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P Mason

Publications and source records attributed to P Mason.

At least 73 records · Page 4Linked to original sources
PubMed1996

Crystallization of rat procathepsin B.

Rat procathepsin B has been expressed in the yeast Pichia pastoris. To facilitate crystallization of the proform two mutations were introduced: Cys29Ser to avoid self-processing and Ser115Ala to eliminate an N-glycosylation site. The recombinant protein was purified and crystallized by vapor diffusion against mother liquor containing 100 mM KSCN, 100 mM phosphate buffer, pH 6.5 and polyethylene glycol (PEG) 3350 as a precipitating agent. Crystal size was increased by multiple macroseeding. At a 16% PEG concentration trigonal crystals were obtained, with the space group P3(1)21 and a = 99.6, c = 141.4 A, gamma = 120 degrees. They diffract to 2.8 A resolution using a rotating-anode source. At a concentration of 11% PEG, rod-shaped crystals were grown. They are monoclinic, space group P2(1), a = 62.8, b = 67.9, c = 100.4 A, beta = 98.2 degrees and diffract to approximately 3.5 A.

Journal Article↗
PubMed1996

Potency and selectivity of the cathepsin L propeptide as an inhibitor of cysteine proteases.

The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cerevisiae using a human procathepsin L/alpha-factor fusion construct containing a stop codon at position -1 (the C-terminal amino acid of the proregion). Since the yield after purification was very low, the cathepsin L propeptide was also obtained by an alternate procedure through controlled processing of an inactive mutant of procathepsin L (Cys25Ser/Thrl10Ala) expressed in Pichia pastoris, by small amounts of cathepsin L. The peptide resulting from the cleavage of the proenzyme (phcl-1) was then purified by HPLC. The purified propeptides were characterized by N-terminal sequencing and mass spectrometry and correspond to incomplete forms of the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively, compared to 96 aa for the complete cathepsin L propeptide). The two peptides were found to be potent and selective inhibitors of cathepsin L at pH 5.5, with Ki values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2. The Ki for inhibition of cathepsin S was much higher (44.6 nM with phcl-1), and no inhibition of cathepsin B or papain could be detected at up to 1 microM of the propeptide. The inhibitory activity was also found to be strongly pH-dependent. Two synthetic peptides of 75 and 44 aa corresponding to N-terminal truncated versions of the propeptide were also prepared by solid phase synthesis and displayed Ki values of 11 nM and 2900 nM, respectively, against cathepsin L. The data obtained for the 4 propeptide derivatives of various lengths indicate that the first 20 residues in the N-terminal region of the propeptide are more important for inhibition than the C-terminal region which contributes little to the overall inhibitory activity.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of the carboxyl-domain of human hexokinase type III expressed as fusion protein.

In mammalian tissues hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) exists as four isoenzymes encoded by distinct genes. These proteins are homologous and are organized in two homologous domains, with the exception of hexokinase type IV which has only one. This organization is believed to be the result of a duplication and tandem fusion event involving the gene encoding for the ancestral hexokinase. In this study, we cloned the carboxyl-domain of human hexokinase type III and expressed it in Escherichia coli as a glutathione S-transferase fusion protein, using the pGEX-2T expression vector. The recombinant protein showed catalytic activity. A comparative study of the kinetic properties of the expressed carboxyl-domain and the enzyme partially purified from human lymphocytes is also shown. The results now allow a better understanding of the role of the carboxyl-domain in determining the catalytic properties of the enzyme.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of human procathepsin L.

Human procathepsin L has been expressed in the yeast Pichia pastoris and its inactive (Cys25Ser) and unglycosylated (Thr110Ala) mutant purified, concentrated to 4 mg/ml, and crystallized by vapor diffusion against solution containing 1.4 M (Na,K)PO4 buffer, pH 7.8. Crystal size was increased by multiple macroseeding. The crystals are orthorhombic, of space group P2 1 2 1 2 1, with cell dimensions of a = 40.2 A, b = 88.4 A, and c = 94.9 A. A 2.2 A native data set was collected using synchrotron radiation. Although molecular replacement solution for the mature portion of the enzyme was easily found, the resulting maps could not be interpreted in the proregion. Heavy-atom derivative search is in progress.

Cathepsin L↗
PubMed1996

Predicting the long-term outcome of schizophrenia.

