Intra-articular injection of magnesium chloride attenuates osteoarthritis progression in rats.

OBJECTIVE: To explore the effects of Mg2+ on the expression of osteoarthritic markers in human cartilage and synovium tissue explants. To investigate the therapeutic effect of intra-articular injection of Mg2+ in an established rat OA (Osteoarthritis) model of anterior cruciate ligament transection with partial medial meniscectomy (ACLT + PMM). DESIGN: Human cartilage and synovium explants were collected from total knee replacement surgeries and incubated with MgCl2 (20 mmol/L) in vitro. A rat OA model was established by ACLT + PMM surgery in 450-500 g male Sprague Dawley (SD) rats. To select the optimal dose, intra-articular injections of MgCl2 (0.05, 0.5, 5 mol/L) were performed at 4 weeks after the surgery every 3 days for 2 weeks. The effect of optimized MgCl2 was further determined by histology, immunohistochemistry, and quantitative real-time polymerase chain reaction. RESULTS: The expressions of osteoarthritic markers in human cartilage and synovium explants were inhibited by Mg2+in vitro. Immunohistochemical analysis further suggested the inhibitory effects of Mg2+ on the expression of MMP-13 and IL-6 in the human tissue explants. Cartilage degeneration and synovitis in ACLT + PMM rats were significantly improved by intra-articular injections of Mg2+ (0.5 mol/L). Immunohistochemical analysis also showed the regulatory effects of Mg2+ on osteoarthritic markers in both cartilage and synovium in rats, consistent with in vitro results. CONCLUSION: Intra-articular injections of Mg2+ at 0.5 mol/L attenuate the progression of OA in the ACLT + PMM rat model. Such effect was at least in part explained by the promotion of cartilage matrix synthesis and the suppression of synovial inflammation.

Structure of the Golgi apparatus is not influenced by a GAG deletion mutation in the dystonia-associated gene Tor1a.

Autosomal-dominant, early-onset DYT1 dystonia is associated with an in-frame deletion of a glutamic acid codon (ΔE) in the TOR1A gene. The gene product, torsinA, is an evolutionarily conserved AAA+ ATPase. The fact that constitutive secretion from patient fibroblasts is suppressed indicates that the ΔE-torsinA protein influences the cellular secretory machinery. However, which component is affected remains unclear. Prompted by recent reports that abnormal protein trafficking through the Golgi apparatus, the major protein-sorting center of the secretory pathway, is sometimes associated with a morphological change in the Golgi, we evaluated the influence of ΔE-torsinA on this organelle. Specifically, we examined its structure by confocal microscopy, in cultures of striatal, cerebral cortical and hippocampal neurons obtained from wild-type, heterozygous and homozygous ΔE-torsinA knock-in mice. In live neurons, the Golgi was assessed following uptake of a fluorescent ceramide analog, and in fixed neurons it was analyzed by immuno-fluorescence staining for the Golgi-marker GM130. Neither staining method indicated genotype-specific differences in the size, staining intensity, shape or localization of the Golgi. Moreover, no genotype-specific difference was observed as the neurons matured in vitro. These results were supported by a lack of genotype-specific differences in GM130 expression levels, as assessed by Western blotting. The Golgi was also disrupted by treatment with brefeldin A, but no genotype-specific differences were found in the immuno-fluorescence staining intensity of GM130. Overall, our results demonstrate that the ΔE-torsinA protein does not drastically influence Golgi morphology in neurons, irrespective of genotype, brain region (among those tested), or maturation stage in culture. While it remains possible that functional changes in the Golgi exist, our findings imply that any such changes are not severe enough to influence its morphology to a degree detectable by light microscopy.

Attenuation of subchondral bone abnormal changes in osteoarthritis by inhibition of SDF-1 signaling.

