EELS: Autonomous snake-like robot with task and motion planning capabilities for ice world exploration.

Ice worlds are at the forefront of astrobiological interest because of the evidence of subsurface oceans. Enceladus in particular is unique among the icy moons because there are known vent systems that are likely connected to a subsurface ocean, through which the ocean water is ejected to space. An existing study has shown that sending small robots into the vents and directly sampling the ocean water is likely possible. To enable such a mission, NASA's Jet Propulsion Laboratory is developing a snake-like robot called Exobiology Extant Life Surveyor (EELS) that can navigate Enceladus' extreme surface and descend an erupting vent to capture unaltered liquid samples and potentially reach the ocean. However, navigating to and through Enceladus' environment is challenging: Because of the limitations of existing orbital reconnaissance, there is substantial uncertainty with respect to its geometry and the physical properties of the surface/vents; communication is limited, which requires highly autonomous robots to execute the mission with limited human supervision. Here, we provide an overview of the EELS project and its development effort to create a risk-aware autonomous robot to navigate these extreme ice terrains/environments. We describe the robot's architecture and the technical challenges to navigate and sense the icy environment safely and effectively. We focus on the challenges related to surface mobility, task and motion planning under uncertainty, and risk quantification. We provide initial results on mobility and risk-aware task and motion planning from field tests and simulated scenarios.

Loss of Autophagy Disrupts Stemness of Ameloblast-Lineage Cells in Aging.

Autophagy is one of the intracellular degradation pathways and maintains cellular homeostasis, regulating the stress response, cell proliferation, and signal transduction. To elucidate the role of autophagy in the maintenance of dental epithelial stem cells and the subsequent enamel formation, we analyzed autophagy-deficient mice in epithelial cells (Atg7f/f;KRT14-Cre mice), focusing on the influence of aging and stress environments. We also performed in vitro cell and organ culture experiments with an autophagy inhibitor. In young Atg7f/f;KRT14-Cre mice, morphological change was not obvious in maxillary incisors, except for the remarkable cell death in the stratum intermedium of the transitional stage. However, under stress conditions of hyperglycemia, the incisor color changed to white in diabetes Atg7f/f;KRT14-Cre mice. Regarding dental epithelial stem cells, the shape of the apical bud region of the incisor became irregular with age, and odontoma was formed in aged Atg7f/f;KRT14-Cre mice. In addition, the shape of apical bud culture cells of Atg7f/f;KRT14-Cre mice became irregular and enlarged atypically, with epigenetic changes during culture, suggesting that autophagy deficiency may induce tumorigenesis in dental epithelial cells. The epigenetic change and upregulation of p21 expression were induced by autophagy inhibition in vivo and in vitro. These findings suggest that autophagy is important for the regulation of stem cell maintenance, proliferation, and differentiation of ameloblast-lineage cells, and an autophagy disorder may induce tumorigenesis in odontogenic epithelial cells.

Functional Expression of Sodium-Dependent Glucose Transporter in Amelogenesis.

Glucose is an essential source of energy for mammalian cells and is transported into the cells by glucose transporters. There are 2 types of glucose transporters: one is a passive glucose transporter, GLUT (SLC2A), and the other is a sodium-dependent active glucose transporter, SGLT (SLC5A). We previously reported that the expression of GLUTs during tooth development is precisely and spatiotemporally controlled and that the glucose uptake mediated by GLUT1 plays a crucial role in early tooth morphogenesis and tooth size determination. This study aimed to clarify the localization and roles of SGLT1 and SGLT2 in murine ameloblast differentiation by using immunohistochemistry, immunoelectron microscopy, an in vitro tooth organ culture experiment, and in vivo administration of an inhibitor of SGLT1/2, phloridzin. SGLT1, which has high affinity with glucose, was immunolocalized in the early secretory ameloblasts and the ruffle-ended ameloblasts in the maturation stage. However, SGLT2, which has high glucose transport capacity, was observed in the stratum intermedium, papillary layer, and ameloblasts at the maturation stage and colocalized with Na+-K+-ATPase. The inhibition of SGLT1/2 by phloridzin in the tooth germs induced the disturbance of ameloblast differentiation and enamel matrix formation both in vitro (organ culture) and in vivo (mouse model). The expression of SGLT1 and SGLT2 was significantly upregulated in hypoxic conditions in the ameloblast-lineage cells. These findings suggest that the active glucose uptake mediated by SGLT1 and SGLT2 is strictly regulated and dependent on the intra- and extracellular microenvironments during tooth morphogenesis and that the appropriate passive and active glucose transport is an essential event in amelogenesis.

