Biomedical subjects

D J Morton

Publications and source records attributed to D J Morton.

At least 19 recordsLinked to original sources

PubMed2001

Vitamin C supplement use and bone mineral density in postmenopausal women.

Vitamin C is known to stimulate procollagen, enhance collagen synthesis, and stimulate alkaline phosphatase activity, a marker for osteoblast formation. Studies of dietary vitamin C intake and the relation with bone mineral density (BMD) have been conflicting, probably because of the well-known limitations of dietary nutrient assessment questionnaires. The purpose of this study was to evaluate the independent relation of daily vitamin C supplement use with BMD in a population-based sample of postmenopausal women. Subjects were 994 women from a community-based cohort of whom 277 women were regular vitamin C supplement users. Vitamin C supplement use was validated. Daily vitamin C supplement intake ranged from 100 to 5,000 mg; the mean daily dose was 745 mg. Average duration of use was 12.4 years; 85% had taken vitamin C supplements for more than 3 years. BMD levels were measured at the ultradistal and midshaft radii, hip, and lumbar spine. After adjusting for age, body mass index (BMI), and total calcium intake, vitamin C users had BMD levels approximately 3% higher at the midshaft radius, femoral neck, and total hip (p < 0.05). In a fully adjusted model, significant differences remained at the femoral neck (p < 0.02) and marginal significance was observed at the total hip (p < 0.06). Women taking both estrogen and vitamin C had significantly higher BMD levels at all sites. Among current estrogen users, those also taking vitamin C had higher BMD levels at all sites, with marginal significance achieved at the ultradistal radius (p < 0.07), femoral neck (p < 0.07), and total hip (p < 0.09). Women who took vitamin C plus calcium and estrogen had the highest BMD at the femoral neck (p = 0.001), total hip (p = 0.05), ultradistal radius (p = 0.02), and lumbar spine. Vitamin C supplement use appears to have a beneficial effect on levels of BMD, especially among postmenopausal women using concurrent estrogen therapy and calcium supplements.

Age Factors↗

PubMed2000

Birth weight as a predictor of adult bone mass in postmenopausal women: the Rancho Bernardo Study.

Understanding the determinants of adult bone mass may help to identify women for prevention of osteoporosis. We postulated that birth weight would predict low adult bone mass in old age. Subjects were 305 postmenopausal Caucasian women (mean age 70 years). Bone mineral content (BMC) and bone mineral density (BMD) were measured at the wrist, forearm, hip and lumbar spine. Birth weight was assessed by self-report. Birth weight was positively correlated with BMC at the forearm (r = 0.15), hip (r = 0.12) and lumbar spine (r = 0.18), and the age-adjusted mean BMC increased significantly from the lowest to the highest birth weight tertile. Adjusting for adult weight diminished this association at the forearm and hip, but not at the spine. Adjustment for multiple other covariates, including height, did not materially change these associations. Adult weight and height were significantly correlated with birth weight (r = 0.19 and r = 0.24, respectively). Birth weight was not independently correlated with BMD. Birth weight was thus positively correlated with adult weight and BMC 70 years later. These findings suggest that low birth weight may be a marker for future low bone mass and that different mechanisms exist for establishing the adult bone envelope (estimated by BMC) versus its density (estimated by BMD).

Absorptiometry, Photon↗

PubMed2000

Lower extremity arterial disease in older women: the Rancho Bernardo Study.

