The development of novel and selective p56lck tyrosine kinase inhibitors

Early T-cell receptor mediated signal transduction involves the activation of several tyrosine protein kinases. One of these tyrosine kinases, p56lck, is expressed primarily in T-cells and Natural Killer (NK) cells and has been shown to be critical for their proliferative and effector functions. Indandiones have been identified as a potent and selective chemical class that inhibits p56lck.     

The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans

In an attempt to identify proteins that might underlie membrane trafficking processes in ciliates, calcium-dependent, phospholipid-binding proteins were isolated from extracts of Paramecium tetraurelia. The major protein obtained, named copine, had a mass of 55 kDa, bound phosphatidylserine but not phosphatidylcholine at micromolar levels of calcium but not magnesium, and promoted lipid vesicle aggregation. The sequence of a 920-base pair partial cDNA revealed that copine is a novel protein that contains a C2 domain likely to be responsible for its membrane active properties. Paramecium was found to have two closely related copine genes, CPN1 and CPN2. Current sequence data bases indicate the presence of multiple copine homologs in green plants, nematodes, and humans. The full-length sequences reveal that copines consist of two C2 domains at the N terminus followed by a domain similar to the A domain that mediates interactions between integrins and extracellular ligands. A human homolog, copine I, was expressed in bacteria as a fusion protein with glutathione S-transferase. This recombinant protein exhibited calcium-dependent phospholipid binding properties similar to those of Paramecium copine. An antiserum raised against a fragment of human copine I was used to identify chromobindin 17, a secretory vesicle-binding protein, as a copine. This association with secretory vesicles, as well the general ability of copines to bind phospholipid bilayers in a calcium-dependent manner, suggests that these proteins may function in membrane trafficking.     

Functional MRI of the lumbar spine in erect position in a superconducting open-configuration MR system: preliminary results

High-level penicillin resistance in pneumococci is due to alterations in penicillin-binding proteins (PBPs) 2X, 2B, and 1A. We have sequenced the penicillin-binding domain of PBP 1A from penicillin-resistant South African pneumococcal isolates and have identified amino acid substitutions which are common to all the resistant isolates analyzed. Site-directed mutagenesis was then used to determine whether particular amino acid substitutions at specific positions in PBP 1A mediate penicillin resistance. PCR was used to isolate PBP 2X, 2B, and 1A genes from clinical isolate 8303 (penicillin MIC, 4 micrograms/ml). These wild-type PBP genes were cloned into pGEM-3Zf and were used as the transforming DNA. Susceptible strain R6 (MIC, 0.015 microgram/ml) was first transformed with PBP 2X and 2B DNA, resulting in PBP 2X/2B-R6 transformants for which MICs were 0.25 microgram/ml. When further transformed with PBP 1A DNA, 2X/2B/1A-R6 transformants for which MICs were 1.5 micrograms/ml were obtained. Site-directed mutagenesis of the PBP 1A gene from isolate 8303 was then used to reverse particular amino acid substitutions, followed by transformation of PBP 2X/2B-R6 transformants with the mutagenized PBP 1A DNA. For PBP 2X/2B/1A-R6 transformants, the introduction of the reversal of Thr-371 by Ser or Ala in PBP 1A decreased the MIC from 1.5 to 0.5 micrograms/ml, whereas the reversal of four consecutive amino acid substitutions (Thr-574 by Asn, Ser-575 by Thr, Gln-576 by Gly, and Phe-577 by Tyr) decreased the MIC from 1.5 to 0.375 micrograms/ml. These data reveal that amino acid residue 371 and residues 574 to 577 of PBP 1A are important positions in PBP 1A with respect to the interaction with penicillin and the development of resistance.     

Phosphatidylinositol-dependent membrane fusion induced by a putative fusogenic sequence of Ebola virus

