Characterization of indigenous fat-tailed and fat-rumped hair sheep in Kenya: diversity in blood proteins.

This paper reports the variation in five blood proteins from five populations of sheep found in Kenya. Blood samples were collected from a total of 309 adult sheep of both sexes in Kwale, Makueni and Kakamega districts for the fat-tailed sheep and in Isiolo district for the fat-rumped hair sheep. Fine-wooled Merino sheep were used in this study as the reference population. Transferrin, esterase-A and esterase-C were polymorphic in all the populations investigated, while albumin was monomorphic for the S allele in the fat-tailed sheep and haemoglobin was fixed for the B allele in the Kwale, Makueni and Isiolo populations. Phylogenies derived from the pairwise genetic distance estimates showed a clear separation between the indigenous sheep populations and the exotic Merino. However, the topology of the former showed rather poor consistency with their morphological classification based on the localization of their fat deposits, namely fat-tailed or fat-rumped hair sheep.

Centric fusion polymorphisms in waterbuck (Kobus ellipsiprymnus).

Twenty-six captive individuals of the ellipsiprymnus subspecies group of Kobus ellipsiprymnus were found to have chromosomal complements of 2n = 50-52 (FN = 61-62), and 26 of the defassa subspecies group, including three specimens from Lake Nakuru National Park, Kenya, had complements of 2n = 53-54 (FN = 62). G-banded karyotypes that were numbered according to the standard karyotype of Bos taurus revealed that variation in diploid number was the result of polymorphism for two independent centric (Robertsonian) fusions. The ellipsiprymnus group was polymorphic for a 7;11 centric fusion. Both elements of chromosome pairs 7 and 11 were fused in fusion homozygotes (2n = 50); in fusion heterozygotes (2n = 51), only one element of each pair was fused. The 7;11 fusion was lacking in specimens with 2n = 52. The defassa group was polymorphic for a 6;18 centric fusion; individuals were either heterozygous for the fusion (2n = 53) or lacking it (2n = 54). There were no defassa group individuals that were homozygous for the 6;18 fusion (2n = 52), but this may be a sampling artifact. The 6;18 fusion was fixed in the ellipsiprymnus group, whereas the 7;11 fusion was absent in the defassa group. In G- and C-banded karyotypes, all autosomal arms and the X chromosomes of the two subspecies groups appeared to be completely homologous. However, the Y chromosome was acrocentric in the ellipsiprymnus group and submetacentric in the defassa group, possibly the result of a pericentric inversion. Fixed chromosomal differences between the two subspecies groups reflect a period of supposed geographic isolation during which time they diverged genetically and phenotypically, and the centric fusion polymorphisms raise the possibility of reduced fertility in hybrids. These data, in conjunction with phenotypic and mitochondrial DNA data, suggest to us that populations of the ellipsiprymnus and defassa groups should be managed separately.

Extreme genetic differences among populations of Gazella granti, Grant's gazelle in Kenya.

Mitochondrial DNA (mtDNA) control region sequences from six Kenyan Grant's gazelle (Gazella granti) populations were highly divergent among locations. Neighbouring populations not separated by geographical or vegetational barriers exhibited and nucleotide sequence divergence about 14 per cent. A similar level of divergence separates Grant's gazelles from a closely related species, the Soemmering's gazelle (G. soemmeringii). Nuclear microsatellite repeat number variation at two loci also indicated substantial population genetic differentiation. Despite high levels of sequence divergence populations of Grant's gazelles were more closely related to each other than to Soemmering's and Thompson's gazelles (G. thomsoni) as measured by nucleotide sequence divergence at the mtDNA protein coding cytochrome b gene and the nuclear alpha-lactalbumin gene. This pattern of extensive differentiation is hypothesized to have resulted from recently established contacts between formerly allopatric populations.

Characterization of a native and recombinant Schistosoma haematobium serine protease inhibitor gene product.