Most studies of lower extremity arterial disease (LEAD) have not included women. To study the frequency of LEAD and its association with cardiovascular disease risk factors and estrogen use in community-dwelling postmenopausal women, we conducted a cross-sectional study of LEAD in 826 women whose average age was 74 years. Cardiovascular disease risk factors and medical history, body mass index (BMI), blood pressure, glucose tolerance, lipids and lipoproteins, and current and past medication use were determined using a standard protocol. Ankle-brachial artery index (ABI) of systolic blood pressure was measured by a trained technician using Doppler ultrasound. LEAD was defined as ABI <0.8. LEAD prevalence increased with age from nearly 5% in the 60-69-year-old group to >25% in women aged 90 and older. In age-adjusted analyses, women with LEAD had significantly lower levels of high-density lipoprotein (HDL) cholesterol, were less likely to exercise regularly, and were less likely to have ever used estrogen replacement therapy. They also had significantly higher levels of blood pressure, low-density lipoprotein (LDL) cholesterol, triglycerides, glucose, and insulin. In multivariate analyses, high HDL cholesterol, regular exercise, and estrogen use were each associated with a reduced risk of LEAD, whereas age, high blood pressure, and abnormal glucose tolerance were each associated with increased risk. Few women (6%) were smokers, but they had twice the risk of LEAD compared with nonsmokers. Estrogen was independently associated with LEAD in a model containing all covariates except LDL and HDL, and this association was no longer significant in a second model adjusting for these lipoproteins. LEAD is common in older women and associated with modifiable risk factors. The apparent protection associated with estrogen should be studied in clinical trials.

Age Distribution↗

PubMed1999

Distribution of a family of Haemophilus influenzae genes containing CCAA nucleotide repeating units.

A family of genes containing lengths of CCAA nucleotide repeating units directly following the sequence encoding the leader peptide has been identified in Haemophilus influenzae. The length of the CCAA repeats ranges from 6 to 43 and all of the identified genes encode proteins or predicted proteins with a significant homology to bacterial iron- or heme-related outer membrane proteins. We have previously shown that two of these gene products, HgpA and HgpB, bind hemoglobin and the hemoglobin-haptoglobin complex. Studies were performed to define the species distribution of the five identified genes and the CCAA repeats. We show that both the CCAA motif and the structural genes for hemoglobin and hemoglobin-haptoglobin binding are widely distributed among H. influenzae strains.

Bacterial Outer Membrane Proteins↗

PubMed1999

Quality of care for patients hospitalized with heart failure at academic medical centers.

BACKGROUND: The purpose of this study was to determine the standard of care provided by academic medical centers for the management of congestive heart failure (CHF). METHODS AND RESULTS: The standard of care was estimated by assessing adherence to the treatment guidelines published by the US Agency for Health Care Policy and Research among 522 patients hospitalized at 7 university hospitals with a diagnosis of CHF. Data were abstracted by retrospective chart review. Of the 522 patients analyzed, 435 (83%) had a left ventricular ejection fraction (LVEF) measured or documented. Among these patients, 192 were considered "ideal" candidates for angiotensin-converting enzyme (ACE) inhibitor therapy (ie, with systolic dysfunction [LVEF <40%] and no contraindications to ACE inhibitors). In this cohort of "ideal" candidates, 138 (72%) were receiving ACE inhibitors at hospital discharge, including 60 (44%) who were prescribed doses recommended in large clinical trials. Compliance with patient education guidelines was assessed in all 487 patients who were alive at the time of discharge. Of these patients, 365 (75%) received dietary counseling, 404 (83%) were educated about exercise, 54 (11%) were instructed to follow daily weights, and 468 (96%) were counseled regarding medication compliance. Among the 87 smokers who were alive at time of discharge, 8 (9%) had documented advice to quit smoking. CONCLUSIONS: This study indicates that academic medical centers performed fairly well on the assessment of LVEF, the prescription of ACE inhibitors at discharge, and on education regarding diet, exercise, and compliance with medications. However, the results suggest opportunities for improvement in ACE inhibitor dosing and patient education regarding the importance of monitoring daily weights and smoking cessation.

Academic Medical Centers↗

PubMed1999

Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b.

Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from the hgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation of hgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpC complete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.

Amino Acid Sequence↗

PubMed1999

Role of CCAA nucleotide repeats in regulation of hemoglobin and hemoglobin-haptoglobin binding protein genes of Haemophilus influenzae.

