The morphology of internal elastic lamina corrugations in arteries under physiological conditions.

BACKGROUND: In elastic and resistance arteries, an elastin-rich membrane, the Internal Elastic Lamina (IEL), separates the tunica intima from the underlying tunica media. The IEL often appears wrinkled or corrugated in histological images. These corrugations are sometimes ascribed to vessel contraction ex vivo, and to fixation artifacts, and therefore regarded as not physiologically relevant. We examine whether the IEL remains corrugated even under physiological conditions. METHODS: The diameters of carotid arteries of anesthetized pigs were measured by ultrasound. The arteries were then excised, inflated within a conical sleeve, fixed, and imaged by confocal microscopy. The conical sleeve allows fixing each artery across a wide range of diameters, which bracket its ultrasound diameter. Thus the study was designed to quantify how corrugations change with diameter for a single artery, and test whether corrugations exist when the fixed artery matches the ultrasound diameter. RESULTS: At diameters below the ultrasound diameter (i.e. when the artery was constricted as compared to ultrasound conditions), the IEL corrugations were found to decrease significantly with increasing diameter, but not fully flatten at the ultrasound diameter. The contour length of the IEL was found to be roughly 10% larger than the circumference of the artery measured by ultrasound. The physiological diameter is likely to be even smaller than the ultrasound diameter since ultrasound was conducted with the animal under general anesthesia, which leads to vasodilation, suggesting a higher level of corrugation under physiological conditions. For arterial cross sections constricted below the ultrasound diameter, the IEL contour length decreased roughly with the square root of the diameter. CONCLUSION: The primary conclusions of this study are: a) the IEL is corrugated when the artery is constricted and flattens as the artery diameter increases; b) the IEL is corrugated under physiological conditions and has a contour length at least 10% more than the physiological arterial diameter; and c) the IEL despite being relatively stiffer than the surrounding arterial layers, does not behave like an inextensible membrane.

Adaptive Remodeling in the Elastase-induced Rabbit Aneurysms.

BACKGROUND: Rupture of brain aneurysms is associated with high fatality and morbidity rates. Through remodeling of the collagen matrix, many aneurysms can remain unruptured for decades, despite an enlarging and evolving geometry. OBJECTIVE: Our objective was to explore this adaptive remodeling for the first time in an elastase induced aneurysm model in rabbits. METHODS: Saccular aneurysms were created in 22 New Zealand white rabbits and remodeling was assessed in tissue harvested 2, 4, 8 and 12 weeks after creation. RESULTS: The intramural principal stress ratio doubled after aneurysm creation due to increased longitudinal loads, triggering a remodeling response. A distinct wall layer with multi-directional collagen fibers developed between the media and adventitia as early as 2 weeks, and in all cases by 4 weeks with an average thickness of 50.6 ± 14.3 µm. Collagen fibers in this layer were multi-directional (AI = 0.56 ± 0.15) with low tortuosity (1.08 ± 0.02) compared with adjacent circumferentially aligned medial fibers (AI = 0.78 ± 0.12) and highly tortuous adventitial fibers (1.22 ± 0.03). A second phase of remodeling replaced circumferentially aligned fibers in the inner media with longitudinal fibers. A structurally motivated constitutive model with both remodeling modes was introduced along with methodology for determining material parameters from mechanical testing and multiphoton imaging. CONCLUSIONS: A new mechanism was identified by which aneurysm walls can rapidly adapt to changes in load, ensuring the structural integrity of the aneurysm until a slower process of medial reorganization occurs. The rabbit model can be used to evaluate therapies to increase aneurysm wall stability.

Multiphoton intravital microscopy of the transplanted mouse kidney.

