Barrett's oesophagus and HLA-B*0702/HLA-B*0706.
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Asian People↗
Biomedical subjects
S Murad
Publications and source records attributed to S Murad.
Separation of gas mixtures using a range of zeolite membranes: a molecular-dynamics study.
Gas separation efficiencies of three zeolite membranes (Faujasite, MFI, and Chabazite) have been examined using the method of molecular dynamics. Our investigation has allowed us to study the effects of pore size and structure, state conditions, and compositions on the permeation of two binary gas mixtures, O(2)N(2) and CO(2)N(2). We have found that for the mixture components with similar sizes and adsorption characteristics, such as O(2)N(2), small-pore zeolites are not suited for separations, and this result is explicable at the molecular level. For mixture components with differing adsorption behavior, such as CO(2)N(2), separation is mainly governed by adsorption and small-pore zeolites separate such gases quite efficiently. When selective adsorption takes place, we have found that, for species with low adsorption, the permeation rate is low, even if the diffusion rate is quite high. Our results further indicate that loading (adsorption) dominates the separation of gas mixtures in small-pore zeolites, such as MFI and Chabazite. For larger-pore zeolites such as Faujasite, diffusion rates do have some effect on gas mixture separation, although adsorption continues to be important. Finally, our simulations using existing intermolecular potential models have replicated all known experimental results for these systems. This shows that molecular simulations could serve as a useful screening tool to determine the suitability of a membrane for potential separation applications.
Journal Article↗
Human leucocyte antigen determinants of susceptibility to Barrett's oesophagus in Asians--a preliminary study.
BACKGROUND: Characteristic immune profiles have been demonstrated in gastro-oesophageal reflux disease. However, the genetic basis of gastro-oesophageal reflux disease remains unclear. AIM: To investigate whether certain human leucocyte antigen genes are associated with Barrett's oesophagus. METHODS: Asian patients of Malay, Chinese and Indian descent with Barrett's oesophagus (n = 59) and those without reflux symptoms and a normal oesophagus (n =60) were recruited prospectively using endoscopic and histopathological criteria. Human leucocyte antigen class I and II typing was performed using a polymerase chain reaction sequence-specific primers method. RESULTS: The HLA-B7 allele was present in 17% (10 of 59) of patients with Barrett's oesophagus when compared with 0% (zero of 60) of controls [P = 0.0006, corrected P = 0.0171, OR = 25.67]. Subgroup analysis revealed that the HLA-B7 allele was confined almost exclusively to Indians with Barrett's oesophagus, 43% (nine of 21) vs. 0% (zero of 19) Indian controls (P = 0.0014, corrected P = 0.0406, OR = 29.64). No class II associations, protective human leucocyte antigens or extended haplotypes for disease susceptibility were identified. CONCLUSIONS: Barrett's oesophagus in Asians, particularly Indians, is strongly positively associated with HLA-B7; reinforcing a genetic component to gastro-oesophageal reflux disease. A larger sample size and different ethnic populations should be genotyped to further confirm this association and identify possible additional risk factors in the human leucocyte antigen locus.
Asian People↗
Ethnic differences in stage of presentation of adults newly diagnosed with HIV-1 infection in south London.
