A new mathematical model to study bone turnover in growing rats.

A new mathematical model for the study of bone turnover in growing rats was developed. The model predicts a linear relationship between bone mineral content (BMC) and biochemical markers (BMK) of bone turnover assuming that rats are growing, bone turnover is profoundly affected by skeletal maturation, and resorption and formation are physiologically balanced. The model validation was performed by measuring galactosyl-hydroxylysine (GHYL) and hydroxyproline (HYP) in urines. This mathematical evidence supports our proposed use of the specific bone resorption marker GHYL to predict bone mineral content. Further studies on bone turnover will be possible by the application of the same approach.

High predictivity of galactosyl-hydroxylysine in urine as an indicator of bone metastases from breast cancer.

We measured the urinary excretion of galactosyl-hydroxylysine (GH) and hydroxyproline in two groups of women with breast cancer, with (M+, n = 24) and without (Mo, n = 30) clinical, scintigraphic, or radiological evidence of bone metastases. Both these compounds are excreted in larger amounts in the M+ group than in the Mo patients. However, GH, which is a specific marker for bone collagen, provides better predictivity for bone metastases than does hydroxyproline: 92% sensitivity and 90% specificity vs 74% and 79%, respectively, for hydroxyproline.

High-performance liquid chromatographic preparation of galactosyl-hydroxylysine, a specific bone collagen marker.

Galactosyl-hydroxylysine, a specific bone collagen marker, has been prepared directly from human urine samples by high-performance liquid chromatography (HPLC) on a preparative column. The compound is the didansyl derivative, as proved by HPLC and mass spectrometry under fast atom bombardment conditions. Since this compound is not commercially available, the procedure reported appears to be the simplest way to prepare it, which is necessary to measure the urinary excretion of this collagen metabolite by HPLC.

Determination of galactosyl hydroxylysine in urine as a means for the identification of osteoporotic women.

A sensitive and specific method is proposed to follow bone collagen degradation. The procedure consists of the measurement of galactosyl hydroxylysine (GH) in urine by HPLC. The aim of the work is to assess the predictive values of the method for the identification of post-menopausal osteoporotic women. By assuming the value of 12 mumol/g creatinine as the threshold value, the sensitivity of the test is 87% and the specificity 60%. Individuals with a GH/creatinine ratio of 12 or below are not likely to be at risk of bone fractures: an equivalent predictive value is provided by the measurement of bone density by quantitative computed tomography. This biochemical method is however simple and not invasive and may be frequently repeated.

Failure of trypanosomal membrane antigens to induce protection against tsetse-transmitted Trypanosoma vivax or T. brucei in goats and rabbits.

A purified protein, relative molecular weight 83 kilodalton (kD), and plasma membranes from Trypanosoma brucei were tested as potential vaccines against tsetse-transmitted T. vivax and T. brucei in goats and rabbits. The 83 kD protein was found in lysates of all clones of T. brucei examined, as well as in lysates of T. vivax, T. congolense and T. rhodesiense. Rabbits and goats were immunized with various amounts of antigen in Freund's complete adjuvant and boosted twice with antigen in Freund's incomplete adjuvant. Two weeks after the last inoculation, the goats were challenged with T. vivax-infected and the rabbits with T. brucei-infected Glossina morsitans morsitans. Although high antibody levels were detected in all the animals immunized with either antigen as measured by radioimmunoassay and immunodiffusion, they became infected and the course of disease was the same as that in unimmunized controls.

Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes.

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol-dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.

Biosynthesis of Trypanosoma brucei variant surface glycoproteins - analysis of carbohydrate heterogeneity and timing of post-translational modifications.

The variant surface glycoproteins from two cloned populations of Trypanosoma brucei brucei which were known to migrate as multiple bands on SDS gels have been studied. The heterogeneity present was located in those oligosaccharide side chains the addition of which is tunicamycin-sensitive. The time required for the trypanosome to synthesize and express a variant surface glycoprotein molecule in vitro was found, from pulse-chase and limited trypsinisation experiments, to be approximately 40 min. In the light of these data, pulse-chase experiments on the two antigens known to have heterogeneity in their oligosaccharide side chains demonstrated that the heterogeneity probably arose by two different mechanisms. Pulse-chase experiments on three different clones of trypanosomes have also been used to investigate the timing of cleavage of the carboxyl-terminal extension, known to be encoded on variant surface glycoprotein mRNA. Similar pulse-chase experiments followed by immunoprecipitation using affinity purified antiserum have been used to investigate the addition of the cross-reacting determinant. The timing of both these events has been discussed in relation to the time necessary for the synthesis and expression of the variant surface glycoprotein on the surface of the trypanosome.

Antitrypanosomal activity of sinefungin.

Sinefungin, a naturally occurring antifungal antibiotic nucleoside containing an ornithine residue, linked by a C-C bond to C-5' of adenosine, cures mice infected with Trypanosoma brucei brucei, T. congolense, or T. vivax; the effect of the drug is more pronounced towards T. congolense. Anti-trypanosomal activity of sinefungin could be the result of the inhibition of transmethylation reactions or of polyamine biosynthesis--or both--in parasites.

Identification and characterisation of two N-acetylglucosaminyltransferases associated with Trypanosoma Brucei microsomes.

