Hemoglobin Parchman: double crossover within a single human gene.

Structural analysis of a new variant hemoglobin revealed tryptic peptides with the amino acid composition of normal delta-globin, except for two internal peptides, which had the compositions of normal beta-globin. The most likely explanation for these findings is that a double, nonhomologous crossover between the delta-and beta-globin genes had occurred.

Biosynthesis of hemoglobin Ann Arbor: evidence for catabolic and feedback regulation.

Hemoglobin Ann Arbor, in which arginine replaces leucine in position 80 of the a chain, occurs in aflected individuals in low proportion to hemoglobin A. Biosynthetic studies were perforined on reticulocytes of a patient heterozygous for this hemoglobin. These studies suggested that the low percentage of hemoglobin Ann Arbor is prinlarily due to preferential destruction of the abnormal component. The reduced concentration of alpha Ann Arbor chains was also reflected in a decreased synthesis of normal beta chains.

Hemoglobin Indianapolis (beta 112[G14] arginine). An unstable beta-chain variant producing the phenotype of severe beta-thalassemia.

Hemoglobin (Hb) Indianapolis is an extremely labile beta-chain variant, present in such small amounts that it was undetectable by usual techniques. Clinically, it produces the phenotype of severe beta-thalassemia. Biosynthetic studies showed a beta:alpha ratio of 0.5 in reticulocytes and about 1.0 in marrow after a 1-h incubation. These results, similar to those seen in typical heterozygous beta-thalassemia, suggested that betaIndianapolis was destroyed so rapidly that its net synthesis was essentially zero. To examine the kinetics of globin synthesis, reticulocyte incubations of 1.25--20 min were performed with [3H]leucine. The betaIndianapolis:beta A ratio at 1.25 min was 0.80 suggesting that beta Indianapolis was synthesized at a near normal rate. At 20 min, this ratio was 0.46 reflecting rapid turnover of beta Indianapolis. The erythrocyte ghosts from these incubations contained only betaIndianapolis and alpha-chains, and the proportion of betaIndianapolis decreased with time, indicating loss of betaIndianapolis. Pulse-chase studies showed little change in beta A:alpha ratio and decreasing betaIndianapolis:alpha and betaIndianapolis:beta A with time. The half-life of betaIndianapolis in the soluble hemoglobin was approximately equal to 7 min. There was also rapid loss of beta Indianapolis from the erythrocyte membrane. From these results, it may be inferred that betaIndianapolis is rapidly precipitated from the soluble cell phase to the membrane, where it is catabolized. Heterozygotes for beta 0-thalassemia usually have minimal hematologic abnormalities, whereas heterozygotes with betaIndianapolis, having a similar net content of beta-chain, have severe disease. The extremely rapid precipitation and catabolism of betaIndianapolis and the resulting excess of alpha-chains, both causing membrane damage, may be responsible for the severe clinical manifestations associated with this variant. It seems likely that other, similar disturbances in the primary sequence of globin polypeptide chains may produce clinical findings similar to those seen with hemoglobin Indianapolis and thus produce the phenotype of severe beta-thalassemia.

Molecular basis for nondeletion alpha-thalassemia in American blacks. Alpha 2(116GAG----UAG).

An American black woman was found to have the phenotype of moderately severe alpha-thalassemia normally associated with the loss of two to three alpha-globin genes despite an alpha-globin gene map that demonstrated the loss of only a single alpha-globin gene (-alpha/alpha alpha). Several individuals in her kindred with normal alpha-globin gene mapping studies (alpha alpha/alpha alpha) had mild alpha-thalassemia hematologic values consistent with the loss of one to two alpha-globin genes. These data suggested the presence of a nondeletion alpha-thalassemia defect in this family which segregates with the intact alpha alpha gene cluster. An abnormally migrating and highly unstable alpha-globin gene product was demonstrated by in vitro translation of the reticulocyte mRNA from the proposita and this mutant alpha-globin protein was mapped to the alpha 2-globin gene by hybrid-selected translation. The abnormal alpha 2-globin gene was cloned and sequenced. A single base mutation that results in a premature termination codon was identified at codon 116 (GAG----UAG). The defined alpha-globin genotype of the proposita (-alpha/alpha 116UAG alpha) and the positioning of this nonsense mutation at the alpha 2-globin gene locus are fully consistent with the observed alpha-thalassemia phenotype.

Hemoglobin Mississippi (beta 44ser----cys). Studies of the thalassemic phenotype in a mixed heterozygote with beta +-thalassemia.

