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J P Murphy

Publications and source records attributed to J P Murphy.

At least 37 records · Page 2Linked to original sources
PubMed1996

Amplified expression and large-scale purification of protein L'.

The gene fragment (PPL') encoding the functional unit of peptostreptococcus protein L was isolated using PCR and expressed in E. coli. As the gene fragment lacked its own promoter, the 5' PCR primer was designed to incorporate an Nde1 restriction site (CATATG) into the gene. This enabled the gene to be cloned in frame into an Nde1 restriction site immediately downstream of a trp promoter. To prevent read through, a stop codon was introduced into the 3' primer. Expression of PPL' was up to 27% total cell protein which compares favourably to the 0.1% total soluble cell protein obtained from the original clone of peptostreptococcus. Following a heat step homogeneous PPL' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in yields of 90%.

Bacterial Proteins↗
PubMed1995

Crystallization and X-ray analysis of a single fab binding domain from protein L of Peptostreptococcus magnus.

Protein L is a multidomain cell wall constituent of certain strains of Peptostreptococcus magnus which binds to the variable domain of immunoglobulin kappa-light chains. A single immunoglobulin-binding domain of M(r) = 9000 from this protein has been isolated and crystallized. The crystals are of space group P4(2)2(1)2, with cell dimensions a = b = 66.9 A, c = 68.3 A, and diffract to at least 2.2 A resolution. The asymmetric unit of the crystal contains two molecules of the protein L domain, related by a noncrystallographic 2-fold axis, as revealed by a self-rotation function calculated with native diffraction data.

Bacterial Proteins↗
PubMed1995

Total and respirable dust in swine confinement buildings: the benefit of respiratory protective masks and effect of recirculated air.

Caretakers and pigs in dusty environments inhale particles and toxic gases which can cause subclinical illness. We determined the reduction in sampled dust elicited by respiratory masks mounted on glass funnels. Open-faced filters or British cyclones were sampled to measure quantities of dust which the masks had trapped. Respiratory masks reduced the sampled total suspended particulates (open-faced filters) by > or = 75% with NIOSH/MSHA certified protectant (2-tie) masks and > or = 50% with NIOSH/MSHA non-certified comfort (1-tie) masks. Respirable particulates (British cyclones) were reduced by > or = 45% with NIOSH/MSHA certified protectant (2-tie) masks. These data suggest properly worn respiratory protective masks afford significant protection against both total suspended and respirable particulates in swine confinement facilities. Penetration of 3-25% of total aerosol mass through masks allowing only 1% penetration of a silica aerosol with an aerodynamic diameter of 0.6-1.0 microns suggested that measurable portions of the aerosol mass in these confinement houses behaved as if they were less than 1 micron in diameter. Because of the small size of the aerosol, NIOSH/MSHA certified respiratory protective masks should be worn when working in those facilities. We also studied concentrations of ammonia, endotoxin and total and respirable dust particles to determine effects of a recirculation fan which increased the nominal air flow capacity of the building by 10%. Recirculated air had minimal effects on ammonia, total airborne endotoxin or total particulate mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Air Conditioning↗
PubMed1995

Cloning, expression and purification of Ppl-1, a kappa-chain binding protein, based upon protein L from Peptostreptococcus magnus.

Protein L is a multi-domain, cell wall constituent of certain strains of Peptostreptococcus magnus, which binds to the variable domain of the L-chains of Ig. A gene fragment which codes for a single Ig-binding domain of protein L (Ppl-1) was cloned into a modified pKK223-3 vector and over-expressed in E. coli JM103. A rapid protein purification protocol is described. In these studies, purified Ppl-1 was immobilised on to an agarose gel and tested against an array of Igs and Ig fragments. It was found that Ppl-1-bound Igs from a number of different sources via interactions with the L kappa-chain. An enzyme linked immunosorbant assay has been developed to assay the binding of Ppl-1 to IgG. The incubation of Ppl-1 with human serum does not produce an immunoprecipitate, thus suggesting one unique interaction per binding domain which has been confirmed by ELISA. These experiments demonstrate the potential value of Ppl-1 as an immunological tool and as an affinity chromatography ligand for the purification of Igs.