Epitope analysis of Ara h 2 and Ara h 6: characteristic patterns of IgE-binding fingerprints among individuals with similar clinical histories.

BACKGROUND: Ara h 2 and Ara h 6 are moderately homologous and highly potent peanut allergens. OBJECTIVE: To identify IgE-binding linear epitopes of Ara h 6, compare them to those of Ara h 2, and to stratify binding based on clinical histories. METHODS: Thirty highly peanut-allergic subjects were stratified by clinical history. Sera were diluted to contain the same amount of anti-peanut IgE. IgE binding to overlapping 20-mer peptides of Ara h 2 and Ara h 6 was assessed using microarrays. RESULTS: Each subject had a unique IgE-binding fingerprint to peptides; these data were coalesced into epitope binding. IgE from subjects with a history of more severe reactions (n = 19) had a smaller frequency of binding events (BEs) for both Ara h 2 (52 BEs of 152 (19X8epitopes) possible BEs and Ara h 6 (13 BEs of 133 (19X7 epitopes) possible BEs) compared to IgE from those with milder histories (n = 11) (Ara h 2: 47 BEs of 88 (11X8 epitopes) possible BEs, P < 0.01; Ara h 6: 25 BEs of 77 (11X7 epitopes) possible BEs, P < 0.001). Using an unsupervised hierarchal cluster analysis, subjects with similar histories tended to cluster. We have tentatively identified a high-risk pattern of binding to peptides of Ara h 2 and Ara h 6, predominantly in subjects with a history of more severe reactions (OR = 12.6; 95% CI: 2.0-79.5; P < 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: IgE from patients with more severe clinical histories recognize fewer linear epitopes of Ara h 2 and Ara h 6 than do subjects with milder reactions and bind these epitopes in characteristic patterns. Close examination of IgE binding to epitopes of Ara h 2 and Ara h 6 may have prognostic value.

p38α (MAPK14) critically regulates the immunological response and the production of specific cytokines and chemokines in astrocytes.

In CNS lesions, "reactive astrocytes" form a prominent cellular response. However, the nature of this astrocyte immune activity is not well understood. In order to study astrocytic immune responses to inflammation and injury, we generated mice with conditional deletion of p38α (MAPK14) in GFAP+ astrocytes. We studied the role of p38α signaling in astrocyte immune activation both in vitro and in vivo, and simultaneously examined the effects of astrocyte activation in CNS inflammation. Our results showed that specific subsets of cytokines (TNFα, IL-6) and chemokines (CCL2, CCL4, CXCL1, CXCL2, CXCL10) are critically regulated by p38α signaling in astrocytes. In an in vivo CNS inflammation model of intracerebral injection of LPS, we observed markedly attenuated astrogliosis in conditional GFAPcre p38α(-/-) mice. However, GFAPcre p38α(-/-) mice showed marked upregulation of CCL2, CCL3, CCL4, CXCL2, CXCL10, TNFα, and IL-1ß compared to p38αfl/fl cohorts, suggesting that in vivo responses to LPS after GFAPcre p38α deletion are complex and involve interactions between multiple cell types. This finding was supported by a prominent increase in macrophage/microglia and neutrophil recruitment in GFAPcre p38α(-/-) mice compared to p38αfl/fl controls. Together, these studies provide important insights into the critical role of p38α signaling in astrocyte immune activation.

Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.

