ABSTRACT
The pathogenesis and management of lateral epicondylalgia, or tennis elbow, a common ailment affecting middle-aged subjects of both genders continue to provoke controversy. Currently it is thought to be due to local tendon pathology, pain system changes, and motor system impairment. Its diagnosis is usually clinical, based on a classical history, as well as symptoms and signs. In selected cases, additional imaging (X-rays, ultrasound, and magnetic resonance imaging) can help to confirm the diagnosis. Different treatment modalities have been described, including the use of orthotics, non-steroidal anti-inflammatory drugs, steroid injections, topical glyceryl trinitrate, exercise therapy, manual therapy, ultrasound therapy, laser therapy, extracorporeal shockwave therapy, acupuncture, taping, platelet-rich plasma injections, hyaluronan gel injections, botulinum toxin injections, and surgery. Nevertheless, evidence to select the best treatment is lacking and the choice of therapy depends on the experience of the management team, availability of the equipment and expertise, and patient response. This article provides a snapshot of current medical practice for lateral epicondylalgia management.
Subject(s)
Tennis Elbow/therapy , Acupuncture Therapy , Administration, Topical , Athletic Tape , Botulinum Toxins/therapeutic use , Braces , Exercise Therapy , Glucocorticoids/therapeutic use , High-Energy Shock Waves , Humans , Hyaluronic Acid/therapeutic use , Injections, Intra-Articular , Laser Therapy , Massage , Muscle Strength/physiology , Neurotoxins/therapeutic use , Nitroglycerin/therapeutic use , Orthopedic Procedures , Pain Perception/physiology , Physical Examination/methods , Platelet-Rich Plasma , Tennis Elbow/diagnosis , Tennis Elbow/physiopathology , Ultrasonic Therapy , Vasodilator Agents/therapeutic use , Viscosupplements/therapeutic useABSTRACT
OBJECTIVES: To delineate the epidemiology of Charcot foot in Hong Kong Chinese diabetic patients, and to provide baseline data for benchmarking the clinic service for this special group of patients. DESIGN: Retrospective cohort study. SETTING: Regional hospital, Hong Kong. PATIENTS: Diabetic patients with Charcot foot and age- and sex-matched diabetic foot clinic attendees between 1995 and 2007. MAIN OUTCOME MEASURES: Clinical presentations were compared in patients with Charcot foot and the controls. RESULTS: Twenty-five patients were diagnosed with Charcot foot over 12 years; 60% were male. At the time of diagnosis, the mean age was 59 (standard deviation, 14; range, 38-85) years, with diabetes being diagnosed for a mean of 11 (standard deviation, 8; range, 0-30) years. Retinopathy was noted in 36% (n=9) and nephropathy in 20% (n=5) of the Charcot foot patients. No patient had peripheral vascular disease. This finding was statistically significant. Delayed presentation occurred in 11 patients. Presentation was usually unilateral. In the minority (n=3, 12%) with bilateral involvement, presentation was sequential. Charcot arthropathy affected the mid-foot in 64% of the patients. Superimposed infection was common (61%). Recurrent ulceration occurred in 11%, all of whom presented late. Only one patient underwent major amputation, but the 5-year mortality of Charcot foot patients could be up to 33%. CONCLUSION: Charcot foot was uncommon in this population. Late presentation was common and might be related to superimposed infection; such patients were prone to recurrent ulcers.
Subject(s)
Arthropathy, Neurogenic/epidemiology , Diabetic Foot/epidemiology , Adult , Aged , Aged, 80 and over , Arthropathy, Neurogenic/ethnology , China/ethnology , Diabetic Foot/ethnology , Diabetic Neuropathies/epidemiology , Diabetic Neuropathies/ethnology , Female , Hong Kong/epidemiology , Humans , Male , Middle Aged , RecurrenceABSTRACT
OBJECTIVE: To explore public awareness of osteoporosis and willingness to manage the problem, with reference to a variety of socio-economic factors. DESIGN: Cross-sectional questionnaire study. SETTING: A public hospital and a private health care clinic in Hong Kong. PATIENTS: Two hundred and fifty postmenopausal women consisting of five equal cohorts recruited at random. The cohorts consisted of: patients with fragile fracture, their next-of-kin, patients (without fragile fractures) from a government primary health care clinic, patients from a government orthopaedic clinic, and patients from a private primary health care clinic. RESULTS: Only 81% of those interviewed had heard of the disease. Among these, 92% believed that the government was responsible for managing osteoporosis. Most (83%) were willing to self-finance treatment; a higher percentage were willing to do so among those with relatives having osteoporotic fractures. Most (87%) of the subjects underestimated the cost. Less than 40% expected to pay more than HK$ 1,200 annually. Given the current market price, only 66% would still consider undertaking the treatment. Notably, 99% of interviewees would commence treatment provided the cost was lower. CONCLUSION: Direct costs of managing osteoporosis deter the public from commencing treatment. If the cost of treatment could be lowered and publicised, a dramatic increase in self-financed treatment can be anticipated.
