The Reactions of Hemoglobin in Various Media

The rate constants for the association of oxygen and of carbon monoxide with reduced hemoglobin and for the dissociation of oxygen from hemoglobin were studied in a variety of media. All three of these rates were essentially independent of diffusion rate and dielectric constant throughout a wide range. These results are very different from those found for other heme proteins whose rates are diffusion-controlled in very viscous solvents.

The interpretation of the average measured rates described herein in terms of the four rate constants discussed by Gibson and Roughton indicates that our measurements reflect most strongly the constants for the first reacting molecule in each case. For the purposes of these reactions, the reactants may be regarded as neutral molecules.

     

A three-dimensional computational simulation of some sources of measurement artifact in microvascular pulsed ultrasound Doppler velocimetry

Recent applications of 20 MHz pulsed ultrasound Doppler velocimetry (PUDVM) in microsurgical research have necessarily employed piezoelectric crystals whose diameter is not negligible compared to the lumen size (1-2 mm) of many vessels of interest. A three-dimensional numerical model was developed to explore relationships between actual and detected flow field parameters, for (steady) Poiseuille flow, when appreciable velocity gradients exist within the PUDVM sample volume. Validation studies showed that highly accurate velocity profiles could be obtained in the limiting case of a very small sample volume (0.1 mm radius), but that for currently employed crystals (approximately equal to 0.5 mm radius) there was appreciable underestimation of the centersteam velocity, and appreciable overestimation of the flow stream diameter. Errors in perceived velocity and flow rate were found to be relatively insensitive to perturbations in the sample volume thickness, in the size of the sampling range increment, or in the angle of insonation beam divergence. By contrast, these apparent flow parameters were found to be very sensitive to perturbations of sample volume diameter or of the Doppler angle. Small variations in the degree of partial sample volume overlap of the flowstream periphery were shown to be capable of causing large fluctuations in apparent flow stream diameter.     

Regression of human melanoma xenografts in nude mice injected with methotrexate linked to monoclonal antibody 225.28 to human high molecular weight-melanoma associated antigen

Summary Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG_2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 × 10⁶ cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotypematched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-M _r MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.     

TWEAK affects keratinocyte G2/M growth arrest and induces apoptosis through the translocation of the AIF protein to the nucleus

The soluble TNF-like weak inducer of apoptosis (TWEAK, TNFSF12) binds to the fibroblast growth factor-inducible 14 receptor (FN14, TNFRSF12A) on the cell membrane and induces multiple biological responses, such as proliferation, migration, differentiation, angiogenesis and apoptosis. Previous reports show that TWEAK, which does not contain a death domain in its cytoplasmic tail, induces the apoptosis of tumor cell lines through the induction of TNFα secretion. TWEAK induces apoptosis in human keratinocytes. Our experiments clearly demonstrate that TWEAK does not induce the secretion of TNFα or TRAIL proteins. The use of specific inhibitors and the absence of procaspase-3 cleavage suggest that the apoptosis of keratinocytes follows a caspase- and cathepsin B-independent pathway. Further investigation showed that TWEAK induces a decrease in the mitochondrial membrane potential of keratinocytes. Confocal microscopy showed that TWEAK induces the cleavage and the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus, thus initiating caspase-independent apoptosis. Moreover, TWEAK induces FOXO3 and GADD45 expression, cdc2 phosphorylation and cdc2 and cyclinB1 degradation, resulting in the arrest of cell growth at the G2/M phase. Finally, we report that TWEAK and FN14 are normally expressed in the basal layer of the physiological epidermis and are greatly enhanced in benign (psoriasis) and malignant (squamous cell carcinoma) skin pathologies that are characterized by an inflammatory component. TWEAK might play an essential role in skin homeostasis and pathology.     

The ecology of 3-D space use in a sexually dimorphic mammal

The distribution of animals is the result of habitat selection according to sex, reproductive status and resource availability. Little is known about how marine predators investigate their 3-dimensional space along both the horizontal and vertical axes and how temporal variation affects space use. In this study, we assessed the spatio-temporal movement of a sexually dimorphic marine mammal, the grey seal Halichoerus grypus by 1) determining seasonal home range size, 2) testing whether space use of seals was affected by water depth, and 3) investigating the vertical movement of seals according to the maximum depth of each dive. Between 1993 and 2005, we fitted 49 grey seals in the Gulf of St. Lawrence with satellite transmitters. We estimated seasonal 95% fixed-kernel home ranges for each individual. For each seal, we tested for selectivity and preference for 4 water depth classes at the home range scale and within the home range. We also evaluated the proportional number of dives made in each water depth classes according to the maximum depth of each dive. Home ranges were 10 times larger in winter than in summer. Seals generally selected habitats <50 m deep. They also mainly dove to depths of 40 m or less. At both scales of selection, preference for shallow areas decreased in winter. We also observed that adults used shallow habitats more than juveniles to establish their home range. A spatial segregation based on sex also occurred at the finer scale of selection where females were more concentrated in the shallowest parts of their home range than males. Segregation in space use according to age and sex classes occurred at both the horizontal and vertical scales. Our results emphasise the importance of studying habitat selection of marine predators in 3-dimensional space, in addition to the temporal scale.     

The extraordinarily complex but highly structured organization of intestinal mucus-gel unveiled in multicolor images

The mucus that coats the gastrointestinal tract of all mammals is a dynamic and sticky gel layer and represents the first protective barrier between the host and the hostile environment. There is, however, a lack of detailed knowledge about the mucus gel organization because of the high water content and the complexity of MUC2, the main gel-forming molecule in the intestine. Histological staining and a multilabel immunofluorescence method were used to examine mucus blankets and Muc2 in mouse colon and ileum samples fixed in Carnoy's solution, unveiling an extraordinarily complex but highly structured mucus gel organization. The inner firmly adherent mucus blanket consists of alternating layers. The thicker outer loosely adherent mucus blanket in the colon is made of alternating laminated layers and loose curl-like structures. The layers consist of Muc2 molecules with different fucosylation states and glycoforms remain unmixed in the mucus. Importantly, distinct goblet cell subpopulations throughout the ileum along the crypt-to-villus axis with an alternation of goblet cells secreting fucosylated and non-fucosylated Muc2 are observed. A better understanding of the mucus structure should contribute to improve the efficiency of DNA and drug delivery and will allow for a better understanding and treatment of inflammatory and infectious intestinal diseases.     

Signalling pathways from NADPH oxidase-4 to idiopathic pulmonary fibrosis

This review focuses on the roles of NADPH oxidase/NOX proteins in idiopathic pulmonary fibrosis (IPF) pathophysiology and in the signalling pathways involved in IPF. NOX proteins are membrane-associated multi-unit enzymes that catalyze the reduction of oxygen using NADPH as an electron donor. Recent studies indicate that NOX4 is induced in pulmonary fibroblasts in response to TGF-β. TGF-β or PDGF induce myofibroblast proliferation, differentiation, migration, contractility and extracellular matrix production, through NOX4 and reactive oxygen species dependent SMAD2/3 phosphorylation. NOX4 is increased in pulmonary fibroblasts from IPF patients and deletion of Nox4 in mice prevents bleomycin-induced pulmonary fibrosis. These data strongly suggest that targeting of NOX4 could be a step forward in the treatment of fibrotic lung diseases, by specifically targeting myofibroblasts, a major player in this disease.