Amino Acid Sequence↗
PubMed1994

The functional units of a peptostreptococcal protein L.

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence of the gene for peptostreptococcal protein L.

A gene bank of Peptostreptococcus magnus DNA was established using an E. coli host-vector system. Western blot analysis identified a clone expressing protein L which bound to the light chain of human immunoglobulins. DNA sequence determination and analysis revealed an open reading frame of 992 amino acids, giving a theoretical secreted protein of 106 kD with a pl of 4.67.

Amino Acid Sequence↗
PubMed1993

Molecular evolution of bacterial cell-surface proteins.

The cell-surface proteins of the infective bacteria Streptococcus and Staphylococcus are probably involved in the process of infection. These proteins share many features including secretion signal peptides, cell-wall spanning regions, membrane anchor domains and repeated domains of various functions. These common features may have evolved by gene duplication and swapping of gene fragments.

Amino Acid Sequence↗
PubMed1993

Characterization of particles, ammonia and endotoxin in swine confinement operations.

To define the atmospheres swine confinement operations, we measured concentrations of total and respirable dust particles, ammonia and endotoxin in the nursery and grower areas of 4 swine confinement houses. Increased ventilation in spring-summer relative to that in winter reduced concentrations of large dust particles more rapidly than it did smaller particles or ammonia. The greater decrease in large particles correlating to increased room air velocity may reflect larger particles' momentum causing impaction on surfaces. There was significant spatial variation in the concentration of airborne endotoxin within individual swine rooms and pens reflecting different mixing of large feed and smaller manure particles. Smaller particles had 4-fold higher concentrations of endotoxin than did larger particles, suggesting they had higher fecal material concentrations. Total airborne endotoxin and total suspended particulates correlated to the fraction of functional endotoxin contained in large particles, suggesting that small particles (0.5-2.0 micrometers) collide with large particles (50 micrometers). These data suggest that large non-respirable particles remove smaller respirable particles from indoor atmospheres due to kinematic coagulation.

Air Pollution, Indoor↗
PubMed1992

Follicular lymphoma with t(8;14)(q24;q32): a distinct clinical and molecular subset of t(8;14)-bearing lymphomas.

The presence of the translocation t(8;14)(q24;q32) has not been well described in follicular lymphoma (FL). In a consecutive series of 278 karyotypically abnormal non-Hodgkin's lymphomas (NHL), six patients with FL showing a t(8;14) without a t(14;18)(q32;q21) were identified. They ranged in age from 45 to 73 years. The cell type was mixed in four patients, small-cleaved in one, and large-cleaved in one; four cases also contained diffuse areas. All cases tested displayed monoclonal surface Ig. The clinical courses were consistent with the histologic subtypes, being less aggressive than other t(8;14)-bearing NHL. In five cases, frozen tissue was available for Southern blotting. The BCL2 gene showed a germline configuration when studied with the MBR, MCR, and 5' cDNA probes. The MYC gene also appeared unrearranged using an exon-1 probe with EcoRI or HindIII digestion. Analysis of the Ig heavy chain (IgH) gene with a JH region probe and BamHI or EcoRI digestion showed only one rearranged band in all cases, indicating that the 14q32 breakpoint did not lie in either the J or switch-mu (SM) regions. In four cases, the exon-1/intron-1 border of the MYC gene, a target area for point mutations in cases of t(8;14) that do not display rearrangements of the MYC gene, was enzymatically amplified and sequenced; no point mutations were identified. The indolent behavior of our six cases, and the finding that the molecular structure of the t(8;14) in these cases does not follow the pattern of breakpoint sites and point mutations defined in other histologic subtypes of NHL with this translocation, suggest that the t(8;14) in these cases is cytogenetically and molecularly distinct from the t(8;14) seen in high-grade NHLs, and is relatively ineffectual in terms of MYC deregulation, or that other genetic elements at these chromosomal sites may be involved. Further analysis of these tumors may provide insights into MYC deregulation and BCL2-independent FL.

