Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the site of regulatory phosphorylation

Phosphorylation of glycogen phosphorylase at residue Ser14 triggers a conformational transition that activates the enzyme. The N-terminus of the protein, in response to phosphorylation, folds into a 310 helix and moves from its location near a cluster of acidic residues on the protein surface to a site at the dimer interface where a pair of arginine residues form charged hydrogen bonds with the phosphoserine. Site-directed mutagenesis was used to replace Ser14 with Asp and Glu residues, analogs of the phosphoserine, that might be expected to participate in ionic interactions with the arginine side chains at the dimer interface. Kinetic analysis of the mutants indicates that substitution of an acidic residue in place of Ser14 at the site of regulatory phosphorylation partially activates the enzyme. The S14D mutant shows a 1.6-fold increase in Vmax, a 10-fold decrease in the apparent dissociation constant for AMP, and a 3-fold decrease in the S0.5 for glucose 1-phosphate. The S14E mutant behaves similarly, showing a 2.2-fold increase in Vmax, a 6-fold decrease in the apparent dissociation constant for AMP, and a 2-fold decrease in the S0.5 for glucose 1-phosphate. The ability of the mutations to enhance binding of AMP and glucose 1-phosphate and to raise catalytic activity suggests that the introduction of a carboxylate side chain at position 14 promotes docking of the N-terminus at the subunit interface and concomitant stabilization of the activated conformation of the enzyme. Like the native enzyme, both mutants show significant activity only in the presence of the activator, AMP. Full activation, analogous to that provided by covalent phosphorylation of the enzyme, likely is not achieved because of differences in the charge and the geometry of ionic interactions at the phosphorylation site.     

Treatment and prognosis of post-transplant lymphoproliferative disease

Post-transplant lymphoproliferative diseases (PTLDs) are a clinically and morphologically heterogeneous group of lymphoid proliferations. They represent a life-threatening complication of solid organ transplantation. The mechanisms of their pathogenesis are not yet fully understood. A combination of impaired immunity, oncogenic consequences of immunosuppressive therapy and EBV infection may play a role. Studies on incidence, treatment and prognosis are difficult because of the small number of cases occurring at each transplant center and the lack of reliable classification. Overall mortality remains high even though 25% of patients require no other measure than reduction in immunosuppression which must be the first step of treatment. Several treatments are currently used but more adequate classification as well as multicenter studies are urgently needed because many questions remain with regard to therapeutic strategy. Late-onset monoclonal tumors may be treated by conventional chemotherapy, while EBV-positive PTLDs may benefit from other approaches such as antiviral therapies or immunologic modulation of tumor functions.     

Anticentromere antibodies in rheumatologic practice are not consistently associated with scleroderma

Anticentromere antibodies identified by indirect immunofluorescence are a valuable aid to the diagnosis and prognosis of patients with systemic sclerosis since they are associated in 50% to 80% of cases with limited cutaneous systemic sclerosis, a pattern usually associated with a good prognosis. We studied clinical presentations in rheumatology patients with anticentromere antibodies by indirect immunofluoresence and by ELISA and/or Western blot, but without scleroderma or Raynaud's phenomenon. Eight of 34 (23.5%) rheumatology clinic patients with centromere antibodies met these criteria, seven women and one man, with a median symptom duration of six years (range 1-20 years). Four had Sj?gren's syndrome, one had isolated xerostomia, one systemic lupus erythematosus, one seronegative symmetric polyarthritis and one primary biliary cirrhosis with arthralgia. The mean anticentromere antibody titer in these eight patients was similar to that in the patients who had at least Raynaud's phenomenon. Given the low incidence of scleroderma, these data illustrate the poor predictive value of anticentromere antibodies for the diagnosis of scleroderma in rheumatology clinic patients.     

