Bile acid synthesis in rat liver peroxisomes: metabolism of 26-hydroxycholesterol to 3 beta-hydroxy-5-cholenoic acid

Rat liver peroxisomes have been found to oxidize 26-hydroxycholesterol, the product of cholesterol C-26 hydroxylation to 3 beta-hydroxy-5-cholenoic acid. Peroxisomes were purified by differential and equilibrium density centrifugation in a steep linear metrizamide gradient to greater than 95% purity. Purity of peroxisomes was determined by measurement of specific marker enzymes. The activities of cytochrome oxidase (a mitochondrial marker) and acid phosphatase (a lysosomal marker) in the purified peroxisome fractions were below the level of detection. Esterase activity indicated a 2-4% microsomal contamination. Subsequent to incubation of peroxisomes with [16,22-3H]-26-hydroxycholesterol, the reaction products were extracted, methylated, acetylated, and subjected to thin-layer, high pressure liquid, and gas-liquid chromatographic analyses. 3 beta-Hydroxy-5-cholenoic acid was the major identifiable metabolite of 26-hydroxycholesterol. Incubations of pure microsomal fractions (greater than 99%) with 26-hydroxycholesterol under the same conditions demonstrated that the production of 3 beta-hydroxy-5-cholenoic acid by peroxisomes was not attributable to microsomal contamination. This study demonstrates that peroxisomes participate in the side-chain oxidation of intermediates in bile acid synthesis.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi.

Allophanate lyase can be induced by urea or acetamide 20-40-fold within 4 h in NH4 + -deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to the light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

Zonal heterogeneity of peroxisome proliferation and morphology in rat liver after gemfibrozil treatment

The effect of gemfibrozil on the fine structure of peroxisomes across the rat liver lobule was investigated by light and electron microscopy using the alkaline diaminobenzidine (DAB) medium for the visualization of catalase peroxidatic activity. The oral administration of gemfibrozil for 2 weeks induces a striking heterogeneity in the lobular distribution of peroxisomes. The size and shape of peroxisomes, variety of matrix modifications, catalase content, and position within the cell, are functions of the zonal localization of the hepatocytes. The largest and most numerous peroxisomes were found in the centrilobular region indicating that these cells are most sensitive to peroxisome proliferation. On the other hand, the greatest variety of peroxisome shapes and matrix alterations (tubules and plates) was seen more peripherally in the mid-zonal and periportal regions. The larger, round centrilobular peroxisomes stained less intensely than the elongated peroxisomes found more peripherally, indicating a discrepancy between peroxisome size and catalase content. A distinct population of small irregularly shaped peroxisomes, lacking matrix specializations and containing variable catalase content, was found in the mid-zonal region. Peroxisomes in the centrilobular region were located within areas of the cell containing SER and glycogen while those in the more peripheral region were relegated to areas of the cytoplasm separate from RER and SER. In addition to modifications of peroxisomes, gemfibrozil treatment resulted in a proliferation and formation of whorled configurations of SER. This was particularly evident in the mid-zonal region, where single peroxisomal profiles could be seen surrounded by whorls of SER membranes. The results suggest that rat liver hepatocytes of the centrilobular region are the most sensitive to peroxisome proliferation and those of the periportal area are most susceptible to peroxisome matrix alterations after gemfibrozil treatment.     

Young patients with colorectal cancer have poor survival in the first twenty months after operation and predictable survival in the medium and long-term: Analysis of survival and prognostic markers

Introduction

Male breast cancer (MBC) is a rare, yet potentially aggressive disease. Although literature regarding female breast cancer (FBC) is extensive, little is known about the etiopathogenesis of male breast cancer. Studies from our laboratory show that MBCs have a distinct immunophenotypic profile, suggesting that the etiopathogenesis of MBC is different from FBCs. The aim of this study was to evaluate and correlate the immunohistochemical expression of cell cycle proteins in male breast carcinoma to significant clinico-biological endpoints.

Methods

75 cases of MBC were identified using the records of the Saskatchewan Cancer Agency over 26 years (1970-1996). Cases were reviewed and analyzed for the immunohistochemical expression of PCNA, Ki67, p27, p16, p57, p21, cyclin-D1 and c-myc and correlated to clinico-biological endpoints of tumor size, node status, stage of the disease, and disease free survival (DFS).