Haemophilus influenzae utilizes hemoglobin and hemoglobin-haptoglobin as heme sources. The H. influenzae hemoglobin- and hemoglobin-haptoglobin binding protein genes, hgpA, hgpB, and hgpC, contain lengths of tetrameric CCAA repeats. Using an hgpA-lacZ translational gene fusion, we demonstrate phase-variable expression of lacZ associated with alteration in the length of the CCAA repeat region.

Bacterial Outer Membrane Proteins↗

PubMed1998

Construction of antibiotic resistance cassettes with multiple paired restriction sites for insertional mutagenesis of Haemophilus influenzae.

Insertional mutagenesis of cloned genes coupled with site specific recombination into the genome of the parent organism is an ideal method for characterizing gene function. In this paper we describe the production and utility of two antibiotic resistance cassettes for use in Haemophilus influenzae. The mutagenic elements encode resistance to chloramphenicol or spectinomycin. Multiple paired restriction enzyme sites bound both cassettes. Use of these constructs to create mutants in H. influenzae demonstrated that the cassettes are readily incorporated into the genome in single copy and allow easy detection of mutant constructs. The insertions are stable following repeated in vitro passage. In addition, the elements are compatible with each other and allow the construction of multiple mutations within a single strain.

Chloramphenicol O-Acetyltransferase↗

PubMed1998

Serum albumin and bone mineral density in healthy older men and women: the Rancho Bernardo Study.

Serum albumin has been found to be positively correlated with bone mass in small studies of ambulatory men or women with diagnosed osteoporosis. In this study the relation between serum albumin and bone mineral density (BMD) was examined in 1593 white, community-dwelling men and women aged 50-95 years. BMD was determined using single-photon absorptiometry (SPA) at the ultradistal radius and the midshaft radius, and using dual-energy X-ray absorptiometry (DXA) at the hip and spine. Albumin was measured from a fasting blood sample using the Technicon SMA 12 autoanalyzer. Mean albumin levels in both men and women decreased significantly with increasing age. All but four values were within the normal range (3.5-5.0 g/dl). BMD decreased with increasing age at all sites. In both sexes there was weak positive correlation between serum albumin and BMD in the unadjusted model (Pearson's r values < 0.3, p values < 0.005). After age adjustment, however, the relationship was no longer significant (Pearson's r values < 0.05, p values > 0.18). Men and women were divided into three sex-specific categories--osteoporotic, osteopenic and normal--based on World Health Organization criteria in relation to young adult means (normal, BMD > -1 SD; osteopenia, BMD between -1 SD and -2.5 SD; osteoporosis, BMD < -2.5 SD). Mean albumin values did not differ significantly across the three BMD categories in men or women. BMD levels stratified for albumin levels and calcium supplement status (a marker for osteoporosis awareness) also did not differ. Albumin levels were also not associated with a history of low-trauma fractures. In summary, there was no age-independent association between serum albumin within the normal range and low BMD or fractures in community-dwelling healthy older adults. We conclude that previously reported associations most likely reflect inadequate adjustment for the age-related decrease in albumin levels and the selection of very frail osteoporotic subjects.

Age Factors↗

PubMed1998

hgpB, a gene encoding a second Haemophilus influenzae hemoglobin- and hemoglobin-haptoglobin-binding protein.

Haemophilus influenzae requires heme for growth and can utilize both hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified a hemoglobin- and hemoglobin-haptoglobin-binding protein, HgpA, in H. influenzae HI689. Mutation of hgpA did not affect binding or utilization of either heme source. The hgpA mutant exhibited loss of a 120-kDa protein and increased expression of a 115-kDa protein. These data suggested that at least one other gene product is involved in binding of these heme sources by H. influenzae. A 3.2-kbp PCR product derived from HI689 was cloned. The nucleotide sequence indicated a separate, distinct gene with high homology to hgpA, which would encode a 115-kDa protein. Primers were designed for directional cloning of the structural gene in the correct reading frame. Sonicates of induced Escherichia coli harboring the cloned open reading frame bound both hemoglobin and hemoglobin-haptoglobin. An insertion/deletion mutant of H. influenzae at the newly identified locus, designated hgpB, was constructed. The 115-kDa protein was not detected in the mutant after affinity purification using biotinylated hemoglobin. An hgpA hgpB double-mutant strain exhibited a reduced ability to utilize hemoglobin-haptoglobin, although it was unaltered in the ability to utilize hemoglobin. Affinity isolation of hemoglobin-binding proteins from the double mutant resulted in isolation of an approximately 120-kDa protein. Internal peptide sequencing revealed this protein to be a third distinct protein, highly homologous to HgpA and HgpB. In summary a second hemoglobin- and hemoglobin-haptoglobin-binding protein of H. influenzae has been identified and characterized, and the presence of an additional protein of similar function has been revealed.