Graft outcomes after kidney transplantation continue to be adversely affected by ischemia-reperfusion injury and rejection. High-resolution, real-time imaging of the transplanted kidney could shed valuable insights into these dynamic processes, but such methodology has not been established. Here we describe a technique for intravital imaging of the transplanted mouse kidney using multiphoton fluorescence microscopy. The technique enabled real-time, high-resolution imaging and quantitation of renal filtration, cell death, leukocyte adhesion and capillary blood flow after transplantation. Using this technique, we found that brief graft ischemia associated with the transplantation procedure led to a rapid decline in renal filtration accompanied by a significant increase in microvascular leakage and renal tubular epithelial cell death within the first 3 h after transplantation. No significant changes in leukocyte adhesion or capillary blood flow were observed during the same time period. This report establishes multiphoton fluorescence microscopy as a sensitive tool for simultaneously studying functional and structural perturbations that occur in the mouse kidney after transplantation and for investigating the migration of leukocytes to the graft.

DNA-based immunization by in vivo transfection of dendritic cells.

Delivery of antigen in a manner that induces effective, antigen-specific immunity is a critical challenge in vaccine design. Optimal antigen presentation is mediated by professional antigen-presenting cells (APCs) capable of taking up, processing and presenting antigen to T cells in the context of costimulatory signals required for T-cell activation. Developing immunization strategies to optimize antigen presentation by dendritic cells, the most potent APCs, is a rational approach to vaccine design. Here we show that cutaneous genetic immunization with naked DNA results in potent, antigen-specific, cytotoxic T lymphocyte-mediated protective tumor immunity. This method of immunization results in the transfection of skin-derived dendritic cells, which localize in the draining lymph nodes. These observations provide a basis for further development of DNA-based vaccines and demonstrate the feasibility of genetically engineering dendritic cells in vivo.

Propagation of dendritic cell progenitors from normal mouse liver using granulocyte/macrophage colony-stimulating factor and their maturational development in the presence of type-1 collagen.

Within 1 wk of liquid culture in granulocyte/macrophage colony-stimulating factor (GM-CSF), normal B10 BR (H-2k I-E+) mouse liver nonparenchymal cells (NPC) formed loosely adherent myeloid cell clusters that have been shown to contain dendritic cell (DC) progenitors in similar studies of mouse blood or bone marrow. Mononuclear cell progeny released from these clusters at and beyond 4 d exhibited distinct dendritic morphology and were actively phagocytic. After 6-10 d of culture, these cells strongly expressed CD45, CD11b, heat stable antigen, and CD44. However, the intensity of expression of the DC-restricted markers NLDC 145, 33D1, and N418, and the macrophage marker F4/80, intercellular adhesion molecule 1, and Fc gamma RII was low to moderate, whereas the cells were negative for CD3, CD45RA, and NK1.1. Splenocytes prepared in the same way also had a similar range and intensity of expression of these immunophenotypic markers. Unlike the splenic DC, however, most of the GM-CSF-propagated putative liver DC harvested at 6-10 d expressed only a low level of major histocompatibility complex (MHC) class II (I-Ek), and they failed to induce primary allogeneic responses in naive T cells, even when propagated additionally in GM-CSF and tumor necrosis alpha and/or interferon gamma-supplemented medium. However, when 7-d cultured GM-CSF-stimulated liver cells were maintained additionally for three or more days on type-1 collagen-coated plates in the continued presence of GM-CSF, they exhibited characteristics of mature DC: MHC class II expression was markedly upregulated, mixed leukocyte reaction stimulatory activity was increased, and phagocytic function was decreased. Similar observations were made when Ia+ cells were depleted from the GM-CSF-propagated cells before exposure to collagen. Further evidence that the GM-CSF-stimulated class IIdim or class II-depleted hepatic NPC were immature DC was obtained by injecting them into allogeneic B10 (H-2b I-E-) recipients. They "homed" to T cell-dependent areas of lymph nodes and spleen where they strongly expressed donor MHC class II antigen 1-5 d later. These observations provide insight into the regulation of DC maturation, and are congruent with the possibility that the migration of immature DC from normal liver and perhaps other organ allografts may help explain their inherent tolerogenicity.

Sexual and marital trajectories and HIV infection among ever-married women in rural Malawi.

