Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Seasonal activity patterns of female polar bears (Ursus maritimus) in the Canadian Arctic as revealed by satellite telemetry

We examined seasonal variations in activity level and mobility of female polar bears inhabiting the Canadian Arctic archipelago. The sea-ice habitat consisted primarily of landfast, multi-year ice except from July through October when loose pack-ice predominated. The proportion of the day during which bears were active, and the distance travelled, were documented for 18 bears during 20 months with the aid of satellite telemetry. A peak of activity and mobility occurred in May-July regardless of the reproductive status of tracked bears. The period of elevated activity coincided with a period when seals were especially vulnerable to bear predation. Winter months were characterized by low activity levels and mobility, a response probably related to a reduced access to seals and to inclement weather. During the period preceding den entry (May through September), pregnant females tended to be more mobile in May-June, and less active in August September, compared to non-pregnant, solitary females. Early den entry by pregnant females and facultative use of dens by other members of the population are viewed as a means to conserve energy for individuals with adequate fat reserves but experiencing conditions unfavourable for hunting seals. We conclude that seasonal variations in activity and mobility of polar bears seem closely linked to the temporal dynamics of bear-seal interactions.     

Mechanisms in bradykinin stimulated arachidonate release and synthesis of prostaglandin and platelet activating factor

Regulatory mechanisms in bradykinin (BK) activated release of arachidonate (ARA) and synthesis of prostaglandin (PG) and platelet activating factor (PAF) were studied in bovine pulmonary artery endothelial cells (BPAEC). A role for GTP binding protein (G-protein) in the binding of BK to the cells was determined. Guanosine 5-O- (thiotriphosphate), (GTPtauS), lowered the binding affinity for BK and increased the Kd for the binding from 0.45 to 1.99 nM. The Bmax remained unaltered at 2.25 x 10(-11) mole. Exposure of the cells to aluminium fluoride also reduced the affinity for BK. Bradykinin-induced release of ARA proved pertussis toxin (PTX) sensitive, with a maximum sensitivity at 10 ug/ml PTX. GTPtauS at 100 muM increased the release of arachidonate. The effect of GTPtauS and BK was additive at suboptimal doses of BK up to 0.5 nM but never exceeded the levels of maximal BK stimulation at 50 nM. PTX also inhibited the release of ARA induced by the calcium ionophore, A23187. Phorbol 12-myristate 13-acetate or more commonly known as tetradecanoyl phorbol acetate (TPA) itself had little effect on release by the intact cells. However, at 100 nM it augmented the BK activated release. This was downregulated by overnight exposure to TPA and correlated with down-regulation of protein kinase C (PKC) activity. The down-regulation only affected the augmentation of ARA release by TPA but not the original BK activated release. TPA displayed a similar, but more potent amplification of PAF synthesis in response to both BK or the calcium ionophore A23187. These results taken together point to the participation of G-protein in the binding of BK to BPAEC and its activation of ARA release. Possibly two types of G-protein are involved, one associated with the receptor, the other activated by Ca(2+) and perhaps associated with phospholipase A(2) (PLA(2)). Our results further suggest that a separate route of activation, probably also PLA(2) related, takes place through a PKC catalysed phosphorylation.     

A hypothetical model for the peptide binding domain of hsp70 based on the peptide binding domain of HLA

The sequences of the peptide binding domains of 33 70 kd heat shock proteins (hsp70) have been aligned and a consensus secondary structure has been deduced. Individual members showed no significant deviation from the consensus, which showed a beta 4 alpha motif repeated twice, followed by two further helices and a terminus rich in Pro and Gly. The repeated motif could be aligned with the secondary structure of the functionally equivalent peptide binding domain of human leucocyte antigen (HLA) class I maintaining equivalent residues in structurally important positions in the two families and a model was built based on this alignment. The interaction of this domain with the ATP domain is considered. The overall model is shown to be consistent with the properties of products of chymotryptic cleavage.     

Cell signalling through phospholipid breakdown

There is much evidence that G-proteins transduce the signal from receptors for Ca²⁺-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein -subunits. It is proposed that this protein is the a-subunit of the G-protein that regulates the phospholipase in this tissue.Ca²⁺-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca²⁺ protein kinase C and perhaps growth factor receptor tyrosine kinases.     