Subject(s)
Health Knowledge, Attitudes, Practice , Osteoporosis, Postmenopausal/psychology , Aged , Aged, 80 and over , Analysis of Variance , Cross-Sectional Studies , Female , Hong Kong/epidemiology , Humans , Middle Aged , Osteoporosis, Postmenopausal/epidemiology , Regression Analysis , Surveys and QuestionnairesABSTRACT
OBJECTIVES: To evaluate the application of a translated version of an established self-administered questionnaire for carpal tunnel syndrome on Chinese patients in Hong Kong. DESIGN: Evaluation of an instrument tool. SETTING: Department of Orthopaedics and Traumatology, Queen Mary Hospital, Hong Kong; Holistic Medical Centre, Aberdeen, Hong Kong. PARTICIPANTS: Patients with carpal tunnel syndrome, translators. MAIN OUTCOME MEASURES: The adaptation was based on forward-backward translation from English to Chinese (Hong Kong) and vice versa. Meetings with translators, investigators, and patients were organised to generate an acceptable version of the questionnaire. A pilot study was carried out on 20 patients and subsequently minor adjustments were added. Fifty patients were recruited to validate the reliability and internal consistency of the questionnaire. RESULTS: The ordinality of response agreed with the original instrument. Test-retest reproducibility showed no significant difference between tests. The Pearson correlation coefficient ranged from 0.83 to 0.93. Internal consistency was good, at 0.85. CONCLUSION: Through the validation of the Hong Kong Chinese version of the questionnaire, we are able to produce an assessment tool for the local patients. Furthermore, we are able to create a platform for: (i) a cross-national and cross-cultural epidemiological comparison as well as a means of (ii) evaluating different types of treatments.
Subject(s)
Carpal Tunnel Syndrome/diagnosis , Severity of Illness Index , Surveys and Questionnaires , Adult , Aged , Aged, 80 and over , Cross-Cultural Comparison , Female , Hong Kong , Humans , Language , Male , Middle Aged , Pilot Projects , Reproducibility of ResultsABSTRACT
OBJECTIVES: To study the relationship between hospitalisation for diabetic foot complications, non-traumatic major lower limb amputations, and seasonal variation in humidity and temperature. DESIGN: Retrospective study. SETTING: Regional hospital, Hong Kong. PATIENTS: Diabetic patients hospitalised for lower limbs infection during the inclusive period 1995 to 2004. MAIN OUTCOME MEASURES: Monthly incidence of admissions for diabetic foot sepsis and non-traumatic non-neoplastic major lower limb amputations correlated with the monthly average humidity and temperature. RESULTS: During the study period, a total of 770 patients contributed to 1285 episodes of hospitalisation and ensued 208 corresponding major lower limb amputations. Poisson regression study showed that the monthly incidences of hospitalisation as well as amputations were related to the monthly average temperature (P<0.001 and =0.0012, respectively) but not the monthly average humidity (P=0.1560 and 0.6332, respectively). CONCLUSION: The warm and humid weather of Hong Kong exerts a seasonal variation on the diabetic foot infection presentations. Warm temperature aggravates the severity of infection and precipitates amputation. More intensive patient education and clustering of medical services in late winter and spring might reduce the incidence of diabetic foot amputation, which is a preventable complication.
