Intracranial elastance is increased in idiopathic intracranial hypertension.

BACKGROUND AND PURPOSE: To date, no pathophysiological model has sufficiently accounted for all the findings encountered in patients with idiopathic intracranial hypertension (IIH). Intracranial elastance is an index of volume-buffering capacity known to play a role in certain disorders of cerebrospinal fluid (CSF) dynamics, which has not been previously investigated in relation to IIH patients. METHODS: This was a single-center retrospective cohort study from 1 July 2011 to 1 July 2016. Values for opening pressure (PO ), closing pressure (PC ) and volume (V) of CSF removed were collected, as well as demographic and clinical covariates. Intracranial elastance (E) and pressure-volume index (PVI) were calculated according to established equations: E = (PO -PC )/V and PVI = V/log10 (PO /PC ), respectively. Those with an alternative central nervous system pathology, including meningitis, encephalitis and normal pressure hydrocephalus were excluded. Eligible patients were subdivided into two groups based on final diagnosis: a control group and an IIH group. RESULTS: In our cohort (n = 49), a significant association of both E (P < 0.0001) and PVI (P = 0.005) with a diagnosis of IIH was observed. Median E was 0.45 [interquartile range (IQR) 0.29-0.63] in the control group and 1 (IQR 0.59-1.29) in the IIH group, and median PVI was 98.07 (IQR 59.92-135.86) in the control group and 64.1 (IQR 42.4-91.7) in the IIH group. Neither E nor PVI were significantly associated with age, gender or body mass index. PVI was independent of opening pressure. CONCLUSIONS: As calculated by clinically accessible indices, our study provides evidence that intracranial elastance is increased in IIH, reflecting a novel insight into disease pathogenesis.

Coordinate expansion of murine hematopoietic and mesenchymal stem cell compartments by SHIPi.

Promoting the expansion of adult stem cell populations offers the potential to ameliorate radiation or chemotherapy-induced bone marrow failure and allows for expedited recovery for patients undergoing these therapies. Previous genetic studies suggested a pivotal role for SH2 domain-containing inositol-5-phosphatase 1 (SHIP1) in limiting the size of the hematopoietic stem cell (HSC) compartment. The aim of this study was to determine whether our recent development of small molecule SHIP1 inhibitors offers the potential for pharmacological expansion of the HSC compartment in vivo. We show here that treatment of mice with aminosteroid inhibitors of SHIP1 (SHIPi) more than doubles the size of the adult mesenchymal stem cell (MSC) compartment while simultaneously expanding the HSC pool sixfold. Consistent with its ability to target SHIP1 function in vivo, SHIPi also significantly increases plasma granulocyte colony-stimulating factor (G-CSF) levels, a growth factor that supports proliferation of HSC. Here, we show that SHIPi-induced G-CSF production mediates HSC and MSC expansion, as in vivo neutralization of G-CSF abrogates the SHIPi-induced expansion of both the HSC and MSC compartments. Due to its expansionary effect on adult stem cell compartments, SHIPi represents a potential novel strategy to improve declining stem cell function in both therapy induced and genetically derived bone marrow failure syndromes.

Developing a national 'low risk' febrile neutropenia framework for use in children and young people's cancer care.

PURPOSE: A Delphi study was undertaken to develop a framework guidance that would rationalise and standardise the care of children with febrile neutropenia (FNP) across the UK. METHODS: A mailed Delphi survey was undertaken with health professionals working in children's cancer units. The survey employed two rounds of feedback on 22 practice statements drawn from a systematic review of clinical evidence. Consensus was assumed for any statement where 80+ % of respondents indicated that they "agreed" or "strongly agreed". RESULTS: Consensus was reached on 21 of the 22 practice statements in round 1 that were categorised into six areas: definition of fever and neutropenia, initial management and choice of antibiotic, defining low-risk patients, strategy in low-risk patients and alternative approaches. Consensus could not be reached on whether patients needed to be afebrile to be suitable for discharge and the required length of outpatient antibiotic treatment. CONCLUSIONS: A Delphi survey allowed the successful development of a national framework for identification and management of children with FNP. The use of an existing well-functioning professional network was key in this project's success.

