Purification and characterization of heparan sulfate from human primary osteoblasts

Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts-soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non-identical by a number of parameters, and that these differences have significant ramifications for their ligand-binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS-dependent factors between the ECM and the cell surface.

Cloning and characterization of a novel human gene related to vascular endothelial growth factor

This paper describes the cloning and characterization of a new member of the vascular endothelial growth factor (VEGF) gene family, which we have designated VRF for VEGF-related-factor. Sequencing of cDNAs from a human fetal brain library and RT-PCR products from normal and tumor tissue cDNA pools indicate two alternatively spliced messages with open reading frames of 621 and 564 bp, respectively. The predicted proteins differ at their carboxyl ends resulting from a shift in the open reading frame. Both isoforms show strong homology to VEGF at their amino termini, but only the shorter isoform maintains homology to VEGF at its carboxyl terminus and conserves all 16 cysteine residues of VEGF165. Similarity comparisons of this isoform revealed overall protein identity of 48% and conservative substitution of 69% with VEGF189. VRF is predicted to contain a signal peptide, suggesting that it may be a secreted factor. The VRF gene maps to the D11S750 locus at chromosome band 11q13, and the protein coding region, spanning approximately 5 kb, is comprised of 8 exons that range in size from 36 to 431 bp. Exons 6 and 7 are contiguous and the two isoforms of VRF arise through alternate splicing of exon 6. VRF appears to be ubiquitously expressed as two transcripts of 2.0 and 5.5 kb; the level of expression is similar among normal and malignant tissues.

Compilation of somatic mutations of the CDKN2 gene in human cancers : non-random distribution of base substitutions

The CDKN2 gene, encoding the cyclin-dependent kinase inhibitor p16, is a tumour suppressor gene that maps to chromosome band 9p21-p22. The most common mechanism of inactivation of this gene in human cancers is through homozygous deletion; however, in a smaller proportion of tumours and tumour cell lines intragenic mutations occur. In this study we have compiled a database of over 120 published point mutations in the CDKN2 gene from a wide variety of tumour types. A further 50 deletions, insertions, and splice mutations in CDKN2 have also been compiled. Furthermore, we have standardised the numbering of all mutations according to the full-length 156 amino acid form of p16. From this study we are able to define several hot spots, some of which occur at conserved residues within the ankyrin domains of p16. While many of the hotspots are shared by a number of cancers, the relative importance of each position varies, possibly reflecting the role of different carcinogens in the development of certain tumours. As reported previously, the mutational spectrum of CDKN2 in melanomas differs from that of internal malignancies and supports the involvement of UV in melanoma tumorigenesis. Notably, 52% of all substitutions in melanoma-derived samples occurred at just six nucleotide positions. Nonsense mutations comprise a comparatively high proportion of mutations present in the CDKN2 gene, and possible explanations for this are discussed.

The CDKN2A (p16) gene and human cancer

CDKN2A, the gene encoding the cell-cycle inhibitor p16CDKN2A, was first identified in 1994. Since then, somatic mutations have been observed in many cancers and germline alterations have been found in kindreds with familial atypical multiple mole/melanoma (FAMMM), also known as atypical mole syndrome. In this review we tabulate the known mutations in this gene and discuss specific aspects, particularly with respect to germline mutations and cancer predisposition.

Food(ing): between human-computer and human-food-experience

This workshop is a continuation and extension to the successful past workshops exploring the intersection of food, technology, place, and people, namely 2009 OZCHI workshop, Hungry 24/7? HCI Design for Sustainable Food Culture and Sustainable Interaction with Food, Technology, and the City [1] and 2010 CHI panel Making Food, Producing Sustainability [3]. The workshop aims to bring together experts from diverse backgrounds including academia, government, industry, and non-for-profit organisations. It specifically aims to create a space for discussion and design of innovative approaches to understanding and cultivating sustainable food practices via human-computer-interaction (HCI) as well as addressing the wider opportunities for the HCI community to engage with food as a key issue for sustainability The workshop addresses environmental, health, and social domains of sustainability in particular, by looking at various conceptual and design approaches in orchestrating sustainable interaction of people and food in and through dynamic techno-social networks.

