Rapid identification of protein phosphatase 1-binding proteins by mixed peptide sequencing and data base searching. Characterization of a novel holoenzymic form of protein phosphatase 1

Microcystin-affinity chromatography was used to purify 15 protein phosphatase 1 (PP1)-binding proteins from the myofibrillar fraction of rabbit skeletal muscle. To reduce the time and amount of material required to identify these proteins, proteome analysis by mixed peptide sequencing was developed. Proteins are resolved by SDS-polyacrylamide gel electrophoresis, electroblotted to polyvinylidene fluoride membrane, and stained. Bands are sliced from the membrane, cleaved briefly with CnBr, and applied without further purification to an automated Edman sequencer. The mixed peptide sequences generated are sorted and matched against the GenBank using two new programs, FASTF and TFASTF. This technology offers a simple alternative to mass spectrometry for the subpicomolar identification of proteins in polyacrylamide gels. Using this technology, all 15 proteins recovered in PP-1C affinity chromatography were sequenced. One of the proteins, PP-1bp55, was homologous to human myosin phosphatase, MYPT2. A second, PP-1bp80, identified in the EST data bases, contained a putative PP-1C binding site and a nucleotide binding motif. Further affinity purification over ATP-Sepharose isolated PP-1bp80 in a quaternary complex with PP-1C and two other proteins, PP-1bp29 and human p20. Recombinant PP-1bp80 also bound PP-1C and suppressed its activity toward a variety of substrates, suggesting that the protein is a novel regulatory subunit of PP-1.     

Diffusion of monomers from bonding resin-resin composite combinations through dentine in vitro

OBJECTIVES: Previous work has demonstrated diffusion of the monomer triethylene glycol dimethacrylate (TEGDMA) from resin composite through dentine in vitro. The objective of the present work was to examine monomer diffusion from bonding resins and resin composites used in combination. METHODS: Occlusal cavities were prepared in tooth crowns and restored with bonding resin-resin composite combinations. Aqueous samples from the 'pulpal chamber' of each tooth were removed at timed intervals for analysis by reversed phase-high performance liquid chromatography and mass spectrometry. RESULTS: Bonding resins contributed to monomer diffusion. A TEDGMA-containing bonding resin used in combination with a TEGDMA-containing resin composite hastened and increased TEGDMA diffusion through dentine. A 2-hydroxyethyl methacrylate (HEMA)-containing bonding resin used in combination with a TEGDMA-containing resin composite reduced TEGDMA diffusion only slightly compared with the resin composite alone and added substantial diffusion of HEMA. CONCLUSION: The bonding resins tested contributed to monomer passage to the pulp space and did not prevent movement of monomer from resin composites to the pulp.     

From data properties to evidence

The problem of making decisions among propositions based on both uncertain data items and arguments which are not certain is addressed. The primary knowledge discovery issue addressed is a classification problem: which classification does the available evidence support? The method investigated seeks to exploit information available from conventional database systems, namely, the integrity assertions or data dependency information contained in the database. This information allows ranking arguments in terms of their strengths. As a step in the process of discovering classification knowledge, using a database as a secondary knowledge discovery exercise, latent knowledge pertinent to arguments of relevance to the purpose at hand is explicated. This is called evidence. Information is requested via user prompts from an evidential reasoner. It is fed as evidence to the reasoner. An object-oriented structure for managing evidence is used to model the conclusion space and to reflect the evidence structure. The implementation of the evidence structure and an example of its use are outlined     