BACKGROUND: Current conservative treatments for osteoarthritis (OA) are largely symptoms control therapies. Further understanding on the pathological mechanisms of OA is crucial for new pharmacological intervention. OBJECTIVE: In this study, we investigated the role of Stromal cell-derived factor-1(SDF-1) in regulating subchondral bone changes during the progression of OA. METHODS: Clinical samples of different stages of OA severity were analyzed by histology staining, micro-CT, enzyme-linked immunosorbent assay (ELISA) and western blotting, to compare SDF-1 level in subchondral bone. The effects of SDF-1 on human mesenchymal stem cells (MSCs) osteogenic differentiation were evaluated. In vivo assessment was performed in an anterior cruciate ligament transaction plus medial meniscus resection in the SD rats. The OA rats received continuous infusion of AMD3100 (SDF-1 receptor blocker) in osmotic mini-pump implanted subcutaneously for 6 weeks. These rats were then terminated and subjected to the same in vitro assessments as human OA samples. RESULTS: SDF-1 level was significantly elevated in the subchondral bone of human OA samples. In the cell studies, the results showed SDF-1 plays an important role in osteogenic differentiation of MSCs. In the OA animal studies, there were less cartilage damage in the AMD3100-treated group; microCT results showed that the subchondral bone formation was significantly reduced and so did the number of positive Nestin or Osterix cells in the subchondral bone region. CONCLUSIONS: Higher level of SDF-1 may induce the subchondral bone abnormal changes in OA and inhibition of SDF-1 signaling could be a potential therapeutic approach for OA.

Minimal Change in the cytoplasmic calcium dynamics in striatal GABAergic neurons of a DYT1 dystonia knock-in mouse model.

DYT1 dystonia is the most common hereditary form of primary torsion dystonia. This autosomal-dominant disorder is characterized by involuntary muscle contractions that cause sustained twisting and repetitive movements. It is caused by an in-frame deletion in the TOR1A gene, leading to the deletion of a glutamic acid residue in the torsinA protein. Heterozygous knock-in mice, which reproduce the genetic mutation in human patients, have abnormalities in synaptic transmission at the principal GABAergic neurons in the striatum, a brain structure that is involved in the execution and modulation of motor activity. However, whether this mutation affects the excitability of striatal GABAergic neurons has not been investigated in this animal model. Here, we examined the excitability of cultured striatal neurons obtained from heterozygous knock-in mice, using calcium imaging as indirect readout. Immunofluorescence revealed that more than 97% of these neurons are positive for a marker of GABAergic neurons, and that more than 92% are also positive for a marker of medium spiny neurons, indicating that these are mixed cultures of mostly medium spiny neurons and a few (~5%) GABAergic interneurons. When these neurons were depolarized by field stimulation, the calcium concentration in the dendrites increased rapidly and then decayed slowly. The amplitudes of calcium transients were larger in heterozygous neurons than in wild-type neurons, resulting in ~15% increase in cumulative calcium transients during a train of stimuli. However, there was no change in other parameters of calcium dynamics. Given that calcium dynamics reflect neuronal excitability, these results suggest that the mutation only slightly increases the excitability of striatal GABAergic neurons in DYT1 dystonia.

Synaptic vesicle recycling is enhanced by torsinA that harbors the DYT1 dystonia mutation.

Early-onset generalized dystonia, DYT1, is caused by a mutation in the gene encoding the evolutionarily conserved AAA+ ATPase torsinA. Synaptic abnormalities have been implicated in DYT1 dystonia, but the details of the synaptic pathophysiology are only partially understood. Here, we demonstrate a novel role for torsinA in synaptic vesicle recycling, using cultured hippocampal neurons from a knock-in mouse model of DYT1 dystonia (ΔE-torsinA) and live-cell imaging with styryl FM dyes. Neurons from heterozygous ΔE-torsinA mice released a larger fraction of the total recycling pool (TRP) during a single round of electrical stimulation than did wild-type neurons. Moreover, when the neurons were subjected to prior high activity, the time course of release was shortened. In neurons from homozygous mice, these enhanced exocytosis phenotypes were similar, but in addition the size of the TRP was reduced. Notably, when release was triggered by applying a calcium ionophore rather than electrical stimuli, neither a single nor two ΔE-torsinA alleles affected the time course of release. Thus, the site of action of ΔE-torsinA is at or upstream of the rise in calcium concentration in nerve terminals. Our results suggest that torsinA regulates synaptic vesicle recycling in central neurons. They also indicate that this regulation is influenced by neuronal activity, further supporting the idea that synaptic abnormalities contribute to the pathophysiology of DYT1 dystonia.

Using faecal DNA to determine consumption by kangaroos of plants considered palatable to sheep.

