Blood alcohol levels (BAL) were maintained at high levels (overall mean +/- S.D. achieved in 14 alcoholic rats was 216.0 +/- 120.1 mg%) in male Wistar rats for 15 to 85 days by continuous intragastric infusion of ethanol and nutritionally defined low fat liquid diet. The ethanol intake was progressively increased from 32% of total calories up to 41.4% in order to maintain high BAL. Pair-fed animals received isocaloric glucose solution and the liquid diet. Despite the low level of dietary fat (4.9% of total calories), histopathological evaluation of the liver revealed severe and progressive fatty infiltration in the alcoholic rats. In addition, following 30 days of intoxication, one third of the animals showed focal necrosis with mononuclear cell infiltration in centrilobular areas of the livers. This was correlated with the markedly elevated levels of SGOT and SGPT in these animals. Pair-fed controls showed no abnormality in the morphology of liver or blood chemistry. Chemical quantitation of liver triglycerides confirmed the histological observation, with triglyceride levels of 61.51 +/- 16.45 and 89.61 +/- 5.94 mg per gm at 30 and 85 days, respectively. Most importantly, the degree of steatosis was tightly and significantly correlated with the mean BAL achieved (r = 0.80, p less than 0.001). These data represent the first confirmation of the hypothesis that continuously high BAL correlate with the severity of alcohol-induced liver pathology.

Abstract. The micromeres that arise at the fourth cell division in developing sea urchin embryos give rise to primary mesenchyme, which in turn differentiates and produces calcareous endoskeletal spicules. These spicules have been isolated and purified from pluteus larvae by washing in combinations of ionic and nonionic detergents followed by brief exposure to sodium hypochlorite. The spicules may be demineralized and the integral matrix dissolves. The matrix is composed of a limited number of glycoproteins rich in aspx, glux, gly, ser, and ala, a composition not unlike that found in matrix proteins of biomineralized tissues of molluscs, sponges, and arthropods. There is no evidence for collagen as a component of the matrix. The matrix contains N-linked glycoproteins of the complex type. The matrix arises primarily from proteins synthesized from late gastrulation onward, during the time that spicule deposition occurs. The mixture of proteins binds calcium and is an effective immunogen. Electrophoresis of the glycoproteins on SDS-conraining acrylamide gels, followed by blotting and immunocytochemical detection, reveals major components of ~47, 50, 57, and 64 kD, and several minor components. These same components may be detected with silver staining or fluorography of amino acid-labeled proteins. In addition to providing convenient molecular marker for the study of the development of the micromere lineage, the spicule matrix glycoproteins provide an interesting system for investigations in biomineralization.W E report here our initial results on purification and characterization of the organic matrix of the endoskeletal spicule of the sea urchin embryo. The pluteus larva has calcareous rods, which are intracellular deposits of calcite (mainly CaCO3 and some MgCO3 on an organic ground substance; Benson et al., 1983). The matrix of the skeletal element is of interest because of its probable role in the biomineralization process and because of the obvious parallel between biomineralization of hydroxylapatite in vertebrates and calcite in marine invertebrates. An equally important consideration is the opportunity the spicule presents to study the determination and differentiation of the micromere lineage, a group of cells whose developmental history is very well known. At the fourth cell division in the sea urchin embryo four micromeres arise at the vegetal pole. These micromeres give rise to primary mesenchyme cells, which in turn differentiate into the skeletal spicules (reviewed by Wilt et al., 1985). Micromeres may be isolated from the embryo and will differentiate autonomously in culture to form spicules (Okazaki, 1975). Hence, information on the matrix of the spicules may be useful in the study of tissuespecific differentiation in sea urchin embryos. We have devised ways to purify the matrix of the spicule, and the data support the conclusion that the matrix is composed of a small number of soluble N-linked glycoproteins that have a strong biochemical similarity to acidic proteins present in calcareous structur...

Rats fed ethanol from 21 to 130 days were subjected to one or more episodes of hypoxia (6% O2) in order to determine if ethanol predisposed to centrilobular liver necrosis induced by hypoxia. Pair-fed control rats were fed the diet regimen in parallel with the ethanol-fed rats through an indwelling gastric cannula. The diet and ethanol were fed continuously 24 hr per day so as to maintain high blood alcohol levels in the ethanol-fed rats. Serum enzyme levels, SGOT and SGPT were measured before and after the hypoxic episodes as an indicator of centrilobular necrosis. Animal livers were studied for centrilobular necrosis by light and electron microscopy. Necrosis was documented to be present when flocculent densities were found in hepatocytic mitochondria or the plasma membrane permitted lanthanum entrance into the cell. The results showed that ethanol feeding to maintain high blood alcohol levels did increase the propensity of the liver to undergo centrilobular necrosis when the rats were subjected to hypoxia (1 hr 45 min to 5 hr 30 min). Centrilobular necrosis was observed in the ethanol-fed rats only. Serum enzyme levels (SGPT and SGOT) rose to very high levels in these rats when they were permitted to die of hypoxia. Serum sediment from the ethanol-fed rats contained numerous cell fragments and free organelles. Since the plasma membranes were missing along the sinusoidal face of centrilobular hepatocytes and microbodies were present, it was concluded that the cell fragments in the blood had originated from necrotic hepatocytes.

Four developmental stages of sea urchin embryos were labeled with colloidal gold in an attempt to elucidate the intracellular trafficking patterns within the cells that produce the glycoprotein matrix of the embryonic spicule. The primary mesenchyme cells (PMCs) form a syncytium and secrete an organic matrix on which calcium carbonate is laid down to form an endoskeletal spicule. The organic matrix has been isolated and characterized as glycoprotein consisting of four major bands. Polyclonal antibodies to these glycoproteins were used to label embryos from the mesenchyme blastula, early gastrula, late gastrula, and plutei stages of development. The label is concentrated in the Golgi complex and associated vesicles, in secretory vesicles, and in the organic matrix. The density of the labeling increases as development proceeds.