OBJECTIVES: To establish whether there were ethnic differences in demographic characteristics, the stage at HIV diagnosis and reasons for and location of HIV testing between 1998 and 2000 in a large ethnically diverse HIV-1-infected clinic population in south London in the era of highly active antiretroviral therapy. METHODS: A retrospective review was carried out of all persons >18 years old attending King's College Hospital with a first positive HIV-1 test between 1 January 1998 and 31 October 2000, and of a random sample of patients attending St Thomas' hospital with a first positive HIV-1 test in the same period. Demographic data, details of reasons for and site of HIV test, clinical stage, CD4 lymphocyte count and HIV-1 viral load at HIV diagnosis were abstracted from the local database and medical records. Comparisons were made according to ethnic group (white, black African and black Caribbean) and over time (1998, 1999 and 2000). RESULTS: Of the 494 patients with new HIV-1 diagnoses between January 1998 and December 2000, 179 (36.2%) were white, 270 (54.7%) were black African and 45 (9.1%) were black Caribbean. There were significant differences across the ethnic groups in HIV risk group, reasons for and site of HIV testing, and clinical and CD4 stage at diagnosis. Among whites, 72.6% were men who had sex with men, 3.4% injecting drug users and 21.2% heterosexuals, compared to 2.2%, 0.4% and 93.3% among black Africans, and 28.9%, 0% and 68.9% among black Caribbeans (P<0.001). Black Africans were more likely to present with an AIDS diagnosis (21.3%) and a lower CD4 cell count [223 cells/microL; interquartile range (IQR) 88-348] compared to both whites (9.9%; 358 cells/microL; IQR 151-508) and black Caribbeans (17.9%; 294 cells/microL; IQR 113-380), who were intermediate between whites and black Africans in their stage of presentation. There was a statistically nonsignificant trend with time, between 1998 and 2000, towards earlier diagnosis based on the CD4 cell count in whites (323 and 403 cells/microL) and black Caribbeans (232 and 333 cells/microL), but a later diagnosis in black Africans (233 and 175 cells/microL). The majority of black Africans were HIV-tested as a result of suggestive symptoms or antenatal screening (58.4%) rather than because of perceived risk (40.5%), in contrast to the situation in whites (24.1% vs. 71.7%, respectively) or black Caribbeans (34.5% vs. 65.5%, respectively) (P<0.001). We found no significant differences across ethnic groups in age, HIV-1 viral load or year of HIV diagnosis. CONCLUSIONS: Black Africans continue to present with more advanced HIV disease than whites or black Caribbeans, with no evidence of any trend towards earlier diagnosis. Future educational campaigns designed to promote the uptake of HIV testing among black Africans and black Caribbeans will need to address the multiple barriers to testing, including misperception of risk, stigma and ready access to testing.
AIDS Serodiagnosis↗
Prevalence of hepatitis C in an ethnically diverse HIV-1-infected cohort in south London.
OBJECTIVES: There is limited information on the prevalence of and risk factors for hepatitis C virus (HCV) infection among HIV-1-infected patients in the UK. Our objective was to determine the prevalence of HCV infection among an ethnically diverse cohort of HIV-infected patients in south London, and to extrapolate from these data the number of co-infected patients in the UK. METHODS: A total of 1017 HIV-1-infected patients who had attended King's College Hospital HIV clinic between September 2000 and August 2002 were screened for HCV antibody using a commercial enzyme-linked immunosorbent assay (ELISA). Positive results were confirmed by polymerase chain reaction (PCR) or recombinant immunoblot assay. Demographic, clinical and laboratory data were obtained from the local computerized database and medical records. We applied our HCV prevalence rates in the different HIV transmission groups to the estimated number of HIV-infected persons in these groups in the UK, to obtain a national estimate of the level of HIV-HCV co-infection. RESULTS: Of the 1017 HIV-1-infected patients, 407 (40%) were white men, 158 (15.5%) were black African men, 268 (26.3%) were black African women, and 61 (6%) and 26 (2.6%) were black Caribbean men and women, respectively. Heterosexual exposure was the most common route of HIV acquisition (53.5%), followed by men having sex with men (36.9%), and current or previous injecting drug use (IDU) (7.2%). The overall prevalence of HCV co-infection was 90/1017 (8.9%), but this varied substantially according to route of transmission, from 82.2% among those with a history of IDU (which accounted for 67% of all HCV infections), to 31.8% in those who had received blood products, to 3.5% and 1.8% in those with homosexually and heterosexually acquired infection, respectively. Multivariate logistic regression analysis identified several independent risk factors for HCV infection: a history of IDU [odds ratio (OR) = 107.2; 95% confidence interval (CI) = 38.5-298.4], having received blood products (OR = 16.5; 95% CI = 5.1-53.7), and either being from a white ethnic group (OR = 4.3; 95% CI = 1.5-12.0) or being born in Southern Europe (OR = 6.7; 95% CI = 1.5-30.7). Based on the 35,473 known HIV-1-infected persons in the UK and the 10 997 estimated to be unaware of their status, we projected that there are at least 4136 HIV-HCV co-infected individuals in the UK and 979 who are unaware of their status. CONCLUSIONS: Overall, 9% of our cohort was HIV-HCV co-infected. The prevalence was highest among intravenous drug users (82%), who accounted for most of our HCV cases, and lowest among heterosexual men and women from sub-Saharan Africa and the Caribbean [< 2%]. Our estimate that a significant number of co-infected persons may be unaware of their HIV and HCV status, highlights an urgent need to increase the uptake of HCV and HIV testing, particularly among injecting drug users, to reduce the risk of onward transmission.