OBJECTIVE: To explore how sexual and marital trajectories are associated with HIV infection among ever-married women in rural Malawi. METHODS: Retrospective survey data and HIV biomarker data for 926 ever-married women interviewed in the Malawi Diffusion and Ideational Change Project were used. The associations between HIV infection and four key life course transitions considered individually (age at sexual debut, premarital sexual activity, entry into marriage and marital disruption by divorce or death) were examined. These transitions were then sequenced to construct trajectories that represent the variety of patterns in the data. The association between different trajectories and HIV prevalence was examined, controlling for potentially confounding factors such as age and region. RESULTS: Although each life course transition taken in isolation may be associated with HIV infection, their combined effect appeared to be conditional on the sequence in which they occurred. Although early sexual debut, not marrying one's first sexual partner and having a disrupted marriage each increased the likelihood of HIV infection, their risk was not additive. Women who both delayed sexual debut and did not marry their first partner are, once married, more likely to experience marital disruption and to be HIV-positive. Women who marry their first partner but who have sex at a young age, however, are also at considerable risk. CONCLUSIONS: These findings identify the potential of a life course perspective for understanding why some women become infected with HIV and others do not, as well as the differentials in HIV prevalence that originate from the sequence of sexual and marital transitions in one's life. The analysis suggests, however, the need for further data collection to permit a better examination of the mechanisms that account for variations in life course trajectories and thus in lifetime probabilities of HIV infection.

Gene expression in visceral endoderm: a comparison of mutant and wild-type F9 embryonal carcinoma cell differentiation.

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.

The subcellular distribution of chromosome 6-encoded dystrophin-related protein in the brain.

Chromosome 6-encoded dystrophin-related-protein (DRP) shows significant structural similarities to dystrophin at the carboxyl terminus, though the two proteins are encoded on different chromosomes. Both DRP and dystrophin are expressed in muscle and brain and show some similarity in their subcellular localization. For example, in skeletal muscle both are expressed at neuromuscular and myotendinous junctions. However, while dystrophin is absent or severely reduced in Duchenne/Becker muscular dystrophy, DRP continues to be expressed. Within the brain, dystrophin is enriched at the postsynaptic regions of specific subsets of neurons, while the distribution of DRP is yet to be described. In this study we demonstrate a distinct though highly specific pattern of distribution of DRP in the brain. DRP is enriched in the choroid plexus, pia mater, intracerebral vasculature, and ependymal lining. Within the parenchyma proper, DRP is located at the inner plasma face of astrocytic foot processes at the abluminal aspect of the blood-brain barrier. The distribution of DRP is conserved across a large evolutionary distance, from mammals to elasmobranchs, suggesting that DRP may play a role in the maintenance of regional specializations in the brain.

The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle.

We use a highly specific and sensitive antibody to further characterize the distribution of dystrophin in skeletal, cardiac, and smooth muscle. No evidence for localization other than at the cell surface is apparent in skeletal muscle and no 427-kD dystrophin labeling was detected in sciatic nerve. An elevated concentration of dystrophin appears at the myotendinous junction and the neuromuscular junction, labeling in the latter being more intense specifically in the troughs of the synaptic folds. In cardiac muscle the distribution of dystrophin is limited to the surface plasma membrane but is notably absent from the membrane that overlays adherens junctions of the intercalated disks. In smooth muscle, the plasma membrane labeling is considerably less abundant than in cardiac or skeletal muscle and is found in areas of membrane underlain by membranous vesicles. As in cardiac muscle, smooth muscle dystrophin seems to be excluded from membrane above densities that mark adherens junctions. Dystrophin appears as a doublet on Western blots of skeletal and cardiac muscle, and as a single band of lower abundance in smooth muscle that corresponds most closely in molecular weight to the upper band of the striated muscle doublet. The lower band of the doublet in striated muscle appears to lack a portion of the carboxyl terminus and may represent a dystrophin isoform. Isoform differences and the presence of dystrophin on different specialized membrane surfaces imply multiple functional roles for the dystrophin protein.

Hereditary pituitary dwarfism in mice affects skeletal and cardiac myosin isozyme transitions differently.

