A new method for the isolation of fresh hepatocytes from periportal and pericentral regions of the liver lobule

A simple method which avoids the use of perfusion with calcium free buffer, hydrolytic enzymes and detergents has been developed to obtain fresh hepatocytes from periportal and pericentral regions of the liver lobule. Cylindrical plugs (200 x 500 microns) of periportal and pericentral areas of the rat liver lobule weighing about 1 mg were collected with a micropunch from fresh or perfused liver. Ninety percent of cells were intact as assessed from trypan blue staining. Glutamine synthetase activity was detected predominantly (ca. 85%) in plugs isolated from pericentral regions indicating that this method allows selective harvesting of pure sublobular zones of the liver lobule. Rates of oxygen uptake measured at 25 degrees C by plugs from livers perfused in the anterograde direction were 56 +/- 5 and 33 +/- 7 mumol/g/h by periportal and pericentral plugs, respectively, values similar to data obtained from the intact organ. This method provides new opportunities to study the regulation of basic metabolic processes in cells from sublobular areas under nearly physiological conditions.     

Differential effect of glucagon on gluconeogenesis in periportal and pericentral regions of the liver lobule.

The effect of glucagon on gluconeogenesis was measured in periportal and pericentral regions of the liver lobule by monitoring changes in rates of O2 uptake on the surface of the perfused liver with miniature O2 electrodes after infusion of lactate. When lactate (2 mM) was infused into livers from starved rats perfused in the anterograde direction, O2 uptake was increased 2.5-fold more in periportal than in pericentral regions, reflecting increased energy demands for glucose synthesis. Under these conditions, glucagon infusion in the presence of lactate increased O2 uptake exclusively in periportal regions of the liver lobule. Thus, when perfusion is in the physiological anterograde direction, the metabolic actions of glucagon predominate in periportal regions of the liver lobule under gluconeogenic conditions in the starved state. When livers were perfused in the retrograde direction, however, glucagon stimulated O2 uptake exclusively in pericentral regions. Thus glucagon only stimulates gluconeogenesis in 'upstream' regions of the liver lobule irrespective of the direction of flow.     

Isolation of a copper complex and its rate of appearance in roots of Mimulus guttatus

A Cu-complex was isolated from the roots of copper-tolerant Mimulus guttatus. The elution volume of the complex determined by gel permeation chromatography was similar to that of rat-liver cadmium thionein. The complex was heat stable, had a relatively high ratio of absorbance at 254 nm: 280 nm and incorporated ³⁵S. The complex, purified using a combination of gel permeation chromatography and anion-exchange chromatography, contained more glutamine/glutamic acid and glycine residues than mammalian metallothioneins. The amount of the complex in roots increased after 5 h growth in a solution containing 16 M Cu. Induction was preceded by an increase in the concentrations in root tissue of unknown compounds containing sulphur which may serve as precursors. The availability of these compounds appeared to regulate the rate of synthesis of this Cu-complex.Abbreviations CuBP copper-binding protein - HPLC highperformance liquid chromatography - MT metallothionein - Th thionein - Tris 2-amino-2-(hydroxymethyl)1-3-propanediol     

Cell surface changes and enzyme release during hypoxia and reoxygenation in the isolated, perfused rat liver

We examined the effects of hypoxia and reoxygenation in isolated, perfused rat livers. Hypoxia induced by a low rate of perfusion led to near anoxia confined to centrilobular regions of the liver lobule. Periportal regions remained normoxic. Within 15 min, anoxic centrilobular hepatocytes developed surface blebs that projected into sinusoids through endothelial fenestrations. Periportal hepatocytes were unaffected. Both scanning and transmission electron microscopy suggested that blebs developed by transformation of preexisting microvilli. Upon reoxygenation by restoration of a high rate of perfusion, blebs disappeared. Other changes included marked shrinkage of hepatocytes, enlargement of sinusoids, and dilation of sinusoidal fenestrations. There was also an abrupt increase in the release of lactate dehydrogenase and protein after reoxygenation, and cytoplasmic fragments corresponding in size and shape to blebs were recovered by filtration of the effluent perfusate. We also studied phalloidin and cytochalasin D, agents that disrupt the cytoskeleton. Both substances at micromolar concentrations caused rapid and profound alterations of cell surface topography. We conclude that hepatic tissue is quite vulnerable to hypoxic injury. The morphological expression of hypoxic injury seems mediated by changes in the cortical cytoskeleton. Reoxygenation causes disappearance of blebs and paradoxically causes disruption of cellular volume control and release of blebs as cytoplasmic fragments. Such cytoplasmic shedding provides a mechanism for selective release of hepatic enzymes by injured liver tissue.     

The bactericidal action of beta-lactam antibiotics on an autolysin-deficient strain of Bacillus subtilis

An autolysin-deficient mutant of Bacillus subtilis was completely tolerant to 5 h incubation with 50-100 micrograms cycloserine ml-1 whereas the wild-type was rapidly lysed and killed by 12 micrograms ml-1. Lysis also did not occur when low concentrations of beta-lactams were added to exponentially growing cultures of the mutant, but over 90% of the bacteria were killed within 90-120 min. Protein, lipid and peptidoglycan synthesis as well as growth were inhibited after about 60 min. At this time, but not earlier, small amounts of these three cell components appeared in culture supernatants. Earlier, at about 20-30 min, the intracellular pools of amino acids started to decline rapidly and there was a temporary apparent increase in the rate of lipid synthesis. Neither of the latter phenomena occurred with cycloserine, with which protein and lipid synthesis declined only slowly and the rate of peptidoglycan synthesis was 80% inhibited within 30 min. Only occasional cells with damaged walls were seen 30-90 min after addition of either beta-lactams or cycloserine to the cultures. It thus seems unlikely that wall hydrolysis or penetration by residual autolysins in the mutant are responsible for mass cell death caused by the beta-lactams.     

The degradation of canavanine by jack bean cotyledons

Summary Germinating jack bean cotyledons liberated ¹⁴CO₂ when fed ¹⁴C-guanidoxy-canavanine but did not accumulate any ¹⁴C-compounds other than the applied canavanine. This suggested that the canavanine was being degraded by the action of canavanase to canaline and urea, the urea then being converted to ammonia and carbon dioxide by the action of urease. Hydroxyurea and acetohydroxamic acid (both inhibitors of urease activity) strongly inhibited the liberation of ¹⁴CO₂ from ¹⁴C-guanidoxy-canavanine by the cotyledons but neither compound induced the accumulation of ¹⁴C-urea within the tissues. This inhibitory action of hydroxyurea on ¹⁴CO₂ output was thought to be due at least in part, to this inhibition of canavanase activity.