Comparative analysis with collagen type II distinguishes cartilage oligomeric matrix protein as a primary TGFß-responsive gene.

OBJECTIVE: This study aims to investigate the regulation of expression of Cartilage oligomeric matrix protein (COMP), which is predominately expressed by chondrocytes and functions to organize the extracellular matrix. Mutations in COMP cause two skeletal dysplasias: pseudoachondroplasia and multiple epiphyseal dysplasia. The mechanism controlling COMP expression during chondrocyte differentiation is still poorly understood. DESIGN: Primary human bone marrow-derived stem cells were induced to differentiate into chondrocyte by pellet cultures. We then compared the temporal expression of COMP with the well-characterized cartilage-specific Type II collagen (Col2a1), and their response to transforming growth factor (TGF)ß and Sox trio (Sox5, 6, and 9) stimulation. RESULTS: COMP and Col2a1 expression are differentially regulated by three distinct mechanisms. First, upregulation of COMP mRNA precedes Col2a1 by several days during chondrogenesis. Second, COMP expression is independent of high cell density but requires TGF-ß1. Induction of COMP mRNA by TGF-ß1 is detected within 2h in the absence of protein synthesis and is blocked by specific inhibitors of the TGFß signaling pathway; and therefore, COMP is a primary TFGß-response gene. Lastly, while Col2a1 expression is intimately controlled by the Sox trio, overexpression of Sox trio fails to activate the COMP promoter. CONCLUSION: COMP and Col2a1 expression are regulated differently during chondrogenesis. COMP is a primary response gene of TGFß and its fast induction during chondrogenesis suggests that COMP is suitable for rapidly accessing the chondrogenic potential of stem cells.

Decision process to assess medical equipment for hyperbaric use.

There are very few items of medical equipment specifically designed for hyperbaric use; and little information is available about medical equipment already tested for hyperbaric use. Hyperbaricists are usually left to their own devices in making a determination about the safe and effective use of standard medical equipment in the hyperbaric setting. This article proposes a logical and systematic process to arrive at this determination. The process involves seven steps beginning with a need assessment and ending with endorsement by appropriate individuals. The discussion of decision steps includes identifying risk elements, compliance with safety standards, testing, and documentation.

The role of cartilage oligomeric matrix protein (COMP) in skeletal disease.

Cartilage oligomeric matrix protein is a non-collagenous extracellular matrix protein expressed primarily in cartilage, ligament, and tendon. Cartilage oligomeric matrix protein has been studied extensively because mutations in the gene cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. Pseudoachondroplasia is a disproportionate dwarfing condition associated with joint abnormalities, while multiple epiphyseal dysplasia is less severe. Both of these skeletal dysplasias have a characteristic chondrocyte pathology that consists of intracellular retention of cartilage oligomeric matrix protein and other extracellular matrix proteins in an enlarged rough endoplasmic reticulum. This toxic intracellular retention of extracellular matrix proteins causes chondrocyte cell death thereby decreasing linear bone growth. Additionally, when cartilage oligomeric matrix protein and the other co-retained proteins are not exported to the extracellular matrix, the resulting matrix is abnormal and easily erodes with normal physical activity. Cartilage oligomeric matrix protein is also a marker for joint destruction associated osteoarthritis, rheumatoid arthritis, joint trauma, and intense activity. Serum cartilage oligomeric matrix protein levels are higher in aggressive cases of arthritis and levels are used to predict future disease progression. Recent studies have identified molecular functions of cartilage oligomeric matrix protein that may contribute to its role in skeletal disease. These molecular functions include: binding other ECM proteins, catalyzing polymerization of type II collagen fibrils, and regulation of chondrocyte proliferation. Here, we review cartilage oligomeric matrix protein's role in skeletal disease and potential molecular mechanisms.

Model systems for studying skeletal dysplasias caused by TSP-5/COMP mutations.

Cartilage oligomeric matrix protein, also known as thrombospondin-5 (TSP-5), is an extracellular matrix protein found primarily in cartilage and musculoskeletal tissues. TSP-5 is of interest because mutations in the gene cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Both PSACH and EDM1 have a characteristic chondrocyte phenotype distinguished by giant rough endoplasmic reticulum (rER) cisternae containing TSP-5 and other extracellular matrix proteins such as type IX collagen and matrilin-3. The accumulation of proteinaceous material in the rER compromises cellular function and leads to premature chondrocyte death. Both in vitro and in vivo models have been generated with varying degrees of success to study the cellular mechanisms of the disease process. Here we review and discuss in vitro and in vivo PSACH and MED model systems and describe two transgenic mouse lines expressing human mutant TSP-5 protein. These model systems have revealed several important features of the PSACH cellular pathology: unfolded protein response activation, upregulation of apoptosis and inappropriate assembly of matrix network in the rER. Some of these models are valuable reagents that may be of use in testing therapeutic interventions. (Part of a Multiauthor Review).

Survey of transcripts in the adult Drosophila brain.