Adult↗
Molecular dynamics simulations of gas separations using faujasite-type zeolite membranes.
Gas separations with faujasite zeolite membranes have been examined using the method of molecular dynamics. Two binary mixtures are investigated, oxygen/nitrogen and nitrogen/carbon dioxide. These mixtures have been found experimentally to exhibit contrasting behavior. In O(2)/N(2) mixtures the ideal selectivity (pure systems) is higher than the mixture selectivity, while in N(2)/CO(2) the mixture selectivity is higher than the ideal selectivity. One of the key goals of this work was to seek a fundamental molecular level understanding of such divergent behavior. Our simulation results (using previously developed intermolecular models for both the gases and zeolites investigated) were found to replicate this experimental behavior. By examining the loading of the membranes and the diffusion rates inside the zeolites, we have been able to explain such contrasting behavior of O(2)/N(2) and N(2)/CO(2) mixtures. In the case of O(2)/N(2) mixtures, the adsorption and loading of both O(2) and N(2) in the membrane are quite competitive, and thus the drop in the selectivity in the mixture is primarily the result of oxygen slowing the diffusion of nitrogen and nitrogen somewhat increasing the diffusion of oxygen when they pass through the zeolite pores. In N(2)/CO(2) systems, CO(2) is rather selectively adsorbed and loaded in the zeolite, leaving very little room for N(2) adsorption. Thus although N(2) continues to have a higher diffusion rate than CO(2) even in the mixture, there are so few N(2) molecules in the zeolite in mixtures that the selectivity of the mixture increases significantly compared to the ideal (pure system) values. We have also compared simulation results with hydrodynamic theories that classify the permeance of membranes to be either due to surface diffusion, viscous flow, or Knudsen diffusion. Our results show surface diffusion to be the dominant mode, except in the case of N(2)/CO(2) binary mixtures where Knudsen diffusion also makes a contribution to N(2) transport.
Journal Article↗
Atopic sensitization of children with rhinitis in Malaysia.
Atopy is defined as the genetic propensity to develop immunoglobulin E antibodies in response to exposure to allergens and assessed by skin prick test (SPT) responses to common allergens, which may contribute to the development of the clinical disorders (phenotype). Although it is generally agreed that atopy is an important risk factor for allergic diseases such as asthma, rhinitis, and eczema, the extent to which atopy accounts for these diseases is controversial. One hundred forty one children (up to 12 years) were skin prick tested to evaluate 16 foods common to the Malaysian diet and 4 common aeroallergens. Eighty-five percent of patients had positive SPT reactivity. The most commonly implicated aeroallergen and food allergen was house dust mite (HDM) and Prawn. Seventy percent had positive SPT reactivity results to HDM and 24.8% to prawns. Fifty five percent were positive to more than one allergen and 17.7% positive to single aeroallergen. The prevalence of atopy in children with history of eczema was 90%. The incidence of HDM and food allergy especially crabs and prawns, is significantly greater in Malaysian Children with rhinitis symptoms.