Hemoglobin Mississippi (HbMS: beta 44ser----cys) has anomalous properties that include disulfide linkages with normal beta-, delta-, gamma-, and alpha-chains, and the formation of high molecular weight multimers. While heterozygotes for HbMS are clinically and hematologically normal and carriers of the beta +-thalassemia gene in our family had mild microcytic anemia, the proband with HbMS-beta +-thalassemia had a hemoglobin level of 7 g/dl, mean corpuscular volume (MCV) of 68 fl, reticulocytes of 2-6%, HbF of 18%, marked anisocytosis and poikilocytosis, and splenomegaly, all features of thalassemia intermedia. With oxidant stress, her erythrocytes developed multiple dispersed Heinz bodies, but HbMS was only mildly unstable. HbMS was susceptible to proteolytic degradation in the presence of ATP. The unexpectedly severe clinical findings in HbMS-beta +-thalassemia may result from the proteolytic digestion of HbMS, as well as the excessive alpha-chains characteristic of beta +-thalassemia, which combined provide the increment of cellular damage that results in the phenotype of thalassemia intermedia.

Two missense mutations in the beta-globin gene can cause severe beta thalassemia. Hemoglobin Medicine Lake (beta 32[B14]leucine-->glutamine; 98 [FG5] valine-->methionine).

We studied the molecular basis of transfusion-dependent hemolytic anemia in an infant who rapidly developed the phenotype of beta thalassemia major. DNA sequence of one beta-globin gene of the proband revealed two mutations, one for the moderately unstable hemoglobin (Hb) Köln and another for a novel codon 32 cytosine-thymidine-guanine-->cytosine-adenine-guanine transversion encoding a leucine-->glutamine mutation. A hydrophilic glutamine residue at beta 32 has an uncharged polar side chain that could potentially distort the B helix and provoke further molecular instability. This new hemoglobin was called Hb Medicine Lake. Biosynthesis studies showed a deficit of beta-globin synthesis with early loss of beta-globin chains. An abnormal unstable hemoglobin, globin chain, or tryptic globin peptide was not present, demonstrating the extreme lability of this novel globin. Hb Medicine Lake mRNA was present, but an aberrantly spliced message was not. Absence of an abnormal beta-globin gene in the mother makes it likely that a de novo mutation occurred in the proband. The molecular pathogenesis of Hb Medicine Lake illustrates a mechanism whereby the phenotype of a genetic disorder, like the mild hemolytic anemia associated with a hemoglobinopathy, can be modulated by a coincident mutation in the same gene.

Effects of alpha-thalassemia and sickle polymerization tendency on the urine-concentrating defect of individuals with sickle cell trait.

A defect in urine concentrating ability occurs in individuals with sickle cell trait (HbAS). This may result from intracellular polymerization of sickle hemoglobin (HbS) in erythrocytes, leading to microvascular occlusion, in the vasa recta of the renal medulla. To test the hypothesis that the severity of the concentrating defect is related to the percentage of sickle hemoglobin present in erythrocytes, urinary concentrating ability was examined after overnight water deprivation, and intranasal desmopressin acetate (dDAVP) in 27 individuals with HbAS. The HbAS individuals were separated into those who had a normal alpha-globin genotype (alpha alpha/alpha alpha), and those who were either heterozygous (-alpha/alpha alpha) or homozygous (-alpha/-alpha) for gene-deletion alpha-thalassemia, because alpha-thalassemia modulates the HbS concentration in HbAS. The urinary concentrating ability was less in the alpha alpha/alpha alpha genotype than in the -alpha/alpha alpha or -alpha/-alpha genotypes (P less than 0.05). After dDAVP, the urine osmolality was greater in patients with the -alpha/-alpha genotype than with the -alpha/alpha alpha genotype (882 +/- 37 vs. 672 +/- 38 mOsm/kg H2O) (P less than 0.05); patients with the -alpha/alpha alpha genotype had greater concentrating ability than individuals with a normal alpha-globin gene arrangement. There was an inverse linear correlation between urinary osmolality after dDAVP and the percentage HbS in all patients studied (r = -0.654; P less than 0.05). A linear correlation also existed for urine concentrating ability and the calculated polymerization tendencies for an oxygen saturation of 0.4 and O (r = -0.62 and 0.69, respectively). We conclude that the severity of hyposthenuria in HbAS is heterogeneous. It is determined by the amount of HbS polymer, that in turn is dependent upon the percentage HbS, which is itself related to the alpha-globin genotype.

Molecular analysis of the beta-thalassemia phenotype associated with inheritance of hemoglobin E (alpha 2 beta2(26)Glu leads to Lys).