Subject(s)
Amputation, Surgical , Diabetic Foot/surgery , Seasons , Adult , Aged , Aged, 80 and over , China/ethnology , Diabetic Foot/epidemiology , Diabetic Foot/ethnology , Female , Hong Kong/epidemiology , Hospitalization/statistics & numerical data , Hospitals , Humans , Humidity , Male , Middle Aged , Poisson Distribution , Regression Analysis , Retrospective Studies , Sepsis/etiology , TemperatureABSTRACT
PURPOSE: To evaluate clinical and cosmetic outcomes of reconstruction in thumb polydactyly and prognostic value of the Wassel classification. METHODS: Between 1993 and 2000 inclusive, out of the patients with thumb polydactyly (involving 80 thumbs) operated on, 34 patients (36 thumbs) were available for review and underwent clinical and radiological assessment. Outcomes in terms of the Tada score and complications were recorded. RESULTS: The mean age of patients at the time of operation was 2.8 (range, 0.6-47) years. The mean follow-up period was 5 (range, 2.4-10) years. According to the Wassel classification, 12 were type-II thumb polydactyly, 3 type-III, 11 type-IV, 6 type-V, one type-VI, and 3 type-VII. There was no perioperative mortality or wound infection. More than 88% of the patients were satisfied or very satisfied with functional and cosmetic outcomes. Postoperative complications such as scar hypertrophy, pulp atrophy, joint deformity, and instability were common but minor. Ridge nail deformity after the Bilhaut Cloquet procedure was amenable to secondary corrective procedures. All types of operated thumb polydactyly achieved similar mean Tada scores (14.7- 16.6 out of 20). The Wassel classification category, age, and surgical procedures were found to have no prognostic value with regard to the Tada score and presence of complications. CONCLUSION: Surgery on thumb polydactyly is rewarding. The Wassel classification category can be used as a guide for treatment, although it fails to predict the occurrence of postoperative complications or Tada scores. Our patients' results can serve as guidelines of expected outcomes after reconstructive procedures in different sub-types of thumb polydactyly.
Subject(s)
Polydactyly/surgery , Thumb/abnormalities , Thumb/surgery , Adolescent , Adult , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Plastic Surgery ProceduresABSTRACT
Acupuncture is often regarded as innocuous. However, its complication can be serious and deadly if unattended. We report a case of iatrogenic bilateral pneumothorax after acupuncture therapy. Setting up a government regulatory body and using needles with safety design can prevent further inadvertent incidences from occurring.
Subject(s)
Acupuncture Therapy/adverse effects , Pneumothorax/etiology , Adult , Female , Humans , Pneumothorax/diagnostic imaging , Pneumothorax/therapy , RadiographyABSTRACT
Irreducible dislocation of the hallucal interphalangeal joint is a rare condition, with only 41 cases reported in the literature. We present a patient with a Miki type 2 irreducible dislocation of the hallucal interphalangeal joint and review literature pertinent to this condition.
Subject(s)
Hallux/injuries , Joint Dislocations/surgery , Toe Joint/surgery , Adolescent , Hallux/diagnostic imaging , Humans , Joint Dislocations/diagnostic imaging , Radiography , Toe Joint/diagnostic imagingABSTRACT
OBJECTIVE: To determine the reliability and validity of a dedicated assessment tool for lateral epicondylitis after translation into Hong Kong Chinese. DESIGN: Cross-sectional study. SETTING: District hospital, Hong Kong. PATIENTS: Seventy-four patients, 12 of whom were bilingual, were recruited (total of 82 elbows). MAIN OUTCOME MEASURES: Translation equivalence and reliability were measured with the test-retest method. Validity was measured against the Roles and Maudsley outcome score and mean maximal grip strength. RESULT: The Patient-rated Forearm Evaluation Questionnaire had high English-Chinese equivalence (Spearman's rho correlation coefficient=0.926; P<0.001). It was also very reliable (intraclass correlation coefficient=0.99; P<0.001). Validity according to the Roles and Maudsley outcome score and mean of maximal grip strength was significant (P<0.01). CONCLUSION: The Hong Kong Chinese version of Patient-rated Forearm Evaluation Questionnaire is a reliable and valid assessment tool for chronic lateral epicondylitis. Its equivalence to the original English version makes outcome assessment across cultural barrier feasible.
Subject(s)
Surveys and Questionnaires/standards , Tennis Elbow/diagnosis , Adult , Aged , Cross-Sectional Studies , Hong Kong , Humans , Language Arts , Middle Aged , Reproducibility of ResultsABSTRACT
OBJECTIVE: To assess the predictive power of various parameters on the final outcome of ulcerated diabetic feet among the Hong Kong Chinese population. DESIGN: Retrospective cohort study. SETTING: Regional public hospital, Hong Kong. PATIENTS: Medical records of 340 diabetic patients with foot ulcers (535) who were referred to the Diabetic Foot Clinic between July 1995 and June 2000 were reviewed. MAIN OUTCOME MEASURES: Demographic and clinical data, including assessment of the foot and blood parameters. RESULTS: Increasing age, wound depth, the presence of ischaemia, a low albumin level, and the lack of simultaneous ulceration were determined by stepwise logistic regression analysis to be the most significant independent predictors of an unfavourable outcome. CONCLUSIONS: Major amputation is more likely to occur in elderly patients, with progressive wound depth, and in the presence of ischaemia. A low albumin level was also noted to be an independent predictor of major amputation in the population studied.