BACKGROUND AND OBJECTIVE: Some clinical cases of hypoplastic tooth root are congenital. Because the formation of Hertwig's epithelial root sheath (HERS) is an important event for root development and growth, we have considered that understanding the HERS developmental mechanism contributes to elucidate the causal factors of the disease. To find integrant factors and phenomenon for HERS development and growth, we studied the proliferation and mobility of the cervical loop (CL). MATERIAL AND METHODS: We observed the cell movement of CL by the DiI labeling and organ culture system. To examine cell proliferation, we carried out immunostaining of CL and HERS using anti-Ki67 antibody. Cell motility in CL was observed by tooth germ slice organ culture using green fluorescent protein mouse. We also examined the expression of paxillin associated with cell movement. RESULTS: Imaging using DiI labeling showed that, at the apex of CL, the epithelium elongated in tandem with the growth of outer enamel epithelium (OEE). Cell proliferation assay using Ki67 immunostaining showed that OEE divided more actively than inner enamel epithelium (IEE) at the onset of HERS formation. Live imaging suggested that mobility of the OEE and cells in the apex of CL were more active than in IEE. The expression of paxillin was observed strongly in OEE and the apex of CL. CONCLUSION: The more active growth and movement of OEE cells contributed to HERS formation after reduction of the growth of IEE. The expression pattern of paxillin was involved in the active movement of OEE and HERS. The results will contribute to understand the HERS formation mechanism and elucidate the cause of anomaly root.

Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth.

BACKGROUND AND OBJECTIVE: It is well known that tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS). However, relatively little is known about the regulatory mechanisms involved in root development. As hepatocyte growth factor (HGF) is one of the mediators of epithelial-mesenchymal interactions in rodent tooth, the objective of this study was to examine the effects of HGF on the root development of mouse molars. MATERIAL AND METHODS: The HERS of mouse molars and HERS01a, a cell line originated from HERS, were used in this study. For detection of HGF receptors in vivo and in vitro, we used immunochemical procedures. Root development was assessed by implanting molar tooth germs along with HGF-soaked beads into kidney capsules, by counting cell numbers in HERS01a cell cultures and by performing a 5'-bromo-2'-deoxyuridine (BrdU) assay in an organ-culture system. RESULTS: HGF receptors were expressed in the enamel epithelium of molar germs as well as in HERS cells. HGF stimulated root development in the transplanted tooth germs, the proliferation of HERS01a cells in culture and HERS elongation in the organ-culture system. Examination using BrdU revealed that cell proliferation in HERS was increased by treatment with HGF, especially that in the outer layer of HERS. This effect was down-regulated when antibody against HGF receptor was present in the culture medium. CONCLUSION: Our results raise the possibility that HGF signaling controls root formation via the development of HERS. This study is the first to show that HGF is one of the stimulators of root development.

The role of autophagy in the heart.

Autophagy has evolved as a conserving process that uses bulk degradation and recycling of cytoplasmic components, such as long-lived proteins and organelles. In the heart, autophagy is important for the turnover of organelles at low basal levels under normal conditions and it is upregulated in response to stresses such as ischemia/reperfusion and in cardiovascular diseases such as heart failure. Cardiac remodeling involves increased rates of cardiomyocyte cell death and precedes heart failure. The simultaneously occurring multiple features of failing hearts include not only apoptosis and necrosis but also autophagy as well. However, it has been unclear as to whether autophagy is a sign of failed cardiomyocyte repair or is a suicide pathway for failing cardiomyocytes. The functional role of autophagy during ischemia/reperfusion in the heart is complex. It has also been unclear whether autophagy is protective or detrimental in response to ischemia/reperfusion in the heart. In this review, we will summarize the role of autophagy in the heart under both normal conditions and in response to stress.

Identification of rab7 as a melanosome-associated protein involved in the intracellular transport of tyrosinase-related protein 1.