Predictors of long-term (13 year) outcome of schizophrenia are reported for a representative cohort of 'treated incidence' patients ascertained on their first contact with Nottingham psychiatric services between 1978-80. An initial (baseline) model including previously reported predictors of 2-year outcome (age, gender, ever married, acuteness of onset) and length of untreated illness was used to predict a range of outcome measures covering the domains of disability, psychopathology, hospitalization, employment, social activity, and global outcome. This model demonstrated significant prognostic ability across all non-hospitalization outcomes under both ICD-10 and ICD-9 diagnoses of schizophrenia, but was attenuated under broad (ICD-9 and CATEGO S, P or O) and restrictive (S+) diagnostic classifications. Female gender predicted more favourable outcome under all diagnostic classifications except S+. In an extended analysis, the addition of initial 2-year course type substantially increased the prognostic ability of the model under all diagnostic classifications and enabled over 30% of the variance in global ratings of disability and symptoms to be predicted. In this extended model female gender predicted more favourable outcome over and above the effect of course type, across most domains under ICD-10, and for disability and psychopathology under other diagnostic classifications. The inclusion of measures of psychopathology at the time of first assessment, pre-morbid functioning, and duration of index admission conferred only marginal additional predictive ability for respective outcomes in the domains of psychopathology, social activity, employment and hospitalization. Hospitalization during the past year was the most difficult outcome to predict under any model suggesting that resource utilization represents the 'administrative outcome' of schizophrenia and serves as a poor proxy for broader concerns in the era of community care. These data demonstrate that key demographic variables and the mode of onset influence the long-term course of schizophrenia, but that early course type is a particularly strong predictor.

Adult↗
PubMed1996

Antagonism of the antinocifensive action of halothane by intrathecal administration of GABAA receptor antagonists.

BACKGROUND: The hind brain and the spinal cord, regions that contain high concentrations of gamma-aminobutyric acid (GABA) and GABA receptors, have been implicated as sites of action of inhalational anesthetics. Previous studies have established that general anesthetics potentiate the effects of gamma-aminobutyric acid at the GABAA receptor. It was therefore hypothesized that the suppression of nocifensive movements during anesthesia is due to an enhancement of GABAA receptor-mediated transmission within the spinal cord. METHODS: Rats in which an intrathecal catheter had been implanted 1 week earlier were anesthetized with halothane. Core temperature was maintained at a steady level. After MAC determination, the concentration of halothane was adjusted to that at which the rats last moved in response to tail clamping. Saline, a GABAA, a GABAB, or glycine receptor antagonist was then injected intrathecally. The latency to move in response to application of the tail clamp was redetermined 5 min later, after which the halothane concentration was increased by 0.2%. Response latencies to application of the noxious stimulus were measured at 7-min intervals during the subsequent 35 min. To determine whether these antagonists altered baseline response latencies by themselves, another experiment was conducted in which the concentration of halothane was not increased after intrathecal administration of GABAA receptor antagonists. RESULTS: Intrathecal administration of the GABAA receptor antagonists bicuculline (0.3 micrograms) or picrotoxin (0.3, 1.0 micrograms) antagonized the suppression of nocifensive movement produced by the small increase in halothane concentration. In contrast, the antinocifensive effect of the increase in halothane concentration was not attenuated by the GABAB receptor antagonist CGP 35348 or the glycine receptor antagonist strychnine. By themselves, the GABAA receptor antagonists did not alter response latency in rats anesthetized with sub-MAC concentrations of halothane. CONCLUSIONS: Intrathecal administration of bicuculline or picrotoxin, at doses that do not change the latency to pinch-evoked movement when administered alone, antagonized the suppression of noxious-evoked movement produced by halothane concentrations equal to or greater than MAC. These results suggest that enhancement of GABAA receptor-mediated transmission within the spinal cord contributes to halothane's ability to suppress nocifensive movements.

Analgesics↗
PubMed1996

Spectral analysis of arterial blood pressure and raphe magnus neuronal activity in anesthetized rats.

Recent evidence suggests that nociceptive modulatory cells in the nucleus raphe magnus (RM) and adjacent nucleus reticularis magnocellularis (NRMC) may participate in the modulation of autonomic processing. Therefore, spectral analyses were used to determine component frequencies common to both arterial blood pressure and the activity of RM/NRMC neurons in rats lightly anesthetized with isoflurane. These analyses detected powerful, extremely low frequency (period length: 6.4-18.5 min) oscillations in arterial blood pressure and in the activity of two classes of RM/NRMC neurons, ON and OFF cells. All ON cells discharged during periods of low blood pressure, whereas all OFF cells discharged during periods of high blood pressure. In contrast, the discharge of NEUTRAL and REGULAR cells did not have a consistent relationship to blood pressure. The role of these cells in nociceptive modulation is also unclear. The results presented indicate that ON and OFF cells may participate in the modulation of both autonomic and nociceptive processing.

