Founding Amerindian mitochondrial DNA lineages in ancient Maya from Xcaret, Quintana Roo.

Ancient DNA from the bone remains of 25 out of 28 pre-Columbian individuals from the Late Classic-Postclassic Maya site of Xcaret, Quintana Roo, was recovered, and mitochondrial DNA (mtDNA) was amplified by using the polymerase chain reaction. The presence of the four founding Amerindian mtDNA lineages was investigated by restriction analysis and by direct sequencing in selected individuals. The mtDNA lineages A, B, and C were found in this population. Eighty-four percent of the individuals were lineage A, whereas lineages B and C were present at low frequencies, 4% and 8%, respectively. Lineage D was absent from our sample. One individual did not possess any of the four lineages. Six skeletons out of 7 dated from the Late Classic period were haplotype A, whereas 11 skeletons out of 16 dated from the Postclassic period were also haplotype A. The distribution of mtDNA lineages in the Xcaret population contrasts sharply with that found in ancient Maya from Copán, which lack lineages A and B. On the other hand, our results resemble more closely the frequencies of mtDNA lineages found in contemporary Maya from the Yucatán Peninsula and in other Native American contemporary populations of Mesoamerican origin.

Copolymerization of normal type I collagen with three mutated type I collagens containing substitutions of cysteine at different glycine positions in the alpha 1 (I) chain.

Previous observations with type I collagen from a proband with lethal osteogenesis imperfecta demonstrated that type I collagen containing a substitution of cysteine for glycine alpha 1-748 copolymerized with normal type I collagen (Kadler, K. E., Torre-Blanco, A., Adachi, E., Vogel, B. E., Hojima, Y., and Prockop, D. J. (1991) Biochemistry 30, 5081-5088). Here, three preparations containing normal type I procollagen and type I procollagen with a substitution of cysteine for glycine alpha 1-175, glycine alpha 1-691, or glycine alpha 1-988 were purified from cultured skin fibroblasts from probands with osteogenesis imperfecta. The procollagens were then used as substrates in a system for assaying the self-assembly of type I collagen into fibrils. The cysteine-substituted collagens in all three preparations were incorporated into fibrils. The cysteine alpha 1-175 and cysteine alpha 1-691 collagens were shown to increase the lag time and decrease the propagation rate constant for fibril assembly. All three preparations containing cysteine-substituted collagens formed fibrils with diameters that were two to four times the diameter of fibrils formed under the same conditions by normal type I collagen. Also, the three preparations containing cysteine substituted collagens had higher solubilities than normal type I collagen. The results, therefore, demonstrated that the three cysteine-substituted collagens copolymerized with normal type I collagen. The effects of the mutated collagens on fibril assembly can be understood in terms of a recently proposed model of fibril growth from symmetrical tips by assuming that the mutated monomers partially inhibit tip growth but not lateral growth of the fibrils. Of special interest was the observation that the Cys alpha 1-175 collagen from a proband with a non-lethal variant of osteogenesis imperfecta had quantitatively less effect on several parameters of fibril assembly at 37 degrees C than cysteine-substituted collagens from three probands with lethal variants of the disease.

Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils.

Previous observations suggested that incubating fibroblasts at elevated temperature caused over-modification of type I procollagen by post-translational enzymes because of a delay in folding of the collagen triple helix. Here, human skin fibroblasts were incubated at 40.5 instead of 37 degrees C, and the type I procollagen secreted into the medium was isolated. Analysis of the protein indicated that there was an increase of about 5 residues of hydroxylysine/alpha chain and about 1 residue of glycosylated hydroxylysine/alpha chain. Assays with procollagen N-proteinase indicated that the N-propeptide of the over-modified collagen was cleaved at a decreased rate, apparently because the over-modification altered the conformation-dependent cleavage site for the enzyme. Assays in a system for assembly of collagen into fibrils demonstrated that the over-modified protein had a higher critical concentration for self-assembly. Also, the fibrils formed from the over-modified collagen at 31 and 29 degrees C had smaller diameters than fibrils formed from normal type I collagen. The results provide direct evidence for earlier suggestions that post-translational over-modification of a fibrillar collagen can alter the morphology of the fibrils formed. The results also indicate that some of the biological consequences of the mutations in type I procollagen causing heritable disorders must be ascribed to the effects of post-translational over-modifications that frequently occur as secondary consequences of changes in the primary structure of the protein.