Disagreement exists within the scientific community with regards to the level of competition for feed between sheep and kangaroos in the Australian rangelands. The greatest challenge to solving this debate is finding effective means of determining the composition of the diets of these potential grazing competitors. An option is to adopt a non-invasive approach that combines faecal collection and molecular techniques that focus on faecal DNA as the primary source of dietary information. As proof-of-concept, we show that a DNA reference data bank on plant species can be established. This DNA reference data bank was then used as a library to identify plant species in kangaroo faeces collected in the southern rangelands of Western Australia. To enhance the method development and to begin the investigation of competitive grazing between sheep and kangaroos, 16 plant species known to be palatable to sheep were initially targeted for collection. To ensure that only plant sequences were studied, PCR amplification was performed using a universal primer pair previously shown to be specific to the chloroplast transfer RNA leucine (trnL) UAA gene intron. Overall, genus-specific, single and differently sized amplicons were reliably and reproducibly generated; enabling the differentiation of reference plants by PCR product length heterogeneity. However, there were a few plants that could not be clearly differentiated on the basis of size alone. This prompted the adoption of a post-PCR step that enabled further differentiation according to base sequence variation. Restriction endonucleases make sequence-specific cleavages on DNA to produce discrete and reproducible fragments having unique sizes and base compositions. Their availability, affordability and simplicity-of-use put restriction enzyme sequence (RES) profiling as a logical post-PCR step for confirming plant species identity. We demonstrate that PCR-RES profiling of plant and faecal matter is useful for the identification of plants included in the diet of kangaroos. The limitations, potential and the opportunities created for researchers interested in investigating the diet of competing herbivores in the rangelands are discussed.

Membrane hydrocarbon thickness modulates the dynamics of a membrane transport protein.

Nitroxide spin labels were incorporated into selected sites within the beta-barrel of the bacterial outer-membrane transport protein BtuB by site-directed mutagenesis, followed by chemical modification with a methanethiosufonate spin label. The electron paramagnetic resonance lineshapes of the spin-labeled side chain (R1) from these sites are highly variable, and have spectral parameters that reflect secondary structure and local steric constraints. In addition, these lineshape parameters correlate with crystallographic structure factors for Calpha carbons, suggesting that the motion of the spin label is modulated by both the local modes of motion of the spin label and the local dynamics of the protein backbone. Experiments performed as a function of lipid composition and sample temperature indicate that nitroxide spin labels on the exterior surface of BtuB, which face the membrane hydrocarbon, are not strongly influenced by the phase state of the bulk lipids. However, these spectra are modulated by membrane hydrocarbon thickness. Specifically, the values of the scaled mobility parameter for the R1 lineshapes are inversely proportional to the hydrocarbon thickness. These data suggest that protein dynamics and structure in BtuB are directly coupled to membrane hydrophobic thickness.

MAD2DeltaC induces aneuploidy and promotes anchorage-independent growth in human prostate epithelial cells.

The mitotic arrest deficient 2 (MAD2) is suggested to play a key role in a functional mitotic checkpoint because of its inhibitory effect on anaphase-promoting complex/cyclosome (APC/C) during mitosis. The binding of MAD2 to mitotic checkpoint regulators MAD1 and Cdc20 is thought to be crucial for its function and loss of which leads to functional inactivation of the MAD2 protein. However, little is known about the biological significance of this MAD2 mutant in human cells. In this study, we stably transfected a C-terminal-deleted MAD2 gene (MAD2DeltaC) into a human prostate epithelial cell line, Hpr-1 and studied its effect on chromosomal instability, cell proliferation, mitotic checkpoint control and soft agar colony-forming ability. We found that MAD2DeltaC was able to induce aneuploidy through promoting chromosomal duplication, which was a result of an impaired mitotic checkpoint and cytokinesis, suggesting a crucial role of MAD2-mediated mitotic checkpoint in chromosome stability in human cells. In addition, the MAD2DeltaC-transfected cells displayed anchorage-independent growth in soft agar after challenged by 7,12-dimethylbenz[A]anthracene (DMBA), demonstrating a cancer-promoting effect of a defective mitotic checkpoint in human cells. Furthermore, the DMBA-induced transformation was accompanied by a complete loss of DNA damage-induced p53 response and activation of the MAPK pathway in MAD2DeltaC cells. These results indicate that a defective mitotic checkpoint alone is not a direct cause of tumorigenesis, but it may predispose human cells to carcinogen-induced malignant transformation. The evidence presented here provides a link between MAD2 inactivation and malignant transformation of epithelial cells.

