The inactivation of single-chain urokinase-type plasminogen activator by thrombin on cultured human endothelial cells

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by thrombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge about cell-mediated inactivation of scu-PA. We have studied whether scu-PA bound to cultured human umbilical vein endothelial cells (HUVEC) could be inactivated by thrombin. High molecular weight scu-PA was bound to HUVEC and incubated with increasing amounts of thrombin for 30 min at 37 degrees C. Cell-bound urokinase-type plasminogen activator (u-PA) was released and levels of scu-PA, tcu-PA/T and active two-chain u-PA were measured using sensitive bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombin. Fifty percent inactivation of scu-PA occurred at about 0.2 nM thrombin. In the presence of monoclonal anti-urokinase receptor IgG, at least 50% of the binding of scu-PA to HUVEC was inhibited. The relative amount of tcu-PA/T that was generated by thrombin was not affected by the monoclonal antibody. These results indicated that scu-PA bound to HUVEC via the urokinase receptor can be inactivated by thrombin. The efficient inactivation of cell-bound scu-PA suggests that a cofactor for thrombin may be involved, like thrombomodulin or glycosaminoglycans. It is concluded that scu-PA bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on u-PA-mediated local fibrinolysis and extracellular proteolysis during processes in which thrombin is also involved.     

Vasa protein expression and localization in the zebrafish

Primordial germ cell (PGC) development in the zebrafish is poorly understood. The expression of vasa RNA, the only molecular marker so far found to be expressed in fish PGCs, suggests its function in the establishment of the germline. The protein product of vasa is present throughout the life cycle in the germline of Drosophila, Caenorhabditis and Xenopus. The expression pattern of the Vasa protein in zebrafish, is still unresolved. We generated an anti-Vasa polyclonal antibody and show that it is maternally expressed initially throughout the embryo. Interestingly, from the two-cell- to the 1000-cell stage the protein is highly concentrated in two 'dots' near the center of the blastomeres and as such remains longest detectable in the animal pole blastomeres. The first distinct cell-specific expression occurs at 60% epiboly on one side of the blastoderm margin. The Vasa protein in the PGCs is organized in a subcellular granular-like conformation which is dynamic throughout development.     

Asymmetry in histone H3 variants and lysine methylation between paternal and maternal chromatin of the early mouse zygote

In mammalian fertilization, the paternal genome is delivered to the secondary oocyte by sperm with protamine compacted DNA, while the maternal genome is arrested in meiotic metaphase II. Thus, at the beginning of fertilization, the two gametic chromatin sets are strikingly different. We elaborate on this contrast by reporting asymmetry for histone H3 type in the pre-S-phase zygote when male chromatin is virtually devoid of histone H3.1/3.2. Localization of the histone H3.3/H4 assembly factor Hira with the paternal chromatin indicates the presence of histone H3.3. In conjunction with this, we performed a systematic immunofluorescence analysis of histone N-tail methylations at position H3K4, H3K9, H3K27 and H4K20 up to the young pronucleus stage and show that asymmetries reported earlier are systematic for virtually all di- and tri-methylations but not for mono-methylation of H3K4 and H4K20, the only marks studied present in the early male pronucleus. For H4K20 the expanding male chromatin is rapidly mono-methylated. This coincides with the formation of maternally derived nucleosomes, a process which is observed as early as sperm chromatin decondensation occurs. Absence of tri-methylated H3K9, tri-methylated H4K20 and presence of loosely anchored HP1-beta combined with the homogenous presence of mono-methylated H4K20 suggests the absence of a division of the paternal chromatin in eu- and heterochromatin. In summary the male, in contrast to female G1 chromatin, is uniform and contains predominantly histone H3.3 as histone H3 variant.     

Localization-dependent oskar protein accumulation; control after the initiation of translation

The appearance of Oskar protein occurs coincident with localization of oskar mRNA to the posterior pole of the Drosophila oocyte, and earlier accumulation of the protein is prevented by translational repression. We find that the nascent polypeptide-associated complex (NAC) is required for correct localization of oskar mRNA. The timing of the defects suggests that, if NAC acts directly via an interaction with nascent Oskar protein, oskar mRNA should be undergoing translation prior to its localization. Polysome analysis confirms that oskar mRNA is associated with polysomes even in the absence of localization of the mRNA or accumulation of Oskar protein. Thus, the mechanisms that prevent accumulation of Oskar protein until it can be secured at the posterior pole of the oocyte include regulated degradation or inhibition of translational elongation.     

Intratumour temperature distributions during hyperthermia

Temperature gradients as high as 2.2°C/cm were observed within Walker 256 mammary carcinoma in vivo, when the tumours were heated at one end by a diathermic lamp. When the blood supply to the tumour was interrupted, the temperature gradients increased as expected, due to lack of cooling effect caused by blood. A mathematical model described these data adequately.     

A Validated Smartphone-Based Assessment of Gait and Gait Variability in Parkinson’s Disease

Background

A well-established connection exists between increased gait variability and greater fall likelihood in Parkinson’s disease (PD); however, a portable, validated means of quantifying gait variability (and testing the efficacy of any intervention) remains lacking. Furthermore, although rhythmic auditory cueing continues to receive attention as a promising gait therapy for PD, its widespread delivery remains bottlenecked. The present paper describes a smartphone-based mobile application (“SmartMOVE”) to address both needs.