The dwarf mutation in mice interferes with the development of those anterior pituitary cells responsible for production of thyroid stimulating hormone, growth hormone, and prolactin. Myosin isozyme transitions in both cardiac and skeletal muscle were also found to be affected in this mutant. Electrophoresis of native myosins demonstrated that the fetal (V3) to adult (V1) ventricular cardiac isozyme transition was completely blocked in dwarf mice; in contrast, the neonatal to adult fast myosin transition in hind limb skeletal muscle was slowed but not totally inhibited. The persistence of neonatal myosin heavy chain for up to 55-75 d after birth in dwarf mice, as compared with 16 d in normal mice, was directly demonstrated by polypeptide and immunopolypeptide mapping. Morphological examination of 18-36-d-old dwarf skeletal muscles by optical and electron microscopy revealed a relative immaturity, but no signs of gross pathology were evident. Immunocytochemical analysis showed that the abnormal persistence of neonatal myosin occurs in most of the fibers. Multiple injections of thyroxine restored a normal isozyme complement to both cardiac and skeletal muscles within 11-15 d. Therefore, the effects of the dwarf mutation on myosin isozymes can be explained by the lack of thyroid hormone in these animals. Because the synthesis of growth hormone is not stimulated by thyroid hormone in dwarf mice as it would be in normal animals, these results demonstrate that thyroid hormone promotes myosin isozyme transitions independent of growth hormone production.

Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin.

We have characterized a protein immunologically related to dystrophin, the protein product of the Duchenne muscular dystrophy gene. We identify this related protein as a fast-twitch glycolytic isoform (mouse extensor digitorum longus-specific) of myofibrillar alpha-actinin. This specific isoform of alpha-actinin exhibits a more restricted pattern of expression in skeletal muscle than fast-twitch-specific isoforms of both myosin and Ca2+-ATPase. Our results provide evidence that dystrophin and myofibrillar alpha-actinin are related proteins, reinforcing the previous data concerning the sequence homologies noted between nonmuscle cytoskeletal alpha-actinin and dystrophin. In addition, we describe the first antisera directed against a specific myofibrillar skeletal muscle isoform of alpha-actinin.

Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta.

We have studied the interaction of 125I-antithrombin (125I-AT) with microvascular endothelial cells (RFPEC) to localize the cellular site of anticoagulantly active heparan sulfate proteoglycans (HSPG). The radiolabeled protease inhibitor bound specifically to the above HSPG with a Kd of approximately 50 nM. Confluent monolayer RFPEC cultures exhibited a linear increase in the amount of AT bound per cell for up to 16 d, whereas suspension RFPEC cultures possessed a constant number of protease inhibitor binding sites per cell for up to 5 d. These results suggest that monolayer RFPEC cultures secrete anticoagulantly active HSPG, which then accumulate in the extracellular matrix. This hypothesis was confirmed by quantitative light and EM level autoradiography which demonstrated that the AT binding sites are predominantly located in the extracellular matrix with only small quantities of protease inhibitor complexed to the cell surface. We have also pinpointed the in vivo position of anticoagulantly active HSPG within the blood vessel wall. Rat aortas were perfused, in situ, with 125I-AT, and bound labeled protease inhibitor was localized by light and EM autoradiography. The anticoagulantly active HSPG were concentrated immediately beneath the aortic and vasa vasorum endothelium with only a very small extent of labeling noted on the luminal surface of the endothelial cells. Based upon the above data, we propose a model whereby luminal and abluminal anticoagulantly active HSPG regulate coagulation mechanism activity.

Growth and muscle defects in mice lacking adult myosin heavy chain genes.

The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying null mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both null strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb null mutants are generally milder than in the MyHC-IId/x null strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for null expression of the two genes. Most striking is that while both null strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb null mice has significantly reduced ability to generate force while IId null mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.

Negative inotropic effects of cytokines on the heart mediated by nitric oxide.

