Primate hemoglobins: Some sequences and some proposals concerning the character of evolution and mutation

During a multipurpose survey we examined electrophoretic mobilities of major (A, i.e., ₂₂) and minor (A₂, i.e., ₂₂) adult hemoglobins from populations of nine primate genera representing a total of 440 New World monkeys and apes. Sequences of hemoglobin chains were inferred from differences in amino acid composition between homologous tryptic peptides supplemented by detailed placement of more than 270 residues. Beta sequences were thus analyzed in five genera (Aotus, Ateles, Hylobates, Saimiri, and Saguinus) and sequences in seven (foregoing plus Gorilla and Pan). In most genera, sequences from several individuals, often from several species, were delineated. Fifteen kinds of intraspecies mutants were detected; 10 of these were precisely characterized. Five of the 15 mutants form electrophoretically detected genetic polymorphisms of ; none such occur in . Six electrophoretically detected mutants, four in and two in , are uncommon. One of these represents the complete absence of minor component. Three kinds of variants, two in and one in , are electrophoretically neutral and chance findings during sequence analysis of the equivalent of 38 allele products. Two of the neutral variants are not especially common; one may have polymorphic frequency. Several general conclusions stem from these and supplementary findings. First, comparisons of sequences suggest that and genes in all primates either arose from a single event in a common ancestor or from two approximately coincident events. Either assumption allows reconstruction of a reasonably accurate archetype sequence that is effectively common to all descendants. Second, there is a pancellular quantitative disproportion between major and minor hemoglobins ranging from 16:1 to 220:1 in species studied. Delta is consequently presumed to be functionally and adaptively less vital than . When these premises are adopted, is expected to be relatively invisible to natural selection, and, where darwinism is the principal arbiter of evolution and polymorphism, is expected to show fewer fixed changes and fewer genetic polymorphisms than . The opposite is observed. Delta exhibits as many or more changes from archetype than . This finding and the comparative abundance of polymorphism are attributed to nonadaptive factors which are thus considered the source of much evolutionary change. Third, particular sequence positions in various species are the site of recurrent mutations in both and . One such area is occupied by the majority of genetic polymorphisms found in man and other primates. The overall distribution of mutations arising in evolution is remarkably nonrandom in , , and a pool of both. These results are quite unlike most other observations in higher organisms. The sources of such nonrandomness are either selection and/or differential mutability. We rely on our prior assumption of relative selective invisibility for and, in part, ascribe the nonrandom distribution of changes to microzones of enhanced mutability. Fourth, the six uncommon electrophoretically detected mutants provide an estimate of heterozygosity (1/73) at hemoglobin loci that is tenfold greater than observed in man. Fifth, the unprecedented chance detection of three kinds of electrophoretically neutral intraspecies mutants among the equivalent of 38 characterized allele products suggests that neutral changes are as common as electrostatically active ones and at least tenfold more common than expected in extrapolation from human variant surveys. Sixth, analyses from three kinds of gibbon (Hylobates) hemoglobin suggest that one of these is a potentially unchanged relict of the ancient archetype and, further, indicate a degree of homozygous diversity within a species that nearly equals the difference between gibbon and man.This investigation received support from grants to S.H.B., HD-02508-04 and K3-GM-6308-03, from the National Institutes of Health.     

Characteristics of the endoglucanase encoded by a cel gene from Bacteroides succinogenes expressed in Escherichia coli

A cel gene from Bacteroides succinogenes inserted into the vector pUC8 coded for an enzyme which exhibited high hydrolytic activity on carboxymethylcellulose, p-nitrophenylcellobioside, and lichenan and low activity on laminarin and xylan. The enzyme was not synthesized by the Escherichia coli host when cells were cultured in complex medium containing added glucose. In the absence of added glucose, the endoglucanase and cellobiosidase activities synthesized were partitioned into the periplasmic space during growth, and practically all enzyme was located in the periplasm when the stationary phase of growth was reached. The enzyme exhibited 17- and sixfold higher Km values for the hydrolysis of carboxymethylcellulose and lichenan, respectively, than did the extracellular endoglucanase complex from B. succinogenes. The Cel endoglucanase had a pH optimum similar to that of the B. succinogenes enzyme except that the range was narrower, and the Cel endoglucanase was more readily inactivated on exposure to high temperature, detergents, and certain metals. Its activity was stimulated by calcium and magnesium. Nondenaturing polyacrylamide gel electrophoresis at different acrylamide concentrations revealed the presence of three endoglucanase components, two with molecular weights of 43,000 and one with a molecular weight of 55,000.     