The melanosome is a unique secretory granule of the melanocyte in which melanin pigments are synthesized by tyrosinase gene family glycoproteins. Melanogenesis is a highly regulated process because of its inherent toxicity. An understanding of the various regulatory mechanisms is important in delineating the pathophysiology involved in pigmentary disorders and melanoma. We have purified and analyzed the total melanosomal proteins from B16 mouse melanoma tumors in order to identify new proteins that may be involved in the control of the melanogenesis process. Melanosomal proteins were resolved by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, a predominant spot (27 kDa with isoelectric point 5.8-6.4) was excised and digested with cyanogen bromide, and the fragments were sequenced. Synthetic oligonucleotide primers were synthesized corresponding to the peptide sequences, and reverse transcriptase polymerase chain reaction amplification of total RNA from B16 cells was carried out. Sequencing of one of the polymerase-chain-reaction-mediated clones demonstrated 80%-97% sequence homology of 200 bp nucleotide with GTP-binding proteins at the 3'-untranslated region. GTP-binding assay on two-dimensional gels of melanosomal proteins showed the presence of several (five to six) small GTP-binding proteins, suggesting that small GTP-binding proteins are associated with the melanosome. Among the known GTP-binding proteins with similar molecular weight and isoelectric point ranges, rab3, rab7, and rab8 were found to be present in the melanosomal fraction by immunoblotting. Confocal immunofluorescence microscopy showed that rab7 is colocalized with the tyrosinase-related protein 1 around the perinuclear area as well as, in part, in the perikaryon, thereby suggesting that rab7 might be involved in the intracellular transport of tyrosinase-related protein 1. Tyrosinase-related protein 1 transport was blocked by the treatment of B16 cells with antisense oligonucleotide to rab7. We suggest (i) that rab7 is a melanosome-associated molecule, (ii) that tyrosinase-related protein 1 is present in late-endosome delineated granules, and (iii) that rab7 is involved in the transport of tyrosinase-related protein 1 from the late-endosome delineated granule to the melanosome.

Intracellular calcium level required for calpain activation in a single myocardial cell.

We have hypothesized that calpain mediates myocardial injury induced by Ca(2+)overload. However, in vitro study demonstrated that the calcium requirement for calpain activation is around 10 microm, which is difficult to reach without the cell collapsing. Furthermore, because calpastatin is abundant in the myocardial cell, calpain may not be activated in physiological conditions. To elucidate whether calpain is activated by the calcium concentration reachable in myocardial living cells, we measured the calpain activity and the calcium concentration simultaneously in isolated guinea-pig cardiomyocytes. t-Butoxycarbonyl-Leu-Met-7-amino-4chlorimethylcoumarin (Boc-Leu-Met-CMAC), a fluorescent substrate of calpain, and/or fura red, a calcium indicator, were loaded into isolated cardiomyocytes together, and their fluorescence were measured separately. Intracellular Ca overload was induced by changing the superfusate from normal Tyrode solution to a sodium-free one. After changing the solution, fluorescence intensity of fura red and Boc-Leu-Met-CMAC did not change for a while, then fluorescence intensity of fura red began to rise. This was followed by the fluorescence intensity of Boc-Leu-Met-CMAC starting to rise 160+/-45 s after [Ca(2+)](i)increase. The relative fluorescence intensity of fura red increased to 1.37+/-0.32 folds of the control at the point that calpain became active. The calcium concentration at this point was estimated as 451 n m. These results indicate that calpain is activated by the slight rise of Ca concentration in intact cardiomyocytes.

Differential regulation of liver-specific and ubiquitously-expressed genes in primary rat hepatocytes by the extracellular matrix.

