forming abilities and the inter-strand fluctuation strength of double-stranded DNA for several DNA sequences.     

Molecular characterization of an adenylate cyclase gene of the cyanobacterium Spirulina platensis

A cyaA gene, encoding an adenylate cyclase, was isolated from a filamentous cyanobacterium, Spirulina platensis, by functional complementation of a cya mutant of Escherichia coli, defective in adenylate cyclase activity. The predicted gene product of cyaA contains a signal peptide-like domain, a putative sensor domain similar to the gene product of vsrA of Pseudomonas solanacearum, a putative membrane-spanning domain and an adenylate cyclase-like catalytic domain. Two other positive clones that complemented the E. coli mutant were isolated from the same cyanobacterium, suggesting that several cya genes are functioning in S. platensis.     

Acceleration of the oxygen reaction in CuA-deficient Nitrosomonas europaea cytochrome c oxidase as revealed by the flow-flash measurement.

The oxygen reaction of Nitrosomonas europaea cytochrome c oxidase containing either 2Cu or 1Cu per two heme a molecules was investigated by the flow-flash technique at 20 degrees C. The reaction profiles of the bacterial enzyme were essentially the same as those of bovine heart cytochrome c oxidase, although the rate of the primary oxygen compound formation was much slower. The 1Cu enzyme exhibited higher rates for both primary oxygen compound formation and intramolecular electron transfer than the 2Cu enzyme. This result clearly indicates that CuA is not essential functionally for the oxidation of ferrous heme a moieties, and suggests its structural importance in maintaining the molecular integrity of N. europaea cytochrome oxidase.