Subject(s)
Diabetic Foot/diagnosis , Diabetic Foot/epidemiology , Adult , Age Distribution , Aged , Aged, 80 and over , Amputation, Surgical/methods , Amputation, Surgical/statistics & numerical data , Chi-Square Distribution , Cohort Studies , Diabetic Foot/surgery , Female , Hong Kong/epidemiology , Humans , Incidence , Logistic Models , Male , Middle Aged , Predictive Value of Tests , Probability , Retrospective Studies , Risk Factors , Severity of Illness Index , Sex DistributionABSTRACT
PURPOSE: To correlate the radiographic measurement, cord diameter shown on magnetic resonance imaging (MRI), and clinical hand sign of cervical myelopathic patients. METHODS: Patients with clinical cervical myelopathy who had had MRI in Kwong Wah Hospital between January 2001 and December 2002 were enlisted. Their cervical spine radiographs and clinical records were reviewed. RESULTS: Of 36 patients with a complete set of MRI films, cervical spine radiographs, and clinical notes; 18% did not have Hoffman's sign, 47% had normal supinator reflex, 39% had unimpaired 10-second test, and 45% showed no finger escape sign. The presence of myelopathic hand signs was not correlated to any radiological assessment, cord diameter, or presence of myelomalacia at any level. CONCLUSION: Cervical spine radiography cannot predict the level and degree of cervical spinal cord compression. Myelopathic hand signs are not diagnostically fail-safe and cannot predict the level and degree of cord compression.
Subject(s)
Cervical Vertebrae , Magnetic Resonance Imaging , Spinal Cord Compression/diagnosis , Tomography, X-Ray Computed , Adult , Aged , Aged, 80 and over , Chi-Square Distribution , Female , Humans , Male , Middle Aged , ROC Curve , Spinal Cord Compression/diagnostic imagingABSTRACT
PURPOSE: Major amputation of the lower limb is considered the last resort when limb salvage is impossible. The aim of this study is to determine the morbidity, mortality, and rehabilitation outcome of patients that underwent a lower-limb amputation. METHODS: A retrospective cohort study was conducted among 100 elderly patients who underwent a total of 120 lower-limb amputations in a regional hospital in Hong Kong from 1996 to 2001. RESULTS: The mean age of the amputees was 77.9 years; 58 were female. 95% of the amputations were performed because of infection with or without vascular compromise; 55 transfemoral and 60 transtibial amputations contributed 96% of the case mix. Some 43% of patients experienced early complications and 12% required re-amputation. The early (30-day) mortality rate was 15%. Only 55% of the amputees survived after 4 years. A 44% return-home rate was achieved. However, only 11% of the amputees could walk without help from other people. Although prostheses were issued to 42% of the survivors, compliance was only 53%; 24% of the survivors lost their remaining leg within 2 years. CONCLUSION: The outcome of major lower-extremity amputation remains poor. Efforts should be made to retain these limbs. When it is proven impossible, one should strive to preserve the knee joint whenever feasible.
Subject(s)
Amputation, Surgical/methods , Amputation, Surgical/rehabilitation , Asian People/statistics & numerical data , Aged , Aged, 80 and over , Amputation, Surgical/statistics & numerical data , Cohort Studies , Female , Follow-Up Studies , Geriatric Assessment , Hong Kong/epidemiology , Humans , Lower Extremity , Male , Perioperative Care/methods , Postoperative Complications/epidemiology , Retrospective Studies , Risk Assessment , Socioeconomic Factors , Survival Analysis , Treatment OutcomeABSTRACT
With advances in the treatment of patients with chronic renal failure, their life expectancy has increased. In turn, the prevalence of osteitis fibrosa cystica, a manifestation of secondary hyperparathyroidism, and beta2 microglobulin amyloidosis, a result of long-term haemodialysis, has risen. While both conditions share similar radiological features, their management is very different. We present the case of a patient with renal failure who had been receiving haemodialysis for over 20 years. Lytic lesions had been observed in the proximal part of both femurs for ten years. A presumptive diagnosis of osteitis fibrosa cystica was made. However, no regression of the lesions occurred after parathyroidectomy. The patient subsequently developed sequential pathological fractures through the lesions, for which bilateral total hip replacements were performed. Histology of the lesions revealed that the patient was in fact suffering from amyloidosis. In patients with chronic renal failure, osseous amyloidosis is a highly probable differential diagnosis, especially if no regression of a lytic lesion is observed after parathyroidectomy.
