Purification and properties of the hemoglobins of the platyhelminth Isoparorchis hypselobagri (Trematoda: Isoparorchidae) and its host Wallagu attu (catfish).

1. The hemoglobins of the trematode Isoparorchis hypselobagri and of its host Wallagu attu (catfish) were isolated and purified. 2. SDS-polyacrylamide gel electrophoresis showed both to consist of single, 15-17 kDa chains, having different electrophoretic mobilities. 3. Isoelectric focusing showed the trematode hemoglobin to be homogeneous with a pI of 4.2 and the host hemoglobin to consist of several components. 4. Gel filtration of freshly prepared trematode hemoglobin revealed one peak corresponding to M(r) approximately 17 kDa; gel filtration of a preparation which had been stored for 2-3 months demonstrated the presence of two peaks, whose elution volumes corresponded to M(r) of ca 35 and 17 kDa, respectively. 5. Reversed-phase chromatography of carboxymethylated 35 and 17 kDa peaks on a C8 column, gave a single peak a and two peaks b and c, respectively. 6. Edman degradation of peaks a, b and c obtained provided identical sequences of 27 amino acid residues for peaks a and c and another sequence differing at 10 of the 27 positions, for peak b. Edman degradation of the freshly prepared Isoparorchis hemoglobin provided the first 15 amino acid residues found for peaks a and c. The host hemoglobin gave an N-terminal sequence completely different from the trematode sequences. 7. Since gel filtration of the 35 and 17 kDa peaks showed no sign of an interconversion equilibrium, it appears that the 35 kDa peak and peak a represent a disulfide-bonded dimer of a monomer globin chain which shares the 27 N-terminal residues with chain c, the major monomer globin component of the 17 kDa peak.(ABSTRACT TRUNCATED AT 250 WORDS)

The amino acid sequence of hemoglobin III from the symbiont-harboring clam Lucina pectinata.

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clam Lucina pectinata consists of 152 amino acid residues, has a calculated Mm of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66. Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnson et al., J. Prot. Chem. 10, 609-622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 A of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the two Lucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the two Lucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36-49% when compared to the two groups of mollusc globins. The highest homology appears to exist between the Lucina globins and the cytoplasmic hemoglobin of Busycon canaliculatum.

Bicycle helmet law for children: a case study of activism in injury control.

In 1990, the first regulation requiring the use of helmets for bicyclists younger than 16 years of age was passed in Howard County, Maryland. This unexpected injury control measure resulted from the convergence of multiple factors and efforts: the bicycle-related deaths of two children from the same middle school, creative students and teachers motivated by these deaths, a responsive legislator to introduce the legislation, available surveillance and research statistics supporting the need and efficacy for helmet use, increased national awareness of the importance of helmet use to prevent bicycle-related head injuries, and organized national and local public health groups to support the legislation. This case study of activism in injury control illustrates the importance of supporting research, of well-organized public health coalitions and groups, and of creative community activists motivated by local circumstances.

Comparison of the hemoglobins of the platyhelminths Gastrothylax crumenifer and Paramphistomum epiclitum (Trematoda: Paramphistomatidae).

1. Gastrothylax crumenifer and Paramphistomum epiclitum parasitize the water buffalo Bubalus bubalis. 2. Gastrothylas hemoglobin consisted of two fractions of ca 30,000 and ca 18,000 by gel filtration. SDS-electrophoresis showed both to be single, ca 15,000 chains. 3. Paramphistomum hemoglobin was ca 16,000 by both gel filtration and SDS-electrophoresis. 4. Reversed-phase chromatography of carboxymethylated trematode and buffalo globins gave single peaks and two peaks, respectively. Although Paramphistomum hemoglobin provided and N-terminal sequence, Gastrothylax hemoglobin did not, suggesting blocked N-terminals. The buffalo sequences were found to be identical to the sequences of the alpha and beta chains of bovine hemoglobin. 5. Although Paramphistomum hemoglobin consists of only one chain, Gastrothylax hemoglobin consists either of one chain which aggregates to a dimer or of two different chains, only one of which aggregates to a dimer.

Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii.