Characterisation of monoclonal and polyclonal antibodies to 19-linked conjugates of testosterone with corresponding radioligands

Monoclonal antibodies to testosterone T were produced using testosterone 19-O-carboxymethyl ether (T19C) and testosterone 19-hemisuccinate (T19H) immunogens. All antibodies were characterised with iodinated derivatives of both T19C and T19H. Monoclonal antibodies derived from the T19C immunogen had similar titres and assay sensitivities with both T19-tracers. In contrast antibodies derived from the T19H immunogen bound the homologous but not the heterologous tracer. Individual antibodies showed a wide variation in cross-reactivity with 5 alpha-dihydrotestosterone, DHT (4.4-100%), androstenedione AN (0.5-100%) and progesterone, Po (0.08-5.4%). One antibody 3F11 derived from a T19C immunogen gave 50% displacement of tracer with 180 pgT/tube and low cross-reactivity of 12% with DHT, 3.0% with AN and 1.1% with Po. In general, assay sensitivity and antibody specificity were poorer with an [125I]-histamine conjugate of T-3-carboxymethyloxime than with T19 tracers. Radioimmunoassays for T in extracted human serum were developed with [125I]T19C as tracer and monoclonal antibody 3F11 (T19C immunogen) and rabbit antiserum T19H3R1 (T19H immunogen). Sensitivities of the extracted assays were 43 and 20 pg/tube respectively and results correlated well with those obtained after chromatographic separation of testosterone (r = 0.97 for both antibodies). We conclude that 19-linked derivatives of T are highly immunogenic for the production of specific testosterone antibodies. Selection of the appropriate iodinated tracer is essential to achieve optimal titre, assay sensitivity and specificity, since these characteristics vary widely with individual monoclonal antibodies, and classical bridge recognition is not observed.     

Estimation of human fetal-placental unit metabolic rate by application of the Bohr principle

The Bohr Principle via continuous indirect calorimetry was used to estimate human fetal-placental unit metabolic rate in 12 normal women undergoing elective caesarean section under continuous lumbar epidural anaesthesia. Maternal oxygen consumption decreased after umbilical cord clamping and after placental removal. Fetal-placental unit oxygen consumption was 10.7 +/- 1.3 ml/min per kg (mean +/- SEM). Fetal oxygen consumption was 6.8 +/- 1.4 ml/min per kg. Placental oxygen consumption was 37 +/- 12 ml/min per kg. Fetal-placental unit carbon dioxide production was 9.2 +/- 1.2 ml/min per kg. These mean values agree favourably with measurements of uterine and fetal metabolism from other mammalian species. Maternal minute ventilation decreased with removal of the fetal-placental unit, and this decrease was found to be linearly related to the fetal-placental unit carbon dioxide production.     

Identification of ecdysone 22-long-chain fatty acyl esters in newly laid eggs of the cattle tick Boophilus microplus.

The five major apolar ecdysone esters present in newly laid eggs of the cattle tick Boophilus microplus have been purified by h.p.l.c. The quantities of the apolar esters present in the eggs were increased by administration of ecdysone to the mature females. G.c.-m.s. analysis, as their methyl esters, of the fatty acids released from the apolar ecdysone derivatives by alkali, coupled with positive-ion fast-atom-bombardment m.s. of the intact ecdysone esters, showed that the compounds consisted of a series of fatty acyl esters of ecdysone. The position of esterification of the ecdysone was established by p.m.r. spectroscopy. The combined data show that the novel apolar derivatives of ecdysone consist of the 22-palmitate, -palmitoleate, -stearate, -oleate, and -linoleate esters respectively. Confirmation was obtained by comparison with synthetic ecdysone 22-palmitate. The significance of the ecdysone fatty acyl esters as a possible source of free hormone during embryogenesis is discussed.     

General Surgical Pre-admission Clinic

Ninety-nine patients from a non-urgent general surgical waiting list were randomly selected for either direct admission to a hospital bed or review at a preadmission clinic. A considerable reduction in subsequent bed occupancy was shown in the latter group. The findings suggest that more detailed review of patients in the outpatient department would result in the more efficient use of hospital facilities.     

Pulmonary Oil Deposition in Patients Subjected to Lymphography: Detection by Thoracic Photoscan and Sputum Examination

During clinical trials of intralymphatic therapy with radioiodinated ethiodized oil (Lipiodol Ultra-Fluid; Ethiodol) [LUF-I¹³¹] for malignant disease involving lymph nodes, significant pulmonary deposition of radioactive material was demonstrated by thoracic scan in each of five cases treated. Radioactivity was detected in sputum obtained from two cases. Induced sputum specimens were subsequently obtained from patients undergoing lymphography. Fat demonstrated in sputum was confirmed as Lipiodol in one of six patients tested. Sputum examination and use of tracer doses of LUF-I¹³¹ plus photoscanning are suggested as sensitive methods of assessing the incidence of oil deposition in the lungs of patients undergoing lymphography. Despite limitation of the volume of oil injected, monitoring of the infusion, and absence of radiographic evidence of contrast medium in the lungs, some degree of pulmonary oil deposition appears to be an inevitable result of lymphography. Further study of lung dosimetry is being undertaken by the authors before clinical usage of endolymphatic radioisotope therapy is expanded.