Subject(s)
Amyloidosis/diagnosis , Hyperparathyroidism, Secondary/diagnostic imaging , Kidney Failure, Chronic/therapy , Osteitis Fibrosa Cystica/diagnostic imaging , Parathyroidectomy/adverse effects , Renal Dialysis/adverse effects , Amyloidosis/complications , Diagnosis, Differential , Fatal Outcome , Femur Neck/diagnostic imaging , Humans , Male , Middle Aged , Osteitis Fibrosa Cystica/etiology , Radiography , beta 2-Microglobulin/metabolismABSTRACT
STUDY DESIGN: Experimental study evaluated electrophysiological and histologic changes in the paraspinal muscle after spinal fusion. OBJECTIVE: To determine the effect of posterior spinal fusion on paraspinal muscle by means of electrophysiological and histologic evaluation, using a rabbit model. SUMMARY OF BACKGROUND DATA: Posterior lumbar spinal fusion is a common spinal surgery. Paraspinal muscular dysfunction is postulated to be due to injury inflicted by the surgery, either during exposure or from disuse of the fused segment. The effect of spinal fusion on paraspinal muscular function remains unclear. METHODS: Thirty New Zealand white rabbits were divided into 2 groups: 2-level posterior spinal fusion with instrumentation (group F) and the sham control group (group S). Preoperative and follow-up electromyography tests, as well as histologic assessments, were performed in 6-month intervals. RESULTS: In group F, 14 of 15 surgeries resulted in a solid fusion, as judged by postmortem examination. In group S, the root mean square of electromyography did not present a significant difference at 6-month follow-up when compared with preoperative status (P > 0.05). At 6-month follow-up, group F presented a significantly lower root mean square in the fused region than in adjacent regions (P < 0.05). Group S did not demonstrate significant differences in the median frequency between the preoperative and postoperative periods (P < 0.05). Group F resulted in a significant decrease of median frequency in the fusion region. However, increases were observed in the adjacent caudal region and the cranial region. The histologic analysis demonstrated a significant reduction in muscle fiber size (P < 0.05) in the fusion region of group F, when compared with the preoperative status, whereas group S did not present any significant differences (P > 0.05). CONCLUSION: These results demonstrated that spinal fusion resulted in atrophy and reduced adjacent paraspinal muscle activity. Muscular activity was greater in the adjacent regions after spinal fusion, which may indicate muscle hypertrophy.
Subject(s)
Electromyography , Lumbar Vertebrae/surgery , Muscle, Skeletal/physiopathology , Muscular Atrophy, Spinal/etiology , Spinal Fusion/adverse effects , Animals , Hypertrophy , Models, Animal , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/innervation , Muscle, Skeletal/pathology , Muscular Atrophy, Spinal/pathology , Muscular Atrophy, Spinal/physiopathology , Rabbits , Time FactorsABSTRACT
Extracts of Escherichia coli K12 degrade AMP to hypoxanthine, adenine, adenosine, and inosine. Degradation experiments with mutants which lack purine nucleoside phosphorylase or both purine nucleoside phosphorylase and adenosine deaminase demonstrate that hypoxanthine formation is dependent on purine nucleoside phosphorylase. These findings are consistent with an absence of adenine deaminase activity in E. coli. Adenine is formed from AMP in extracts of the E. coli mutants as well as the wild type cells. This activity is due to AMP nucleosidase. Purified, homogeneous AMP nucleosidase gives a subunit Mr = 52,000 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 280,000 by comparative gel filtration. Kinetic studies with this enzyme give cooperative initial rate curves with AMP as substrate, with MgATP2- as an activator, and with Pi as an inhibitor. Phosphate inhibition is competitive with McATP2- (Ki = 0.2 mM) and reverses the activation by MgATP2-. In the absence of MgATP2-, the apparent S0.5 for AMP is 15 mM and decreases to 90 microM at saturating MgATP2-. The maximum rate of AMP hydrolysis is not affected by MgATP2-. Kinetics of MgATP2- activation give a constant for half-maximum activation varying from 120 microM in the presence of low AMP to approximately 2 microM when AMP is present at near saturation. Formycin 5'-PO4 is a powerful competitive inhibitor with respect to AMP, giving a Kis of 72 nM and a Km/Kis ratio of 1,200. Adenylate degradation experiments indicate that AMP nucleosidase is the major enzyme of AMP catabolism in E. coli. The kinetic properties of the purified enzyme indicate that regulation occurs by the intracellular MgATP2- /Pi ratio and the concentration of AMP.