Embryonal and Alveolar Rhabdomyosarcoma in Adults: Real-Life Data From a Tertiary Sarcoma Centre.

AIMS: Embryonal and alveolar rhabdomyosarcoma (ERMS, ARMS) are subtypes of RMS that mainly occur in children, with relatively good outcomes. The incidence in adults is extremely low and survival is significantly worse compared with children. Data are scarce and literature generally combines all RMS subtypes, including pleomorphic RMS, which primarily occurs in adults and behaves more like undifferentiated pleomorphic sarcoma. The aim of this study was to evaluate patient and tumour characteristics, outcome and prognostic factors in adult patients with ERMS and ARMS. MATERIALS AND METHODS: All adult (18 years or older) ERMS and ARMS patients (presenting 1990-2016) were identified from a prospectively maintained database and were included in this analysis. RESULTS: Overall, 66 patients were included (42 men, 24 women). The median age at presentation was 28 years (range 18-71). The median overall survival for all ARMS (n = 42) and ERMS (n = 24) patients was 18 months, with a 5-year overall survival rate of 27%. Patients presenting with localised disease (n = 38, 58%) and metastatic disease (n = 25, 42%), had a 5-year overall survival rate of 36% and 11%, respectively. In univariate analysis we found alveolar subtype, fusion gene positivity, infiltrative tumour and metastatic presentation to be negative prognostic factors. CONCLUSION: Survival in adult ERMS and ARMS patients is poor and the current data may be useful in the design of trials with novel agents. Ideally, paediatric and adult oncologists should set up trials together to get a better understanding of biological, genetic and clinically relevant factors in this disease.

Comparison of the antilipolytic effects of an A1 adenosine receptor partial agonist in normal and diabetic rats.

INTRODUCTION AND AIMS: Elevated plasma free fatty acid (FFA) concentrations play a role in the pathogenesis of type 2 diabetes (2DM). Antilipolytic agents that reduce FFA concentrations may be potentially useful in the treatment of 2DM. Our previous observation that CVT-3619 lowered plasma FFA and triglyceride concentrations in rats and enhanced insulin sensitivity in rodents with dietary-induced forms of insulin resistance suggested that it might be of use in the treatment of patients with 2DM. The present study was undertaken to compare the antilipolytic effects of CVT-3619 in normal (Sprague Dawley, SD) and Zucker diabetic fatty (ZDF) rats. RESULTS: ZDF rats had significantly higher fat pad weight, glucose, insulin and FFA concentrations than those of SD rats. EC(50) values for forskolin-stimulated FFA release from isolated adipocytes from SD and ZDF rats were 750 and 53 nM, respectively (p < 0.05). Maximal forskolin stimulation of FFA release was significantly (p < 0.01) less in ZDF rats (133 +/- 60 microM) compared with SD rats (332 +/- 38 microM). EC(50) values for isoproterenol to increase lipolysis in adipocytes from SD and ZDF rats were 2 and 7 nM respectively. Maximal isoproterenol-stimulated lipolysis was significantly (p < 0.01) lower in adipocytes from ZDF rats (179 +/- 23 microM) compared with SD rats (343 +/- 27 microM). Insulin inhibited lipolysis in adipocytes from SD rats with an IC(50) value of 30 pM, whereas adipocytes from ZDF rats were resistant to the antilipolytic actions of insulin. In contrast, IC(50) values for CVT-3619 to inhibit the release of FFA from SD and ZDF adipocytes were essentially the same (63 and 123 nM respectively). CVT-3619 inhibited lipolysis more than insulin in both SD (86 vs. 46%, p < 0.001) and ZDF (80 vs. 13%, p < 0.001) adipocytes. In in vivo experiments, CVT-3619 (5 mg/kg, PO) lowered FFA to a similar extent in both groups. Plasma concentrations of CVT-3619 were not different in SD and ZDF rats. There was no significant difference in the messenger RNA expression of the A(1) receptors relative to beta-actin expression in adipocytes from SD (0.98 +/- 0.2) and ZDF rats (0.99 +/- 0.3). CONCLUSION: The antilipolytic effects of CVT-3619 appear to be independent of insulin resistance and animal model.