The direct effects of pro-inflammatory cytokines on the contractility of mammalian heart were studied. Tumor necrosis factor alpha, interleukin-6, and interleukin-2 inhibited contractility of isolated hamster papillary muscles in a concentration-dependent, reversible manner. The nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) blocked these negative inotropic effects. L-Arginine reversed the inhibition by L-NMMA. Removal of the endocardial endothelium did not alter these responses. These findings demonstrate that the direct negative inotropic effect of cytokines is mediated through a myocardial nitric oxide synthase. The regulation of pro-inflammatory cytokines and myocardial nitric oxide synthase may provide new therapeutic strategies for the treatment of cardiac disease.

Neuronal peptide release is limited by secretory granule mobility.

Neuropeptides are slowly released from a limited pool of secretory granules. To visualize this process, GFP-tagged preproatrial natriuretic factor (ANF) was expressed in nerve growth factor-treated PC12 cells. Biochemical and microfluorimetric experiments demonstrate that proANF-EGFP is packaged in granules that accumulate at neurite endings and is released in a Ca2+-dependent manner by secretagogs. Confocal microscopy shows that secretion is associated with depletion of granules distributed throughout the terminal. Fluorescence recovery after photobleaching and time-lapse particle tracking reveal that only a subpopulation of cytoplasmic secretory granules, similar in size to the releasable pool, can move quickly enough (D = 6 x 10(-11) cm2/s) to support release. Therefore, sustained secretory responses are limited by the number of mobile granules and their slow rate of diffusion.

Inducible nitric oxide synthase suppresses the development of allograft arteriosclerosis.

In cardiac transplantation, chronic rejection takes the form of an occlusive vasculopathy. The mechanism underlying this disorder remains unclear. The purpose of this study was to investigate the role nitric oxide (NO) may play in the development of allograft arteriosclerosis. Rat aortic allografts from ACI donors to Wistar Furth recipients with a strong genetic disparity in both major and minor histocompatibility antigens were used for transplantation. Allografts collected at 28 d were found to have significant increases in both inducible NO synthase (iNOS) mRNA and protein as well as in intimal thickness when compared with isografts. Inhibiting NO production with an iNOS inhibitor increased the intimal thickening by 57.2%, indicating that NO suppresses the development of allograft arteriosclerosis. Next, we evaluated the effect of cyclosporine (CsA) on iNOS expression and allograft arteriosclerosis. CsA (10 mg/kg/d) suppressed the expression of iNOS in response to balloon-induced aortic injury. Similarly, CsA inhibited iNOS expression in the aortic allografts, associated with a 65% increase in intimal thickening. Finally, we investigated the effect of adenoviral-mediated iNOS gene transfer on allograft arteriosclerosis. Transduction with iNOS using an adenoviral vector suppressed completely the development of allograft arteriosclerosis in both untreated recipients and recipients treated with CsA. These results suggest that the early immune-mediated upregulation in iNOS expression partially protects aortic allografts from the development of allograft arteriosclerosis, and that iNOS gene transfer strategies may prove useful in preventing the development of this otherwise untreatable disease process.

The effect of non-insulin-dependent diabetes mellitus and obesity on glucose transport and phosphorylation in skeletal muscle.

Defects of glucose transport and phosphorylation may underlie insulin resistance in obesity and non-insulin-dependent diabetes mellitus (NIDDM). To test this hypothesis, dynamic imaging of 18F-2-deoxy-glucose uptake into midthigh muscle was performed using positron emission tomography during basal and insulin-stimulated conditions (40 mU/m2 per min), in eight lean nondiabetic, eight obese nondiabetic, and eight obese subjects with NIDDM. In additional studies, vastus lateralis muscle was obtained by percutaneous biopsy during basal and insulin-stimulated conditions for assay of hexokinase and citrate synthase, and for immunohistochemical labeling of Glut 4. Quantitative confocal laser scanning microscopy was used to ascertain Glut 4 at the sarcolemma as an index of insulin-regulated translocation. In lean individuals, insulin stimulated a 10-fold increase of 2-deoxy-2[18F]fluoro-D-glucose (FDG) clearance into muscle and significant increases in the rate constants for inward transport and phosphorylation of FDG. In obese individuals, the rate constant for inward transport of glucose was not increased by insulin infusion and did not differ from values in NIDDM. Insulin stimulation of the rate constant for glucose phosphorylation was similar in obese and lean subjects but reduced in NIDDM. Insulin increased by nearly twofold the number and area of sites labeling for Glut 4 at the sarcolemma in lean volunteers, but in obese and NIDDM subjects translocation of Glut 4 was attenuated. Activities of skeletal muscle HK I and II were similar in lean, obese and NIDDM subjects. These in vivo and ex vivo assessments indicate that impaired glucose transport plays a key role in insulin resistance of NIDDM and obesity and that an additional impairment of glucose phosphorylation is evident in the insulin resistance of NIDDM.