BACKGROUND: Classic methods of identifying genes involved in neural function include the laborious process of behavioral screening of mutagenized flies and then rescreening candidate lines for pleiotropic effects due to developmental defects. To accelerate the molecular analysis of brain function in Drosophila we constructed a cDNA library exclusively from adult brains. Our goal was to begin to develop a catalog of transcripts expressed in the brain. These transcripts are expected to contain a higher proportion of clones that are involved in neuronal function. RESULTS: The library contains approximately 6.75 million independent clones. From our initial characterization of 271 randomly chosen clones, we expect that approximately 11% of the clones in this library will identify transcribed sequences not found in expressed sequence tag databases. Furthermore, 15% of these 271 clones are not among the 13,601 predicted Drosophila genes. CONCLUSIONS: Our analysis of this unique Drosophila brain library suggests that the number of genes may be underestimated in this organism. This work complements the Drosophila genome project by providing information that facilitates more complete annotation of the genomic sequence. This library should be a useful resource that will help in determining how basic brain functions operate at the molecular level.

Methicillin-resistant Staphylococcus aureus in the community: a hospital-based study.

To determine the proportion of methicillin-resistant Staphylococcus aureus (MRSA) among patients presenting for hospitalization and to assess risk factors for MRSA carriage, we conducted a study for 13 months at five Pittsburgh-area hospitals. Of 504 S aureus identified, 125 (25%) were MRSA. Independent risk factors for MRSA included organ transplantation, employment in a healthcare facility, pressure sores, tube feeding, and hospitalization within the preceding year.

DNA recognition properties of the N-terminal DNA binding domain within the large subunit of replication factor C.

Replication Factor C (RFC) is a five-subunit protein complex required for eukaryotic DNA replication and repair. The large subunit within this complex contains a C-terminal DNA binding domain which provides specificity for PCNA loading at a primer-template and a second, N-terminal DNA binding domain of unknown function. We isolated the N-terminal DNA binding domain from Drosophila melanogaster and defined the region within this polypeptide required for DNA binding. The DNA determinants most efficiently recognized by both the Drosophila minimal DNA binding domain and the N-terminal half of the human large subunit consist of a double-stranded DNA containing a recessed 5' phosphate. DNA containing a recessed 5' phosphate was preferred 5-fold over hairpined DNA containing a recessed 3' hydroxyl. Combined with existing data, these DNA binding properties suggest a role for the N-terminal DNA binding domain in the recognition of phosphorylated DNA ends.

Triple cage olfactometer for evaluating mosquito (Diptera: Culicidae) attraction responses.

A triple cage olfactometer for evaluating mosquito attraction responses is described. The olfactometer is designed for easy access for interior cleaning, has a mechanism that allows synchronous operation of the port doors on each cage, and requires 0.8 m2 of floor space. It is constructed of clear acrylic, contains 3 test chambers in a tiered configuration, has paired removable sleeves and mosquito traps on each cage, and is equipped with a filtered external air supply system that has temperature and relative humidity control.

Mosquito density, biting rate and cage size effects on repellent tests.

Mosquito biting rates and the mean duration of protection (in hours) from bites (MDPB) of Aedes aegypti and Anopheles quadrimaculatus, using the repellent 'deet' (N,N-diethyl-3-methylbenzamide) on a 50 cm2 area of healthy human skin, were observed in small (27 l), medium (approximately 65 l) and large (125 l) cages containing low, medium or high densities of mosquitoes: respectively, 640, 128 or 49 cm3 of cage volume per female. At the initial treatment rate of approximately 0.4 microliter/cm2 (1 ml of 25% deet in ethanol on 650 cm2 of skin), the MDPB for deet against Ae. aegypti ranged from 4.5 to 6.5 h and was significantly less (5.0 +/- 0.8 h) in large cages compared with medium (6.2 +/- 0.9 h) and small (6.2 +/- 0.8 h) cages, regardless of the density. Against An. quadrimaculatus the MDPB for deet 0.4 microliter/cm2 was 1.5-8.0 h, less in small (3.7 +/- 2.3 h) and large (2.2 +/- 1.1 h) cages at medium (3.7 +/- 2.3 h) and high (2.5 +/- 1.7 h) mosquito densities, and was longest in medium cages (6.2 +/- 2.6 h) at low mosquito densities (5.8 +/- 2.8 h). With equinoxial photoperiodicity (light on 06.00-18.00 hours) the biting rate was influenced by the time of observation (08.00, 12.00, 16.00 hours) for Ae. aegypti but not for An. quadrimaculatus. For both species, the biting rate was inversely proportional to mosquito density and the MDPB. The shortest MDPBs were obtained in large cages with high densities of mosquitoes and longest protection times occurred in medium sized cages with low mosquito densities.

Acute hemorrhagic leukoencephalitis: a cause of acute brainstem dysfunction.

A 37-year-old female physician was admitted to the hospital with severe headache, facial and hand paresthesias, dysarthria, and ataxia. Neurologic examination disclosed signs of brain stem dysfunction. There was rapid neurologic deterioration, and she died in 28 hours. Postmortem studies showed the characteristic features of acute hemorrhagic leukoencephalitis.