Inheritance of the gene for betaE-globin is associated with hypochromia and microcytosis, reminiscent of typical heterozygous beta-thalassemia. Patients with hemoglobin (Hb)E-beta-thalassemia exhibit clinical phenotypes of severe beta-thalassemia, a circumstance not encountered in other compound heterozygous states for structural beta-chain mutations and beta-thalassemia. We have analyzed the kinetics of globin synthesis and the levels of globin messenger (m) RNA accumulation in patients with Hb E-beta-thalassemia and Hb E trait. The initial rate of beta-globin synthesis (betaE/alpha=0.20-0.34) was less than expected on the basis of gene dosage, or comparable studies of other compound heterozygous states for beta-thalassemia and structurally abnormal beta-chains. betaE-globin synthesis was not only reduced during short-term incubations (1-5 min), but also remained relatively unchanged during long-term pulse or chase incubations up to 5h. Analysis of globin mRNA by cell-free translation and molecular hybridization confirmed that the unexpectedly low levels of betaE-globin synthesis were associated with comparable reduction in the levels of beta-globin mRNA. In Hb E-beta-thalassemia the betaA + betaE (alpha globin nRNA ratio observed were substantially lower than those obtained from reticulocytes of patients with heterozygous beta-thalassemia, or Hb S-betaO-thalassemia, while in Hb E trait, the betaA + betaE/alpha mRNA ratio was in the ranged observed for beta-thalassemia trait. The betaE-globin gene specifies reduced accumulation of betaE-globin mRNA, a property characteristic of other forms of beta-thalassemia. The beta-thalassemia phenotype associated with inheritance of Hb E is thus determined at the level of beta-globin mRNA metabolism.

Sulfonamide-induced hemolytic anemia and hemoglobin Hasharon.

A patient with Hb Hasharon had severe hemolytic anemia after several days of daily ingestion of 2 gm of sulfisoxazole. After recovery, her erythrocytes were incubated with the drug, leading to preferential oxidation and precipitation of the abnormal hemoglobin. Since carboxyhemoglobin and cyanmethemoglobin Hasharon were as stable in the heat stability test as identically liganded Hb A, we conclude that the substitution of the hydrophilic aspartate residue by histidine on the surface of the molecule at alpha47 has led by a still unknown mechanism to an interaction of hemoglobin with the drug that labilized the heme-globin bond. Since Hb Hasharon has been found in several unrelated families, the risk of drug-induced hemolytic anemia in such carriers deserves emphasis.

Usefulness of preoperative laboratory assessment of patients undergoing elective herniorrhaphy.

Preoperative laboratory utilization was evaluated in a retrospective review of 169 adults undergoing elective inguinal herniorrhaphy at a county/university hospital and at a private/community hospital. Tests monitored included a complete blood cell count, urinalysis, serum electrolytes, chest roentgenography, and electrocardiography. Abnormal results and results that altered the patients' treatment were sought. Two groups of patients were evaluated. Group 1 (n = 105) had no disease except for the inguinal hernia; group 2 (n = 64) had evidence of another disease process. Preoperative evaluation of patients in group 1 was similar at the county/university hospital and at the private/community hospital except for increased electrolyte screening at the county/university hospital among patients younger than age 40 years. No differences between hospitals were present among patients in group 2. Abnormal results not predicted by medical history or physical examination were found in 1% of patients in group 1 and 1.4% of patients in group 2. Only four patients (2%) had their treatment altered by these findings. Preoperative laboratory evaluation of these patients revealed that an abnormal test result not predicted by history and physical examination is rare. Routine preoperative laboratory testing is of little value in this patient population.

System contributions to error.

An unacceptably high rate of medical error occurs in the emergency department (ED). Professional accountability requires that EDs be managed to systematically eliminate error. This requires advocacy and leadership at every level of the specialty and at each institution in order to be effective and sustainable. At the same time, the significant operational challenges that face the ED, such as excessive patient care requirements, should be recognized if error reduction efforts are to remain credible. Proper staffing levels, for example, are an important prerequisite if medical error is to be minimized. Even at times of low volume, however, medical error is probably common. Engineering human factors and operational procedures, promoting team coordination, and standardizing care processes can decrease error and are strongly promoted. Such efforts should be coupled to systematic analysis of errors that occur. Reliable reporting is likely only if the system is based within the specialty to help ensure proper analysis and decrease threat. Ultimate success will require dedicated effort, continued advocacy, and promotion of research.

Effect of lead and ethanol upon gamma-globin synthesis in sickle reticulocytes.

