Co-cultivation of rat pneumocytes and bovine endothelial cells on a liquid-air interface.

The blood-air barrier is a most important functional element of the lung but little information is available about the cells constituting this barrier in vivo. The aim of the present study was to create an in vitro model of the blood-air barrier that would allow investigation of cellular interactions and alveolar metabolism, and would be suitable for in vitro drug screening. Rat pneumocytes and bovine microvascular endothelial cells were grown on opposite sides of microporous polycarbonate filters, as immersion, perfusion and liquid-air interface (LAI) cultures. The effects of culture conditions on cell morphology were examined by light and transmission electron microscopy. For immersion and perfusion co-cultures, both compartments were supplied with culture medium. In contrast, for liquid-air interface studies, only the endothelial cell compartment was continuously supplied with serum-free medium, whilst the type II pneumocytes were ventilated with air. The pneumocytes lost their morphological characteristics when using immersion or perfusion co-cultures. Under liquid-air interface conditions, they retained most of their characteristic morphological features when compared to the intact blood-air barrier. A subset of primary type II pneumocytes retained its differentiated phenotype, with cuboidal morphology, lamellar bodies and apical microvilli. These type II pneumocytes appeared to be connected by tight junctions to cells expressing morphological characteristics of type I pneumocytes. As shown herein, the liquid-air interface co-culture possesses many morphological characteristics of the intact blood-air barrier. In summary, this article describes the design of an artificial blood-air barrier, in which rat pneumocytes were cultivated with bovine microvascular endothelial lung cells on opposing sides of a microporous polycarbonate filter. We conclude that it might be a promising in vitro model for studies of molecular transport via the blood-air barrier, the investigation of repair mechanisms after alveolar injury, or as an in vitro screening system.

A H1 hemagglutinin of a human influenza A virus with a carbohydrate-modulated receptor binding site and an unusual cleavage site.

Two receptor binding variants of the influenza virus A/Tübingen/12/85 (H1N1) were separated by their different plaque formation in MDCK cells. Hemagglutination of variant I was restricted to red blood cells of guinea pigs, whereas variant II also hemagglutinated chicken cells. The variants differed also in their ability to bind to alpha 2,6-linked sialic acid. Evidence is presented that this difference is determined by a complex carbohydrate side chain at asparagine131 near the receptor binding site which is absent in variant II. With both variants, the arginine found at the cleavage site of all other human isolates analyzed so far was replaced by lysine.

Effect of functional stimulation on ascorbate content in phagocytes under physiological and pathological conditions.

Neutrophil polymorphonuclear leucocytes and macrophages contain 10-40 times increased intracellular ascorbate concentrations compared to plasma. A slight decrease of ascorbate content could be observed in total white blood cells and in monocytes upon stimulation with opsonized zymosan. These decreases were more pronounced in peritoneal and alveolar macrophages from rats. In patients with rheumatoid disease whose phagocytes are exposed to a constant challenge, significantly lowered intracellular ascorbate contents were found in neutrophils and mononuclear cells. Surgical and thermal trauma in rats depressed intracellular ascorbate levels in peritoneal macrophages. These results are indicative of an essential role ascorbic acid plays in phagocytic cells.

The role of ascorbic acid in carcinogenesis.

Vitamin C is an essential nutrient whose protective role in carcinogenesis has been discussed for more than 50 years. Epidemiologic studies suggest that the consumption of vitamin C-rich foods is associated with a lower risk of cancers of the esophagus and stomach. The observation that cancer patients have low leukocyte vitamin C levels led to therapeutic trials the results of which are controversial; the hypothesis that vitamin C acts like a drug must be questioned. On the other hand, ascorbic acid interacts with various tumor-inducing compounds, such as the precursors of N-nitroso compounds, to prevent the formation of tumors. Experiments with animals and cell cultures indicate that ascorbic acid can interfere with the metabolism of tumor promoters. It has also been postulated that ascorbic acid helps to prevent cancer by enhancing cellular immunity. In general, evidence suggests that vitamin C can inhibit the formation of some carcinogens.

The failure of ascorbic acid therapy to alter the induction or remission of murine amyloidosis.

It has been claimed that ascorbic acid enhances the in vitro degradation of AA amyloid fibrils. This raises the possibility that ascorbic acid may be of benefit in systemic AA amyloidosis, a condition with serious morbidity and mortality for which there is as yet no specific treatment. The effect was therefore tested of oral or injected supplements of ascorbic acid on the induction of AA amyloidosis in mice. Amyloid was induced either by repeated injections of casein or by injection of 'amyloid enhancing factor' and silver nitrate. Mice with established amyloidosis were also treated with additional ascorbic acid. Despite the fact that plasma ascorbic acid levels were significantly higher in orally supplemented mice than in controls there was no demonstrable effect on the induction, the extent and distribution or the progression of amyloidosis.

alpha 1-Microglobulin from normal and pathological urines.

alpha 1-Microglobulin was purified from normal and pathological urines. Significant differences were found in the amino acid compositions of the alpha 1-microglobulin isolated from these two sources. In addition electrofocusing of alpha 1-microglobulin from normal urine gave rise to two peaks of equal intensity with rather acidic isoelectric points (3.8 and 4.2), whilst alpha 1-microglobulin from pathological urine showed two peaks in a 1:5 ratio with less acidic isoelectric points (4.2 and 4.7). Further charge heterogeneity was also observed in the second peaks from both sources. The sugar compositions were also established, as well as the N-terminal sequences of the alpha 1-microglobulin of both peaks isolated from normal and pathological urines.

Structural and functional similarities between mitochondrial malate dehydrogenase and L-3-hydroxyacyl CoA dehydrogenase.

Pig heart mitochondrial malate dehydrogenase (EC 1.1.1.37), which has been obtained free of electrophoretic subforms, has been shown to have a molecular weight of 67,000 and to be composed of two polypeptide chains. Comparison of these and other properties, such as amino-acid composition, isoelectric point, and keto-substrate inhibition, with those of (L)-3-hydroxyaeyl CoA dehydrogenase (EC 1.1.1.35), another NAD(+)-dependent dehydrogenase of mitochondrial origin, suggests structural similarities of the type associated with proteins possessing common evolutionary origins. This conclusion is supported by immunological crossreactivity. In view of these observations, the dissimilarity in the stereospecificity of hydrogen transfer from cofactor to substrate catalyzed by the two enzymes is attributed to 180 degrees rotation in the binding orientation of the nicotinamide moiety of the NAD(+), rather than to gross differences in the geometry of the active site of the two enzymes.