Methods

The accuracy of smartphone-based gait analysis (utilizing the smartphone’s built-in tri-axial accelerometer and gyroscope to calculate successive step times and step lengths) was validated against two heel contact–based measurement devices: heel-mounted footswitch sensors (to capture step times) and an instrumented pressure sensor mat (to capture step lengths). 12 PD patients and 12 age-matched healthy controls walked along a 26-m path during self-paced and metronome-cued conditions, with all three devices recording simultaneously.

Results

Four outcome measures of gait and gait variability were calculated. Mixed-factorial analysis of variance revealed several instances in which between-group differences (e.g., increased gait variability in PD patients relative to healthy controls) yielded medium-to-large effect sizes (eta-squared values), and cueing-mediated changes (e.g., decreased gait variability when PD patients walked with auditory cues) yielded small-to-medium effect sizes—while at the same time, device-related measurement error yielded small-to-negligible effect sizes.

Conclusion

These findings highlight specific opportunities for smartphone-based gait analysis to serve as an alternative to conventional gait analysis methods (e.g., footswitch systems or sensor-embedded walkways), particularly when those methods are cost-prohibitive, cumbersome, or inconvenient.     

Fibrinolytic properties of activated FXII.

Activated factor XII (FXIIa), the initiator of the contact activation system, has been shown to activate plasminogen in a purified system. However, the quantitative role of FXIIa as a plasminogen activator in contact activation-dependent fibrinolysis in plasma is still unclear. In this study, the plasminogen activator (PA) activity of FXIIa was examined both in a purified system and in a dextran sulfate euglobulin fraction of plasma by measuring fibrinolysis in a fibrin microtiter plate assay. FXIIa was found to have low PA activity in a purified system. Dextran sulfate potentiated the PA activity of FXIIa about sixfold, but had no effect on the PA activity of smaller fragments of FXIIa, missing the binding domain for negatively charged surfaces. The addition of small amounts of factor XII (FXII) to FXII-deficient plasma induced a large increase in contact activation-dependent PA activity, as measured in a dextran sulfate euglobulin fraction, which may be ascribed to FXII-dependent activation of plasminogen activators like prekallikrein. When more FXII was added, PA activity continued to increase but to a lesser extent. In normal plasma, the addition of FXII also resulted in an increase of contact activation-dependent PA activity. These findings suggested a significant contribution of FXIIa as a direct plasminogen activator. Indeed, at least 20% of contact activation-dependent PA activity could be extracted from a dextran sulfate euglobulin fraction prepared from normal plasma by immunodepletion of FXIIa and therefore be ascribed to direct PA activity of FXIIa. PA activity of endogenous FXIIa immunoadsorped from plasma could only be detected in the presence of dextran sulfate. From these results it is concluded that FXIIa can contribute significantly to fibrinolysis as a plasminogen activator in the presence of a potentiating surface.     

The GABAA receptor is an FMRP target with therapeutic potential in fragile X syndrome

Previous research indicates that the GABA_Aergic system is involved in the pathophysiology of the fragile X syndrome, a frequent form of inherited intellectual disability and associated with autism spectrum disorder. However, the molecular mechanism underlying GABA_Aergic deficits has remained largely unknown. Here, we demonstrate reduced mRNA expression of GABA_A receptor subunits in the cortex and cerebellum of young Fmr1 knockout mice. In addition, we show that the previously reported underexpression of specific subunits of the GABA_A receptor can be corrected in YAC transgenic rescue mice, containing the full-length human FMR1 gene in an Fmr1 knockout background. Moreover, we demonstrate that FMRP directly binds several GABA_A receptor mRNAs. Finally, positive allosteric modulation of GABA_A receptors with the neurosteroid ganaxolone can modulate specific behaviors in Fmr1 knockout mice, emphasizing the therapeutic potential of the receptor.     

Stepped changes of monovalent ligand-binding force during ligand-induced clustering of integrin alphaIIB beta3

Recent evidence demonstrated that conformational changes of the integrin during receptor activation affected its binding to extracellular matrix; however, experimental assessment of ligand-receptor binding following the initial molecular interaction has rarely been carried out at a single-molecule resolution. In the present study, laser tweezers were used to measure the binding force exerted by a live Chinese hamster ovary cell that expressed integrin alphaIIb beta3 (CHO alphaIIb beta3), to the bead carrier coated with the snake venom rhodostomin that served as an activated ligand for integrin alphaIIb beta3. A progressive increase of total binding force over time was noticed when the bead interacted with the CHO alphaIIb beta3 cell; such an increase was due mainly to the recruitment of more integrin molecules to the bead-cell interface. When the binding strength exerted by a single ligand-receptor pair was derived from the "polyvalent" measurements, surprisingly, a stepped decrease of the "monovalent binding force" was noted (from 4.15 to 2.54 piconewtons (pN)); such decrease appeared to occur during the ligand-induced integrin clustering process. On the other hand, the mutant rhodostomin defective in clustering integrins exhibited only one (1.81 pN) unit binding strength.