Animals↗
PubMed1996

The course of schizophrenia over 13 years. A report from the International Study on Schizophrenia (ISoS) coordinated by the World Health Organization.

BACKGROUND: This paper describes the 13 year course of illness in an epidemiologically defined and representative cohort of patients selected when they were experiencing their first episode of schizophrenia. METHOD: In a 13-year follow-up study of 67 patients with ICD-9 schizophrenia, identified in Nottingham in 1978-80, the course of illness (symptoms, disability and hospitalisation) was assessed using standardised instruments, applied at onset, 1,2, and 13 years. Time to first relapse and first readmission were calculated and plotted as survival curves and patients were assigned to the course types described by Ciompi. RESULTS: The survival curves show that first relapses and first readmissions occur during the first five years. The amount of time spent in psychotic episodes and in hospital is greatest in the first year of follow-up, but stable thereafter. Social adjustment improves from entry to the study to the first follow-up year, but there is a small deterioration in social adjustment between 2 and 13 years. CONCLUSIONS: The findings reported suggest that after the initial episode the course of schizophrenia is relatively stable. The data support neither concepts of progressive deterioration nor progressive amelioration. There was no evidence of a "late recovery'.

Adolescent↗
PubMed1995

Effects of isoflurane concentration on the activity of pontomedullary raphe and medial reticular neurons in the rat.

Neurons in the pontomedullary raphe magnus (RM) and adjacent nucleus reticularis paragigantocellularis pars alpha (NRPG alpha) are thought to participate in the modulation of spinal nociceptive transmission. In order to determine whether these cells also contribute to the suppression of nocifensive reflexes produced by general anesthetics, the spontaneous activity of RM/NRPG alpha cells was recorded in rats anesthetized with isoflurane (IF) at several steady state concentrations, corresponding to depths which are below, equal to, or above the threshold for blocking the motor response to noxious stimuli (minimum alveolar concentration, MAC). Neurons were classified by their spontaneous activity patterns and their responses to noxious stimulation as OFF, ON, REGULAR or NEUTRAL cells. After cell classification, unit activity, arterial blood pressure, heart rate, and EEG activity were simultaneously recorded, in the absence of somatic stimulation, for 1 h at each of two or three concentrations of IF. The concentrations tested were low (1.05-1.25%), medium (1.30-1.45%) and high (1.70-1.90%). ON, OFF and some NEUTRAL cells exhibited alternating periods of inactivity and activity when recorded during periods of low and medium anesthetic concentrations. At high steady state anesthetic concentrations, the mean discharge of most OFF, ON and NEUTRAL cells decreased by greater than 25% from their mean discharge rate at the low concentration. REGULAR cells maintained a uniform firing rate at all steady state anesthetic concentrations studied. Since high concentrations of IF do not activate OFF cells, the putative inhibitory output neuron of the RM/NRPG alpha, it is unlikely that the activity of RM/NRPG alpha neurons contributes to the suppression of nocifensive movement by the general anesthetic, IF.

Animals↗
PubMed1995

Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress.

Glucose 6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme encoded in mammals by an X-linked gene. It has important functions in intermediary metabolism because it catalyzes the first step in the pentose phosphate pathway and provides reductive potential in the form of NADPH. In human populations, many mutant G6PD alleles (some present at polymorphic frequencies) cause a partial loss of G6PD activity and a variety of hemolytic anemias, which vary from mild to severe. All these mutants have some residual enzyme activity, and no large deletions in the G6PD gene have ever been found. To test which, if any, function of G6PD is essential, we have disrupted the G6PD gene in male mouse embryonic stem cells by targeted homologous recombination. We have isolated numerous clones, shown to be recombinant by Southern blot analysis, in which G6PD activity is undetectable. We have extensively characterized individual clones and found that they are extremely sensitive to H2O2 and to the sulfydryl group oxidizing agent, diamide. Their markedly impaired cloning efficiency is restored by reducing the oxygen tension. We conclude that G6PD activity is dispensable for pentose synthesis, but is essential to protect cells against even mild oxidative stress.

Animals↗
PubMed1995

Anesthetic actions within the spinal cord: contributions to the state of general anesthesia.