A type I collagen with substitution of a cysteine for glycine-748 in the alpha 1(I) chain copolymerizes with normal type I collagen and can generate fractallike structures.

Type I procollagen was purified from cultured fibroblasts of a proband with a lethal variant of osteogenesis imperfecta. The protein was a mixture of normal procollagen and mutated procollagens containing a substitution of cysteine for glycine in either one pro alpha 1(I) chain or both pro alpha 1(I) chains, some or all of which were disulfide-linked through the cysteine at position alpha 1-748. The procollagen was then examined in a system for generating collagen fibrils de novo by cleavage of the pCcollagen to collagen with procollagen C-proteinase [Kadler et al. (1987) J. Biol. Chem. 262, 15696-15701]. The mutated collagens and normal collagens were found to form copolymers under a variety of experimental conditions. With two preparations of the protein that had a high content of alpha 1(I) chains disulfide-linked through the cysteine alpha 1-748, all the large structures formed had a distinctive, highly branched morphology that met one of the formal criteria for a fractal. Preparations with a lower content of disulfide-linked alpha 1(I) chains formed fibrils that were 4 times the diameter of control fibrils. The formation of copolymers was also demonstrated by the observation that the presence of mutated collagens decreased the rate of incorporation of normal collagen into fibrils. In addition, the solution-phase concentration at equilibrium of mixtures of mutated and normal collagens was 5-10-fold greater than that of normal collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of antigen B from Taenia solium cysticerci by affinity to mammalian collagen.

A 1-step procedure for the easy and rapid purification of milligram amounts of antigen B from a crude extract of Taenia solium cysticerci is described here. This procedure takes advantage of the property of the antigen B to bind to collagen and is based on antigen adsorption to polymeric collagen.

In vitro hydroxylation of proline in the collagen of the cysticercus of Taenia solium.

1. In vitro hydroxylation of proline in cysticercus collagen was carried out using two different vertebrate enzymes. 2. Chick embryo enzyme is more active on cysticercus collagen than new-born rabbit enzyme. 3. Hydroxylation of cysticercus collagen is more efficient at 40 than at 37 degrees C. 4. No prolyl hydroxylase activity was detected in cysticercus extracts. 5. Collagen from the adult tapeworms Taenia solium and T. saginata lack hydroxyproline. 6. SLS crystallites from T. solium and T. saginata collagen show the same band pattern as cysticercus collagen.

The isolation, purification, and characterization of the collagen of Cysticercus cellulosae.

Insoluble collagen fibrils were obtained from Cysticercus cellulosae after homogenization and treatment with NaCl/mercaptoethanol solutions and were solubilized after limited pepsin digestion. Solubilized Cysticercus collagen shows two different alpha subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 100,000 and is readily degraded by bacterial collagenase. The amino acid composition is characteristic of collagen except that it contains no hydroxyproline. Segment long spacing crystallites measuring 280 nm in length were prepared. These segments showed a band pattern different from that of vertebrate and other invertebrate collagens. The denaturation temperature at neutral pH was 35 degrees C and correlated with the total pyrrolidine content as observed for other collagens. An intrinsic viscosity value of 15.3 dl/g was obtained for this collagen. Its possible evolutionary relationship with other collagens is discussed.

A neutral protease in rheumatoid synovial fluid capable of attacking the telopeptide regions of polymeric collagen fibrils.

Fluorescent-labelled polymeric collagen fibrils have been prepared which contain three fluoresein residues in the telopeptide regions and four fluorescein residues in the helical region of each tropocollagen unit within the polymer. This material has been used as a substrate for the study of enzymes present in the synovial fluid of inflamed rheumatoid joints which are capable of degrading polymeric collagen fibrils. Two enzyme systems were observed, one inhibited by EDTA and having the properties of the known synovial collagenase, the other having the properties of a neutral protease. The neutral protease was found to be present in sonicates of the polymorphonuclear leucocytes in the synovial fluids of inflamed joints. This enzyme attacked the telopeptides of fluorescein-labelled polymeric collagen fibrils and was similar to trypsin in removing two residues of fluorescein-labelled peptides per tropocollagen molecule within the polymeric collagen fibrils but did not depolymerise the polymeric collagen fibrils.