Inducible nitric oxide synthase is an endogenous neuroprotectant after traumatic brain injury in rats and mice.

Nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days and 1. 5 days after injury, respectively. In a model of CCI, knockout mice lacking the iNOS gene (iNOS(-/-)) were compared with wild-type (iNOS(+/+)) mice. Functional outcome (motor and cognitive) during the first 20 days after injury, and histopathology at 21 days, were assessed in both studies. Treatment of rats with either of the iNOS inhibitors after TBI significantly exacerbated deficits in cognitive performance, as assessed by Morris water maze (MWM) and increased neuron loss in vulnerable regions (CA3 and CA1) of hippocampus. Uninjured iNOS(+/+) and iNOS(-/-) mice performed equally well in both motor and cognitive tasks. However, after TBI, iNOS(-/-) mice showed markedly worse performance in the MWM task than iNOS(+/+) mice. A beneficial role for iNOS in TBI is supported.

Elevated levels of hepatocyte nuclear factor 3beta in mouse hepatocytes influence expression of genes involved in bile acid and glucose homeostasis.

The winged helix transcription factor, hepatocyte nuclear factor-3beta (HNF-3beta), mediates the hepatocyte-specific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3beta in regulating these genes remains unknown because homozygous null HNF3beta mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3beta, we created transgenic mice in which the -3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3beta protein. Postnatal transgenic mice exhibit growth retardation, depletion of hepatocyte glycogen storage, and elevated levels of bile acids in serum. The retarded growth phenotype is likely due to a 20-fold increase in hepatic expression of insulin-like growth factor binding protein 1 (IGFBP-1), which results in elevated levels in serum of IGFBP-1 and limits the biological availability of IGFs required for postnatal growth. The defects in glycogen storage and serum bile acids coincide with diminished postnatal expression of hepatocyte genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glycogen synthase) and sinusoidal bile acid uptake (Ntcp), respectively. These changes in gene transcription may result from the disruptive effect of HNF-3beta on the hepatic expression of the endogenous mouse HNF-3alpha,-3beta, -3gamma, and -6 transcription factors. Furthermore, adult transgenic livers lack expression of the canalicular phospholipid transporter, mdr2, which is consistent with ultrastructure evidence of damage to transgenic hepatocytes and bile canaliculi. These transgenic studies represent the first in vivo demonstration that the HNF-3beta transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.

Std1 and Mth1 proteins interact with the glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae.

The Std1 protein modulates the expression of glucose-regulated genes, but its exact molecular role in this process is unclear. A two-hybrid screen for Std1-interacting proteins identified the hydrophilic C-terminal domains of the glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic interactions between STD1, MTH1, SNF3, and RGT2 suggest that the glucose signaling is mediated, at least in part, through interactions of the products of these four genes. Mutations in MTH1 can suppress the raffinose growth defect of a snf3 mutant as well as the glucose fermentation defect present in cells lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in MTH1 is likely to be due to the increased and unregulated expression of hexose transporter genes. In media lacking glucose or with low levels of glucose, the hexose transporter genes are subject to repression by a mechanism that requires the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able to regulate SUC2 gene expression in response to changes in glucose concentration. Finally, studies with green fluorescent protein fusions indicate that Std1 is localized to the cell periphery and the cell nucleus, supporting the idea that it may transduce signals from the plasma membrane to the nucleus.