Air Pollutants↗
HLA-B27 polymorphism in the Malays.
The frequency of HLA-B27 and its subtypes was determined in 878 Malay subjects. Thirty-five of the subjects typed for HLA-A, -B and -DR were found to be positive for HLA-B27. The frequency of this allele in the Malay population was found to be 3.99%. The subtypes observed and their frequencies are: HLA-B*2704 (19.4%), HLA-B*2705 (5.6%), HLA-B*2706 (72.2%) and HLA-B*2707 (2.8%).
HLA-B27 Antigen↗
Epidemiological risk factors for hypersensitivity reactions to abacavir.
We investigated risk factors for hypersensitivity reactions (HSR) to abacavir in a case-control study. In a multivariate analysis, white race [odds ratio (OR), 5.16; 95% confidence interval (CI), 1.16-22.97] and a higher CD8 cell count at initiation of abacavir (>850 vs. < or =850 cells: OR, 3.74; 95% CI, 1.19-11.77) were found to be significantly associated with the development of HSR. Age, gender, stage of disease, prior antiretroviral exposure and type of concurrent antiretroviral therapy were not associated with HSR. Differences in predisposition to HSR according to ethnicity and baseline CD8 cell count may be explained by the reported MHC genetic associations with HSR.
Adult↗
CD4 responses to conserved HIV-1 T helper epitopes show both negative and positive associations with virus load in chronically infected subjects.
Characterization of immune responses to immunodominant CD4 epitopes in HIV-1 that are associated with control of HIV infection could be used to strengthen the efficacy of polyepitope HIV vaccines. We measured both the proliferative and the CD4 interferon (IFN)-gamma and interleukin (IL)-2 cytokine responses specific for 11 previously identified HIV-1 T helper epitopes in 10 HIV-infected non-progressors (LTNPs) (infected for a median of 15 years with a stable CD4 count of >500 cells x 10(6)/l), and seven slow progressors (SPs) (infected for a median of 15 years with a CD4 count that had declined to <500 cells x 10(6)/l). Both groups were antiretroviral treatment-naive at the time of evaluation. The median virus load of SP group was higher than that of the LTNP group (P = 0.0002). The CD4 response to a peptide pool representing all potential CD4 Gag epitopes and to Gag p24 protein was also studied. Compared to SPs, LTNPs had higher numbers of Gag-specific IFN-gamma+IL-2+ CD4s (P = 0.0059). The Gag-specific cytokine and proliferative responses correlated inversely with virus load (P = 0.03 and 0.0002, respectively), highlighting the potential importance of this response in immunity to HIV. A direct correlation was noted between proliferation and the Gag-specific IL-2 (P = 0.0053) rather than IFN-gamma response (P = 0.1336), demonstrating that the proliferation assay reflected the IL-2 rather than the IFN-gamma secreting capacity of CD4 cells. Several subjects with diverse class II DRB1 alleles responded, confirming the 11 selected peptides to be both antigenic and conserved. CD4 cytokine responses to one Gag and two conserved Pol peptides correlated negatively with virus load. The cytokine response to two additional Pol peptides correlated positively with virus load. The data indicate that there is not an absolute correlation between the CD4 immune response to conserved and broadly antigenic helper T cell epitopes in HIV non-progression.
Antigens, Viral↗
Impact of human immunodeficiency virus (HIV) infection on the progression of liver fibrosis in hepatitis C virus infected patients.