There is evidence from both in vivo and in vitro studies that the synthesis of hemoglobin can be modified by posttranslational alterations in the assembly of the tetrameric molecule. Globin biosynthesis in reticulocytes of patients with sickle cell disease was studied to ascertain the effects of lead and ethanol on gamma-globin chain synthesis and hemoglobin assembly. In incubations containing lead (400 micrograms/dl) or ethanol (1.0 M) there were 86.7 +/- 139.7% and 542.7 +/- 397.0% increases in the relative synthesis of the gamma-globin chain. This was associated with a relative reduction in alpha-chain synthesis, as estimated by changes in the alpha/gamma + beta S synthesis ratio, as well as a marked reduction in total globin synthesis.

Interaction between HBS-beta-o-thalassemia and alpha-thalassemia.

We have defined the clinical and laboratory characteristics of a group of patients with HbS-beta o-thalassemia plus alpha-thalassemia, by analysis of erythrocyte indices, hemoglobin A2 and F levels, globin biosynthesis studies and alpha-globin gene mapping. Patients with HbS-beta o + alpha-thalassemia closely resembled individuals with HbS-beta o-thalassemia except for balanced globin synthesis ratios and a lower HbF level. The frequency of painful crises, leg ulceration, aseptic necrosis of bone and acute chest syndrome was similar in HbS-beta o + alpha-thalassemia patients and controls with sickle cell anemia (HbSS), HbSS-alpha-thalassemia and HbS-beta o-thalassemia. These findings are consistent with previous work which failed to show a reduction in the vaso-occlusive severity of sickle cell disease by the coexistence of alpha-thalassemia.

Beta-thalassemia intermedia with exceptionally high hemoglobin A2: relationship to mutations in the beta-gene promoter.

Small deletions of the 5' portion of the beta-globin gene that remove the promoters but stop 3' to the delta-globin gene are recognized as the sole cause of beta-thalassemia with exceptionally high hemoglobin A2 (HbA2) levels. Two patients with beta-thalassemia intermedia and exceptionally high levels of HbA2 (10.4 and 12.0%) were examined. One patient was a combined heterozygote for the -88 C----T and a novel -87 C----A mutation, while the other was homozygous for the -29 A----G beta(+)-thalassemia mutation. The remainder of the beta genes were normal. There was no evidence for deletions involving the 5' portion of the beta gene or the region between the beta and delta genes. Gene mapping studies excluded the possibility of a beta delta-anti-Lepore hemoglobin gene with beta promoters and delta coding sequences. There were no mutations in the promoters of the G gamma or A gamma-globin genes that have been associated with the hereditary persistence of HbF phenotype. The delta-globin gene promoters were normal from codon 17 to position -145 relative to the mRNA capping site. There appears to be considerable heterogeneity of HbA2 and HbF levels in patients who are homozygous or mixed heterozygotes for mutations in the TATA box and other promoter elements of the beta-globin gene. The capacity for proteolysis within the erythrocyte may vary among individuals. The authors hypothesize that in the exceptionally high HbA2 beta-thalassemia intermedia phenotype, proteolysis of superfluous alpha-globin chains is less efficient than in patients with customary levels of HbA2.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective monitoring in abdominal aortic surgery.

Monitoring via a pulmonary artery catheter has been advocated for all patients undergoing abdominal aortic surgery. This study was performed to identify a subgroup of patients who could be safely monitored with a central venous catheter. One hundred twenty-eight consecutive patients undergoing elective infrarenal abdominal aortic surgery were prospectively evaluated for risk of developing perioperative myocardial dysfunction based on criteria determined by the history and physical examination, chest radiography, and electrocardiography. Forty-five patients were identified as having no clinical evidence of coronary artery disease. These patients were then monitored perioperatively using a central venous catheter. All patients monitored via a central venous catheter underwent surgery for abdominal aortic aneurysmal disease (66.7%) or aortoiliac disease (33.3%) without intraoperative complications. There were 15 postoperative complications in 12 (26.7%) patients, comprised mainly of pulmonary (7) and gastrointestinal (3) complications. The cardiac-related morbidity was 4.4 per cent and consisted of congestive heart failure (1) and renal failure (1). No perioperative myocardial infarctions were detected. One (2.2%) postoperative death secondary to aspiration pneumonia occurred. The results of this data suggest that there exists a subgroup of patients undergoing elective infrarenal abdominal aortic surgery that can be monitored safely via a central venous catheter in the perioperative period.

beta-thalassemia-? Selected pseudogenes.