Molecular basis for substrate-dependent transmembrane signaling in an outer-membrane transporter.

Transmembrane signaling events that propagate through receptors and transporters have critical roles in cellular function and regulation. In the Escherichia coli vitamin B(12) transporter, BtuB, substrate binding to the extracellular surface of the protein triggers the unfolding of an energy coupling motif at the periplasmic surface. Here, the molecular interactions mediating this substrate-dependent transmembrane signaling event were investigated in a novel way by combining a two mutant cycle analysis with site-directed spin labeling (SDSL). SDSL was used to monitor the unfolding and conformational equilibrium of the energy-coupling motif, and a thermodynamic two-mutant cycle analysis was used to estimate pair-wise interaction free energies for a pair of charged residues (D316 and R14) within the protein interior. The data indicate that D316 and R14 are critical to this structural transition. Substrate binding is shown to reduce the interaction free energy between these residues, thereby triggering the unfolding of the energy coupling motif of this membrane transporter. The result indicates that SDSL when used in combination with a mutant cycle analysis provides an approach to examine the molecular interactions mediating signaling events in membrane proteins.

Do-not-resuscitate decision: the attitudes of medical and non-medical students.

OBJECTIVES: To study the attitudes of both medical and non-medical students towards the do-not-resuscitate (DNR) decision in a university in Hong Kong, and the factors affecting their attitudes. METHODS: A questionnaire-based survey conducted in the campus of a university in Hong Kong. Preferences and priorities of participants on cardiopulmonary resuscitation in various situations and case scenarios, experience of death and dying, prior knowledge of DNR and basic demographic data were evaluated. RESULTS: A total of 766 students participated in the study. There were statistically significant differences in their DNR decisions in various situations between medical and non-medical students, clinical and preclinical students, and between students who had previously experienced death and dying and those who had not. A prior knowledge of DNR significantly affected DNR decision, although 66.4% of non-medical students and 18.7% of medical students had never heard of DNR. 74% of participants from both medical and non-medical fields considered the patient's own wish as the most important factor that the healthcare team should consider when making DNR decisions. Family wishes might not be decisive on the choice of DNR. CONCLUSIONS: Students in medical and non-medical fields held different views on DNR. A majority of participants considered the patient's own wish as most important in DNR decisions. Family wishes were considered less important than the patient's own wishes.

Electrocardiographical case. A young man with chest pain.

A 31-year-old Chinese man presented with complaint of acute chest pain. 12-lead electrocardiogram (ECG) showed sinus rhythm, with widespread upward concave ST segment elevations. The ECG changes along with a history of acute chest pain in a young man with minimal coronary risk factors are suggestive of acute pericarditis. He subsequently developed a pericardial effusion. Diagnosis, treatment and complications of acute percarditis are discussed.

Effects of general, special, and specific resistance training on throwing velocity in baseball: a brief review.

Throwing velocity is a necessary requirement for success in baseball. All position players, including pitchers, may increase their defensive performance if their throwing velocity is improved. A review of the literature suggests that throwing velocity can be increased by resistance training and/or biomechanical improvement of the throwing motion. This paper reviews the 3 broad categories of resistance-training methods by which throwing velocity is increased. The results of research using general, special, and specific throwing resistance-training exercises are presented. The role and applications of these different exercises for baseball players of different ages are discussed.

Age of seizure onset in adults with Down's syndrome.

In a cohort of 68 adults (35 males and 33 females) with Down's syndrome aged 29-83 years, a history of seizures was found in 26.5%. The overall mean age of onset of seizures was 37 years, males (22 years) being significantly younger than females (51 years). The age of onset was bimodally distributed, with the first peak occurring in the first two decades, and a late-onset peak occurring in the fifth and sixth decades. A strong association between Alzheimer's disease and seizures was confirmed. Of those with a history of seizures, those aged over 45 years were significantly more likely to develop Alzheimer's disease than those younger than 45. It is suggested that late-onset epilepsy in Down's syndrome is associated with Alzheimer's disease, while early-onset epilepsy is associated with an absence of dementia.

Legislative funding of athletic training positions in public secondary schools.