Primary rat hepatocytes were cultured with an extracellular matrix (ECM) overlay, in order to investigate the effect of an ECM on gene expression in hepatocytes. When hepatocytes, isolated by the collagenase-perfusion method, were cultured on type I collagen-coated dishes, the mRNA levels of liver-specific genes (aldolase B, tyrosine aminotransferase and albumin) decreased continuously, while those of ubiquitously-expressed genes (glyceraldehyde 3-phosphate dehydrogenase and beta-actin) increased. When a dilute ECM derived from the Engelbreth-Holm-Swarm mouse sarcoma (an EHS gel) was added to the above hepatocytes 3 days after plating, the mRNA levels of liver-specific genes increased, while those of ubiquitously-expressed genes decreased. The effects of a rat liver biomatrix (a physiological ECM for rat hepatocytes) on gene expression in primary hepatocytes were similar to those of the EHS gel. A nuclear run-on assay, and 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole or actinomycin D treatments revealed that the transcriptional rates of liver-specific genes were enhanced by the EHS gel overlay, while the apparent stability of the corresponding mRNAs were unchanged. In contrast, the transcriptional rates of ubiquitously-expressed genes were not greatly affected by an EHS gel overlay, while the apparent stability of their mRNAs were decreased. These data suggest that the ECM plays an important role in the maintenance of the differentiated characteristics of primary hepatocytes by inducing the transcription of liver-specific genes and, also, by destabilizing the mRNAs of ubiquitously-expressed genes.

[Ca2+ -induced cell injury of cardiomyocyte].

Pathogenesis of ischemia/reperfusion injury involves Ca(2+) -induced cell injury. Elevated intracellular Ca(2+) concentration at the reperfusion activates the Ca(2+) dependent protease, calpain and increases the generation of reactive oxygen species (ROS) in mitochondria, which cause cell injury in ischemia/reperfusion.

Calcineurin inhibitor attenuates left ventricular hypertrophy, leading to prevention of heart failure in hypertensive rats.

BACKGROUND: There is controversy regarding the contribution of calcineurin activation to the development of pressure-overload left ventricular (LV) hypertrophy and heart failure. The aim of this study was to explore whether the inhibition of calcineurin may prevent the transition to heart failure in hypertensive rats and, if so, to clarify in which developmental stage of LV hypertrophy calcineurin plays a key role. METHODS AND RESULTS: Dahl salt-sensitive rats placed on an 8% NaCl diet from the age of 7 weeks (hypertensive rats) were randomized to no treatment (n=6) or treatment with the calcineurin inhibitor FK506 (1 mg x kg(-1) x d(-1)) from 8 weeks (FKE, n=7) or from 17 weeks (FKL, n=7). Rats placed on a 0.3% NaCl diet were defined as control rats (n=6). The administration of FK506 from 8 weeks attenuated, although it did not block, LV hypertrophy observed in the untreated rats and prevented the transition to heart failure. The development of LV fibrosis, however, was not attenuated by the administration of FK506 from 8 weeks. The administration of FK506 from 17 weeks brought no benefit for cardiac remodeling or LV function and failed to prevent heart failure. CONCLUSIONS: Calcineurin inhibition, if started from the initial stage of pressure overload, attenuated the development of LV hypertrophy without any effect on LV fibrosis and prevented the transition to heart failure. The activation of calcineurin is involved in the development of LV hypertrophy but not of LV fibrosis, and this involvement may be crucial at the initial stage.

The involvement of cytokines in the second window of ischaemic preconditioning.

We utilized a rat model of myocardial infarction to investigate whether manganese superoxide dismutase (Mn-SOD), an intrinsic radical scavenger, and tumour necrosis factor- alpha (TNF-alpha) and/or interleukin-1beta (IL-1beta) are involved in the late phase of ischaemic preconditioning (IP). IP was induced in anaesthetized rats by four 3-min left coronary artery (LCA) occlusions, each separated by 10 min of reperfusion. Twenty-four hours after the repetitive brief ischaemia, the LCA was occluded for 20 min followed by reperfusion for 48 h. IP reduced the infarct size by approximately 46% as determined after 48 h of reperfusion. Antisense oligodeoxynucleotides to Mn-SOD inhibited the increases in Mn-SOD content and activity, and abolished the expected decrease in myocardial infarct size. Sense or scrambled oligodeoxynucleotides did not abolish either Mn-SOD induction or tolerance to ischaemia/reperfusion. The simultaneous administration of the antibodies to TNF-alpha (0.5 ml) and IL-1beta (0.5 mg) prior to IP abolished the cardioprotection and the increase in Mn-SOD activity induced by IP. We conclude that the induction and activation of Mn-SOD, mediated by TNF-alpha and IL-1beta after IP, plays an important role in the acquisition of late-phase cardioprotection against ischaemia/reperfusion injury in rats.

