Primary structure of the minor haemoglobins from the sea lamprey (Petromyzon marinus, Cyclostomata)

Erythrocytes of the adult Sea Lamprey Petromyzon marinus contain several haemoglobin species, but only the main constituent has hitherto been sequenced. The present paper describes the determination of the primary structures of the two minor species, whose electrophoretic mobilities are higher and lower than that of the main component. Tryptic peptides from both chains were purified by high-performance liquid chromatography, then sequenced and aligned by homology with the main haemoglobin. The fast and the major components appeared to be very similar, differing in only four positions (pos. 5: Ser----Thr; pos. 33: Thr----Ser; pos. 86: Val----Ala; pos. 99: Gly----Arg). The slow haemoglobin component, however, differed from the main component with respect to 27 amino-acid residues. The position of the three globins of Petromyzon marinus in the phylogenetic tree of haemoglobins is discussed and a relationship with primitive alpha-chains is postulated.     

Conservation of peculiar structural properties by the hemoglobins of anguilloid eels (Teleostei)

The three species investigated, the European eel, Anguilla anguilla (L.), the Mediterranean moray, Muraena helena L., and the conger eel, Conger coner (L.), represent three different superfamilies of the suborder Anguilloidei (infradivision Elopomorpha). Their hemoglobin systems show peculiar structural properties, which distinuish them from all other teleost species studied. They present acidic and basic components differing greatly in their isoelectric points; the basic components have the highest isoelectric points detected in teleost hemoglobins. While there is one major basic component, multiplicity is present in the acidic components in Muraena and Conger. The polyeptides of the acidic comonents show the same electrophoretic mobility in 8 M urea. In the three species, the electroploretic mobility with urea-SDS of the hemoglobin polypeptides shows a shorter β polypeptide in the basic components. These features had never been investigated among Elopomorpha, and are likely to be phylogenetically relevant.     

Comparative study of the erythrocytes and haemoglobins in nototheniid fishes from Antarctica

The blood of seven Antarctic nototheniid species and representatives from three other families contained low haemoglobin concentrations compared with non-polar marine teleosts. Haematocrit values were slightly lower than values from other teleosts, while haemoglobin and erythrocyte counts were substantially reduced by comparison. Interspecific variation in haemoglobin concentration seemed to be a function of activity level. Mean corpuscular haemoglobin concentration of all Antarctic species was strikingly low by comparison with species from lower latitudes and was not correlated with the habits of the species. Haemoglobin componentry was compared using celluslose-acetate electrophoresis and, unlike many temperate species, only one major haemoprotein was isolated from each benthic species, but four components were evident in the pelagic species Trematomus borchgrevinki . The possible functional significance of these findings was discussed in relation to the ecology of each species.     

The natural distribution in angiosperms of anthocyanins acylated with aliphatic dicarboxylic acids

An electrophoretic survey of mainly floral tissues of 282 species from 68 families showed the presence of zwitterionic anthocyanins in 38% of the species and in 30% of the families. Families recognised for the first time as having such pigments include the Alliaceae, Gramineae, Orchidaceae and Ranunculaceae. Families where they are rare or absent include the Boraginaceae, Geraniaceae, Iridaceae, Onagraceae and Umbelliferae. Acylating acids so far identified are malic, malonic and succinic and of these three, malonic seems to be the most common. The significance of this acylation appears to be related to the stabilization of anthocyanins in the acidic environment of the cell sap.     

3 types of sperm proteins in eukaryotes

Basic spermal proteins of various species of hydrobionts attributed to Pisces and Cephalopoda are studied. It is established that chromatin of nine species referring to two Cypriniformes families includes the somatic histones. Histone H1 of Cypriniformes is attributed to the lysine-rich type histones and contains 35% mol. of lysine and 0.7% mol. of tyrosine. Chromatin of 14 species of fish referring to nine families of the percoid fish superorder includes protamines similar to salmin, a typical protamine of salmon. The amino acidic analysis of protamine from the sandre sperma has shown that it contains 59% mol. of arginine and no tyrosine. Chromatin of three species from squid superorder referring to Cephalopoda includes gametones -- proteins differing from histones and protamines both in the electrophoretic mobility and amino acidic composition (75% mol. of arginine, 3% mol. of tyrosine).     

Some biochemical similarities and differences between two jack mackerel species, Trachurus declivis and T. novaezelandiae.