Feeding the fire: Tracing the mass-loading of 107 K galactic outflows with O vi absorption.

Galactic outflows regulate the amount of gas galaxies convert into stars. However, it is difficult to measure the mass outflows remove because they span a large range of temperatures and phases. Here, we study the rest-frame ultraviolet spectrum of a lensed galaxy at z ~ 2.9 with prominent interstellar absorption lines from O i, tracing neutral gas, up to O vi, tracing transitional phase gas. The O vi profile mimics weak low-ionization profiles at low velocities, and strong saturated profiles at high velocities. These trends indicate that O vi gas is co-spatial with the low-ionization gas. Further, at velocities blueward of -200 km s-1 the column density of the low-ionization outflow rapidly drops while the O vi column density rises, suggesting that O vi is created as the low-ionization gas is destroyed. Photoionization models do not reproduce the observed O vi, but adequately match the low-ionization gas, indicating that the phases have different formation mechanisms. Photoionized outflows are more massive than O vi outflows for most of the observed velocities, although the O vi mass outflow rate exceeds the photoionized outflow at velocities above the galaxy's escape velocity. Therefore, most gas capable of escaping the galaxy is in a hot outflow phase. We suggest that the O vi absorption is a temporary by-product of conduction transferring mass from the photoionized phase to an unobserved hot wind, and discuss how this mass-loading impacts the observed circum-galactic medium.

Upper Gastrointestinal Function in Morbidly Obese Adolescents Before and 6 Months After Gastric Banding.

BACKGROUND: The effects of laparoscopic adjustable gastric band (LAGB) placement on upper gastrointestinal tract function in obese adolescents are unknown. Therefore, our aim was to determine the short-term effects of LAGB on esophageal motility, gastroesophageal reflux, gastric emptying, appetite-regulatory hormones, and perceptions of post-prandial hunger and fullness. METHODS: This study was part of a prospective cohort study (March 2009-December 2015) in one tertiary referral hospital. The study included obese adolescents (14-18 years) with a body mass index (BMI) > 40 (or ≥ 35 with comorbidities). Gastric emptying was assessed by 13C-octanoic acid breath test, pharyngeal, and esophageal motor function by high-resolution manometry with impedance (HRIM), and appetite and other perceptions using 100-mm visual analogue scales. Dysphagia symptoms were scored using a Dakkak questionnaire. Data were compared pre- and post-LAGB placement and at a 6-month follow-up. RESULTS: Based upon analysis of 15 adolescents, at the 6-month follow-up, LAGB placement: (i) led to a significant reduction in weight and BMI; (ii) increased fullness and decreased hunger post-meal; (iii) increased symptoms of dysphagia after solid food; and, despite these effects, (iv) caused little or no changes to appetite hormones, while (v) effects on gastric emptying, esophageal motility, esophageal bolus transport, and esophageal emptying were not significant. CONCLUSION: In adolescents, LAGB improved BMI and altered the sensitivity to nutrients without significant effects on upper gastrointestinal tract physiology at the 6-month follow-up.

Modulation by 1,25-dihydroxycholecalciferol of the acute change in cytosolic free calcium induced by thyrotropin-releasing hormone in GH4C1 pituitary cells.