Subject(s)
Adenosine Monophosphate/metabolism , Escherichia coli/enzymology , N-Glycosyl Hydrolases/metabolism , Adenine/metabolism , Adenosine/metabolism , Adenosine Monophosphate/isolation & purification , Hypoxanthines/metabolism , Inosine/metabolism , Kinetics , Molecular Weight , Mutation , N-Glycosyl Hydrolases/isolation & purification , Phosphates/pharmacologyABSTRACT
A mutant of Escherichia coli which contained no detectable AMP nucleosidase activity (EC 3.2.2.4) was produced by treatment with nitrosoguanidine and identified by a colorimetric assay for AMP nucleosidase in individual colonies from agar plates. Conjugation experiments indicated a close linkage between the AMP nucleosidase locus (amn) and his. Assays for AMP nucleosidase in E. coli strains with deletions in the his region established that amn is not located between attP2H and mgl . Transduction experiments with bacteriophage P1 were used to construct a linkage map in the his and amn region of the 100-min chromosome map of E. coli, which places amn at 43.3 min. A Rec- strain of E. coli ( FP4102 ) which carries the uvrC to his region (42.1-44.1 min) as an F' episome was used to confirm this location. The episome from FP4102 was used as the source of DNA for cloning amn. BamHI restriction fragments of the episome were inserted into the homologous site of pBR322 and used to transform the Amn- strain of E. coli to Amn+. The transforming plasmid contained a 9-kb (kilobase) insert. Partial restriction of plasmid with ClaI and religation gave a plasmid with a 6-kb insert which retains the amn gene. Restriction mapping of the plasmid has identified ClaI and PstI sites which appear to be within the amn locus. E. coli (Rec-) which contains the 9-kb plasmid of pBR322 containing amn overproduces AMP nucleosidase when grown in the presence of ampicillin. The specific activity of AMP nucleosidase increases from 0.016 mumol/min/mg of protein to 0.32 mumol/min/mg of protein in extracts of the wild type and the plasmid-bearing strains, respectively. A simple purification procedure yields 10 mg of homogeneous AMP nucleosidase from 25 g of packed cells.
Subject(s)
Genes , N-Glycosyl Hydrolases/genetics , Bacteriophage lambda/enzymology , Chromosome Mapping , Cloning, Molecular , DNA Restriction Enzymes/metabolism , Escherichia coli/enzymology , Phenotype , Transduction, GeneticABSTRACT
Leung, Hazel Barner (University of Pennsylvania School of Medicine, Philadelphia), Alice McGovern Doering, and Seymour S. Cohen. Effect of 9-beta-d-arabinofuranosyladenine on polymer synthesis in a polyauxotrophic strain of Escherichia coli. J. Bacteriol. 92:558-564. 1966.-Adenine-requiring mutants have been obtained from Escherichia coli strain 15 TAU, which also needs thymine, arginine, and uracil for growth. Some of these are killed by 9-beta-d-arabinofuranosyladenine (ara-A) in the absence of exogenous adenine; a particular mutant of this type, designated TAUAd, has been used in our studies. The lethality of ara-A, d-arabinosylhypoxanthine, and the 1-n-oxide of ara-A has been compared; ara-A is equally toxic in the presence or absence of thymine. Although the absence of uracil reduces ara-A toxicity, the lack of arginine almost eliminates lethality. It was found that ara-A completely inhibits deoxyribonucleic acid synthesis without markedly affecting ribonucleic acid (RNA) synthesis. Some inhibition of protein synthesis can be detected. However, the interpretation of these results is complicated because (i) exogenous adenine must be excluded, (ii) endogenous adenine is made available from RNA turnover, and (iii) ara-A is being rapidly converted to only slightly less toxic arabinosylhypoxanthine by the adenosine deaminase of E. coli. A suitable inhibitor for the bacterial deaminase has not yet been found.