OBJECTIVES: To compare the rate of hepatic fibrosis progression in hepatitis C virus (HCV) infected and human immunodeficiency virus (HIV)-HCV coinfected patients, and to identify factors that may influence fibrosis progression. PATIENTS AND METHODS: A total of 153 HCV infected and 55 HCV-HIV coinfected patients were identified from two London hospitals. Eligible patients had known dates of HCV acquisition, were HCV-RNA positive, and had undergone a liver biopsy, which was graded using the Ishak score. Univariate and multivariate logistic regression analyses were used to identify factors associated with fibrosis progression rate and the development of advanced fibrosis (stages 3 and 4). RESULTS: The estimated median fibrosis progression rate was 0.17 units/year (interquartile range (IQR) 0.10-0.25) in HIV-HCV coinfected and 0.13 (IQR 0.07-0.17) in HCV monoinfected patients (p=0.01), equating to an estimated time from HCV infection to cirrhosis of 23 and 32 years, respectively. Older age at infection (p<0.001), HIV positivity (p=0.019), higher alanine aminotransferase (ALT) level (p=0.039), and higher inflammatory activity (p<0.001) on first biopsy were all independently associated with more rapid fibrosis progression. ALT was correlated with histological index (r=0.35, p<0.001). A CD4 cell count < or =250 x 10(6)/l was independently associated with advanced liver fibrosis (odds ratio 5.36 (95% confidence interval 1.26-22.79)) and was also correlated with a higher histological index (r=-0.42, p=0.002). CONCLUSION: HIV infection modifies the natural history of HCV by accelerating the rate of fibrosis progression by 1.4 fold, and the development of advanced fibrosis threefold. A low CD4 cell count was independently associated with advanced disease and correlated with higher histological index, which suggests that early antiretroviral therapy may be of benefit in slowing HCV progression in coinfected patients.
Adult↗
Importance of emergency identification schemes.
BACKGROUND: Millions of people worldwide may have a hidden medical condition that could endanger their life in an emergency. These conditions may include cardiac conditions, severe allergies, or diabetes. Emergency identification schemes such as Medic Alert produce emblems that alert health care professionals to potential problems and can ensure appropriate and prompt treatment. This paper uses mechanical failure of the Björk-Shiley convexo-concave (BSCC) heart valve as an example of a hidden medical condition. These patients have been encouraged to carry information to alert staff in an emergency that they have a BSCC patient in their care and to be alert to the signs and symptoms of acute valve malfunction. OBJECTIVE: To establish awareness and credibility of emergency identification schemes among emergency personnel and to assess if information on specific medical conditions would influence ambulance personnel regarding destination hospitals. METHODS: Questionnaires were sent to senior staff (n=380) of accident and emergency (A&E) departments and operational directors of ambulance headquarters (n=39) throughout the United Kingdom. Hospitals were divided into regional divisions to assess differences in responses across regions. RESULTS: The majority of respondents (99%) had heard of emergency identification schemes and felt that it was important for patients with special conditions to carry some form of identification. Nearly all ambulance respondents (97%) indicated it was routine to search for body worn emblems in contrast with only 71% of A & E staff. However, more than half of ambulance respondents (53.9%) stated information on emblems/cards would not influence their choice of destination hospital. CONCLUSIONS: The importance of how information on pre-existing medical conditions can influence care, is highlighted by the BSCC valve issue, where immediate diagnosis is essential for patient survival. It is vital that all staff routinely search patients for this information and if necessary act upon the information provided.
Emergency Medical Services↗
Maternal periodontal disease and preterm low birthweight: case-control study.
Periodontal disease has been suggested to be an important risk factor for preterm low birthweight (PLBW). Here we report a case-control study of 236 cases (infants < 37 wks and weighing < 2499 g) and a daily random sample of 507 controls (> or = 38 wks and weighing > or = 2500 g). Clinical periodontal indices were measured on the labor wards. Associated risk factors for periodontal disease and PLBW were ascertained by means of a structured questionnaire and maternity notes. The risk for PLBW decreased with increasing pocket depth (odds ratio [OR] 0.83, 95% confidence interval [CI] 0.68 to 1.00). After adjustment for maternal age, ethnicity, maternal education, smoking, alcohol consumption, infections, and hypertension during pregnancy, this decreased further (OR 0.78, 95% CI 0.64 to 0.99). We found no evidence for an association between PLBW and periodontal disease. Our results do not support a specific drive to improve periodontal health of pregnant women as a means of improving pregnancy outcomes.