Receptor-mediated regulation of prolactin synthesis by 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in the pituitary cell strain GH4C1 is dependent on the concentration of extracellular calcium. We have now investigated the actions of 1,25(OH)2D3 on cytosolic free calcium concentrations [( Ca2+]i) in these cells using the fluorescent indicator quin2. Basal resting [Ca2+]i was unchanged in cells treated with 1 nM 1,25(OH)2D3 either acutely (from 0 to 15 min) or for periods of up to 48 h. However, the initial peak of the biphasic change in [Ca2+]i induced by thyrotropin-releasing hormone (TRH) was enhanced more than twofold in cells pretreated for 24 or 48 h with 1,25(OH)2D3. This 1,25(OH)2D3-enhanced calcium response was restricted to the initial phase of TRH action; the secondary plateau phase was unaffected. Neither the affinity nor number of TRH receptors nor the early time course of [3H]MeTRH binding to GH4C1 cells were affected by pretreatment with 1,25(OH)2D3. Because TRH binding was not altered, four sites along the intracellular signal transduction pathway of TRH action were examined. Neither protein kinase C activation nor inositol polyphosphate accumulation were enhanced in response to TRH, in 1,25(OH)2D3 pretreated cells, indicating that phosphatidylinositol hydrolysis was unchanged by pretreatment. A low concentration of ionomycin was used to probe the size of the nonmitochondrial intracellular calcium pool that is sensitive to TRH. Ionomycin was not able to mobilize more calcium from 1,25(OH)2D3 pretreated cells, indicating that TRH-responsive intracellular calcium stores were probably not enhanced by pretreatment. Chelation of extracellular calcium, however, did eliminate enhancement of the TRH response in 1,25(OH)2D3-pretreated cells. We conclude that 1,25(OH)2D3 modulates acute dynamic changes in [Ca2+]i induced by TRH without affecting basal [Ca2+]i. The mechanism of the enhanced response of 1,25(OH)2D3-pretreated cells to TRH appears to depend upon a postreceptor event independent of phosphatidylinositol hydrolysis that involves increased calcium conductance at the level of the plasma membrane. A less likely explanation involves enhancement of intracellular calcium stores in an ionomycin-resistant, EGTA-sensitive, TRH-mobilizable reservoir.

Efficacy of carboplatin given in a phase II window study to children and adolescents with newly diagnosed metastatic soft tissue sarcoma.

AIM: The activity of carboplatin was evaluated in a phase II window study in previously untreated children with metastatic soft tissue sarcoma. METHODS: Children with poor-risk metastatic disease (over 10 years and/or with bone/bone marrow involvement) treated in the SIOP MMT 98 study were scheduled to receive two courses of intravenous carboplatin (area under curve [AUC] of 10), 21 days apart. RESULTS: Sixteen eligible patients were entered into the rhabdomyosarcoma (RMS) group. Response (complete remission or partial remission) was seen in five children (31%, 95% confidence interval (CI) 14-56%). Ten eligible patients with other soft tissue sarcomas were recruited into the non-RMS group. Two responses (20%, 95% CI 6-51%) were seen. Toxicity in both groups was predictable nausea, vomiting and marrow suppression and there were no toxic deaths. CONCLUSION: Single-agent carboplatin at AUC of 10 has an acceptable toxicity profile but only moderate efficacy in poor-risk metastatic soft tissue sarcoma.

Lack of cross-resistance between certain platinum coordination compounds in mouse leukemia.

Two congeners of cis-platinum diamminodichloride, 1,2-diamminocyclohexylplatinum malonate (NSC 224964) and 1,2-cyclohexyldiamminoplatinum sulfate (NSC 250427), show approximately equal inhibitory activity in vitro against leukemia L1210 and a line of L1210 (L1210/PDD) that has developed resistance to cis-platinum diamminodichloride. These compounds are also active against L1210/PDD in vivo. These observations suggest that they be tried clinically in patients whose disease has become resistant to cis-platinum diamminodichloride.

