Adsorption of serum fetuin to hydroxylapatite does not contribute to osteoblast phenotype modifications

Osteoblasts exhibit enhanced differentiation and altered gene profiles when cultured on hydroxyapatite (HA) compared to plastic surfaces. To begin determining mechanisms for this response, we used proteomics to identify proteins predominantly found in osteoblasts on HA but not plastic surfaces. Two-dimensional gel electrophoresis and Western analyses indicate that fetuin is abundant in extracts from HA, but not plastic surfaces. Incubation of HA and plastic surfaces with cell culture medium (containing 10% serum) under cell-free conditions shows that fetuin is predominantly derived from the culture medium serum and readily adsorbs to the HA surface. However, we did detect low levels of fetuin B mRNA in osteoblasts. Serum albumin, actin-beta, apolipoprotein-AI, and vimentin also adsorbed to HA. To determine the role of fetuin in the HA-induced osteoblast phenotype changes, osteoblasts were seeded onto fetuin-coated or uncoated HA under serum-free conditions. Osteoblast morphology was similar on both HA surfaces, suggesting that HA alone (without adsorbed serum proteins) is sufficient for cell attachment and spreading. Similarly, genes previously reported to be modulated by HA (glvr-1, DMP-1, osteoglycin, and proliferin 3) were modulated even in the absence of fetuin or other serum proteins. These data show that HA surface can be enriched selectively with fetuin from serum; however, neither fetuin or other serum proteins are required to mediate HA-induced osteoblast attachment, spreading, or changes in expression of genes examined. This finding suggests that factors intrinsic to HA are required for the response.     

Cytoskeletal changes during neurogenesis in cultures of avian neural crest cells

Summary Neural crest cells are motile and mitotic, whereas their neuronal derivatives are terminally post-mitotic and consist of stationary cell body from which processes grow. The present study documents changes in the cytoskeleton that occur during neurogenesis in cultures of avian neural crest cells. The undifferentiated neural crest cells contain dense bundles of actin filaments throughout their cytoplasm, and a splayed array of microtubules attached to the centrosome. In newly differentiating neurons, the actin bundles are disrupted and most of the remaining actin filaments are reorganized into a cortical layer underlying the plasma membrane of the cell body and processes. Microtubules are more abundant in newly-differentiating neurons than in the undifferentiated cells, and individual microtubules can be seen dissociated from the centrosome. Neuron-specific -III tubulin appears in some crest cells prior to cessation of motility and cell division, and expression increases with total microtubule levels during neurogenesis. To investigate how these early cytoskeletal changes might contribute to alterations in morphology during neurogenesis, we have disrupted the cytoskeleton with pharmacologic agents. Microfilament disruption by cytochalasin immediately arrests the movement of neural crest cells and causes them to round-up, but does not significantly change the morphology of the immature neurons. Microtubule depolymerization by nocodazole slows the movement of undifferentiated cells and causes retraction of processes extended by the immature neurons. These results suggest that changes in the actin and microtubule arrays within neural crest cells govern distinct aspects of their morphogenesis into neurons.     

Prevalence of hearing loss among people aged 65 years and over: screening and hearing aid provision.

The aim of this study was to assess the prevalence of hearing loss among people aged 65 years and over and to offer hearing aids where necessary. The study was carried out in a large health centre practice in Birmingham with a computerized record system. All patients aged 65 years, and a 20% random sample of patients aged 66 years and over were invited for interview. They were questioned about hearing loss and examined audiometrically. All patients with a hearing loss in the better ear of 35 decibels or more over the speech frequencies (0.5-4 kHz) were offered a hearing aid. A total of 322 patients attended (72% of those invited) and of these 34 patients already had a hearing aid. A further 142 patients were identified for whom an aid was recommended and 69 of these accepted. The acceptance rate was higher among men than women (57% compared with 43%). Sixty one patients (19% of those screened) had an asymmetrical hearing loss and of these 24 required consultant assessment; none had serious underlying pathology. There is a large unmet need for hearing aid provision. Simple audiometric assessment in health centres provides an opportunity to meet this need.     

Two novel paradigms for the simultaneous assessment of conditioned taste aversion and food intake effects of anorexic agents

The conditioned taste aversion (CTA) is routinely used to assess the aversive consequences of anorexic agents, including potential pharmacological therapies for obesity. In a typical CTA paradigm, rats briefly sampling a novel tastant (e.g., saccharin) are acutely administered with toxin (e.g., lithium chloride, LiCl). After as few as one taste-toxin pairing, rats will reliably avoid the novel tastant. This paradigm is frequently used for the assessment of possible aversive consequences of drugs that are candidates for pharmacological therapies. The degree to which the drug supports development of a CTA is interpreted as an index of its aversive properties. Difficulties with previous work include the inability to assess affects on food intake and CTA simultaneously, particularly during chronic drug administration. We report here two novel CTA paradigms for the assessment of appetitive and aversive consequences of anorexic agents, simultaneously. In the first experiment, animals receive an intraoral infusion of a novel and highly palatable tastant immediately prior to administration of increasing doses of LiCl. In the second experiment, rats were implanted intraperitoneally with osmotic minipumps that chronically delivered a low dose of LiCl for 7 days. LiCl did not affect short or long term food intake in either experiment. However, LiCl did support the development of a CTA in both paradigms. These results suggest that both the appetitive and aversive consequences of anorexic agents can be assessed simultaneously during either acute or chronic drug administration.     