Aged↗
PubMed1992

Escalator injuries.

A series of 50 patients suffering injuries from falls on escalators attended our accident and emergency department. Of these, 8 (16 per cent) required admission. Alcohol was a factor in 11/15 (74 per cent) of men but in only 2/34 (6 per cent) of women. Of the injuries, 32 (64 per cent) were associated with the practice of walking on the escalator, while only 12 (24 per cent) were associated with stepping onto or off the escalator. Discouraging the practice of walking on one side of the escalator might help to minimize injuries.

Accidents↗
PubMed1992

Congenital diaphragmatic hernia presenting due to gastrointestinal complications.

Congenital diaphragmatic hernia (CDH) presents beyond the first hours of life in 10% to 20% of cases. Presenting symptoms may be quite nonspecific, and are often gastrointestinal rather than respiratory in origin. We have recently had experience with five such cases, one in a newborn and four in older children. All presented with symptoms related to gastrointestinal complications of their diaphragmatic defect. In the newborn, gastric perforation had occurred, a complication of this anomaly not previously reported. The chest radiograph showed loops of bowel in the chest in all cases, allowing correct preoperative diagnoses. Urgent operative intervention was undertaken in each case with good results and no long-term morbidity. The risk of intestinal strangulation in the late-presenting CDH patient warrants emergent surgical management, which should be rewarded by uniform survival with few complications. Although preoperative stabilization may decrease the severity of pulmonary vasospasm in the newborn with respiratory failure, delay may increase the risk of bowel infarction in the older child presenting with gastrointestinal symptoms.

Child, Preschool↗
PubMed1992

Who speaks for mission?

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Community-Institutional Relations↗
PubMed1992

Amplified expression and large-scale purification of protein G'.

PCR was used to isolate the gene fragment coding for Protein G' (SpG'), a truncated bacterial cell surface protein from Streptococcus G148 which binds to the Fc region of IgG and expressed in E. coli [Goward et al. (1990) Biochem. J. 267: 171-177]. The PCR primer was designed to change the TTG initiation triplet to ATG and to incorporate it into an NdeI restriction site (CATATG), allowing the gene to be cloned in frame into an NdeI restriction site immediately downstream of a trp promoter. Expression of SpG' was estimated as about 30% total soluble cell protein which compares very favourably to the less than 1% total soluble cell protein obtained from the original system [Goward, et al. (1990) Biochem. J. 267: 171-177]. Homogeneous SpG' was recovered by a single anion-exchange chromatography step on Q-Sepharose FF in a process which avoided use of an affinity adsorbent. Even though SpG' consists of almost identical repetitive domains from amino acid sequence analysis, different proteolytic sensitivity of each domain was observed indicating their structural dissimilarity.

Amino Acid Sequence↗
PubMed1991

Sequential 1H NMR assignments and secondary structure of an IgG-binding domain from protein G.

Protein G is a member of a class of cell surface bacterial proteins from Streptococcus that bind IgG with high affinity. A fragment of molecular mass 6988, which retains IgG-binding activity, has been generated by proteolytic digestion and analyzed by 1H NMR. Two-dimensional DQF-COSY, TOCSY, and NOESY spectra have been employed to assign the 1H NMR spectrum of the peptide. Elements of regular secondary structure have been identified by using nuclear Overhauser enhancement, coupling constant, and amide proton exchange data. The secondary structure consists of a central alpha-helix (Ala28-Val44), flanked by two portions of beta-sheet (Val5-Val26 and Asp45-Lys62). This is a fundamentally different arrangement of secondary structure from that of protein A, which is made up of three consecutive alpha-helices in free solution (Torigoe et al., 1990). We conclude that the molecular mechanisms underlying the association of protein A and protein G with IgG are different.

Amino Acid Sequence↗