[Animal models of cardiomyopathy].

Animal models of cardiomyopathy have been used in many studies for our understanding of the pathophysiology of this disease. Syrian hamster, BIO 14.6, one of the most widely used models of cardiomyopathy, was reported to be caused by large deletion in the exon 1 and the promoter region of the delta-sarcoglycan gene. Cardiac hypertrophy or heart failure has been induced in dogs, rabbits, rats and many other animals by various manipulations such as drugs, pressure and/or volume overload and chronic rapid pacing. Recently, transgenic or knockout mice were examined to investigate the pathogenesis and development of the disease. It is important to select appropriate models for the aim of the studies.

Involvement of cytokines in the mechanism of whole-body hyperthermia-induced cardioprotection.

BACKGROUND: Hyperthermia increases cardiac tolerance to ischemia/reperfusion injury and activates manganese superoxide dismutase (Mn-SOD), an intrinsic radical scavenger, in myocardium in a biphasic manner. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) induced a biphasic cardioprotection that corresponded to the activation of Mn-SOD. However, a direct association between Mn-SOD activation in myocardium and the acquisition of tolerance to ischemia/reperfusion injury induced by hyperthermia and the involvement of the cytokines in the signal transduction pathway for the hyperthermia-induced cardioprotection have not yet been elucidated. METHODS AND RESULTS: Hyperthermia was induced in anesthetized rats by placement in a temperature-controlled water bath. At 0.5 and 72 hours after hyperthermia, ischemia was induced by occlusion of the left coronary artery for 20 minutes, followed by reperfusion for 48 hours. Inhibition of the increases in Mn-SOD content and activity 72 hours after hyperthermia by the administration of antisense oligodeoxynucleotides to Mn-SOD abolished the expected decrease in myocardial infarct size. The simultaneous administration of neutralizing antibodies to TNF-alpha and IL-1beta before hyperthermia abolished the biphasic cardioprotection and increase in Mn-SOD activity. CONCLUSIONS: The increase in Mn-SOD activity mediated through the production of TNF-alpha and IL-1beta by whole-body hyperthermia is important in the acquisition of early- and late-phase cardioprotection against ischemia/reperfusion injury in rats.

Single-strand conformation polymorphism analysis on the delta-sarcoglycan gene in Japanese patients with hypertrophic cardiomyopathy.

To elucidate the etiology of hypertrophic cardiomyopathy (HC) in humans, we analyzed the delta-sarcoglycan gene (SG), which is reported to be the causal gene for HC in the Syrian hamster BIO14.6. We performed polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses on the delta-SG in 102 patients with HC. SSCP was detected in exon 2 of the gene, but not in the other exons. The direct sequencing analysis of exon 2 revealed a C-->T substitution at nucleotide residue 84 (TAC-->TAT) with no amino acid alteration (Tyr-->Tyr). There were no significant differences in allele frequencies of C/T between the patients with HC and the control group. Patients with HC were classified into 4 subgroups: obstructive HC, nonobstructive HC, apical HC, and familial HC. The allele frequency of C/T polymorphism in each of these groups was compared with that of the control group. The obstructive HC group showed a significantly greater frequency of the allele T than in the control group (31.6% vs 15.1%, RR = 2.6, p = 0.023). No other significant differences were observed. Thus, amino acid alteration in delta-SG may not be a common cause of HC in Japanese patients.

Characterization of the responsive elements to hormones in the rat aldolase B gene.