Immunologic screening of a Schistosoma haematobium cDNA library with species-specific human antisera identified a clone whose predicted amino acid sequence encodes a member of the serine protease inhibitor (serpin) gene family. This cDNA consists of 1397 bp with a single open reading frame that can encode a 409-amino acid protein of 46,261 Da. The native antigen is a 54-58-kDa glycoprotein and is located on the surface of adult worms. Sequence comparison with other serpins predicts the amino acid Phe at the putative reactive center of the protein. Phenylalanine is also found at the corresponding site of a vaccinia serpin that may contribute to the hemorrhagic phenotype of some strains of cowpox virus. Though the human parasites S. haematobium and Schistosoma mansoni demonstrate a close antigenic relationship, the S. haematobium serpin gene product demonstrates marked species-specific immunogenicity. By Northern blot hybridization, however, both species express a 1700-nucleotide mRNA that hybridizes with the S. haematobium serpin cDNA. The intensity of cross hybridization for the S. mansoni mRNA is 10-fold lower than that for S. haematobium. Southern blots of genomic DNA and gene titration experiments indicate that the S. haematobium gene is present in approximately 4-5 copies per haploid genome.

Inhibition of protein synthesis results in super-induction of procyclin (PARP) RNA levels.

Procyclin is an abundant surface antigen found exclusively on the procyclic forms of African trypanosomes. We are interested in the induction of procyclin gene expression during differentiation from bloodstream forms. We find that increased levels of procyclin RNA are evident as early as 15 min after triggering differentiation. The increase in procyclin RNA levels requires the temperature shift from 37 degrees C to 27 degrees C and is aided by addition of the tricarboxylic acid cycle intermediate cis-aconitate. Maximal induction is observed with a combination of three triggers of differentiation: citrate, cis-aconitate and the temperature shift. Protein synthesis does not appear to be required for induction of procyclin RNA during differentiation. In fact, addition of protein synthesis inhibitors results in super-induction of procyclin RNA levels, even under conditions where no induction is normally observed (i.e., at 37 degrees C in the absence of citrate and cis-aconitate). This super-induction was observed with four different protein synthesis inhibitors that affect different stages of translation. Thus, the accumulation of procyclin transcripts may be under the control of a negative regulator whose effective levels are reduced during differentiation from bloodstream to procyclic forms.

Identification of two integral glycosomal membrane proteins in Trypanosoma brucei.

Glycosomal membranes of bloodstream form Trypanosoma brucei were purified to apparent homogeneity by sodium carbonate treatment and found to contain two major integral membrane proteins of 26 kDa (band 10) and 24 kDa (band 11). The procyclic-form glycosomal membranes isolated by the same procedure also resulted in a homogeneous preparation, but each piece of membrane thus purified was associated with an electron-dense granule. Procyclic membranes also contained primarily bands 10 and 11. These two proteins were selectively reduced by protease treatment of intact glycosomes, suggesting surface exposed domain(s) of the two proteins accessible to proteolytic digestion. They and band 8 protein also vanished in glycosomal lysates by apparent proteolysis, implying the presence of a specific protease which degrades the integral membrane proteins upon the loss of membrane integrity. The two proteins, hydrophobic in nature and apparently unglycosylated, have no known biological functions, but their possible involvement in translocating precursor proteins from the cytoplasm into the glycosome of T. brucei is postulated.

An improved purification of glycosomes from the procyclic trypomastigotes of Trypanosoma brucei.

The glycosomes of in vitro grown procyclic trypomastigote forms of Trypanosoma brucei were purified by three different procedures and the results compared by electron microscopy, enzyme assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Centrifugation on a self-forming Percoll gradient followed by a sucrose gradient centrifugation resulted in the least enriched glycosomal preparation. Centrifugation on a pre-formed Nycodenz gradient gave an improved preparation but the most homogeneous preparation of intact glycosomes was obtained after centrifugation on two successive sucrose gradients. Glycosomes purified by both the Nycodenz and double sucrose gradient procedures appeared larger than in situ glycosomes presumably due to an osmotic effect resulting from disruption of the granular matrix of the organelles. Nevertheless, there appears to be no loss of cisternal contents due to the swelling of the organelles. The glycosomes of the bloodstream form trypomastigotes purified by the same procedures show, however, no sign of swelling. A comparison of glycosomes purified from procyclic trypomastigotes and bloodstream form trypomastigotes prepared by the same double sucrose procedure demonstrated that in the glycosome of procyclic trypomastigotes: activities of hexokinase, phosphoglucose isomerase, phosphofructose kinase, aldolase and phosphoglycerate kinase and diminished by 80-100%; activities of glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and glycerol-3-phosphate dehydrogenase remain unchanged or are only slightly reduced; there is an appearance of four major new proteins, among which could be phosphoenol pyruvate carboxykinase and malate dehydrogenase. These observations are in basic agreement with those by Hart et al. (Mol. Biochem. Parasitol. 12, 25-35, 1984).

