Expression of bone morphogenetic proteins in normal human intramembranous and endochondral bones.

Bone morphogenetic proteins (BMPs) are growth and differentiation factors that have been purified and widely accepted to be the most important regulators in the processes of bone formation. The aim of this study was to identify the BMPs that are expressed in normal human bone, and to investigate the specific pattern of BMP2-BMP9 expression in normal human intramembranous and endochondral bone to maintain homeostasis, as well as in ex vivo primary cell culture of human osteoblasts from intramembranous and endochondral bone. Semi-quantitative RT-PCR indicated that 2 types of bone of different embryological origin have distinct patterns of BMP expression. BMP3, 4, 7 and 8 were strongly expressed in normal intramembranous bone compared to endochondral bone, whereas BMP2 and 5 were highly expressed in endochondral bone. The expression of BMP9 and BMP15 in human bone was identified for the first time. From the very similar expression patterns of BMPs in fresh normal bone and ex vivo osteoblastic cell culture, it can be proposed that the different proportions of BMPs in normal human intramembranous and endochondral bone needed to maintain normal homeostasis.

Tuberculosis in Thai prisons: magnitude, transmission and drug susceptibility.

BACKGROUND: Because of the human immunodeficiency virus (HIV) epidemic, tuberculosis has reemerged as a major public health problem in Thailand. Prison inmates are at high risk for developing tuberculosis because of the high prevalence of HIV infection. OBJECTIVES: To determine the magnitude, transmission, and drug susceptibility of tuberculosis in Thai prisons. SETTINGS: Four provincial prisons in Southern Thailand. DESIGN: Cross-sectional, descriptive, clinical and molecular study. RESULTS: Miniature chest roentgenograms were performed on 304 (6.4%) of 4751 inmates screened for a > or = 2 week history of chronic cough and fever. At least 17 (35%) of 49 inmates who had a miniature chest roentgenogram compatible with tuberculosis were HIV-positive. The prevalence of smear-positive pulmonary tuberculosis was 568 per 100,000 inmates, which was eight times higher than that in the general population. Eight (38%) of 21 culture-positive Mycobacterium tuberculosis isolates had DNA fingerprints matching those of another inmate who was housed in the same room or in the same dormitory unit; 39% of the M. tuberculosis isolates were resistant to isoniazid; three of these isolates were also borderline resistant to rifampicin. CONCLUSION: The prevalence of pulmonary tuberculosis in these prisons was high. A substantial proportion were acquired in the prisons. Isoniazid (INH) resistance was common, and theoretically precludes the use of INH-preventive therapy for contacts of these cases. Active case finding should be done and directly observed therapy implemented to prevent the spread of tuberculosis into the community.

Expression of a mammalian alpha 2,6-sialyltransferase gene in Pichia pastoris.

Terminal sialic acid on oligosaccharides of glycoproteins shows several biological functions of the glycoproteins. The yeast Pichia pastoris normally does not contain sialic acid on the oligosaccharides of glycoproteins. A sialyltransferase (ST) gene was transfected into P. pastoris to assess the possibility of using yeast cells as a host to produce sialoglycoproteins. The expression vectors pPIC3.5 and pPIC9 were used as carriers. The recombinant P. pastoris harbouring ST-pPIC3.5 and ST-pPIC9 had sialyltransferase activity of 1.1 and 10.2 mU l(-1) respectively. The ability of the recombinant ST-pPIC3.5 and ST-pPIC9 to transfer the fluoresceinyl-NeuAc into the cell glycoproteins was 36.9 and 20.9 pmol mg -1 protein respectively.

Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha.

A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M(r) of 49400. The encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.

Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint.

DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.