1. Six proteins, haemoglobin (two loci), serum esterase (two loci) and lactate dehydrogenuase (two loci) from two species of jack mackerel, Trachurus declivis and T. novaeselandiae, were compared electrophoretically. All six loci were found to be monomorphic. 2. LDH from red and white skeletal muscle tissue were electrophoretically different, but the red form and the white form were found to be identical in both species. 3. Both species share an electrophoretically identical serum esterase, but each species also has an electrophoretically distinct serum esterase. 4. Both species share an electrophoretically identical haemoglobin, but each species also has an electrophoretically distinct haemoglobin. 5. The otolith iron contents of the two species were compared and found to be identical.     

Biochemische und cytogenetische Untersuchungen zur Natur des Hämoglobin-Polymorphismus bei Chironomus tentans und Chironomus pallidivittatus

The haemoglobin of chironomids is dissolved in the body fluid of the larvae and can be separated electrophoretically in Chironomus tentans into 10, and in C. pallidivittatus into 8, different bands. The molecular weight determined under the electrophoretic conditions was 15,000±1,000 for each haemoglobin band. This means that each haemoglobin band represented a single protein chain. In each species 7 haemoglobins could be characterised as species specific according to their different electrophoretic mobilities, developmental characteristics and the fact that one haemoglobin could be correlated genetically with a specific chromosome inversion. The inheritance of all these species specific haemoglobins was found to be co-dominant. With cytogenetic methods it was possible to define the loci of the species specific haemoglobin genes as being restricted to certain parts of chromosome 3. This finding suggests gene duplication as the most likely mecanism of the evolution of haemoglobins in Chironomus.     

Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH

The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.     

Molecular aspects of embryonic mouse haemoglobin ontogeny.

Embryos from C57BL/6J mice between the gestational ages of 9 and 16 days possess three embryonic haemoglobins EI, EII and EIII, the proportions of which change as a function of gestational age. Component EI, originally present at approx. 65% at day 9, decreases to approx. 20% by day 16, while component EII increases in an inverse manner to that of component EI. During this period component EIII remains essentially constant at approx. 25%. Separation of these species by ion-exchange chromatography has allowed the characterization of the Hill coefficient, Bohr effect, heat of oxygenation and binding of allosterically active organic phosphates for each component. The three components show marked functional heterogeneity and also differ from maternal haemoglobin. Oxygenation curves for whole embryonic blood show distinct deviations from simple binding behaviour. The presence of a high-affinity component within the blood samples may be accounted for by the presence of haemoglobin EI. By using parameters obtained from the study of the isolated components it has been possible to synthesize mathematically the O2-binding curves, obtained experimentally, throughout the gestational period. The characteristics of the isolated haemoglobin components of embryonic mouse blood are discussed in terms of the changing demands for O2 likely to be encountered by the developing embryo.     

Snake relationships revealed by slow-evolving proteins: a preliminary survey

We present an initial evaluation of relationships among a diverse sample of 215 species of snakes (8% of the world snake fauna) representing nine of the 16 commonly-recognized families. Allelic variation at four slow-evolving. protein-coding loci, detected by starch-gel electrophoresis, was found to be informative for estimating relationships among these species at several levels. The numerous alleles detected at these loci [ Arp -2 (42 alleles). Ltlh -2 (43), Mdh -1 (29), Pgm (Z)] provided unexpected clarity in partitioning these taxa. Most congeneric species and several closely-related genera have the same allele at all four loci or differ at only a single locus. At thc other extreme are those species with three or four unique alleles; these taxa cannot be placed in this analysis. Species sharing two or three distinctive alleles are those most clearly separated into clades. Typhlopids, pythonids, viperids, and elapids were resolved into individual clades. whereas bods were separated into boincs and erycines, and colubrids appeared as scveral distinct clades (colubrines, natricines, psammophines, homalopsines, and xenodontines). Viperids were recognized as a major division containing three separate clades: Asian and American crotalines. Pabearctic and Oriental viperines, and Ethiopian causines. The typhlopids were found to be the basal clade, with the North American erycine boid Chrrrino and the West Indian woodsnakes Tropidophi, Y near the base. A number of species and some small clades were not allocated because of uninformative (common, unique, or conflicting) alleles. Of the 21 S species examined, five to eight appear to have been misplaced in the analysis of these electrophoretic data.     

