One hundred questions of importance to the conservation of global biological diversity.

We identified 100 scientific questions that, if answered, would have the greatest impact on conservation practice and policy. Representatives from 21 international organizations, regional sections and working groups of the Society for Conservation Biology, and 12 academics, from all continents except Antarctica, compiled 2291 questions of relevance to conservation of biological diversity worldwide. The questions were gathered from 761 individuals through workshops, email requests, and discussions. Voting by email to short-list questions, followed by a 2-day workshop, was used to derive the final list of 100 questions. Most of the final questions were derived through a process of modification and combination as the workshop progressed. The questions are divided into 12 sections: ecosystem functions and services, climate change, technological change, protected areas, ecosystem management and restoration, terrestrial ecosystems, marine ecosystems, freshwater ecosystems, species management, organizational systems and processes, societal context and change, and impacts of conservation interventions. We anticipate that these questions will help identify new directions for researchers and assist funders in directing funds.

Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells.

Overexpressing StAR (a mitochondrial cholesterol transporter) increases (>5-fold) the rate of 27-hydroxylation of cholesterol and the rates of bile acid synthesis in primary rat hepatocytes; suggesting that the transport of cholesterol into mitochondria is rate-limiting for bile acid biosynthesis via the CYP27A1 initiated 'acidic' pathway. Our objective was to determine the level of StAR expression in human liver and whether changes in StAR would correlate with changes in CYP27A1 activity/bile acid synthesis rates in human liver tissues. StAR mRNA and protein were detected in primary human hepatocytes and HepG2 cells by RT-PCR/Northern analysis and by Western analysis, respectively. In immunocompetition assays, liver StAR was competed away with the addition of purified human adrenal StAR. Overexpressing CYP27A1 in both cell types led to >2-fold increases in liver StAR concentration. StAR protein levels also increased approximately 2-fold with the addition of 27-hydroxycholesterol to HepG2 cell culture medium. Overexpressing StAR increased the rates of 27-hydroxylation of cholesterol/bile acid synthesis in both cell lines and increased intracellular levels of 27-hydroxycholesterol. In conclusion, human liver cells contain regulable StAR protein whose level of expression appears capable of regulating cellular cholesterol homeostasis, representing a potential therapeutic target in the management of hyperlipidemia.

Mitochondrial cholesterol transport: a possible target in the management of hyperlipidemia.

Sterol 27-hydroxylase (CYP27A1) may defend cells against accumulation of excess cholesterol, making this enzyme a possible target in the management of hyperlipidemia. The study objective was to analyze cholesterol homeostatic responses to increases in CYP27A1 activity in HepG2 cells and primary human hepatocytes. Increasing CYP27A1 activity by increasing enzyme expression led to significant increases in bile acid synthesis with compensatory increases in HMG-CoA reductase (HMGR) activity/protein, LDL receptor (LDLR) mRNA, and LDLR-mediated cholesterol uptake. Under these conditions, only a small increase in cellular 27-hydroxycholesterol (27OH-Chol) concentration was observed. No changes were detected in mature sterol regulatory element-binding proteins (SREBP) 1 or 2. Increasing CYP27A1 activity by increasing mitochondrial cholesterol transport (i.e., substrate availability) led to greater increases in bile acid synthesis with significant increases in cellular 27OH-Chol concentration. Mature SREBP 2 protein decreased significantly with compensatory decreases in HMGR protein. No change was detected in mature SREBP 1 protein. Despite increasing 27OH-Chol and lowering SREBP 2 protein concentrations, LDLR mRNA increased significantly, suggesting alternative mechanisms of LDLR transcriptional regulation. These findings suggest that regulation of liver mitochondrial cholesterol transport represents a potential therapeutic strategy in the treatment of hyperlipidemia and atherosclerosis.

A study on the section sensitivity profile in multi-row-detector spiral CT.

The section sensitivity profile (SSP) was well understood in the case of single-row-detector spiral CT. With the introduction of multi-row-detector spiral CT and the transition into cone-beam spiral CT, a revisit to the SSP issue becomes necessary. In this paper, the SSP of multi-row-detector spiral CT is formulated for the half-scan interpolation method at any transverse position. Based on the SSP formula, numerical simulation is performed to quantify the characteristics of the SSP with the number of detector rows up to 40. It is shown that the SSP varies as a function of the pitch and the number of detector rows. Given an appropriate selection of the pitch and the number of detector rows, the SSP does not change very much over the field of view in terms of the mean, the slice thickness, and the skewness of the SSP. Although in general applications the SSP at the gantry iso-center can be used as the representative of the SSP family, for more accurate analyses the spatial variation of the SSP must be taken into account.

Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis.