Amino Acid Sequence↗

PubMed1998

Nonsteroidal anti-inflammatory drugs and bone mineral density in older women: the Rancho Bernardo study.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to inhibit synthesis of prostaglandins and may help prevent bone loss, but no study has shown the differential association of type or dose of NSAID compound with bone mineral density (BMD). The purpose of this study was to determine the relation of NSAIDs by type and dose to BMD. Participants were 932 Caucasian, community-dwelling women aged 44-98 years from southern California. Data were collected from 1988 to 1991 through the use of standardized medical questionnaires. Medication use was validated by a nurse. BMD at the ultradistal and midshaft radii were measured using single-photon absorptiometry, and at the hip and lumbar spine using dual-energy X-ray absorptiometry. Women (mean age, 72 years) were classified into 818 nonusers and 114 regular daily users of NSAIDs, of which 84 used propionic acid NSAIDs and the remainder used acetic acid NSAIDs. Occasional NSAID users were excluded. Women who used propionic acid NSAIDs, but not acetic acid NSAIDs, had higher BMD at all five sites and significantly higher BMD at the midshaft radius and lumbar spine. These differences remained after controlling for known covariates of osteoporosis. When women with self-reported osteoarthritis were excluded from the model, significantly higher BMD in propionic acid NSAID users was also observed at the femoral neck and total hip. Those who concurrently used estrogen and propionic acid NSAIDs had the highest BMD at all sites, suggesting an additive effect. We conclude that regular daily use of propionic acid NSAIDs, with or without simultaneous use of estrogen, may be helpful in preventing bone loss in older women. However, further research is needed to confirm these results before any clinical practice guidelines can be recommended due to the increased risk of serious complications associated with NSAID use.

Absorptiometry, Photon↗

PubMed1997

Timing of postmenopausal estrogen for optimal bone mineral density. The Rancho Bernardo Study.

OBJECTIVE: To determine the effect of the timing of initiation and the duration of postmenopausal estrogen therapy on bone mineral density (BMD). DESIGN: Cross-sectional study. SETTING: White, middle-class to upper middle-class community-dwelling women. PARTICIPANTS: A total of 740 women aged 60 to 98 years who participated in a study of osteoporosis. MEASUREMENTS: Questionnaire, validated medication use, and height and weight. Bone mineral density at the ultradistal radius and midshaft radius using single-photon absorptiometry (SPA) and at the hip and lumbar spine using dual-energy x-ray absorptiometry (DEXA). RESULTS: Of the 740 women, 69% had used oral estrogen after menopause and 30% were current users. Five groups of estrogen use were identified: never users, past users who started at menopause, past users who started after age 60 years, current users who started after age 60 years, and current users who started at menopause. At all 4 bone sites, current users who started at menopause had the highest BMD levels, which were significantly higher than never users or past users who started at menopause (with 10 years' duration of use). These differences persisted after controlling for all major risk factors for osteoporosis. Among current users, there was no significant difference in BMD levels at any site between those who started estrogen at menopause (with 20 years of use) and those who started after age 60 years (with 9 years of use). CONCLUSIONS: Estrogen initiated in the menopausal period and continued into late life is associated with the highest bone density. Nevertheless, estrogen begun after age 60 years and continued appears to offer nearly equal bone-conserving benefit.