Care at the end of life: a novel curriculum module implemented by medical students.

End-of-life (EOL) and palliative care education in medical school curricula stand at a crossroads. Consensus has emerged that these topics merit systematic instruction throughout medical school training, yet curricula all too often consist of sporadic lectures focused on bioethics instead of clinical skills. The medical student authors identified a deficit in their curriculum, and designed and implemented an EOL curriculum module for their colleagues. In early 2000 the authors surveyed senior medical students about their experiences with EOL and palliative education, identifying deficits in clinical training and recommendations for interventions. They then designed a case-based educational module to teach EOL communication skills to medical students commencing clinical training. Faculty with national and local experience with EOL and palliative care reviewed the curriculum. Twelve of these faculty were oriented to the curriculum and then taught it in pairs to groups of 12 to 16 medical students in 2000 and 2001. The curriculum develops skills, attitudes, and knowledge relevant for communicating bad news and establishing treatment options in the EOL setting by utilizing trigger videos, group discussion, role plays, and case discussions. Approximately 75% of the 86 eligible students attended the module in 2000 and 2001. Feedback has guided the curriculum's refinement by the medical student authors. In addition, a standardized patient exercise, introduced in 2001, allowed students to reinforce the skills learned during the module.     

Serological Survey of Toscana Virus Infections in a High-Risk Population in Italy

Toscana virus is the most important agent responsible for meningitis in central Italy. We report a serosurveillance study, using an immunoenzymatic assay, of 360 serum samples harvested from a high-risk population occupationally exposed to Toscana virus in two regions of Italy, Tuscany and Piedmont. The results indicates a seroprevalence of Toscana virus of 77.2% in the forestry workers, particularly in the Tuscany region. This fact is strictly correlated with the ecological niches specific for the survival of Toscana virus arthropod vector.     

Biosynthetic and regulatory role of lys9 mutants of Saccharomyces cerevisiae

Summary Derepression of lysine biosynthetic enzymes of Saccharomyces cerevisiae was investigated in lys9 auxotrophs which lack saccharopine reductase activity. Five enzymes (homocitrate synthase, homoisocitrate dehydrogenase, -aminoadipate aminotransferase, -aminoadipate reductase and saccharopine dehydrogenase) were constitutively derepressed in all lys9 mutants with up to eight-fold higher enzyme levels than in isogenic wild-type cells. Levels of these enzymes in lys2, lys14, and lys15 S mutants were the same or lower than those in wild-type cells. The regulatory property of lys9 mutants exhibited recessiveness to the wild-type gene in heterozygous diploids. Unlike the mating type effect, homozygous diploids resulting from crosses between lys9 auxotrophs exhibited even higher levels of derepressed enzymes than the haploid mutants. Addition of a higher concentration of lysine to the growth medium resulted in reduction of enzyme levels although they were still derepressed. These results suggest that lys9 mutants represent a lesion for the saccharopine reductase and may represent a repressor mutation which in the wild-type cells simultaneously represses unlinked structural genes that encode for five of the lysine biosynthetic enzymes.     

Computer image sperm selection as a novel approach to subzonal insemination in the human

Utilizing real-time computer image analysis, individual spermatozoawere selected using microaspiration. Selection criteria werebased on potential hyperactivation motility characteristics;the amplitude of lateral head displacement >7.5 µm,curvilinear velocity >70 µm/s and linearity of <30%.For this pilot study, 16 patients (eight in each group) wererecruited. Using subzonal insemination (SUZI), up to five (mean= 4.4 ± 0.3) spermatozoa selected using computer-imagesperm selection (CISS) were microinjected, or up to 15 (mean= 12.8 ± 1.3 SD) unselected spermatozoa. In the groupwhich utilized CISS, 28 out of 49 (57%) oocytes were fertilizedcompared with 13 out of 52 (25%) utilizing conventional SUZI(P < 0.04); polyspermy was 20% (n = 10) and 2% (n = 1) respectively.CISS with SUZI showed increased efficiency in achieving fertilizationand is a novel approach to studying individual sperm functionin a sperm egg bioassay where gamete ratios are close to unity.     

Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse

Muscle wasting is a prominent feature of several systemic diseases, neurological damage and muscle disuse. The contribution of calpain proteases to muscle wasting in any instance of muscle injury or disease has remained unknown because of the inability to specifically perturb calpain activity in vivo . We have generated a transgenic mouse with muscle-specific overexpression of calpastatin, which is the endogenous inhibitor of calpains, and induced muscle atrophy by unloading hindlimb musculature for 10 days. Expression of the transgene resulted in increases in calpastatin concentration in muscle by 30- to 50-fold, and eliminated all calpain activity that was detectable on zymograms. Muscle fibres in ambulatory, transgenic mice were smaller in diameter, but more numerous, so that muscle mass did not differ between transgenic and non-transgenic mice. This is consistent with the role of the calpain-calpastatin system in muscle cell fusion that has been observed in vitro . Overexpression of calpastatin reduced muscle atrophy by 30 % during the 10 day unloading period. In addition, calpastatin overexpression completely prevented the shift in myofibrillar myosin content from slow to fast isoforms, which normally occurs in muscle unloading. These findings indicate that therapeutics directed toward regulating the calpain-calpastatin system may be beneficial in preventing muscle mass loss in muscle injury and disease.