The membrane-interacting abilities of three sequences representing the putative fusogenic subdomain of the Ebola virus transmembrane protein have been investigated. In the presence of calcium, the sequence EBO(GE) (GAAIGLAWIPYFGPAAE) efficiently fused unilamellar vesicles composed of phosphatidylcholine, phosphatidylethanolamine, cholesterol, and phosphatidylinositol (molar ratio, 2:1:1:0.5), a mixture that roughly resembles the lipid composition of the hepatocyte plasma membrane. Analysis of the lipid dependence of the process demonstrated that the fusion activity of EBO(GE) was promoted by phosphatidylinositol but not by other acidic phospholipids. In comparison, EBO(EA) (EGAAIGLAWIPYFGPAA) and EBO(EE) (EGAAIGLAWIPYFGPAAE) sequences, which are similar to EBO(GE) except that they bear the negatively charged glutamate residue at the N terminus and at both the N and C termini, respectively, induced fusion to a lesser extent. As revealed by binding experiments, the glutamate residue at the N terminus severely impaired peptide-vesicle interaction. In addition, the fusion-competent EBO(GE) sequence did not associate significantly with vesicles lacking phosphatidylinositol. Tryptophan fluorescence quenching by vesicles containing brominated phospholipids indicated that the EBO(GE) peptide penetrated to the acyl chain level only when the membranes contained phosphatidylinositol. We conclude that binding and further penetration of the Ebola virus putative fusion peptide into membranes might be governed by the nature of the N-terminal residue and by the presence of phosphatidylinositol in the target membrane. Moreover, since insertion of such a peptide leads to membrane destabilization and fusion, the present data would be compatible with the involvement of this sequence in Ebola virus fusion.     

The relation between electric auditory brain stem and cognitive responses and speech perception in cochlear implant users

Electrically evoked brainstem responses (EABR) and event-related cortical potentials were recorded in seven postlingually deaf adults who were experienced users of a Nucleus multichannel cochlear implant. The patients were divided into two subgroups: good performers and moderate performers. Poor EABR were found in two of the moderate performers. The latencies and amplitudes of the cortical N1 P2 complex in the good performers were within the same range as those of subjects with normal hearing, but were deviant in the group of moderate performers. This may indicate disturbed cochleotopical organization of the auditory cortex in the latter group. P300 measurements in the good performers showed normal latencies, whereas in the moderate performers they were prolonged. The results suggest that the outcomes of electrophysiological measurements to assess the integrity of a patient's auditory neural system on a brainstem and a cortical level, are related to the patient's performance with the cochlear implant.     

Computational determination of side chain specificity for pockets in class I MHC molecules

We show that a rapidly executable computational procedure provides the basis for a predictive understanding of antigenic peptide side chain specificity, for binding to class I major histocompatibility complex (MHC) molecules. The procedure consists of a combined search to identify the joint conformations of peptide side chains and side chains comprising the MHC pocket, followed by conformational selection, using a target function, based on solvation energies and modified electrostatic energies. The method was applied to the B pocket region of five MHC molecules, which were chosen to encompass the full range of specificities displayed by anchors at peptide position 2. These were a medium hydrophobic residue (Leu or Met) for HLA-A*0201, a basic residue (Arg or Lys) for HLA-B*2705; a small hydrophobic residue (Val) for HLA-A*6801, an acidic residue (Glu) for HLA-B*4001 and a bulky residue (Tyr) for H-2K(d). The observed anchors are correctly predicted in each case. The agreement for HLA-B40 and H-2K(d) is especially promising, since their structures have not yet been determined experimentally. Because the experimental determination of motifs by elution is difficult and these calculations take only hours on a high speed workstation, the results open the possibility of routine determination of motifs computationally.     

Stability of orthopedic and surgically assisted rapid palatal expansion over time

At the present time no reports are available on the stability between orthopedic and surgically assisted rapid palatal expansion. This study was designed to examine and compare the dental and skeletal changes over time for both orthopedic maxillary expansion and surgically assisted palatal expansion. The study was divided into two groups. Group one was orthopedically expanded and consisted of 14 males and 10 females. The ages ranged from 6 years to 12 years with a mean of 8.5 years. Group two received surgically assisted rapid palatal expansion and consisted of 12 males and 16 females with ages ranging from 13 years to 35 years and a mean age of 19.25 years. All 52 subjects were white, from the same geographic area, and were treated by the same two operators. Dental models and posterior anterior cephalograms were obtained immediately before and after expansion, at removal of the expansion device, and 1 year after removal of the appliance. A repeated measures analysis of variance test was applied to assess changes over time between groups. The surgical and nonsurgical techniques displayed similar trends over time although the surgical group contained a greater quantity of expansion. Both the orthopedic and the surgical groups showed stable results.     

Sepsis and serum cytokine concentrations

Hematological and plasma chemistry parameters were measured in 129 juvenile white storks (Ciconia ciconia), either wild or captive bred, April to June 1994. Wild storks were members of a colony in the Lozoya River Valley, Madrid, Spain. Red blood cells count, packed cell volume and hemoglobin increased significantly with age. White blood cells count, lymphocytes count and platelets decreased with age. Total solids, total proteins, fibrinogen, albumin, alpha, beta, gamma-globulins and urea increased with age. Differences between captive and wild birds were not notable.