Absorptiometry, Photon↗

PubMed1997

Transcription of genes encoding iron and heme acquisition proteins of Haemophilus influenzae during acute otitis media.

Unencapsulated Haemophilus influenzae is the second most common etiologic agent of otitis media in children. H. influenzae requires heme for aerobic growth in vitro and is able to utilize hemoglobin and complexes of heme-hemopexin, heme-albumin, and hemoglobin-haptoglobin and ferritransferrin as sources of iron and heme in vitro. Several of the acquisition mechanisms have been characterized and been shown to be heme repressible in vitro. However, little is known about the expression of heme and/or iron acquisition mechanisms during infections in the middle ear. This study was performed to determine if the genes encoding heme and iron acquisition proteins are transcribed during in vivo growth and to compare these findings with those for samples grown in vitro. Reverse transcriptase PCR (RT-PCR) was used to analyze total RNA fractions derived from in vitro- and in vivo-grown H. influenzae. Genes encoding the transferrin-binding proteins TbpA and TbpB, the 100-kDa hemopexin-binding protein HxuA, and the hemoglobin-binding protein HgpA were transcribed during otitis media. Twelve middle ear fluid samples were analyzed by blind RT-PCR to determine the transcriptional status of these genes in H. influenzae during otitis media. Five isolates had transcripts corresponding to tbpA, tbpB, and hxuA. The presence of hgpA transcripts was variable, depending on the presence of hgpA in the genome of the H. influenzae isolate. Samples without H. influenzae gene transcripts contained other etiologic agents commonly causing otitis media. These data demonstrate that H. influenzae iron and/or heme acquisition genes are transcribed during otitis media and suggest that the microenvironment during acute otitis media starves H. influenzae of heme.

Acute Disease↗

PubMed1997

Clinical characteristics and management of acute stroke in patients with atrial fibrillation admitted to US university hospitals.

UNLABELLED: The optimal evaluation and management of patients with atrial fibrillation who suffer an acute ischemic stroke remains controversial. METHODS: Medical records of 171 consecutive patients with atrial fibrillation and acute stroke at six U.S. university hospitals were reviewed. Data collected included the use of antithrombotic therapy, brain and cardiac imaging, bleeding complications, stroke risk factors, and contraindications to anticoagulation. RESULTS: Mean age was 75.4 years. Cardiovascular risk factors associated with increased stroke risk were present in 87%; 35% had at least one contraindication to anticoagulation. Half of the patients with stroke risk factors and no contraindications to anticoagulation were not receiving any antithrombotic therapy at the time of admission. Of the 22 patients who were treated with warfarin, and had INR values on admission, 16 had levels of < 2.0; only six had INR values between 2.0 and 3.0. Transthoracic echocardiography was performed in 107 patients (63%); intracardiac thrombi were visualized in only 5%. Initial brain imaging revealed hemorrhagic transformation in nine. Heparin was used in 93 patients (54%), usually within 48 hours of stroke onset. Patients who received delayed heparin typically did not have repeat brain imaging prior to starting heparin. One patient had a delayed symptomatic cerebral hemorrhage. Of the survivors, 47% were discharged and treated with warfarin (or warfarin plus aspirin), 28% with ASA, 7% with other antithrombotic therapies, and 18% with no antithrombotic therapy. CONCLUSION: Antithrombotic therapy was underutilized and inadequately monitored in atrial fibrillation patients prior to stroke onset. After hospital admission, a wide range of diagnostic and management strategies, which often did not follow current recommendations, were employed.

Acute Disease↗

PubMed1997

Carbonated beverage consumption and bone mineral density among older women: the Rancho Bernardo Study.

OBJECTIVES: The association between carbonated beverage consumption and bone mineral density was examined in a community-based cohort of older White women. METHODS: One thousand women 44 to 98 years of age had bone mineral density measured at four sites and provided medical and behavioral histories, including type and quantity of carbonated beverages consumed. RESULTS: Bone mineral density levels were not associated with intake of any type of carbonated beverage after adjustment for age, obesity, calcium intake, exercise, and current use of tobacco and alcohol, thiazides, estrogen, or thyroid hormone. CONCLUSIONS: Modest intake of carbonated beverages does not appear to have adverse effects on bone mineral density in older women.