Microsomes forom Trypanosoma brucei contain glycosyltransferases able to incorporate N-[14C]acetylglucosamine into two different types of acceptors. A first transferase catalyzes the transfer of N-[14C]acetylglucosamine 1-phosphate from uridine diphosphate N-[14C]acetylglucosamine into dolichol monophosphate. The enzymatic activity requires Mn2+, is time and temperature dependent, has an optimum pH of 7.4 and is completely inhibited by the antibiotic tunicamycin. Exogenous dolichol monophosphate enhances the glycosyltransferase activity. The kinetics of incorporation are characterized by a Km of 2.6 microM for uridine diphosphate N-acetylglucosaminyltransferase are comparable to those reported for the first enzyme of the dolichol cycle described in several eukaryotes. N-Acetyl-glucosaminylpyrophosphoryl-dolichol is essentially the only product of the reaction. A second type of activity which is responsible for the direct transfer of N[14C]acetylglucosamine from uridine diphosphate N[14C]acetylglucosamine into several endogenous polypeptide acceptors, is also associated with T. brucei microsomes. The reaction, which might be due to more than one enzyme, is dependent on Mn2+, but differs from the other transferase in all other characteristics. Time course and optimal temperature are different, and the optimum pH is 6.5. The reaction is independent of the external addition of dolichol monophosphate and tunicamy cin has no inhibitory effect on the enzymatic activity. AKm of 1.6 microM was calculated fr uridine diphosphate N-acetylglucosamine.

Studies on the biosynthesis of the variant surface glycoproteins of Trypanosoma brucei: sequence of glycosylation.

The variant surface glycoprotein (VSG) of several Trypanosoma brucei clones has been metabolically labeled with [35S]methionine in parasites grown in the presence or in the absence of tunicamycin. Pulse and chase experiments followed by immunoprecipiation with anti-VSG sera and polyacrylamide gel electrophoresis of the immunoprecipitates, have helped to elucidate two steps of the sequence of glycosylation of VSG. Immediately after, or concomitantly with the synthesis of the protein, a first type of oligosaccharide side chain is also synthesized. Tunicamycin, a specific inhibitor of asparagine glycosylation, completely inhibits this reaction. The total amount of VSG found in trypanosomes grown in the presence of tunicamycin is less than in controls. The unglycosylated molecule has an apparent molecular weight 5-10% smaller than the mature form. A subsequent step, occurring after translation, is the synthesis of carbohydrate side chains which contain a cross-reacting antigenic determinant detected in all VSGs so far studied by immunoprecipitations with heterologous antisera. The percentage of total VSG bearing sugars involved in cross-reactions is variable in different clones, increases with time and subsequently decreases, suggesting that some of the carbohydrate might undergo trimming. Polyacrylamide gel electrophoresis of immunoprecipitates suggests that the absence or the incomplete synthesis of this oligosaccharide does not alter the apparent molecular weight of VSG. In four out of the five clones studied, the sugar(s) responsible for cross-reactions seem to be located within oligosaccharides linked to the protein through both N-glycosidic and other unidentified types of linkages. This is suggested by the partial effect of tunicamycin on the extent of VSGs cross-reactivities measured by immunoprecipitations with heterologous antiserum.

Sub-cellular fractionation of Trypanosoma brucei. Isolation and characterization of plasma membranes.

A procedure is described for the isolation of sub-cellular fractions from bloodstream forms of Trypanosoma brucei. The method leaves intact most of the nuclei, mitochondria and microbodies. All the fractions have been chemically characterized and tested for 10 enzymatic markers. About 5% of total cell protein was isolated as a microsomal fraction containing mostly plasma membranes and endoplasmic reticulum vesicles. Plasma membranes were purified by high-speed centrifugation on magnesium-containing Dextran, and on linear sucrose-density gradients. The yield of membranes was approximately 0.3% of the total cell protein. The purified material had a sucrose density of 1.14 g/cm3 and consisted of smooth vesicles. Specific activity of the membrane markers Na+, K+, ouabain-sensitive ATPase and adenylate cyclase were 26- and 20-fold higher, respectively, than in total cells. Neither DNA nor RNA was detected. The sum of the cholesterol and phospholipid content was 0.99 mg/mg protein. The cholesterol/phospholipid molar ratio was 1:2.

A characterization of mRNA activites and their sequence complexities in Trypanosoma brucei: partial purification and properties of the VSSA mRNA.

Polyadenylated RNA isolated from a clone of Trypanosoma brucei was shown to direct the synthesis of a variety of polypeptides in a cell-free system. A predominant 58,000 dalton polypeptide was immunoprecipitated with antisera to the T. brucei variant specific surface antigen (VSSA). The mRNA that directed the synthesis of the VSSA was 2.0 kilobases (kb) long as measured by polyacrylamide gel electrophoresis in 98% formamide. Complementary DNA was prepared with avian myeloblastosis virus reverse transcriptase and the nucleotide sequence complexities of the total polysomal poly(A)+RNA and a gel purified VSSA mRNA were measured. 20% of the total cellular poly(A)+RNA contained abundant sequences with an apparent complexity of 9.6 kb; 42% of the purified VSSA mRNA contained abundant sequences with a complexity of 7.2 kb. Complementary DNA synthesized from gel purified VSSA mRNA was hybridized to total cellular poly(A)+RNA isolated from an unrelated T. brucei clone expressing a different variant antigen. A portion of the low complexity RNA sequence component was absent in the heterologous mRNA population but the same plateau of hybridization was achieved (93%). The abundance of some of the low complexity mRNAs appears to be T. brucei clone specific.