Results

Decreased DFS was observed in the majority of tumors that overexpressed PCNA (98%, p = 0.004). The overexpression of PCNA was inversely correlated to the expression of Ki67 which was predominantly negative (78.3%). Cyclin D1 was overexpressed in 83.7% of cases. Cyclin D1 positive tumors were smaller than 2 cm (55.6%, p = 0.005), had a low incidence of lymph node metastasis (38.2%, p = 0.04) and were associated with increased DFS of >150 months (p = 0.04). Overexpression of c-myc (90%) was linked with a higher incidence of node negativity (58.3%, p = 0.006) and increased DFS (p = 0.04). p27 over expression was associated with decreased lymph node metastasis (p = 0.04). P21 and p57 positive tumors were related to decreased DFS (p = 0.04). Though p16 was overexpressed in 76.6%, this did not reach statistical significance with DFS (p = 0.06) or nodal status (p = 0.07).

Conclusion

Aberrant cell cycle protein expression supports our view that these are important pathways involved in the etiopathogenesis of MBC. Tumors with overexpression of Cyclin D1 and c-myc had better outcomes, in contrast to tumors with overexpression of p21, p57, and PCNA with significantly worse outcomes. P27 appears to be a predictive marker for lymph nodal status. Such observation strongly suggests that dysregulation of cell cycle proteins may play a unique role in the initiation and progression of disease in male breast cancer. Such findings open up new avenues for the treatment of MBC as a suitable candidate for novel CDK-based anticancer therapies in the future.     

A second gene for peroxisomal HMG-CoA reductase? A genomic reassessment

HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonate, the rate-limiting step of eukaryotic isoprenoid biosynthesis, and is the main target of cholesterol-lowering drugs. The classical form of the enzyme is a transmembrane-protein anchored to the endoplasmic reticulum. However, during the last years several lines of evidence pointed to the existence of a second isoform of HMGCR localized in peroxisomes, where mevalonate is converted further to farnesyl diphosphate. This finding is relevant for our understanding of the complex regulation and compartmentalization of the cholesterogenic pathway. Here we review experimental evidence suggesting that the peroxisomal activity might be due to a second HMGCR gene in mammals. We then present a comprehensive analysis of completely sequenced eukaryotic genomes, as well as the human and mouse genome drafts. Our results provide evidence for a large number of independent duplications of HMGCR in all eukaryotic kingdoms, but not for a second gene in mammals. We conclude that the peroxisomal HMGCR activity in mammals is due to alternative targeting of the ER enzyme to peroxisomes by an as yet uncharacterized mechanism.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

Degradation of cholesterol to propionic acid by rat liver peroxisomes

Under standard assay conditions peroxisomes were found to contain less than 5% of the liver's cholesterol degradation activity. The remainder of the activity was localized in the mitochondria. When CaCl₂ was added to the standard assay mixture, peroxisomal cholesterol degradation activity increased to 34%. These results suggest that peroxisomes are capable of cholesterol catabolism, with the assay conditions used invitro determining the relative organelle contribution.     

Disturbed cholesterol homeostasis in a peroxisome-deficient PEX2 knockout mouse model

We evaluated the major pathways of cholesterol regulation in the peroxisome-deficient PEX2(-/-) mouse, a model for Zellweger syndrome. Zellweger syndrome is a lethal inherited disorder characterized by severe defects in peroxisome biogenesis and peroxisomal protein import. Compared with wild-type mice, PEX2(-/-) mice have decreased total and high-density lipoprotein cholesterol levels in plasma. Hepatic expression of the SREBP-2 gene is increased 2.5-fold in PEX2(-/-) mice and is associated with increased activities and increased protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, the upregulated cholesterogenic enzymes appear to function with altered efficiency, associated with the loss of peroxisomal compartmentalization. The rate of cholesterol biosynthesis in 7- to 9-day-old PEX2(-/-) mice is markedly increased in most tissues, except in the brain and kidneys, where it is reduced. While the cholesterol content of most tissues is normal in PEX2(-/-) mice, in the knockout mouse liver it is decreased by 40% relative to that in control mice. The classic pathway of bile acid biosynthesis is downregulated in PEX2(-/-) mice. However, expression of CYP27A1, the rate-determining enzyme in the alternate pathway of bile acid synthesis, is upregulated threefold in the PEX2(-/-) mouse liver. The expression of hepatic ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) involved in cholesterol efflux is not affected in PEX2(-/-) mice. These data illustrate the diversity in cholesterol regulatory responses among different organs in postnatal peroxisome-deficient mice and demonstrate that peroxisomes are critical for maintaining cholesterol homeostasis in the neonatal mouse.