The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaete Eudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27.5 nm and height of 18.5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass (Mm) of 3480 +/- 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0.251 +/- 0.021 wt%, corresponding to a minimum Mm of 22.4 kDa. Mass spectra and nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of approximately 30 kDa and five chains of approximately 16 kDa; the two types of subunits occur in the ratio 0.26:0.74(+/- 0.08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the approximately 16 kDa chains and fraction D consisted of the approximately 30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C provided Mm of 208 +/- 23 kDa and 65 +/- 12 kDa, respectively, in agreement with 191 +/- 13 kDa and 67 +/- 5 kDa obtained by gel filtration. Particles with Mm = 221 +/- 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the approximately 30 kDa linker subunits that have 0.26 to 0.47 heme groups/chain, comprises approximately 65 kDa tetramers and approximately 200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 +/- 4 and 43 +/- 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 +/- 14 Ca2+/mol protein, and was decreased to approximately 35% by the presence of 10 to 25 mM-Ca2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2.5 to 5 mM-Ca2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca2+ exhibited very limited (less than 10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

The amino acid sequence of hemoglobin II from the symbiont-harboring clam Lucina pectinata.

The cytoplasmic hemoglobin II from the gill of the clam Lucina pectinata consists of 150 amino acid residues, has a calculated Mm of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J., J. Biol. Chem. 265, 16054-16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of the Lucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 A of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 in Lucina Hb II.

The cDNA sequences encoding two components of the polymeric fraction of the intracellular hemoglobin of Glycera dibranchiata.

The intracellular hemoglobin of the polychaete Glycera dibranchiata consists of several components, some of which self-associate into a "polymeric" fraction. The cDNA library constructed from the poly(A+) mRNA of Glycera erythrocytes (Simons, P. C., and Satterlee, J. D. (1989) Biochemistry 28, 8525-8530) was screened with two oligodeoxynucleotide probes corresponding to the amino acid sequences MEEKVP and AMNSKV. Each of the two probes identified a full-length positive insert; these were sequenced using the dideoxynucleotide chain termination method. One clone was 630 bases long and contained 36 bases of 5'-untranslated RNA, a reading frame of 441 bases coding for the 147 amino acids of globin P2 including the residues MEEKVP, and a 3'-untranslated region of 153 bases. The other clone was 540 bases long and contained 24 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for globin P3 including the residues AMNSKV, and a 3'-untranslated region of 75 bases. The inferred amino acid sequences of the two globins were in agreement with the partial amino acid sequences obtained by chemical methods. The P2 and P3 globin sequences, together with the previously determined P1 sequence of a complete insert and partial sequences P4, P5, and P6 obtained from partial inserts (Zafar, R. S., Chow, L. H., Stern, M. S., Vinogradov, S. N., and Walz, D. A. (1990) Biochim. Biophys. Acta, in press) suggest that there are at least six components in the polymeric fraction of Glycera hemoglobin, which is in agreement with the results of polyacrylamide gel electrophoresis in Tris/glycine buffer, pH 8.3, 6 M urea. Nothern and dot blot analyses of Glycera erythrocyte poly(A+) mRNA using the foregoing two cDNA probes clearly demonstrated the presence of mature messages encoding both types of globins. Comparison of the polymeric sequences P1, P2, and P3 with the "monomeric" globins M-II and M-IV using the alignment and templates of Bashford et al. (Bashford, D., Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196, 199-216) showed that all five globins have identical residues at 39 positions. At 44 positions, the three polymeric globins share identical residues that differ from the identical residues at the corresponding locations in the monomeric sequences M-II and M-IV including position E7, where the latter have leucine instead of the distal histidine. At 15 positions, there occurs an alteration from polar to nonpolar or from a small nonpolar to a larger nonpolar residue in going from the monomeric to the polymeric globins.(ABSTRACT TRUNCATED AT 400 WORDS)

The heterogeneity of the polymeric intracellular hemoglobin of Glycera dibranchiata and the cDNA-derived amino acid sequence of one component.