Expression of INK4 inhibitors of cyclin D-dependent kinases during mouse brain development

In situ hybridization of mouse embryo sections demonstrated expression of mRNAs encoding two polypeptide inhibitors (p18INK4c and p19INK4d) of cyclin D-dependent kinase (CDK) 4 and CDK6 in the central nervous system. No expression of two other INK4 members, p16INK4a and p15INK4b, was observed. The p19INK4d and p18INK4c proteins formed complexes with either CDK4 or CDK6 in a temporal pattern consistent with the results of in situ hybridization. Expression of INK4c was observed at embryonic day 13.5 in neuroepithelial zones of the developing brain, being restricted to dividing neuroblasts but absent from differentiating postmitotic neurons. In the neocortex, p18INK4c was expressed precisely at those developmental stages when neuroblasts switch from a symmetric to an asymmetric pattern of cell division with concomitant increases in their G1 interval. INK4d RNA was detected from embryonic day 11.5 onward, at higher levels than INK4c and with a distinctly different spatial and temporal pattern. Marked INK4d expression was seen in dorsal root ganglia, spinal cord, and focally throughout the brain, but primarily in postmitotic neurons. Neural expression of INK4d continued postnatally into adulthood in postmitotic cells of the dentate gyrus, the pyramidal layer of the hippocampus, and in discrete regions of the cerebral cortex, cerebellum, thalamus, and brainstem. Downregulation of p19INK4d in the dentate gyrus after kainic acid-induced seizures indicated that its expression could also be modified in nondividing cells by excitotoxic stress. Therefore, p19INK4d may contribute to maintaining the quiescent state, acting as a buffer to prevent reactivation of cyclin D-dependent kinases in terminally differentiated cells.     

Phenotypic characterization of Rambouillet sheep expressing the callipyge gene: II. Carcass characteristics and retail yield

Magnetic resonance imaging (MRI) has received considerable attention in recent years over its potential for providing indices of multiple sclerosis activity and progression in clinical trials of new pharmaceuticals. The perceived advantages of MRI-derived measurements include greater objectivity, sensitivity, and reproducibility when compared with clinical rating scales. Clinical scales are also somewhat biased toward lesions affecting locomotion. However, the myriad permutations of MRI acquisition parameters, analysis methodologies, and disease indices demand careful consideration when employing MRI. Moreover, the use of MRI in research into the basic mechanisms of a disease may have different requirements than its use in a clinical trial setting. Consequently, a conference was held, sponsored by the US and Canadian multiple sclerosis societies, to review the present status of various MRI processing strategies and their potential role in clinical trials. Thirteen laboratories from North America and Europe as well as regulatory agencies and statistical consultants made formal presentations followed by extended discussion. This report presents the conclusions reached and recommendations for further action that emerged from the meeting.     

Killing of Chlamydia trachomatis by novel antimicrobial lipids adapted from compounds in human breast milk

The development of new methods for prevention of sexually transmitted Chlamydia trachomatis infection is a top public health priority. Topical self-administered vaginal microbicides represent one such approach in which the organism is eradicated at the time of initial exposure. To this end, we examined the activity of five synthetic lipids adapted from naturally occurring compounds found in human breast milk. C. trachomatis serovar D or F elementary bodies were added to serial dilutions of the lipids and incubated for various times. Aliquots were then cultured in monolayers of McCoy cells, and inclusions were counted. A 7.5 mM concentration of 2-O-octyl-sn-glycerol completely prevented growth of C. trachomatis after 120 min of contact with the organism. The remaining lipids, 1-O-octyl-, 1-O-heptyl-, 2-O-hexyl-, and 1-O-hexyl-sn-glycerol, showed less activity. On electron microscopic examination, the lipids were shown to have disrupted the chlamydial inner membrane, allowing leakage of the cytoplasmic contents from the cell. Lipid activity was unaffected by the presence of 10% human blood or alterations in pH from 4.0 to 8.0, conditions reflecting those sometimes found in the vagina. Our results suggest that these lipids, especially 2-O-octyl-sn-glycerol, may be effective as topical microbicides in preventing the transmission of C. trachomatis. Further efficacy and toxicity studies with these lipids and assessment of their activity against other sexually transmitted disease pathogens are in progress.     

Far-field analysis of coupled bulk and boundary layer diffusion toward an ion channel entrance

We present a far-field analysis of ion diffusion toward a channel embedded in a membrane with a fixed charge density. The Smoluchowski equation, which represents the 3D problem, is approximated by a system of coupled three- and two-dimensional diffusions. The 2D diffusion models the quasi-two-dimensional diffusion of ions in a boundary layer in which the electrical potential interaction with the membrane surface charge is important. The 3D diffusion models ion transport in the bulk region outside the boundary layer. Analytical expressions for concentration and flux are developed that are accurate far from the channel entrance. These provide boundary conditions for a numerical solution of the problem. Our results are used to calculate far-field ion flows corresponding to experiments of Bell and Miller (Biophys. J. 45:279, 1984).