The behavioral state known as general anesthesia is the result of actions of general anesthetic agents at multiple sites within the neuraxis. The most common end point used to measure the presence of anesthesia is absence of movement following the presentation of a noxious stimulus. The actions of general anesthetics within the spinal cord have been shown to contribute significantly to the suppression of pain-evoked movements, an important component of clinical anesthesia. Studies in the spinal cord are likely to increase our understanding of the pharmacology by which general anesthetics alter the transmission of somatomotor information. It now appears that the pharmacology responsible for the production of anesthesia is agent- and site-selective, and not the result of a unitary mechanism of action.

Anesthesia, General↗
PubMed1995

Modification of S1 subsite specificity in the cysteine protease cathepsin B.

Cysteine proteases of the papain family generally exhibit broad P1 specificity. A notable exception is papaya proteinase IV (PPIV), which only accepts Gly at this position. In all other cysteine proteases the S1 subsite residues 23 and 65 (papain numbering) are absolutely conserved as Gly, while in PPIV they are replaced by Glu and Arg, respectively. These differences appear to underlie both PPIV specificity and its resistance to inhibition by cystatins. To test this hypothesis, the equivalent residues (Gly27 and Gly73) in the mammalian cysteine protease cathepsin B were changed to Glu and Arg, respectively. Relative to the wild-type enzyme, the Gly27Glu and Gly73Arg mutants showed a drastic reduction in activity with substrates containing a P1 Arg. In contrast, substrates having a Gly residue in P1 were hydrolyzed effectively. The double mutant (Gly27Glu:Gly73Arg) exhibited no detectable activity against any substrate studied. Inhibition of the Gly73Arg mutant by E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane] was found to be similar to that of the wild-type enzyme. In contrast, inhibition by cystatin C exhibited a 20,000-fold reduction. These results demonstrate the dramatic influence of side chains at sequence locations 27 and 73 on the S1 subsite specificity of cysteine proteases.

Animals↗
PubMed1995

Obstacles to donor eye procurement and their solutions at the University of Iowa.

Shortages in transplantable corneas are common, yet little appears in the medical literature about patterns of tissue donation and factors affecting procurement. We have analyzed data on eye donations and taken measures to improve procurement rates based on our findings. Fifty consecutive Cardiovascular Intensive Care Unit (CVICU) deaths were reviewed to compare the number of transplant-eligible donors to the amount of tissue received. An anonymous survey of 250 house staff and nurses was undertaken to identify obstacles to donor eye procurement. Although 12 of 50 potential donors in the CVICU met transplant eligibility criteria, only 1 became a donor. A required request policy notwithstanding, the most common reason for nonprocurement was failure to make a request. According to the survey, the most significant impediments to making the request were (a) not thinking to ask, (b) unfamiliarity with eligibility criteria, (c) unfamiliarity with enucleation procedures, (d) feeling that someone else should make the request, and (e) reluctance to impose on a grieving family. Very few cited religious reasons or being too busy. Education based on the specific concerns listed in the survey was undertaken. During the 12 months after this initiative, the number of transplantable corneas donated from our facility doubled, as compared with the same period in 1992. Despite required request laws and regulations, failure to request tissue donation is common in our facility and may be common elsewhere. Systematic analysis of obstacles to donor eye procurement and their solutions may help to improve our country's performance in this area.

Corneal Transplantation↗
PubMed1995

Comparison of Cryptosporidium parvum and Cryptosporidium wrairi by reactivity with monoclonal antibodies and ability to infect severe combined immunodeficient mice.

Twenty-three monoclonal antibodies raised to Cryptosporidium parvum and 12 raised to C. wrairi reacted with equal intensity with the heterologous species. Despite demonstration of a close immunologic relationship between these two species, C. wrairi did not induce persistent infection in severe combined immunodeficient mice as did C. parvum.

Animals↗
PubMed1995

Characteristics of outcome in schizophrenia at 13 years.

BACKGROUND: This paper describes the 13-year outcome of an epidemiologically defined and representative cohort of patients selected when they were experiencing their first episode of schizophrenia. METHOD: In a 13-year follow-up study of a cohort identified in Nottingham in 1978-80, the outcome (symptoms, disability, residence and treatment) was assessed using standardised instruments. RESULTS: Four of the original 67 patients with ICD-9 schizophrenia were lost to follow-up and five were dead: 52% were without psychotic symptoms in the last two years of follow-up, 52% were without negative symptoms and 55% showed good/fair social functioning. However, only 17% were alive at follow-up, without symptoms and disability, and receiving no treatment. CONCLUSIONS: The findings reported are similar to those of other long-term follow-up studies of schizophrenia and also to 5-year follow-up studies. Kraepelin's emphasis on the longitudinal implications of a diagnosis of schizophrenia are supported, but may be over-pessimistic.

Adolescent↗