Differential expression of osteogenic factors associated with osteoinductivity of human osteosarcoma cell lines

Differential expression of multiple osteogenic factors may be responsible for the different osteoinductivity of osteosarcoma cell lines. We compared in vivo osteoinductivity of human osteosarcoma cell lines (Saos-2 vs. U-2 OS) in nude mice, and their in vitro expression of various osteogenic factors of protein level by quantitative immunocytochemistry and mRNA level by RT-PCR and/or in situ hybridization. Saos-2 cells, but not U-2 OS, were osteoinductive in vivo. Significantly higher expression (independent t-test, all p < 0.005) of osteogenic factors were observed in Saos-2 cells compared with U-2 OS, which included bone morphogenetic proteins (particularly BMPs-2, 3, 4, and 7), transforming growth factor-beta (TGF-beta), BMP receptor (BMPR)-1A, receptor-regulated Smads (R-Smads), Smads 1, 2, and 5, and common-mediator Smad (Co-Smad), Smad 4. In contrast, U-2 OS cells expressed higher levels of inhibitory Smad 6 (I-Smad) protein than Saos-2 cells (p < 0.001). These results suggest that a combination of osteogenic factors (BMPs, TGF-beta, BMPRs, and R/Co-Smads) against I-Smad may play important roles in the Saos-2 cell osteoinductivity. This may have a clinical implication in selecting key osteogenic factors for combined therapy for bone defect diseases. The characterized cell lines can be used as positive and negative controls for the assessments of both in vitro and in vivo bone formation capabilities of designed tissues or biomaterials.     

Microsatellite analysis of early stage (Ia-IIb) uterine cervical squamous carcinoma

Cervical cancer is the most common gynecologic malignancy of the developing world. The oncogenic role of human papilloma virus (HPV) is well known. Attention is now focusing on the complicit genetic changes, which allow progression of these tumors. Regarding these changes, deletion of tumor suppressor genes (loss of heterozygosity [LOH]) is the preferred pathway of progression with only a subset manifesting microsatellite instability (MSI). Implicated loci include 3p14.1-22. Several studies suggest that the mutator phenotype in cervical cancer may correlate with higher grade tumors, more advanced disease stage, and poor outcome. Unlike colorectal cancer, in which an inverse relationship has been demonstrated between microsatellite instability and loss of heterozygosity, cervical cancers expressing MSI have been found to coexpress LOH at other loci. In this study we analyzed 8-microsatellite loci including p53, DCC, APC, the MMR gene hMLH1 and 2 regions of interest on chromosome 3 in a high-risk population group in which HPV infection is endemic.     

Mitochondrial genome: defects, disease, and evolution.

Defects of mitochondrial function are often caused by defects of the mitochondrial genome. The hypothesis that defective organelles may spread through syncytial tissues as a result of a process of subcellular Darwinian selection is proposed. Tissues are likely to be involved in mitochondrial disease if they are syncytial, are derived from a few embryonic cells only, have little redundancy of function, and are subject to repeated metabolic stress. These effects, together with the random distribution of genetically heterogeneous mitochondria within the fertilised zygote, may account for the varied clinical pictures of mitochondrial disease. Evolution will have favoured the shift of mitochondrial DNA sequences to the nucleus, once the differentiation of tissues had created body compartments in which defective mitochondria could flourish to the detriment of the organism. This model of mitochondrial disease allows the generation of several predictions, testable using currently available laboratory techniques. Avenues of potential therapeutic value are indicated, including the avoidance of hypoglycaemia and the use of selective mitochondrial toxins.     

Benzylamine oxidase in normal and atherosclerotic human aortae

Assays of serum benzylamine oxidase (BzAO) have led some workers to postulate a relationship between elevated BzAO activity and diseases characterized by proliferating connective tissue. The present study was designed to determine whether BzAO activity of a cellular tissue is also affected. BzAO was assayed in homogenates of normal and atherosclerotic human aortae. Characterization done in normal aortae showed that BzAO is not a classical monoamine, diamine, polyamine, or lysyl oxidase, nor is it a ceruloplasmin. The enzyme is heat stable at 60 degrees C and is associated primarily with the microsomal fraction on density centrifugation. Compared with phenylethylamines and indoleamines, benzylamine is the best substrate. BzAO is sensitive to inhibition by hydrazines and chymotrypsin but not trypsin, and is insensitive to Triton X-100 and sulfhydryl-group blockade. BzAO activity of atherosclerotic plaque (expressed per gram wet weight or per milligram protein) was decreased markedly compared to that in adjacent, nonplaque regions and in normal aortae. However, on a per milligram DNA basis, the BzAO activity of plaque did not differ from that of nonplaque tissue. We conclude that there is a decreased cell population density in plaque, a contention supported by kinetic analysis. Plaque BzAO showed a decreased Vmax with no change in the Km of benzylamine compared with nonplaque tissue. Thus, if a relationship exists between BzAO activity and proliferating connective tissue, it is not apparent at the level of the cellular enzyme in atherosclerotic aortae of man.     

Neural responses to free field and virtual acoustic stimulation in the inferior colliculus of the guinea pig

Virtual auditory space (VAS) stimuli based on outer ear transfer functions became increasingly important in spatial hearing research. However, few studies have investigated the match between responses of auditory neurons to VAS and free-field (FF) stimulation. This study validates acoustic spatial receptive fields (SRFs) of 183 individual midbrain units using both VAS and FF stimuli. The first-spike latency, which varied systematically across SRFs, was 14.9 +/- 8.3 (SD) ms in FF, and 15.1 +/- 8.3 ms in VAS. Spike-count-based SRFs measured 0-20 dB above the neural threshold covered on average 44.5 +/- 18.0% of the recorded sphere in FF and 45.5 +/- 18.7% in VAS. The average deviation of the centroid position of SRFs using FF and VAS stimuli was 7.4 degrees azimuth and 3.3 degrees elevation. The average spike rate remained unchanged. The SRF overlap recorded using FF and VAS stimuli (mean: 71.3 +/- 12.6%) or repeated FF stimuli (70.2 +/- 14.2%) was high and strongly correlated (r = 0.96; P < 0.05). The SRF match observed with FF and VAS stimuli was not significantly altered over a range of stimulus levels (paired t-test P = 0.51; n = 6). Randomized VAS barely affected SRF sizes, centroids, or maximum spike count but decreased the average minimum response to 59% compared with sequential stimulation (paired t-test; P = 0.05; n = 26). SRF recordings in VAS excluding the acoustic distortions of the recording equipment differed from those in VAS incorporating the equipment (paired t-test P = 0.01; n = 5). In conclusion, neurophysiological recordings demonstrate that individualized VAS stimuli provided a good simulation of a FF environment.     

Genetic diversity and identification of human infection by amplification of the chlamydial 60-kilodalton cysteine-rich outer membrane protein gene.

The 60-kDa cysteine-rich outer membrane protein genes of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis have very different 5' ends, but two areas flanking this variable region show absolute sequence conservation. This observation permitted differentiation of the three species of Chlamydia by the polymerase chain reaction (PCR), forming the basis of a diagnostic test for chlamydial infections. The PCR product containing the variable region of the respective 60-kDa CrP genes was also subjected to restriction endonuclease digestion, enabling differentiation of individual type strains of C. psittaci. Differentiation was possible between lymphogranuloma venereum and trachoma isolates of C. trachomatis. The PCR-based diagnostic test was successful with all strains of chlamydiae studied. The PCR primers showed high specificity and did not produce any product with common bacterial pathogens that may share the same sites of infection.