The synthesis of delta-globin is directed by a gene whose inherent characteristics permit only limited expression, a gene resembling in some respects that of beta +-thalassemia. The existence of delta O-thalassemia and the presence of delta-globin genes in this condition recall the molecular findings in most types of beta O-thalassemia. The delta-globin and beta +-thalassemia genes may be evolving pseudogenes. Those for delta O and beta O-thalassemia are, in functional sense, already pseudogenes in that they closely resemble functional genes but lack a discernible protein product. In time they should accumulate sufficient changes in their nucleotide sequences which will make them more analagous to what we now recognize as pseudogenes. the selective pressure of Falciparum malaria infection may help maintain the beta-thalassemia "pseudogene" in many populations.

Role of platelet-activating factor in skeletal muscle ischemia-reperfusion injury.

These studies indicate that PAF, a known stimulator of aggregation, secretory and/or contractile activity of platelets, neutrophils, smooth muscle and other cells, is produced by skeletal muscle during IRI. The maximum increase occurs at 10 to 15 minutes and continues for at least an hour. Infusions of PAF into skeletal muscle subjected to 20 minutes of ischemia and 20 hours of reperfusion resulted in tissue necrosis similar to that produced by 5 hours of ischemia and 20 hours of reperfusion. 25 mg/kg of pentoxifylline, infused immediately prior to reperfusion of ischemic skeletal muscle, decreased PAF production and muscle necrosis. It was also demonstrated that skeletal muscle necrosis can be reduced by infusion of PAF antagonists (WEB-2086) into the muscle immediately prior to reperfusion. Thus, PAF has an important role in skeletal muscle IRI and additional studies of PAF inhibition during IRI are warranted.

Professionalism in emergency medicine.

At its root, medical professionalism is service delivered according to patient's interest. It is essential to reinforce this notion because financial pressures threaten the integrity of the patient-physician relationship. Excessive commercialism directly contrasts the ideals of medical professionalism. This fact necessitates re-examination and reaffirmation of professional behavior. If historical standards of professionalism give way to market-driven incentives, the provision of medical care will become a commodity and the practitioners will be only agents of service delivery. Such a model not only threatens the the physician's identity, but also threatens the patient's interests. Medicine can never succeed as a transaction; it can only succeed as a partnership, a trusting exchange with patients, which is the hallmark of professionalism.

Cardiovascular effects recorded in horses during anaesthesia after treatment with trichlorfon.

Five horses were anaesthetised twice with thiopentone sodium, guaifenesin and halothane. The second anaesthesia was 16 days after the first and two days following oral administration of trichlorfon. Heart rate, carotid arterial, pulmonary arterial and right atrial pressures, cardiac output and blood temperature were measured every 15 minutes for 120 minutes. Heart rate, carotid arterial pressure and cardiac output were similar on both occasions. Pulmonary arterial and right atrial pressures were highest during anaesthesia after treatment with trichlorfon when compared with values obtained before treatment. Pulmonary vascular resistance was significantly decreased at four measurement times during anaesthesia after treatment with trichlorfon. All cardiovascular measurements were within ranges accepted as normal for halothane anaesthesia in horses. In a second experiment, four ponies were anaesthetised with xylazine and ketamine on two occasions one week apart. Two ponies received trichlorfon two days before the second anaesthesia. Heart rate, arterial pressure and respiratory rate recorded during anaesthesia were not different in ponies after organophosphate treatment. The time to standing after the second anaesthesia was significantly increased in all ponies.

Otitis media in a llama.

A 7-month-old female llama was examined because of chronic otitis media and externa of 7 months' duration. Radiographically, the tympanic bullae appeared thicker than normal, and the ventral borders were poorly defined; the left external acoustic meatus (ear canal) appeared to be narrower than the right. The llama was treated with penicillin, and the ear canals were lavaged daily. Contrast radiography was performed on day 15 to determine the shape and size of the ear canals and evaluate the integrity of the tympanic membranes. Contrast medium was visible radiographically in the left tympanic bulla, indicating that the left tympanic membrane was ruptured, but the right tympanic membrane appeared to be intact. The left ear canal was narrower than the right, and the bony ear canals had a well-defined sigmoid shape. The right ear improved with medial treatment alone, but the left ear did not. Therefore, lateral ear canal resection was performed. After surgery, however, exudate was still evident in the left ear canal, and the llama became more lethargic and more reluctant to eat. Lateral bulla osteotomy was attempted, but no purulent material was obtained, and curettage of the bulla resulted in hemorrhage. Because of this and because of the llama's poor physical condition, a decision was made to euthanatize the llama. The sigmoid shape of the bony ear canal and the multicompartmental nature of the tympanic bulla make surgical treatment of otitis media and externa in llamas difficult. Further study of surgical treatments for otitis media in llamas is needed.