Subject(s)
Adenine Nucleotides/pharmacology , Escherichia coli/metabolism , Polymers/biosynthesis , Adenine Nucleotides/metabolism , Aminohydrolases/metabolism , Arginine/metabolism , Bacterial Proteins/biosynthesis , Carbon Isotopes , Culture Media , DNA, Bacterial/biosynthesis , Hypoxanthines/pharmacology , Mutation , RNA, Bacterial/biosynthesis , Radiometry , Thymine/metabolism , Uracil/metabolismABSTRACT
Adenosine 5'-phosphate was synthesized with 3H or 14C label specifically located as [1'-3H]AMP, [1'-14C] AMP, [5'-3H]AMP, and [5'-14C]AMP. The synthesis was accomplished from adenine and glucose or adenine and ribose using enzymes from the pentose pathway and/or from the purine salvage pathways. Structural analysis of the compounds confirmed the locations of the radiolabels. The methods provide a general scheme for the efficient synthesis of adenine nucleotides of high purity with 3H or 14C at any stable position on the ribose ring. Synthesis of [5'-14C]dAMP and [1'-3H] dAMP from the corresponding ribonucleotides was accomplished with ribonucleotide reductase. Labeled inosine was prepared by enzymatic dephosphorylation and deamination of labeled AMP. These compounds have been used to measure the secondary kinetic isotope effects on the acid-catalyzed hydrolysis of the N-glycosidic bond of AMP, dAMP, and inosine and the corresponding primary kinetic isotope effects with AMP. Acid hydrolysis in 0.1 or 0.2 N HCl at 50 degrees C gave 1H/3H secondary kinetic isotope effects of 1.23 +/- 0.01, 1.26 +/- 0.01, and 1.230 +/- 0.003 for AMP, dAMP, and inosine, respectively. The primary kinetic isotope effect for 12C/14C was 1.049 +/- 0.010 for AMP. The apparent rate constants for hydrolysis under these conditions were similar for inosine and AMP and were in the range 10(-6)-10(-5)s-1. Acid hydrolysis of dAMP is approximately 1000-fold faster than AMP but gives a similar 1H/3H kinetic isotope effect. The results of secondary isotope effects indicate that the transition states for the acid-catalyzed hydrolysis of the N-glycosidic bonds of inosine, AMP, and dAMP have similar bonding to 1'-3H in the transition state and have considerable carboxonium character. Results with [1'-14C]AMP demonstrate that a significant primary isotope effect can be measured in the acid solvolysis of the N-glycosidic bond of AMP.
Subject(s)
Adenosine Monophosphate/chemical synthesis , Deoxyadenine Nucleotides/chemical synthesis , Inosine/chemical synthesis , Isotope Labeling/methods , Adenosine Triphosphate/metabolism , Carbon Radioisotopes , Chemical Phenomena , Chemistry , Glucose/metabolism , Hydrolysis , Kinetics , TritiumABSTRACT
The gene for AMP nucleosidase from Escherichia coli (amn) has been sequenced and characterized. The gene codes for a transcript of 1.7 +/- 0.2 kb, and the open reading frame corresponds to a protein of 483 amino acids (Mr = 53848). Amino acid sequences from tryptic peptides of AMP nucleosidase, N-terminal amino acid analysis, and the amino acid composition confirm the gene assignment and the open reading frame of amn. Primer extension studies determined the 5'-end of the amn transcript. The 5'-regulatory region contains overlapping sequences with similarity to the consensus sequences for binding cAMP receptor protein and inorganic phosphate repressor protein. Addition of exogenous cAMP to E. coli deficient in adenylate cyclase resulted in a 3-fold increase in AMP nucleosidase activity. Growth of E. coli on limiting phosphate resulted in an 8-fold increase in the production of AMP nucleosidase. The amn gene was expressed in AMP nucleosidase deficient strains of Azotobacter vinelandii and E. coli. A pUC-amn construct is described that causes approximately 20% of the total protein in E. coli to be produced as AMP nucleosidase. Comparison of the amino acid sequence for AMP nucleosidase with that for yeast AMP deaminase indicates a region in which six of eight amino acids are identical but no other overall homology. The amino acid sequence showed poor agreement with consensus sequences for adenylate binding sites even though the enzyme is known to have a catalytic site for AMP and regulatory sites for MgATP and phosphate.