Polyamine analogue regulation of NMDA MK-801 binding: a structure-activity study

A series of analogues and homologues of spermine were synthesized, and their impact on MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor was evaluated. These tetraamines encompass both linear and cyclic compounds. The linear molecules include norspermine, N1, N11-diethylnorspermine, N1,N12-bis(2,2,2-trifluoroethyl)spermine, homospermine, and N1,N14-diethylhomospermine. The cyclic tetraamines consist of the piperidine analogues N1,N3-bis(4-piperidinyl)-1,3-diaminopropane, N1,N4-bis(4-piperidinyl)-1,4-diaminobutane, N1,N4-bis(4-piperidinylmethyl)-1,4-diaminobutane, and N1,N4-bis[2-(4-piperidinyl)ethyl]-1,4-diaminobutane and the pyridine analogues N1,N3-bis(4-pyridyl)-1,3-diaminopropane, N1,N4-bis(4-pyridyl)-1,4-diaminobutane, N1,N4-bis(4-pyridylmethyl)-1,4-diaminobutane, and N1,N4-bis[2-(4-pyridyl)-ethyl]-1,4-diaminobutane. This structure-activity set makes it possible to establish the importance of charge, intercharge distance, and terminal nitrogen substitution on polyamine-regulated MK-801 binding in the NMDA channel. Four families of tetraamines are included in this set: norspermines, spermines, homospermines, and tetraazaoctadecanes. Calculations employing a SYBYL modeling program revealed that the distance between terminal nitrogens ranges between 12.62 and 19.61 A. The tetraamines are constructed such that within families cyclics and acyclics have similar lengths but different nitrogen pKa's and thus different protonation, or charge, states at physiological pH. The pKa values for all nitrogens of each molecule and its protonation state at physiological pH are described. The modifications at the terminal nitrogens include introduction of ethyl and beta,beta,beta-trifluoroethyl groups and incorporation into piperidinyl or pyridyl systems. The studies clearly indicate that polyamine length, charge, and terminal nitrogen substitution have a significant effect on how the tetraamine regulates MK-801 binding to the NMDA receptor. Thus a structure-activity basis set on which future design of MK-801 agonists and antagonists can be based is now available.     

Acute inferior myocardial infarction complicated by rupture into the coronary sinus: diagnosis by two-dimensional echocardiography

Ventricular rupture is a catastrophic, often fatal complication of myocardial infarction. We present a unique case of left ventricular rupture into the coronary sinus that was diagnosed by two-dimensional Doppler echocardiography in a patient with a recent inferior myocardial infarction. The echocardiographic findings essential to diagnosis were subsequently confirmed at autopsy and are reviewed in detail.     

Necrotizing fasciitis of the face

Necrotizing fasciitis (NF) is a rapidly progressive soft tissue infection with high morbidity and mortality rates. Its occurrence in the head and neck region is uncommon, the majority of reported cases being limited to involvement of the neck, usually from infections of dental or pharyngeal origin. Involvement of the face from NF is rare; only 35 such cases have been reported in the literature since 1960. It is not only associated with a high mortality but can also result in severe disfigurement of the face, posing challenging reconstructive problems. Successful management of facial NF requires early diagnosis, prompt institution of broad spectrum antibiotics, aggressive surgical debridement to control the infection, and reconstruction of the resultant soft tissue defects. This report describes four additional cases of facial necrotizing fasciitis. One of the four patients died as a result of sepsis and multi-organ system failure. Two of the three surviving patients had significant facial disfigurement. A comprehensive review of the facial NF cases reported in the literature is also provided. Based on our experience with facial NF and the results of all previous case reports, the clinical manifestations, pathogenesis, and management of this disease are discussed.     

Focal segmental glomerulosclerosis in primates infected with a simian immunodeficiency virus

Focal and segmental glomerulosclerosis (FSG) with endothelial tubuloreticular inclusions (TRIs) is the typical lesion of human HIV-associated glomerulopathy. Autopsy studies showed the presence of FSG in 3 of 15 macaques dying 15-120 weeks after experimental infection with a simian immunodeficiency virus (SIVMne). Ultrastructural studies generally revealed numerous endothelial TRIs (also present in normals), mesangial expansion, and evidence of mesangial cell injury. One additional animal had a small-vessel polyarteritis with a proliferative and focally crescentic glomerulonephritis; seven animals had mild, multifocal interstitial nephritis. All animals had documented viremia after infection; 14 of 15 developed antibodies to SIV postinoculation. Additional postmortem findings included severe enterocolitis, encephalitis, and opportunistic infections. In contrast, autopsy studies of macaques infected with a type D simian retrovirus (SAIDS-D/Washington, SRV-2) for similar periods of time (n = 40) showed no evidence of FSG. One SRV-infected animal had a mild proliferative glomerulonephritis. These studies indicate SIV-infected primates may provide a relevant model for study of human HIV-associated nephropathy. They also indicate the variable pathology that can be seen in primate infections of distinct retrovirus types, each of which produces a simian immunodeficiency state that resembles human AIDS.