The initial and rate-limiting step in the classic pathway of bile acid biosynthesis is 7alpha-hydroxylation of cholesterol, a reaction catalyzed by cholesterol 7alpha-hydroxylase (CYP7A1). The effect of CYP7A1 overexpression on cholesterol homeostasis in human liver cells has not been examined. The specific aim of this study was to determine the effects of overexpression of CYP7A1 on key regulatory steps involved in hepatocellular cholesterol homeostasis, using primary human hepatocytes (PHH) and HepG2 cells. Overexpression of CYP7A1 in HepG2 cells and PHH was accomplished by using a recombinant adenovirus encoding a CYP7A1 cDNA (AdCMV-CYP7A1). CYP7A1 overexpression resulted in a marked activation of the classic pathway of bile acid biosynthesis in both PHH and HepG2 cells. In response, there was decreased HMG-CoA-reductase (HMGR) activity, decreased acyl CoA:cholesterol acyltransferase (ACAT) activity, increased cholesteryl ester hydrolase (CEH) activity, and increased low-density lipoprotein receptor (LDLR) mRNA expression. Changes observed in HMGR, ACAT, and CEH mRNA levels paralleled changes in enzyme specific activities. More specifically, LDLR expression, ACAT activity, and CEH activity appeared responsive to an increase in cholesterol degradation after increased CYP7A1 expression. Conversely, accumulation of the oxysterol 7alpha-hydroxycholesterol in the microsomes after CYP7A1 overexpression was correlated with a decrease in HMGR activity.

Regulation of sterol 12alpha-hydroxylase and cholic acid biosynthesis in the rat.

BACKGROUND & AIMS: Sterol 12alpha-hydroxylase (CYP8b1) is required for the biosynthesis of cholic acid (CA) and hence helps determine the ratio of CA to chenodeoxycholic acid (CDCA) in bile. This study examined the in vivo regulation of CYP8b1 in the rat by bile acids, cholesterol, and thyroxine. METHODS: The specific activities (SAs), messenger RNA (mRNA) levels, and transcriptional activities of CYP8b1 were determined in intact rats and rats with biliary diversion. RESULTS: CA, CDCA, and deoxycholic acid (DCA), fed as a supplement to the diet, down-regulated CYP8b1 SAs by 99% +/- 0%, 72% +/- 10%, and 98% +/- 1%, respectively. Under these same conditions, mRNA levels decreased by 93% +/- 7%, 60% +/- 11%, and 93% +/- 4%, respectively. Intraduodenal infusion of taurocholate (36 micromol/h. 100 g rat(-1)) decreased SAs and mRNA levels by 63% +/- 8% and 74% +/- 8%, respectively. Ursodeoxycholic acid (UDC) and hyocholic acid (HC) feeding increased CYP8b1 SAs by 119% +/- 21% and 65% +/- 18%, respectively. CA feeding decreased CYP8b1 transcriptional activity by 72%. Complete biliary diversion increased CYP8b1 SAs and mRNA levels by 150% +/- 30% and 287% +/- 51%, respectively. Cholesterol feeding decreased CYP8b1 mRNA by 39% +/- 8%. In intact rats, a single injection of thyroid hormone eliminated CYP8b1 activity. CONCLUSIONS: CYP8b1 is transcriptionally down-regulated by hydrophobic but not hydrophilic bile acids. Cholesterol feeding and a single thyroid hormone injection repressed CYP8b1 in the face of induction of cholesterol 7alpha-hydroxylase (CYP7a1 by the new nomenclature) SAs. These results suggest that cholesterol, thyroid hormone, and hydrophobic bile acids are important regulators of CYP8b1 and consequently of the bile acid pool composition.

Hormonal regulation of cholesterol 7alpha-hydroxylase specific activity, mRNA levels, and transcriptional activity in vivo in the rat.

In primary cultures of rat hepatocytes, transcription of the cholesterol 7alpha-hydroxylase gene is induced synergistically by glucocorticoid and thyroid hormones. The objective of the present study was to evaluate the role of endogenous glucocorticoid and thyroid hormones in the maintenance of cholesterol 7alpha-hydroxylase gene expression in vivo. Male Sprague-Dawley rats underwent adrenalectomy (A), thyroidectomy (T), adrenalectomy + thyroidectomy (A + T), hypophysectomy (H), or sham surgery (paired controls). Ten days post surgery, livers were harvested and choles terol 7alpha-hydroxylase specific activity, steady-state mRNA levels, and transcriptional activity were determined. Serum corticosterone levels were <2% of paired controls in A, A + T, and H rats. Free thyroxine index was <32% of paired controls in rats with T and H. When compared to sham-operated controls, A + T and H led to decreases in cholesterol 7alpha-hydroxylase specific activities of 44 +/- 8% and 57 +/- 3%, respectively (P < 0.03 and < 0.05). Similar changes were observed in cholesterol 7alpha-hydroxylase steady-state mRNA levels, which decreased by 43 +/- 10% (P < 0.001) and 56 +/- 19% (P < 0.05), respectively. Cholesterol 7alpha-hydroxylase transcriptional activity in A + T and H rats decreased by 34 +/- 11% (P < 0.01) and 61 +/- 4% (P < 0.001), respectively. The observed decreases were greater after H than after A + T, suggesting the possibility that another pituitary hormone plays a role in regulation of cholesterol 7alpha-hydroxylase. Thyroidectomy alone led to a decrease in cholesterol 7alpha-hydroxylase specific activity of 37 +/- 7% (P < 0.05) and a trend toward decreased steady-state mRNA levels (21 +/- 12%; P = ns). Adrenalectomy did not significantly decrease cholesterol 7alpha-hydroxylase specific activity or mRNA levels. Neither thyroidectomy nor adrenalectomy alone affected transcriptional activity. We conclude that under physiologic circumstances, full expression of the cholesterol 7alpha-hydroxylase gene requires synergistic action of glucocorticoids and thyroid hormone.