Use of specific monoclonal and polyclonal antibodies to define distinct antigens of the porcine zonae pellucidae

Specific monoclonal and polyclonal antibodies to solubilized porcine and rabbit zonae pellucidae (ZP) and to purified ZP glycoprotein components have been used to define distinct ZP antigens. These studies demonstrate that the individual ZP glycoproteins contain both unique and shared determinants. One monoclonal antibody (R5) has been used to demonstrate that the major porcine ZP glycoprotein, which has multiple charge species ranging in molecular weight from 42,000 to 120,000, is composed of two distinct polypeptide antigens unique to this glycoprotein class. These distinct antigens can be differentiated by immunoblotting after high-resolution two-dimensional polyacrylamide gel electrophoretic separation of trypsin-treated or deglycosylated glycoproteins. The two polypeptides also differ in their staining properties with the silver-based color stain and in their susceptibility to proteolysis. A second monoclonal antibody (PSI) has been used to define a determinant shared by all three major porcine ZP glycoprotein classes. This determinant appears to involve either a carbohydrate moiety or some other molecular feature related to post-translational modification, since the antibody recognizes only the acidic species of each glycoprotein class, and does not recognize the deglycosylated forms of the proteins. This work demonstrates that there are both unique and shared antigenic determinants present in the individual components of the ZP, but that the immunodominant determinants appear to be unique to each glycoprotein.     

ELECTROPHORETIC CHARACTERIZATION OF BASIC PROTEINS IN ACID EXTRACTS OF CENTRAL NERVOUS SYSTEM TISSUE

A technique has been outlined for identification of myelin basic proteins in mixtures of CNS proteins. Myelin basic proteins can be recognized easily by high cathodic mobility at low pH, a unique electrophoretic pattern exhibited at high pH and a characteristic colour when complexed with Amido black. The major protein extracted at pH 3·0 from either brain or spinal cord is myelin basic protein. In the low pH electrophoretic pattern of these extracts it is the most conspicuous component and the component migrating farthest cathodically; it does not appear in comparable electrophoretic patterns of liver extracts. Guinea pig myelin basic protein appears as a single dense blue-green band in low pH electrophoretic patterns, in contrast to the other proteins which are stained greyish-blue or greyish-purple by Amido black. The pattern of rat myelin basic protein is similar except that it consists of a pair of dense blue-green bands. A third characteristic which facilitates the identification of myelin basic proteins in mixtures is a considerable cathodic mobility and electrophoretic heterogeneity at pH 10·6. Most other basic CNS proteins barely penetrate the gel at this pH. We have also examined in detail the behaviour of two other components of pH 3·0 extracts which migrate close to myelin basic protein at low pH. Both are present in pH 3·0 extracts of liver and brain but not of spinal cord, and both stain grey instead of blue-green, a characteristic which readily distinguishes them from myelin basic protein. Neither of these components affects the characteristic pattern of microheterogeneity observed in high pH electrophoretograms of myelin basic proteins. One of these components has been purified and tentatively identified as lysine-rich histone F1.     

Amelogenins. Purification and partial characterization of proteins from developing bovine dental enamel

1. Procedures are described for the purification of amelogenin electrophoretic components and their analysis for homogeneity by polyacrylamide-gel electrophoresis at both acidic and alkaline pH values. 2. Most of these components belonged to two main groups, termed the J group and the C group after their major electrophoretic components. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis indicated that, within each group, proteins were of similar size, but the C-group proteins were larger than those of the J group. 3. By sedimentation-equilibrium ultracentrifugation and amino acid analysis, the four J-group components were found to be very small proteins (mol. wt. 5500-3000) and, except for one, similar in amino acid composition. The components of the C group were found to be proteins of moderate size (mol. wt. 16800-16100) with very similar amino acid compositions. A third minor amelogenin group of intermediate size was also found, but not further analysed. Details of the results of the ultracentrifuge studies are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50014 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. 4. Two of the J-group components were similar to amelogenins isolated by other workers. 5. All amelogenins analysed were rich in proline, glutamic acid, histidine and methionine, and contained no half-cystine. Their amino acid compositions, combined with their molecular weights, serve to distinguish the amelogenins from both collagens and keratins.     

The genetics of an electrophoretic variant of an erythrocytic protein in the house mouse (Mus musculus)

An electrophoretic mobility variant of a protein band anodal to haemoglobin in alkaline starch gels was found in wild house mouse populations in South Australia. Most wild mice and all inbred lines examined are homozygous for the Erp-1 ^a allele at the locus controlling this variation. The rare allele Erp-1 ^b, which has a frequency of about 1% in South Australia, produces a protein band of slower mobility in alkaline gels. Homozygotes show one protein band whilst the heterozygote has three bands. The Erp-1 protein does not appear to be haemoglobin or carbonic anhydrase. The Erp-1 locus is closely linked to Es-1 on chromosome 8 with a recombination fraction of 6.04 ± 1.32%.     