The erythrocytes of the marine polychaete Glycera dibranchiata contain a number of different, single-chain hemoglobins, some of which self-associate into a 'polymeric' fraction. An oligodeoxynucleotide probe was synthesized based on partial amino acid sequences determined by chemical methods, and used to screen a cDNA library constructed from the poly(A+)mRNA of Glycera erythrocytes (Simons, P.C. and Satterlee, J.D. (1989) Biochemistry 28, 8525-8530). The longest positive inserts found were sequenced using the dideoxy nucleotide chain termination method. One complete clone was obtained: clone 5A, 816 bases long, contained 59 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for 147 amino acids and a 3'-untranslated region of 316 bases. The derived amino acid sequence of Glycera globin P1 was in agreement with the partial amino acid sequences obtained by chemical methods. Three additional inserts obtained in the screening were also sequenced: the inferred amino acid sequences proved to be partial globin sequences which were different from each other and from the sequence of P1. Thus, the 'polymeric' fraction of the intracellular hemoglobin of Glycera probably consists of at least four different globin chains much like the 'monomeric' fraction. Comparison of the 'polymeric' sequence with the two known 'monomeric' sequences, M-II and M-IV, shows that they share 54 identical residues. At 74 positions, the identical residues in M-II and M-IV differ from the corresponding residue in P1, including at E-7, where P1 has a distal His, in contrast to Leu in M-II and M-IV. The alignment of Bashford et al. ((1987) J. Mol. Biol. 196, 199-216) and their templates were used to examine the principal differences between the two types of Glycera globin sequences. They appear to consist of uncommon surface amino acid residues at positions C6 (Phe vs. Ala), E10 (Val vs. Lys), E17 (Lys vs. Val), G1 (Arg vs. Lys), G10 (Met vs. Ala) and H5 (Arg vs. Lys). One or more of these residues could be responsible for the self-association exhibited by the 'polymeric' Glycera globins.

Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex.

The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide-bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16,286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 75 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human beta-globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys-Val-Lys at positions 9-11 [Gotoh et al. (1987) Biochem. J. 241, 441-445]. The two cysteine residues, at positions 2 and 131, appear to be disulfide-bonded.

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora.

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)

Mental health care of black clients in two types of healthcare organizations.

This article compares the characteristics of black clients and their mental health treatment in two types of treatment facilities: an outpatient clinic of a community mental health center and a comprehensive medical facility. The comprehensive medical facility provides short-term case management to the less severely ill, while the community mental health center provides longer-term, individual psychotherapy and medications to those with more serious diagnoses. Given these findings and the current shift from traditional health insurance to prepaid healthcare, the evolving role of the community mental health center is discussed.

Diagnosis of a posterior fossa tumour by magnetic resonance imaging.

The application of magnetic resonance imaging as a diagnostic tool for diseases of the central nervous system is described in the case of a 17-year-old girl who presented with neurological symptoms in January 1986. A comparison is made with the results of computed tomography in the diagnosis of the disease, which proved to be a grade-III glioma that appeared as a lesion of the posterior fossa. The patient recovered uneventfully after radiotherapy. It is suggested that MRI should be considered seriously as a first line of investigation in any suspected lesion of the posterior fossa.

Aspartic acid-121 functions at the active site of bovine pancreatic ribonuclease.

The fully active semisynthetic enzyme formed by the non-covalent interaction of residues 1-118 of bovine pancreatic ribonuclease and a synthetic tetradecapeptide containing residues 111-124 of the enzyme has allowed a direct test of the role of aspartic acid-121 in the functioning of the molecule. Replacement of this residue by asparagine results in a derivative that is 4.5% active against cytidine 2',3'-cyclic phosphate at pH 7.0 under standard assay conditions. Further studies with the same substrate at pH 5.8 reveal that the reduced activity results entirely from a diminished catalytic efficiency and not from a decreased affinity for substrate.

The clinical work of the psychiatrist in a community mental health center.

The difficulties community mental health centers have in recruiting and retaining psychiatrists may be related to the type of clinical work psychiatrists perform in the centers. The authors conducted a study examining how and by whom a sample of 87 clients in one mental health center were treated. The findings showed that the psychiatrists tended to treat older, poorly educated, and more severely disturbed patients. Their patients were more likely to receive drug therapy and less likely to receive individual psychotherapy than were the patients of other staff members. The authors conclude that most psychiatrists find the centers to be unattractive work settings because the work differs from what they were trained to expect, to value, and to perform.