Adolescent↗
Respiratory symptoms, asthma, exercise test spirometry, and atopy in schoolchildren from a Lima shanty town.
BACKGROUND: Little is known about the associations between symptoms of asthma, pulmonary function tests, and atopy in developing countries. While asthma in children is often associated with atopy, some studies of wheezing illness have found little or no association, leading to suggestions that there are subgroups of wheezing illness. The ISAAC study recently reported that the prevalence of reported asthma symptoms in Lima, Peru was among the highest in the world, but did not report on the atopic status of the subjects. METHODS: A cross sectional survey was conducted of children aged 8-10 years who had previously participated in a cohort study of respiratory and diarrhoeal illnesses in infancy. Questionnaires were administered asking about respiratory symptoms and asthma diagnoses, pulmonary function tests were performed before and after exercise on a treadmill, and atopy was determined from skin prick tests and specific serum IgE levels. RESULTS: A total of 793 children participated in the survey. The prevalence of asthma related symptoms in the last 12 months was 23.2%, but only 3.8% of children reported a recent asthma attack. The mean differences in pretest percentage predicted forced expiratory volume in one second (FEV(1)) were 8.1% (95% CI 2.4 to 13.8) between children who did and did not report an asthma attack in the last 12 months, and 5.3% (95% CI 2.8 to 7.9) in children who did and did not report respiratory symptoms. The corresponding differences in mean percentage fall in FEV(1) after exercise were 3.1% (95% CI -1 to 7.1) and 5.1% (95% CI 3.4 to 6.8). Recent asthma or respiratory symptoms were not associated with atopy in this population (odds ratios 1.29 (95% CI 0.56 to 2.97) and 0.91 (95% CI 0.61 to 1.37), respectively). CONCLUSIONS: Most asthma in these children was unrecognised and mild. Asthma and asthma symptoms in this population do not seem to be related to atopy.
Asthma↗
Skin prick test reactivity to foods in adult Malaysians with rhinitis.
The aim of the study was to determine the incidence of food and house dust mite (HDM) allergy in patients with nasal congestion and rhinorrhea attending the Otorhinolaryngology Clinic, National University of Malaysia, Kuala Lumpur. This was a prospective matched, controlled study of patients skin prick tested with commercial food and common aeroallergens. The participants were 148 Malaysian adults with symptoms of nasal congestion and rhinorrhea and 113 adult Malaysian control subjects without rhinitis symptoms. The skin prick test (SPT) was used to evaluate 11 foods common to the Malaysian diet and 3 HDM inhalants. Forty-eight percent of the patients with rhinitis had positive SPT results to foods, compared with 4.4% of control subjects (P < 0.05). The most commonly implicated foods were shrimp (48%) and rice (30%), which are common in the Malaysian diet. Seventy-two percent of rhinitis patients had positive SPT results to HDM, compared with 22.2% of control subjects (P < 0.05). Patients with rhinitis also had significantly more gastrointestinal problems than control subjects (P < 0.05). The incidences of HDM and food allergy are significantly greater in Malaysian adults with rhinitis symptoms than in control subjects without rhinitis. The effect of avoidance or immunotherapy awaits further study.
Adolescent↗
Deletion of cysteine 369 in lysyl hydroxylase 1 eliminates enzyme activity and causes Ehlers-Danlos syndrome type VI.