The haemoglobins of the fish Sarotherodon mossambicus (Peters): functional significance and ontogenetic changes

The haemoglobin patterns of adult and young larval stages of the fish species Sarotherodon mossambicus were determined by polyacrylamide–gel electrophoresis. Oxygen affinity determinations of the adult and larval haemolysates and of each haemoglobin component of the adult indicates that the affinity changes with the pH, temperature and osmotic pressure. The possible evolutionary significance of the multiple haemoglobins is discussed. The electrophoretic pattern of adult S. leucostictus , a species closely related to S. mossambicus was also determined and used as a comparative study.     

The Three Cortical Membranes of the Gregarines (Parasitic Protozoa). III. Comparative Studies of the Membrane Proteins Among Different Sporozoan Species During Their Vegetative Phase1

ABSTRACT. The cortical membrane proteins of three gregarine and one coccidian species were compared using sodium dodecyl sulfate/polyacrylamide-gel electrophoresis. About 30 proteins were identified in the ghosts of Gregarina blaberae and G. garnhami and 20 in G. rhyparobiae ghosts and Sarcocystis tenella pellicles. No protein with the electrophoretic mobility of muscular actin was present in the ghosts of the sporozoan species under study. Each species possessed a characteristic electrophoretic pattern; no protein was present simultaneously in the four sporozoan species and only one protein band with a similar electrophoretic mobility was found in the three gregarine species (52 Kd protein). Two G. garnhami subpopulations living in Locusta migratoria and Schistocerca gregaria exhibited the same ghost protein pattern. Thus, large differences were observed between species and not within species, and the protein electrophoretic analysis appears to be a powerful tool for taxonomic investigations in gregarines. Gregarina blaberae and G. garnhami glycoconjugates were compared after periodate/Schiff staining of the polyacrylamide gel slabs. Several glycoconjugates were reported to belong to the cytoplasmic fraction; and, in view of cytochemical and ultrastructural data, a contribution of these glycocomponents to the secretion of a mucus is discussed.     

Distribution of Iron-Containing Granules in Lampreys,with Particular Reference to the Southern Hemisphere Species Geotria australis Gray

Perl's Prussian blue and bathophenanthroline have been used to investigate the sites where iron-containing granules are found within representatives of each of the three extant families of lampreys. Amongst the larvae, granules were present in the columnar cells of the posterior intestine in small amounts in some but not all northern hemisphere species (Petromyzonidae), and in moderate and very large amounts in the southern hemisphere families Geotriidae and Mordaciidae respectively. Granules were only found in large numbers elsewhere in the body of larvae of the Geotriidae, in which they were predominant in the adipose tissue of the nephric fold and fat column. It is suggested that these large iron deposits reflect the intake of an unusually large amount of iron, a feature which in turn could be related to the relatively high plasma iron levels and haemotocrit found in larvae of the Geotriidae. During metamorphosis, iron deposits accumulate in the liver of all species as haemoglobin is broken down.     

Biochemical genetic markers to identify two morphologically similar South African Mastomys species (Rodentia: Muridae)

The two common southern African mice species (Mastomys coucha and M. natalensis) are morphologically almost identical, making field identification impossible at present. Specimens from two localities were collected and tissue and blood samples taken. The habitat type of each locality was studied, and a distribution map compiled. A definite correlation between biome-type and species range was found to be present. Three isozyme markers were identified: glucose phosphate isomerase in liver, and two general (non-specific) protein coding loci in muscle. In addition, we also identified species characteristic haemoglobin components in both species. This is the first study to report genetic variation within, and differentiation between these species. Our results are of medical importance because Mastomys coucha carries bubonic plague and M. natalensis carries Lassa Fever.     

Expression,Characterization, and Cellular Localization of Knowpains,Papain-Like Cysteine Proteases of the Plasmodium knowlesi Malaria Parasite

Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries against knowpains for developing broadly effective compounds active against multiple human malaria parasites.     

The development of two flanking SCAR markers linked to a sex determination locus in Salix viminalis L

Most studies of sex determination systems in plants involve dioecious annuals that have known sex chromosomes. Despite the absence of such structures in the majority of dioecious plants, gender seems to be under relatively strict genetic control in some species. Genetic markers linked to a female sex-determination locus in Salix viminalis L. have been discovered through bulked segregant analysis of three full-sib families using approximately 1,000 arbitrary primers. Two RAPD markers that were present in the common female parent as well as in predominantly female progeny of these families were subsequently sequenced and converted to sequence characterized amplified region (SCAR) markers. The two SCAR markers are correlated with gender in the three full-sib families and are present in 96.4% of the female progeny and 2.2% of the males, providing evidence of linkage to a putative female-specific locus associated with gender determination in S. viminalis. Estimates of recombination suggest that the two markers are flanking a putative sex determination locus, SDL-II, in certain families of S. viminalis.