Synergistic effects of the combination of cis-platinum diamminodichloride and 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine in transplanted mouse leukemias.

cis-Platinum diamminodichloride has been studied in combination with 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine on an every-4-day schedule in various lines of mouse leukemia. This combination is synergistic in leukemias L1210 and P388 and sublines made resistant to 5-fluorouracil or methotrexate. There is no cross-resistance between cis-platinum diamminodichloride and 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine, but the combination is no more effective against lines of leukemia made resistant to cis-platinum diamminodichloride or to 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine than either single active compound alone. Since these compounds have no cross-resistance, act by quite different mechanisms of action, and have different limiting toxicity, the combination is now being evaluated clinically.

Endocrine disorders among long-term survivors of childhood head and neck rhabdomyosarcoma.

PURPOSE: Head and neck rhabdomyosarcoma (HNRMS) survivors are at increased risk of developing pituitary dysfunction as an adverse event of radiotherapy. Our aim was to investigate the frequency and risk factors for pituitary dysfunction in these survivors. Secondly, we aimed to compare the prevalence of pituitary dysfunction between survivors treated with external beam radiation therapy (EBRT) and survivors treated with the ablative surgery, moulage technique after loading brachytherapy, and surgical reconstruction (AMORE) procedure. METHODS: Eighty HNRMS survivors treated in London (EBRT based) and Amsterdam (AMORE based: AMORE if feasible, otherwise EBRT) in the period 1990-2010 and alive ≥ 2 years post-treatment were evaluated. Survivors were evaluated in multidisciplinary late-effects clinics, with measurement of linear growth, determination of thyroid function, and growth hormone parameters. Additional data, such as baseline characteristics, anthropometrics, pubertal stage, and the results of additional laboratory investigations, were retrieved from patient charts. RESULTS: Pituitary dysfunction was diagnosed in 24 in 80 (30%) survivors, after a median follow-up time of 11 years. Median time to develop pituitary dysfunction after HNRMS diagnosis was 3.0 years. Risk factors were EBRT-based therapy (odds ratio [OR] 2.06; 95% confidence interval [CI] 1.79-2.46), parameningeal tumour site (OR 1.83; 95% CI 1.60-2.17) and embryonal RMS histology (OR 1.49; 95% CI 1.19-1.90). CONCLUSIONS: Radiotherapy used for the treatment of HNRMS confers a significant risk of the development of pituitary dysfunction. AMORE-based treatment in children with HNRMS resulted in less pituitary dysfunction than treatment with conventional EBRT. Our findings underscore the importance of routine early endocrine follow-up in this specific population.

Fatty acid binding protein in flight muscle of the locust, Schistocerca gregaria.

The cytosol of flight muscle from the adult desert locust, Schistocerca gregaria, contains a fatty acid binding protein (FABP). Locust FABP has a molecular weight of 15,000 and an isoelectric point of 5.2. It binds fatty acids stoichiometrically in a 1:1 ratio. Its molecular characteristics, tissue specificity and electrophoretic behavior are reminiscent of mammalian M-FABP. Compared to its mammalian counterpart, the cytosolic concentration is much higher, reflecting the high rate of fatty acid oxidation observed during locust flight. Our discovery, showing for the first time an FABP in an invertebrate species, supports the proposed function of muscle FABP as intracellular fatty acid receptor or transport protein.

Accumulation of cholestatic lipoproteins in ANIT-treated human apolipoprotein A-I transgenic rats is diminished through dose-dependent apolipoprotein A-I activation of LCAT.