Static and dynamic assessment of the Biodex dynamometer

The validity and accuracy of the Biodex dynamometer was investigated under static and dynamic conditions. Static torque and angular position output correlated well with externally derived data (r = 0.998 and r greater than 0.999, respectively). Three subjects performed maximal voluntary knee extensions and flexions at angular velocities from 60 to 450 degrees.s-1. Using linear accelerometry, high speed filming and Biodex software, data were collected for lever arm angular velocity and linear accelerations, and subject generated torque. Analysis of synchronized angular position and velocity changes revealed the dynamometer controlled angular velocity of the lever arm to within 3.5% of the preset value. Small transient velocity overshoots were apparent on reaching the set velocity. High frequency torque artefacts were observed at all test velocities, but most noticeably at the faster speeds, and were associated with lever arm accelerations accompanying directional changes, application of resistive torques by the dynamometer, and limb instability. Isokinematic torques collected from ten subjects (240, 300 and 400 degrees.s-1) identified possible errors associated with reporting knee extension torques at 30 degrees of flexion. As a result of tissue and padding compliance, leg extension angular velocity exceeded lever arm angular velocity over most of the range of motion, while during flexion this compliance meant that knee and lever arm angles were not always identical, particularly at the start of motion. Nevertheless, the Biodex dynamometer was found to be both a valid and an accurate research tool; however, caution must be exercised when interpreting and ascribing torques and angular velocities to the limb producing motion.     

Functional torque-velocity and power-velocity characteristics of elite athletes

Technical limitations of some isokinetic dynamometers have called into question the validity of some data on human muscle mechanics. The Biodex dynamometer has been shown to minimize the impact artefact while permitting automatic gravity correction. This dynamometer was used to study quadriceps muscle torque and power generation in elite power (n = 6) and elite endurance (n = 7) athletes over 12 randomly assigned isokinetic velocities from 30 degrees.s-1 to 300 degrees.s-1. The angle at peak torque varied as a negative, linear function of angular velocity, with the average angle across test velocities being 59.5 degrees (SD 10.2 degrees). Power athletes developed greater peak torque at each angular velocity (P less than 0.05) and experienced a 39.7% decrement in torque over the velocity range tested. Endurance athletes encountered a 38.8% decline in peak torque. Torques measured at 60 degrees of knee flexion followed a similar trend in both groups; however the greatest torques were recorded at 60 degrees.s-1 rather than at 30 degrees.s-1. Leg extensor muscle power increased monotonically with angular velocity in both power (r2 = 0.728) and endurance athletes (r2 = 0.839); however these curves diverged significantly so that the power athletes produced progressively more power with each velocity increment. These inter group differences probably reflected a combination of natural selection and training adaptation.     

A family of muscle gene promoter element (CArG) binding activities in Xenopus embryos: CArG/SRE discrimination and distribution during myogenesis.

The CArG box is an essential promoter sequence for cardiac muscle actin gene expression in Xenopus embryos. To assess the role of the CArG motif in promoter function during Xenopus development, the DNA-binding activities present in the embryo that interact with this sequence have been investigated. A family of four Embryo CArG box1 Factors (ECFs) was separated by a 2-step fractionation procedure. These factors were distinct from the previously described C-ArG box binding activity Serum Response Factor (SRF). ECF1 was the most prominent binding activity in cardiac actin-expressing tissues, and bound the CArG box in preference to a Serum Response Element (SRE). SRF was also detectable in muscle, but it bound preferentially to an SRE. The properties of ECF3 were similar to those of ECF1, but it was much less prominent in cardiac actin-expressing tissues. The properties of the two other factors were distinctive: ECF2 was of relatively low affinity and high abundance, whilst ECF4 bound non-specifically to ends of DNA. The binding activity (or activities) that interacted with the CArG box was found to be influenced by both the concentrations of the other CArG box binding activities and the sequence of the site. Although there was no evidence for a muscle-specific CArG box binding activity, the properties of ECF1 suggest that it could play a role in the expression of the cardiac actin gene during Xenopus development.