This study describes the relative contribution of the 10 cysteine residues in lysyl hydroxylase 1 (LH1) to enzyme activity. We have identified a novel mutation of a 15-bp deletion in exon 11 in one LH1 allele, that codes for amino acids 367-371 (DLCRQ), in two unrelated compound heterozygous patients with Ehlers-Danlos type VI. The mutations in their other alleles were a C1119T change (exon 10) and a predicted Q49X (exon 2). We confirmed that the loss of cysteine 369 in the deleted sequence contributed to the diminished enzyme activity by structure/function analysis of mutant LH1 constructs, in which C369 and the nine other cysteines were individually mutated to serine by site-directed mutagenesis of a normal pAcGP67/LH1cDNA construct. Following their expression in an Sf9 insect cell/baculovirus system, SDS-PAGE and Western analysis showed that equivalent levels of correctly-sized (85-kDa) products were secreted. The mutation of residues C369 and also C375, C552 and C687 virtually eliminated LH activity, whereas mutations of C267, C270, and C680 had an intermediate effect. In contrast, the C204S, C484S and C566S constructs had normal activity. Although disulfide bond formation may affect the relative contribution of each cysteine to LH activity, catalytic activity does not appear to be directly related to dimerization of the enzyme.
Alleles↗
A common duplication in the lysyl hydroxylase gene of patients with Ehlers Danlos syndrome type VI results in preferential stimulation of lysyl hydroxylase activity and mRNA by hydralazine.
Patients with Ehlers Danlos Syndrome type VI (EDS VI) are biochemically characterized by a deficiency of lysyl hydroxylase (LH), an enzyme that hydroxylates lysine residues required in the formation of stable crosslinks in collagen biosynthesis. Recently, in 19% of 35 EDS VI families, a duplication of seven exons in the LH gene has been identified as a common cause of EDS VI. We have observed that in fibroblasts from patients with this duplication mutation, administration of hydralazine, an iron-chelating agent, and ascorbate, a cofactor for LH activity, stimulates LH activity and its mRNA significantly more than in other EDS VI patients who do not have this duplication. Administration of these reagents, either singly or in combination, to fibroblasts from five patients homozygous for the duplication stimulated the low basal level of LH activity (<20% of normal) by five- to sixfold (hydralazine +/- ascorbate) and by twofold (ascorbate alone) at 72 h. This paralleled the increase in the steady-state level of mRNA for LH measured in similarly treated fibroblasts from four of these patients. In contrast, the activity of LH in fibroblasts from six other EDS VI patients and the mRNA from four of these patients who did not have the duplication were increased only three- to fourfold by hydralazine +/- ascorbate. The mechanism for the preferential stimulation of LH activity and mRNA by hydralazine in the EDS VI cells carrying the duplication is unknown, but it could be attributed to the presence of, for example, an enhancer sequence within the duplicated region of the LH gene.
2,2'-Dipyridyl↗
The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system.
This study reports the expression of functional human lysyl hydroxylase (LH), a post-translational modifying enzyme that catalyzes the hydroxylation of the lysine residues essential for cross-linking in collagen biosynthesis. We have developed a novel baculovirus system for the expression of LH, a protein that exists normally within the lumen of the endoplasmic reticulum, using a powerful baculovirus signal sequence for secretion. The supernatant from Sf9 cells infected with the viral recombinant showed significant LH activity that increased linearly with supernatant concentration, whereas there was no detectable LH activity in the cell pellet. Silver staining of the fractions purified from the active supernatant by concanavalin A Sepharose chromatography and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated an 85-kDa protein (the expected size of the LH subunit) that was most prominent in those fractions with the highest LH activity. N-terminal amino acid sequencing verified that the N-terminal primary structure of this 85-kDa protein was identical to human LH. Moreover, the activity of the expressed protein was shown to be dependent on the presence of Fe++, ascorbate, and alpha-ketoglutarate, three essential cofactors for LH activity. We have therefore successfully developed a novel expression system that produces functional human LH and enables this normally nonsecretory enzyme to be secreted, facilitating its separation from the intracellular proteins of insect cells. Future applications should allow characterization of the LH active site by crystallographic studies and site-directed mutagenesis for structure-function comparison.
Amino Acid Sequence↗