Transcription of the aldolase B gene, AldB, in the liver is regulated by hormones such as insulin and glucagon. To characterize the elements that are responsive to these hormones in the upstream region of AldB, plasmids carrying various length of the upstream region of this gene were constructed and transfected to primary cultured rat hepatocytes. The promoter activities were gradually increased by progressive deletion of the 5'-upstream region, and high activities were observed for constructs carrying the sequence between -408 and -85 bp, suggesting the presence of suppressive element(s) in the upstream region of -409 bp. The transcription activities of the mutants containing the sequences between -228 and -85 bp were enhanced by insulin, and glucagon suppressed the transcription activities of those containing the sequence between -764 and -85 bp. Two sequence elements similar to the cAMP-responsive element (CRE), one from -89 to -82 bp and another from +13 to +20 bp, were found in the upstream sequence of the gene. The latter element is not functional because its deletion did not affect either the transcription efficiency or glucagon response. However, the deletion of the former element diminished both functions. A gel retardation assay showed that the nuclear factor binds to the former element, which was competitive with authentic CRE oligonucleotide but not with the mutant CRE one. These results suggest that the CRE-like element in the promoter region is prerequisite for both fundamental transcription efficiency of the gene and suppression by glucagon in hepatocytes.

Sandwich configuration of type I collagen suppresses progesterone production in primary cultured porcine granulosa cells by reducing gene expression of cytochrome P450 cholesterol side-chain cleavage enzyme.

When porcine granulosa cells were cultured on type I collagen (TIC)-coated dishes, progesterone was continuously secreted in the culture medium. However, when cells were overlaid with a TIC gel, progesterone production was decreased to 34% (day 3) and 16% (day 4) of the value measured for cells without the overlay. The effect of TIC gel overlay on cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), which catalyzes the conversion of cholesterol to pregnenolone and is thought to be the rate-limiting enzyme in the conversion of cholesterol to progesterone, was examined. P450scc gene expression in cells overlaid with a TIC gel was decreased to 62% (day 3) and 36% (day 4) of the value measured for cells without the overlay. Amounts of P450scc were also reduced in the cells overlaid with a TIC gel. When pregnenolone, the direct precursor of progesterone, was added to the culture medium, the increase in progesterone production by cells overlaid with a TIC gel was much greater than that for cells without a TIC gel and a statistical difference in progesterone production was no longer observed between the two groups of cells. Treatment of the cells with human FSH (hFSH) enhanced progesterone production in a dose-dependent manner, irrespective of the presence of a TIC gel overlay. Moreover, hFSH induced P450scc gene expression in cells with and without a TIC gel overlay. These results indicate that a TIC gel overlay reduces progesterone production in granulosa cells via the suppression of P450scc gene expression. This supports the possibility that the existence of a TIC gel on the apical side of granulosa cells prevents the spontaneous luteinization of granulosa cells cultured on TIC-coated dishes. The fact that hFSH overcomes the suppressive effect of the TIC gel overlay on progesterone production may explain the mechanism for the subtle rise in serum progesterone concentration in the late follicle phase of the "in vitro fertilization" program.

Cholesterol feeding exacerbates myocardial injury in Zucker diabetic fatty rats.

We measured infarct size after coronary occlusion (30 min) and reperfusion (24 h) in genetic non-insulin-dependent Zucker diabetic fatty (ZDF) rats with and without 4-wk cholesterol feeding. Infarct size was similar in ZDF rats and lean control rats but was significantly larger in cholesterol-fed diabetic rats than in cholesterol-fed lean rats (P < 0.05). Plasma levels of glucose, insulin, and triglycerides were significantly higher in diabetic rats and were not influenced by cholesterol feeding. The increase in total plasma cholesterol induced by cholesterol feeding was significantly greater in diabetic rats than in lean rats (P < 0.05). A significant positive correlation was found between total plasma cholesterol and infarct size (P < 0.05). Myeloperoxidase activity, as an index of neutrophil accumulation, was significantly higher and expression of P-selectin was more marked in the ischemic myocardium of cholesterol-fed diabetic rats than of cholesterol-fed lean rats. Acetylcholine-induced endothelium-dependent relaxation (EDR) of aortic rings was markedly impaired in cholesterol-fed diabetic rats. Thus cholesterol feeding significantly exacerbated myocardial injury produced by coronary occlusion-reperfusion in non-insulin-dependent diabetic rats, possibly because of enhanced expression of P-selectin and impairment of EDR in the coronary bed.