Restricted role for methionine synthase reductase defined by subcellular localization.

Methionine synthase reductase (MSR; gene name MTRR) is responsible for the reductive activation of methionine synthase. Cloning of the MTRR gene had revealed two major transcription start sites which, by alternative splicing, allows for two potential translation products of 698 and 725 amino acids. While the shorter protein was expected to target the cytosol where methionine synthase is located, the additional sequence in the longer protein was consistent with a role as a mitochondrial leader sequence. The possibility that MSR might target mitochondria was also suggested by the work of Leal et al. [N.A. Leal, H. Olteanu, R. Banerjee, T.A. Bobik, Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase, J. Biol. Chem. 279 (2004) 47536-47542.] who showed that it can act as the reducing enzyme in combination with MMAB (ATP:Cob(I)alamin adenosyltransferase) to generate adenosylcobalamin from cob(II)alamin in vitro. Here we examined directly whether MSR protein is found in mitochondria. We show that, while two transcripts are produced by alternative splicing, the N-terminal segment of the putative mitochondrial form of MSR fused to GFP does not contain a sufficiently strong mitochondrial leader sequence to direct the fusion protein to the mitochondria of human fibroblasts. Further, antibodies to MSR protein localized MSR to the cytosol, but not to the mitochondria of human fibroblasts or the human hepatoma line Huh-1, as determined by Western blot analysis and immunofluorescence of cells in situ. These data confirm that MSR protein is restricted to the cytosol but, based on the Leal study, suggest that a similar protein may interact with MMAB to reduce the mitochondrial cobalamin substrate in the generation of adenosylcobalamin.

The captive bubble method for the evaluation of pulmonary surfactant: surface tension, area, and volume calculations.

For measuring the properties of lung surfactant, we provide formulas for calculating the surface tension, area, and volume of captive air bubbles in aqueous media. Only measurements of bubble height (h) and diameter (d) are required. Data processing has been automated using standard video capture hardware and software, and our own image-processing and -analyzing programs. Our polynomials in h/d describe the ratios of actual bubble surface area and volume to that of a spherical bubble having the same diameter. For surface tension, a polynomial in h/d describes the ratio of the surface tension of a flat semi-infinite bubble to the surface tension of the measured bubble of the same height. Coefficients for the area and volume polynomials were obtained from h and d, and measured areas and volumes of revolution of calibrating bubbles. Coefficients for the surface tension polynomial were obtained analytically from a published polynomial in h/d [3]. Results using these polynomials agree satisfactorily with those obtained independently [4] using bubble perimeter measurements.

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos.

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatous and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 +/- 1.5 (S.D.) days compared to 27.3 +/- 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 +/- 0.7 (S.D.) days compared to 48.5 +/- 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.

Measurement of lung water dynamics and lung aeration in the perinatal period by use of digital X-ray fluoroscopy.

A method has been developed for the measurement of lung water dynamics and regional aeration of the lung in anesthetized newborn lambs by use of X-ray fluoroscopy, video recording, and digital image processing. After cesarean section and before the first breath fetal lambs under halothane-oxygen were placed on an X-ray table and connected to a volume-cycled respirator. X-ray fluoroscopy commenced before the initiation of respiration, and the images were recorded on video tape. X-ray transmission through the thorax increased as the lung was aerated. The enhanced transmission was compared with the values obtained from a calibration water wedge from which an equivalent path length through water can be estimated. In testing this method, it was demonstrated that X-ray transmission was linearly related to the wet lung weight-to-body weight ratio and to the product of the wet-to-dry weight ratio multiplied by anatomic thickness of frozen lung blocks. Calibrated values were also linearly related to this product and to the actual measured height of the fluid and tissue in the fluid-filled lung.

Lung aeration and lung water dynamics in artificially ventilated newborn lambs.

To the present lung liquid dynamics in the immediate neonatal period have been measured mainly by gravimetric techniques. This paper explores lung aeration and lung water dynamics in seven fetal lambs between 139 and 142 days of gestation delivered by caesarean section and ventilated on a constant-volume respirator. After caesarean section and instrumentation, fetal lambs were quickly transferred to a warm X-ray table and connected to a volume-cycled respirator. X-ray fluoroscopic images of the chest commenced before the first breath and were recorded on video tape. After 1 h of ventilation, measurements were made of pulmonary blood volume, and lung samples were taken for wet weight and dry weight analysis. Fluoroscopic image brightness was calibrated by comparison with images obtained from a water wedge, which extended across the X-ray field. Thus image brightness was related to "equivalent water path length" through the thorax. Within a defined lung field, image brightness increases as the amount of lung water in the path of the X-ray beam is reduced. This occurs rapidly at first and then more slowly over the remaining hour. There was considerable variation between the reduction of liquid in the lung fields examined within the one animal, as well as the absolute amount of fluid that had been cleared during the 1st h.

Afferent projections related to attack sites in the ventral midbrain tegmentum of the cat.

Quiet attack was elicited by electrical stimulation of the ventrolateral midbrain tegmentum of the cat. The paw was not used other than to position or hold the rat during the bite. Bites were directed toward the head and neck region and were not accompanied by autonomic responses other than pupillary dilation and sometimes slight piloerection on the back. Horseradish peroxidase was deposited at the attack sites. Cells labeled with the peroxidase reaction product were located in gyrus propreus, gyrus genualis, nucleus accumbens, bed nucleus of the stria terminalis, bed nucleus of the anterior commissure, nucleus of the diagonal band, substantia innominata, anterior amygdaloid area, ventromedial hypothalamic area, paraventricular nucleus, perifornical hypothalamic area, lateral hypothalamic area, dorsal hypothalamic area, field of Forel, midbrain reticular formation, superior colliculus, ventral central grey, lateral central gray, locus coeruleus, parabrachial nuclei, nucleus of the lateral lemniscus, oral pontine reticular nucleus, and the dorsal raphe. Other regions were less prominently labeled. Previous studies have shown most of these sites to have some involvement in attack.