Effects of different bile salts on steady-state mRNA levels and transcriptional activity of cholesterol 7 alpha-hydroxylase.

Cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in the bile acid synthesis pathway, is down-regulated by taurocholate by way of negative feedback control at the level of gene transcription. The molecular basis of regulation of cholesterol 7 alpha-hydroxylase by other hydrophobic bile salts and under more physiological conditions is not known. The aim of this study was to investigate the molecular basis of regulation of cholesterol 7 alpha-hydroxylase by several naturally occurring bile salts in rats with intact enterohepatic circulation. Male Sprague-Dawley rats were pair-fed for 14 days normal chow (control), cholestyramine (5% of diet), cholic acid (1%), chenodeoxycholic acid (1%) or deoxycholic acid (0.25%). When rats were killed, livers were harvested and HMG-CoA reductase specific activity and cholesterol 7 alpha-hydroxylase specific activities, steady-state mRNA levels and transcriptional activity were determined and compared with those of control rats fed normal chow. Compared with results in paired controls, cholestyramine feeding led to an approximate threefold increase in HMG-CoA reductase specific activity. Feeding of hydrophobic bile salts profoundly decreased the specific activity of HMG-CoA reductase. Cholestyramine led to a three-fold increase in cholesterol 7 alpha-hydroxylase specific activity, steady-state mRNA levels and gene transcriptional activity. The feeding of cholic (1%), chenodeoxycholic (1%) and deoxycholic acid (0.25%) led to significant decreases in cholesterol 7 alpha-hydroxylase specific activities (62%, 84% and 97%, respectively), steady-state mRNA levels (72%, 29% and 61%, respectively) and transcriptional activities (44%, 43% and 54%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatocellular phenotype in vitro is influenced by biophysical features of the collagenous substratum.

Hepatocytes maintained on different substrata in vitro possess strikingly different morphological and biochemical features. Rounded, multicellular aggregates of hepatocytes are seen if the cells are plated onto Matrigel, a reconstituted basement membrane, whereas a flattened, monolayer of hepatocytes is observed with Vitrogen. Hepatocellular protein synthesis is much greater on the Matrigel, although collagen biosynthesis appears selectively enhanced on Vitrogen-grown hepatocytes. We determined that denatured type I collagen could be substituted for Matrigel as the substratum, with the hepatocytes remaining the same both morphologically and biochemically. This suggested that the cells respond to the biophysical state of the extracellular matrix not only to protein sequences that determine a binding site. Measurement of steady-state messenger RNA levels within cells cultured onto different matrices indicated that the fluid substrate of either Matrigel or denatured type I collagen were facilitative for induction of cytochrome P-450b/e, which was not seen with the rigid type I collagen substrata. In contrast the messenger RNA level for the cytoskeletal protein actin was decreased on the fluid matrices, suggesting that the rounded cells had a lower requirement for this protein. These findings indicate that hepatocytes are responsive to the biophysical state of the extracellular matrix, which can lead to significant changes in gene expression by the cells.

Induction of prolyl hydroxylase activity in a nonadherent population of human leukocytes.

A nonadherent population of human monocytes has been shown to express the collagen hydroxylating enzyme prolyl hydroxylase in vitro. Enzyme levels present in freshly isolated nonadherent cells were induced 300% during the first 72 hours of culturing, which could be suppressed by cycloheximide. Maximum induction required both a feeder layer of adherent leukocytes, and 10-15% autologous plasma. Biosynthesis of Clq, a protein which also is hydroxylated by prolyl hydroxylase, by the nonadherent cells was significantly less than the adherent monocytes. Therefore, this collagen biosynthetic marker enzyme was not associated with Clq synthesis, which suggests that the enzyme is present for collagen biosynthesis.