In 1991, approximately 21 000 student athletes were actively participating in organized athletics in Hawaii's 61 (38 public and 23 private) secondary schools. Of the 61 schools, only 5 (all private) employed full-time, NATABOC-certified athletic trainers (ATCs) to facilitate the sports health care of their respective student athletes. In an attempt to convince the state legislature that providing funding to hire ATCs was a primary health and safety issue in the state, a community-based educational platform was established and a twofold needs-assessment study was implemented statewide. The educational platform was aimed at parents, coaches, athletic directors, and school administrators. The needs-assessment studies consisted of a 30-question survey on the current practices of sports health care and a year-long injury surveillance survey within the 38 public secondary schools. There were significant differences between the public and private schools with respect to the practice of sports health care. The public school student athletes demonstrated a normative incidence of injury rate. These findings definitively quantified and qualified the need to hire ATCs in the public secondary schools. In July of 1993, the State of Hawaii funded a 2-year athletic training pilot program for approximately $1.2 million, following an extensive lobbying effort and media campaign.

Pre-participation examination: a new form for Hawaii.

A recent study examining the adequacy of the existing pre-participation physical examination (PPE) form in the State of Hawaii suggested that the form be modified and expanded. The standards for a comprehensive PPE indicate that the screening should include an extensive medical history, assessment of height, weight, blood pressure, pulses, vision, cardiopulmonary (heart, and lungs), maturation, skin, abdominal, genitalia, and musculoskeletal function. Pursuant to the recommendation of this recent study and the accepted standards of the American Academy of Family Physicians, American Academy of Pediatrics, American Medical Society for Sports Medicine, American Orthopedic Society for Sports Medicine, and the American Osteopathic Academy of Sports Medicine, the PPE form utilized by the Hawaii High School Athletic Association has been drastically modified. The new form includes an expanded medical history, a maturational assessment (Tanner Stage), a complete musculoskeletal examination, and a participation clearance and recommendation.

Adequacy of a pre-participation examination form: a study of Hawaii physicians.

Many states currently require a medical screening prior to participation in organized sports. The purpose of this study was to examine the adequacy of the existing pre-participation examination form in Hawaii. One hundred forty-eight physicians who perform school health/pre-participation physical examinations were surveyed. The results indirectly suggest that these physicians agreed that the form should be modified and improved (p, .001).

Prevention of hepatitis B virus in athletic training.

Hepatitis B virus (HBV) infection is highly communicable and is the leading cause of acute and chronic liver disease worldwide. In recognition that 10,000 to 15,000 health care workers are becoming infected with HBV annually, the Occupational Health and Safety Administration has instituted strict regulations and guidelines concerning the handling of blood-borne pathogens. Due to the exposure to blood-borne pathogens and potentially infectious materials, athletic training is an allied health care profession that has an increased risk of exposure to HBV. Therefore, it is essential that athletic trainers employ extensive preventive strategies to decrease the exposure to this health-and life-threatening infection.

Multi-adjustable post-operative orthosis for congenital muscular torticollis.

A multi-adjustable torticollis orthosis is described for the post-operative bracing of patients after surgical correction of congenital muscular torticollis. The orthosis can be put on in the early post-operative period and the head and neck position can be maintained in the corrected, and later over-corrected position by the built-in multi-adjustable joint-mechanism. The details of the manufacturing are described. Twenty-five patients (13 girls and 12 boys) from age 1 to 22 with congenital muscular torticollis were fitted with the orthosis post-operatively for an average duration of 10 weeks. Satisfactory compliance with the orthosis was found in 23 cases. Complications were minimal (3 cases) and were related to scalp irritation which improved after minor adjustments of the halo.

Sleep apnoea syndrome--a study of 5 cases.

Sleep apnoea syndrome (SAS) is common in the West but its prevalence is uncertain in Southeast Asia. Five Chinese patients seen in a Sleep Assessment Unit in Hong Kong are presented to illustrate the spectrum of clinical features and treatment methods involved in obstructive and central sleep apnoea. The first patient is a 45-year old woman with severe obstructive SAS and cardiopulmonary complications who improved significantly after tracheostomy. The second patient is a 43-year old man who improved with weight reduction and protriptyline. The third is a 42-year old man whose SAS did not improve with uvulopalatopharyngoplasty but with continuous positive airway pressure (CPAP). The fourth is a 12-year old girl with obstructive SAS who improved significantly after tonsillectomy. The last patient is a 52-year old man with central SAS who improved with CPAP.