The osmotic stability of lysosomes from adult and foetal guinea-pig liver tissue

1. Lysosome-rich fractions were obtained from foetal liver tissues as early as 35 days uterine age. Foetal lysosomes showed the same ;structure-linked latency' and acid hydrolytic potentiality characteristic of their adult counterparts. 2. The osmotic stability of lysosome-rich fraction from foetal guinea-pig liver tissue was greater than that of the corresponding adult lysosome fractions, p-nitrophenyl-phosphatase being used as marker enzyme. 3. The observation was confirmed by using beta-glycerophosphatase and phenolphthalein beta-glucuronidase as alternative marker enzymes. p-Nitrophenyl phosphate and beta-glycerophosphate appear to act as substrates for the same enzyme. 4. By using p-nitrophenylphosphatase activity measurements it was shown that the osmotic stability of foetal lysosomal fractions decreased with increasing foetal age, but at no time achieved the degree of osmotic instability characteristic of adult lysosomal fractions. 5. The correlation of these findings with the intracellular environment of lysosomes is discussed.     

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

An isoelectric focusing study of acid phosphohydrolases in rat liver lysosomes.

The differentiation of rat liver lysosomal acid phosphatase, acid ATPase, acid phosphodiesterase, acid ribonuclease, and acid deoxyribonuclease was studied by isoelectric focusing. To prevent autolytic digestion, inhibitors of cathepsins and neuraminidase were used. The proportion of acidic forms of acid phosphatase, acid ATPase and acid phosphodiesterase was increased by the use of extraction medium containing 0.05% Triton X-100. To investigate the identity of acid ATPase and acid phosphodiesterase, the relative activities among the multiple forms of these enzymes, the acid phosphodiesterase/acid ATPase ratio at each activity peak, and the degree of enzyme inhibition by p-chloromercuriphenyl sulfonic acid were estimated. The results suggest that acid ATPase is not identical with acid phosphodiesterase. With extraction medium free of Triton X-100, acid ribonuclease appeared in two forms. However, in addition to these forms, a new form of this enzyme with a more acidic pI (4.22) emerged when extraction medium containing 0.05% Triton X-100 was used. The major peak of acid deoxyribonuclease with pI=8.40-9.39 was obtained regardless of the extracting method.     

Acid hydrolases in early and late endosome fractions from rat liver.

We have examined the distribution of the cation-independent mannose 6-phosphate receptor and five acid hydrolases in early and late endosomes and a receptor-recycling fraction isolated from livers of estradiol-treated rats. Enrichment of mannose 6-phosphate receptor mass relative to that of crude liver membranes was comparable in membranes of early and late endosomes but was even greater in membranes of the receptor-recycling fraction. Enrichment of acid hydrolase activities (aryl sulfatase, N-acetyl-beta-glucosaminidase, tartrate-sensitive acid phosphatase, and cholesteryl ester acid hydrolase) and cathepsin D mass was also comparable in early and late endosomes but was considerably lower in the receptor-recycling fraction. The enrichment of two acid hydrolases, acid phosphatase and cholesteryl ester acid hydrolase, in endosomes was severalfold greater than that of the other three examined, about 40% of that found in lysosomes. Acid phosphatase and cholesteryl ester acid hydrolase were partially associated with endosome membranes, whereas cathepsin D was found entirely in the endosome contents. These findings raise the possibility that lysosomal enzymes traverse early endosomes during transport to lysosomes in rat hepatocytes and suggest that the greater enrichment of some acid hydrolases in endosomes is related to their association with endosome membranes. Despite the substantial enrichment of lysosomal enzymes in hepatocytic endosomes, we found that two, cholesteryl ester acid hydrolase and cathepsin D, did not degrade cholesteryl esters and apolipoprotein B-100 of endocytosed low density lipoproteins in vivo, presumably because they are inactive at the pH within endosomes.     

An isoelectric focusing study of acid phosphohydrolases in liver lysosomes of higher vertebrates.

1. The multiple forms of acid phosphohydrolases in liver lysosomes of Sus scrofa domesticus and Gallus gallus domesticus were studied by use of isoelectric focusing. 2. Acid phosphatase was resolved into two forms in G. gallus domesticus and three forms in S. scrofa domesticus. Especially, two forms of G. gallus domesticus were different from each other in their enzymatic properties. 3. The pI values of acid ATPase agreed with those of acid phosphodiesterase in G. gallus domesticus. According to the data on activity ratios, however, these enzymes seemed not to be identical. 4. Except acid deoxyribonuclease, extraction by Triton X-100 of lysosomes increased the proportions of acidic forms of these enzymes. In particular, a new form of acid ribonuclease with pI 4.5 or 4.9 appeared in both cases of G. gallus domesticus and S. scrofa domesticus.     