Administration of alpha-naphthylisothiocyanate (ANIT) to rats induces changes to plasma lipids consistent with cholestasis. We have previously shown (J. Lipid Res. 37 (1996) 1086) that animals treated with ANIT accumulate large amounts of free cholesterol (FC) and phospholipid (PL)-rich cholestatic lipoproteins in the LDL density range by 48 h. This lipid was cleared by 120 h through apparent movement into HDL with concomitant cholesteryl ester (CE) production. It was hypothesised that the clearance was mediated through the movement of the PL and FC into apolipoprotein A-I (apo A-I) containing lipoproteins followed by LCAT esterification to form CE. To test this hypothesis, rats overexpressing various amounts of human apo A-I (TgR[HuAI] rats) were treated with ANIT (100 mg/kg) and the effect of plasma apo A-I concentration on plasma lipids and lipoprotein distribution was examined. In untreated TgR[HuAI] rats, human apo A-I levels were strongly correlated to plasma PL (r(2)=0. 94), FC (r(2)=0.93) and CE (r(2)=0.90), whereas in ANIT-treated TgR[HuAI] rats, human apo A-I levels were most strongly correlated to CE levels (r(2)=0.80) and an increased CE/FC ratio (r(2)=0.62) and the movement of cholestatic lipid in the LDL to HDL. Since LCAT activity was not affected by ANIT treatment, these results demonstrate that the ability of LCAT to esterify the plasma FC present in cholestatic liver disease is limited by in vivo apo A-I activation of the cholestatic lipid and not by the catalytic capacity of LCAT.

Influenza immunisation in children with solid tumours.

We assessed response to immunisation with trivalent split virus influenza vaccine in children with non-leukaemic malignant disease. Children with solid tumours and lymphoma received one or two doses of influenza vaccine, according to current UK guidelines, in autumn 2001 and/or 2002. Children were currently receiving chemotherapy or were within 6 months of completing chemotherapy. Pre and post vaccination sera were assessed for antibodies to the prevalent influenza strains by haemagglutination inhibition (HI). Sixty six children were assessed prior to 69 episodes of vaccination. In 30% episodes, children were susceptible to all three circulating influenza viruses (65% to H1N1, 42% to H3N2 and 90% to B) and only one patient showed protective titres (HI32) against all three strains. Seroresponse rates (4-fold rise in HI) for H1N1, H3N2 and B were 52%, 33% and 51% in 65 episodes. Following immunisation protective titres to all three viruses were seen in 25 episodes (38%) and protective responses to one or two viruses were seen in a further 12 (19%) episodes. There was no significant difference in response rates among children on treatment and off treatment and by intensity of chemotherapy. Children with solid tumours and lymphoma are highly susceptible to influenza infection. Influenza vaccine was well tolerated in this patient group and children showed a significant response to immunisation. These findings support the recommendation for annual influenza vaccination in these children.

Retinal toxicity during pegylated alpha-interferon therapy for chronic hepatitis C: a multifocal electroretinogram investigation.

BACKGROUND: Ocular side-effects in the form of retinal ischaemia and haemorrhages have been reported in patients undergoing standard alpha-interferon therapy. AIM: To assess the ocular impact of therapy with sustained release pegylated alpha-2a interferon (Pegasys) for chronic hepatitis C. METHODS: Ten patients receiving Pegasys and ribavirin and 10 healthy volunteers were recruited. Patients underwent full ophthalmic investigations and multifocal electroretinogram testing at baseline, at regular intervals during treatment and post-treatment. The multifocal electroretinogram maps retinal function. Responses were compared with sequential recordings from healthy volunteers. RESULTS: All patients had normal clinical ophthalmic investigations at baseline. During therapy a single patient experienced central visual disturbance lasting 24 h with no prolonged ill effect. No other patient was aware of any change in vision. Fundal abnormalities appeared in five patients during treatment. The multifocal electroretinogram showed reductions in retinal function in five patients. Nine of 10 patients exhibited abnormalities on at least one multifocal electroretinogram or fundoscopic investigation. CONCLUSIONS: Subclinical retinal toxicity during anti-viral therapy with pegylated alpha-interferon and ribavirin was frequent in this study and it suggests that patients should be warned of this risk and monitored during therapy.

The detection of horse and donkey using real-time PCR.