The effect of ocular surface conditions on blink rate and completeness

A healthy human would be expected to show periodic blinks, making a brief closure of the eyelids. Most blinks are spontaneous, occurring regularly with no external stimulus. However a reflex blink can occur in response to external stimuli such as a bright light, a sudden loud noise, or an object approaching toward the eyes. A voluntary or forced blink is another type of blink in which the person deliberately closes the eyes and the lower eyelid raises to meet the upper eyelid. A complete blink, in which the upper eyelid touches the lower eyelid, contributes to the health of ocular surface by providing a fresh layer of tears as well as maintaining optical integrity by providing a smooth tear film over the cornea. The rate of blinking and its completeness vary depending on the task undertaken during blink assessment, the direction of gaze, the emotional state of the subjects and the method under which the blink was measured. It is also well known that wearing contact lenses (both rigid and soft lenses) can induce significant changes in blink rate and completeness. It is been established that efficient blinking plays an important role in ocular surface health during contact lens wear and for improving contact lens performance and comfort. Inefficient blinking during contact lens wear may be related to a low blink rate or incomplete blinking and can often be a reason for dry eye symptoms or ocular surface staining. It has previously been shown that upward gaze can affect blink rate, causing it to become faster. In the first experiment, it was decided to expand on previous studies in this area by examining the effect of various gaze directions (i.e. upward gaze, primary gaze, downward gaze and lateral gaze) as well as head angle (recumbent position) on normal subjects’ blink rate and completeness through the use of filming with a high-speed camera. The results of this experiment showed that as the open palpebral aperture (and exposed ocular surface area) increased from downward gaze to upward gaze, the number of blinks significantly increased (p<0.04). Also, the size of closed palpebral aperture significantly increased from downward gaze to upward gaze (p<0.005). A weak positive correlation (R² = 0.18) between the blink rate and ocular surface area was found in this study. Also, it was found that the subjects showed 81% complete blinks, 19% incomplete blinks and 2% of twitch blinks in primary gaze, consistent with previous studies. The difference in the percentage of incomplete blinks between upward gaze and downward gaze was significant (p<0.004), showing more incomplete blinks in upward gaze. The findings of this experiment suggest that while blink rate becomes slower in downward gaze, the completeness of blinking is typically better, thereby potentially reducing the risk of tear instability. On the other hand, in upward gaze while the completeness of blinking becomes worse, this is potentially offset by increased blink frequency. In addition, blink rate and completeness were not affected by lateral gaze or head angle, possibly because these conditions have similar size of the open palpebral aperture compared with primary gaze. In the second experiment, an investigation into the changes in blink rate and completeness was carried out in primary gaze and downward gaze with soft and rigid contact lenses in unadapted wearers. Not surprisingly, rigid lens wear caused a significant increase in the blink rate in both primary (p<0.001) and downward gaze (p<0.02). After fitting rigid contact lenses, the closed palpebral aperture (blink completeness) did not show any changes but the open palpebral aperture showed a significant narrowing (p<0.04). This might occur from the subjects’ attempt to avoid interaction between the upper eyelid and the edge of the lens to minimize discomfort. After applying topical anaesthetic eye drops in the eye fitted with rigid lenses, the increased blink rate dropped to values similar to that before lens insertion and the open palpebral aperture returned to baseline values, suggesting that corneal and/or lid margin sensitivity was mediating the increased blink rate and narrowed palpebral aperture. We also investigated the changes in the blink rate and completeness with soft contact lenses including a soft sphere, double slab-off toric design and periballast toric design. Soft contact lenses did not cause any significant changes in the blink rate, closed palpebral aperture, open palpebral aperture and the percentage of incomplete blinks in either primary gaze or downward gaze. After applying anaesthetic eye drops, the blink rate reduced in both primary gaze and downward gaze, however this difference was not statistically significant. The size of the closed palpebral aperture and open palpebral aperture did not show any significant changes after applying anaesthetic eye drops. However it should be noted that the effects of rigid and soft contact lenses that we observed in these studies were only the immediate reaction to contact lenses and in the longer term, it is likely that these responses will vary as the eye adapts to the presence of the lenses.

Elaborating a tactile-proprioceptive illusion : the kinaesthetic fusion effect

This study investigated the Kinaesthetic Fusion Effect (KFE) first described by Craske and Kenny in 1981. In Experiment 1 the study did not replicate these findings following a change in the reporting method used by participants. Participants did not perceive any reduction in the sagittal separation of a button pressed by the index finger of one arm and a probe touching the other, following repeated exposure to the tactile stimuli present on both unseen arms. This study’s failure to replicate the widely-cited KFE as described by Craske et al. (1984) suggests that it may be contingent on several aspects of visual information, especially the availability of a specific visual reference, the role of instructions regarding gaze direction, and the potential use of a line of sight strategy when referring felt positions to an interposed surface. In addition, a foreshortening effect was found; this may result from a line-of-sight judgment and represent a feature of the reporting method used. Finally, this research will benefit future studies that require participants to report the perceived locations of the unseen limbs. Experiment 2 investigated the KFE when the visual reference was removed and participants made reports of touched position, blindfolded. A number of interesting outcomes arose from this change and may provide clarification to the phenomena.