Absence of substrate channeling in the glycosome of Trypanosoma brucei.

Glycolytic enzymes in the purified glycosomes of Trypanosoma brucei brucei bloodstream forms were crosslinked to form a large protein complex by the bifunctional reagent dimethyl suberimidate [Aman, R.A., Kenyon, G.L. and Wang, C.C. (1985) J. Biol. Chem. 260, 6966-6973]. The crosslinked enzyme complex was found capable of catalyzing the chain reactions leading from glucose to the formation of alpha-glycerophosphate in the presence of ATP and NADH. To determine whether the crosslinked enzyme complex exhibits multiple substrate channelings, these chain reactions were investigated in the crosslinked complex as well as in a preparation of solubilized native glycosomes. When glucose was present at a relatively high concentration (20 mM), production of alpha-glycerophosphate by the crosslinked complex had no apparent lag phase whereas the free enzyme mixture did. However, when the glucose level was lower (0.5-5.0 mM) the difference between the two enzyme preparations disappeared, a lag phase was found in both cases. Formation of sugar phosphates from radiolabeled glucose, followed by high performance liquid chromatographic analysis, showed no significant difference in the diluting powers of exogenous, unlabeled sugar phosphates added to the crosslinked complex or free enzymes. Exogenous fructose 1,6-diphosphatase demonstrated the same effectiveness in disrupting the chain reactions catalyzed by the crosslinked complex and the free enzyme mixture. In situ generation of 2-deoxyglucose-6-phosphate from 2-deoxyglucose and ATP was observed in the crosslinked complex, but the product had no amplified inhibitory effect on the phosphoglucose isomerase activity in the complex. All results suggest an absence of substrate channelings among the glycolytic enzymes in the glycosome of T. b. brucei bloodstream form.

The role of compartmentation and glycerol kinase in the synthesis of ATP within the glycosome of Trypanosoma brucei.

Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3-phosphoglycerate. The multienzyme reaction which produces 2 alpha-glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha-glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions.

Cross-linking of the enzymes in the glycosome of Trypanosoma brucei.

Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.

Discrimination of three types of opioid binding sites in rat brain in vivo.

Opiate receptor sites in the rat brain were defined in vivo by measuring the binding of etorphine, sufentanil, diprenorphine, and naloxone in saturation and cross-competition experiments. The binding data were analyzed simultaneously, using a computerized curve-fitting technique with an extended least-squares nonlinear regression program. Three types of binding sites could be distinguished: site 1 (18 pmoles/g of brain), site 2 (15 pmoles/g of brain), and site 3 (20 pmoles/g of brain). Site 1 is bound selectively by sufentanil (the ratio of the apparent equilibrium dissociation constants K2/K1 approximately equal to 1200), etorphine (K2/K1 approximately equal to 20), and naloxone (K2/K1 approximately equal to 15), and it resembles the mu binding site previously demonstrated in vitro. Diprenorphine binds to both site 1 and site 2 with high affinity and a slight (approximately 3.7-fold) selectivity for site 1 over site 2. The latter site may represent a mixture of the delta and kappa binding sites. The third site displays relatively high affinity for naloxone, but it is clearly different from sites 1 and 2, as it exhibits a lack of affinity for sufentanil, etorphine, and diprenorphine. This binding site population does not resemble any of the known opiate binding sites. Recent in vitro binding studies revealed that site 3 (now termed lambda site) is highly labile in vitro and was, therefore, not previously detected. These results suggest significant differences between in vitro and in vivo opioid receptor binding characteristics.