We have developed real-time PCR assays specific for horse and donkey, applicable to the detection of low levels of horse or donkey meat in commercial products. Primers, designed to the mitochondrial cytochrome b gene, were 3' mismatched to closely related and other commercial species. Amplification of non-target species DNA was prevented by truncation of primers at the 5' position, thereby conferring complete specificity. Both assays were highly sensitive and detected the presence of 1pg of donkey template DNA or 25pg of horse template DNA when assessed using dilutions of DNA in water. Model food samples, spiked with horse or donkey muscle and commercial products containing horse, were successfully tested for the presence of horse or donkey, demonstrating the applicability of the assays to food products.

Classical LCAT deficiency resulting from a novel homozygous dinucleotide deletion in exon 4 of the human lecithin: cholesterol acyltransferase gene causing a frameshift and stop codon at residue 144.

Lecithin: cholesterolacyltransferase (LCAT) transacylates the fatty acid at the sn-2 position of lecithin to the 3beta-OH group of cholesterol forming lysolecithin and the majority of cholesteryl ester found in plasma. LCAT participates in the reverse cholesterol transport pathway in man where it esterifies tissue-derived cholesterol following efflux from peripheral cells into HDL. Only 38 unique mutations in the human LCAT gene have been reported worldwide. Our French female proband presented with corneal opacity and no detectable plasma LCAT activity using either endogenous or exogenous assays. Her total plasma cholesterol and HDL cholesterol were low (2.34 mmol/l and 0.184 mmol/l, respectively) with a very high cholesterol/cholesteryl ester molar ratio (10.9:1). Plasma triglycerides were 0.470 mmol/l with low apo B (40.5 mg/dl), apo A-I (14.7 mg/dl), apo A-II (6.8 mg/dl) and apo E (2.1 mg/dl) levels. Plasma lipoprotein analysis by ultracentrifugation showed very low HDL concentrations and a characteristic shift of the lipoprotein profile towards larger, less dense particles. No proteinuria, renal dysfunction or signs of atherosclerosis were noted at age 45. Sequence analysis of her LCAT gene showed a novel homozygous TG-deletion at residues 138-139 that resulted in a frameshift causing the generation of a stop codon and premature termination of the LCAT protein at amino acid residue 144. Western blotting of the patient's plasma using a polyclonal IgY primary antibody against human LCAT failed to demonstrate the presence of a truncated LCAT protein. A 53 bp mismatched PCR primer was designed to generate an Fsp 1 restriction site in the wild type sequence of exon 4 where the mutation occurred. The 155 bp PCR product from the wild type allele produced a 103 bp and 52 bp fragment with Fsp 1 and no cleavage products with the mutant allele thus permitting rapid screening for this novel mutation.

Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the activity of the mature proteinase and the VPg-proteinase precursor.

The 3C-like proteinase (Pro) from Tomato ringspot virus (genus Nepovirus) is responsible for the processing of the RNA1-encoded (P1) and RNA2-encoded (P2) polyproteins. Cleavage between the VPg and Pro domains is inefficient in vitro and in E. coli, resulting in the accumulation of the VPg-Pro. In this study, we have compared the trans-activity of the Pro and VPg-Pro on various P1- and P2-derived precursors. Recombinant Pro and VPg-Pro were partially purified using an E. coli expression system. A mutation of the VPg-Pro cleavage site was introduced into the VPg-Pro to prevent slow release of the Pro. The Pro was five to ten times more active than the VPg-Pro on two P2 cleavage sites (at the N- and C-termini of the movement protein domain) and was approximately two times more active than the VPg-Pro on the third P2 cleavage site (between the X3 and X4 domains). Neither the Pro nor the VPg-Pro could cleave in trans P1-derived substrates containing the three cleavage sites delineating the X1, X2, putative NTP-binding protein and VPg domains. These results are discussed in light of the possible regulation of the proteinase activity during virus replication.