Studies on morphological features of foetal and adult ovaries in Kano brown goats

In this work we studied the structures of 51 foetal and 14 adult ovaries obtained from slaughtered Kano brown does in Nsukka abattoir. The ages of the adult does were determined by dentition and foetuses by crown rump length method. The foetal and adult ovaries were divided into five different groups using specific age intervals as Gestation day (GD) 50–65, 66–95, 96–125 and 126–145 and adults. For histological studies the ovaries were fixed, processed and routinely stained with H&E. The ovarian follicles were classified into 5 types according to granulosa cell layers surrounding the oocytes. The number of ovarian follicles per microscopic field, number of granulosa cells surrounding type 1 and 1A follicles and diameter of the ovarian follicles were determined for each group at 400× magnification. Grossly the foetal ovaries were like pin head, oval in shape, uniformly smooth and creamy in colour. The adult ovaries had follicles with different sizes. The adult mean ovarian weights were significantly higher (P < 0.01) than those of the foetuses. Microscopically, the GD 50–65 ovaries had no distinct cortex and medulla. Oogonia were numerous among other stromal cells toward the periphery of the ovary. By GD 66–95 the ovaries contained types 1, 1a, 2 and 3 follicles. GD 96–125 ovaries contained type 4 follicles with early antrum formation and those of GD 126–145 comprised type 5 among other follicles. The adult ovaries comprised all the ovarian follicle types. The number of type 1 follicles increased significantly (P < 0.01) with foetal age. It was least in the adults. The diameter of adult follicles was significantly higher (P < 0.01) than those of the foetuses. This result provides baseline information on the morphological development of ovaries in Kano brown goats.     

Distribution of fibronectin in foetal tissues.

The distribution of fibronectin in tissues of four human foetuses (7-14 gestation weeks/GW) and twenty seven pig foetuses (25-114 days of gestation) was investigated using immunofluorescence and avidin/biotin methods. Fibronectin was abundant in the circulatory and gastrointestinal system and its derivates, in reticular stroma of immune organs, and in connective tissues and chorionic villi at all developmental stages.     

Development of fatty acid oxidation in neonatal guinea-pig liver.

The capacity of foetal and neonatal liver to oxidize short-, medium- and long-chain fatty acids was studied in the guinea pig. Liver mitochondria from foetal and newborn animals were unable to synthesize ketone bodies from octanoate, but octanoylcarnitine and palmitoylcarnitine were readily ketogenic. The ketogenic capacity at 24 h after birth was as high as in adult animals. Hepatocytes isolated from term animals were unable to oxidize fatty acids, but at 6 h after birth production of 14CO2, acid-soluble products and acetoacetate from 1-14C-labelled fatty acids was 40-50% of the rates at 24 h. At 12 h of age these rates had already reached the 24 h values and did not change during suckling in the first week of life. The activities of hepatic fatty acyl-CoA synthetases, which were minimal in the foetus or at term, increased to maximal values in 12-24 h. The data show that the capacity for beta-oxidation and ketogenesis develops maximally in this species during the first 6-12 h after birth, and appears to be partly dependent on the development of fatty acid-activating enzyme.     

Biochemical and histochemical studies on p-nitrophenyl phosphate and beta-glycerophosphate hydrolysis at acid pH by rabbit parotid tissue.

Synopsis A biochemical and histochemical study has been made of the characteristics and distribution of acid phosphatases in the parotid gland of the rabbit. The substratesp-nitrophenyl phosphate and -glycerophosphate have been used. Homogenate studies showed that substantial hydrolysis ofp-nitrophenyl phosphate occurred in the presence of tartrate and fluoride ions but that -glycerophosphate hydrolysis was almost abolished by these additives. The pH optima usingp-nitrophenyl phosphate and -glycerophosphate were found to be around pH 5.0 and pH 4.0 respectively. Studies on subcellular fractions revealed that maximalp-nitrophenyl phosphate hydrolysis occurred in the microsomal fraction whereas maximal hydrolysis of -glycerophosphate occurred in the mitochondrial fraction. The histochemical staining patterns produced by the two substrates differed in several respects. Whenp-nitrophenyl phosphate was used heavy plasmalemmal staining occurred, while reactions using -glycerophosphate as substrate produced heavy staining of the acinar cell cytoplasm including many of the zymogen granules. Both substrates resulted in the staining of lysosomes and certain vesicles associated with the Golgi complex. Formaldehyde fixation resulted in an overall reduction in staining intensity and glutaraldehyde fixation used in electron microscopic studies heavily inhibited much of the extralysosomal activity. The conclusion is reached that significant extralysosomal acid hydrolase activity is present in the rabbit parotid gland, much of which is located on smooth microsomes. The data is discussed in the context of the distribution of extralysosomal acid hydrolases and the staining patterns are considered in the light of the limitations inherent in the techniques employed.