Adult↗

PubMed1997

Economic impact of digoxin toxicity.

The costs of digoxin toxicity to the US healthcare system have not been previously reported. Therefore, the 1994 database of US University Health-System Consortium (UHC) was searched for cases of digoxin toxicity using the International Classification of Diseases (9th edition) [ICD-9] codes. In addition, the medical records of 17 patients admitted to the University of Illinois Hospital from September 1994 to July 1995 with a diagnosis of digoxin toxicity were also reviewed. Of the 17 patients, 14 were admitted with a primary diagnosis of digoxin toxicity. Causes of digoxin toxicity were worsening renal function (6 patients), excessive dosage prescribed (4 patients), excessive dosage self-administered (2 patients), multiple prescriptions (2 patients), accidental ingestion (1 patient), drug-drug interaction (1 patient) and unknown (1 patient). Digoxin toxicity could have been prevented in 9 (53%) of the 17 patients. The mean length of stay in the hospital as a result of digoxin toxicity was 3.3 +/- 1.2 days. The mean laboratory cost associated with digoxin toxicity was $US275.54 +/- $US106.57 and the mean hospital bed cost was $US3781.92 +/- $US2572.22. The mean overall cost associated with digoxin toxicity was $US4087.05 +/- $US2659.76. There was a significant correlation between the total cost associated with digoxin toxicity and the serum digoxin concentration on admission (r = 0.73, p < 0.01). From the UHC database, a total of 836 cases of digoxin toxicity in 56 hospitals were identified. This represented the occurrence of digoxin toxicity in 0.07% of all patients admitted to these US academic hospitals. Digoxin toxicity results in considerable costs to the healthcare system. Most cases can be considered readily preventable with proper patient counselling and education.

Adolescent↗

PubMed1996

Status of antithrombotic therapy for patients with atrial fibrillation in university hospitals.

BACKGROUND: The risk of stroke in patients with atrial fibrillation can be significantly reduced with antithrombotic therapy. Despite this, many physicians remain hesitant to prescribe warfarin sodium or aspirin therapy for patients with atrial fibrillation. OBJECTIVE: To assess the use of antithrombotic therapy in patients with atrial fibrillation at 6 academic hospitals in the United States. METHODS: Records were reviewed from consecutive hospital admissions of 309 patients with atrial fibrillation at 6 members of the University Health System Consortium, Oak Brook, III, which is a member driven alliance of 70 academic health centers in the United States. Risk factors for stroke, contraindications to anticoagulant therapy, and use of antithrombotic therapy at admission and discharge were recorded. RESULTS: The mean age of patients was 71.6 years, 54% had chronic, 22% paroxysmal, and 24% new-onset atrial fibrillation. Eighty-two percent of the patients had cardiovascular risk factors that have been associated with increased risk of stroke. At least 1 relative contraindication to anticoagulant therapy was present in 44%. At the time of admission. 32% of the patients with previously diagnosed atrial fibrillation (n = 235) were receiving warfarin (or warfarin plus aspirin), 31% were receiving aspirin alone, and 36% were receiving no antithrombotic therapy. At discharge (n = 230), 41% of these patients were taking warfarin (or warfarin plus aspirin) and 36% were taking aspirin. Forty-four percent of the patients with risk factors for stroke and no contraindications to anticoagulation (n = 134) were discharged on a regimen of warfarin (or warfarin plus aspirin), 34% were discharged on a regimen of aspirin, and 22% received no antithrombotic therapy. CONCLUSIONS: About half of the patients with atrial fibrillation admitted to these academic hospitals had clinical risk factors that are associated with increased risk of stroke and no contraindications to anticoagulation. Antithrombotic therapy was underused in these patients.

Academic Medical Centers↗

PubMed1996

Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae.

Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobin-binding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo.

Amino Acid Sequence↗