Vitamin D3 seems more appropriate than D2 to sustain adequate levels of 25OHD: a pharmacokinetic approach.

BACKGROUND/OBJECTIVES: The superiority of cholecalciferol (D3) over ergocalciferol (D2) in sustaining serum 25-hydroxy vitamin D (25OHD) levels is controversial. To compare D2 with D3 we performed a single-blind, placebo-controlled randomized trial spanning 11 weeks. SUBJECTS/METHODS: Healthy volunteers (n=33, aged 33.4±6 years) were divided into three groups (n=11, each): D2, D3 and placebo. Treatment started with a loading dose (100,000 IU) followed by 4800 IU/day (d) between d7 and d20 and follow-up until d77. Serum samples were obtained at baseline and at days 3, 7, 14, 21, 35, 49, 63 and 77. RESULTS: Baseline 25OHD values in the D2 group were lower than those in the D3 and placebo groups (P<0.01). Placebo 25OHD levels never changed. As after the loading dose both D2 and D3 groups had reached similar 25OHD levels, we tested equivalence of the area under the concentration × time curve (AUC) between d7 and d77. The AUC was 28.6% higher for D3 compared with D2, and both were higher with respect to placebo. At d77, D2 25OHD levels were higher than those at baseline, but similar to placebo; both were lower than D3 (P<0.04). According to raw data, the elimination half-life of 25OHD was 84 and 111 days under D2 and D3 supplementation, respectively; after subtracting the placebo values, the corresponding figures were 33 and 82 days. CONCLUSIONS: D2 and D3 were equally effective in elevating 25OHD levels after a loading dose. In the long term, D3 seems more appropriate for sustaining 25OHD, which could be relevant for classic and non-classic effects of vitamin D.

Relative bioavailability of a 5 mg mosapride/10 mg rabeprazole fixed dose combination tablet versus separate single tablets in healthy volunteers: a single-dose randomized open-label crossover study.

OBJECTIVE: To evaluate the relative bioavailability of a new formulation containing 5 mg mosapride and 10 mg rabeprazole (T) and compare it with the branded formulations of both drugs co-administered in separate tablets (R) to meet the regulatory requirements of bioequivalence in Argentina. METHODS: A randomized-sequence, open-label, two-period crossover study was conducted on 24 healthy Caucasian volunteers in a fasting state. A single oral dose of either T or R formulations was followed by a 7-day washout period. Blood samples for mosapride were collected before administration (baseline) and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 18, and 24 h after administration. Samples for rabeprazole were taken baseline and at 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8 and 10 h after dosing. Mosapride and rabeprazole concentrations were determined using a validated LC-MS/MS method. Adverse events were monitored based on clinical parameters and volunteer reports. RESULTS: The geometric means (90% CI) C(max) for mosapride in T and R were 23.13 (20.05-39.45) and 23.09 (21.69-32.37) ng/mL, the AUC(0-)(t) were 70.80 (66.23-102.37) and 70.81 (66.35-93.26) ng h/mL and the AUC(0-∞) were 74.05 (69.29-106.11) and 74.98 (70.43-97.77) ng h/mL. For rabeprazole T and R the C(max) were 197.42 (186.12-239.91) and 195.50 (186.08-250.07) ng/mL, the AUC(0-)(t) were 294.90 (275.13-374.15) and 296.96 (280.11-387.89) ng h/mL and the AUC(0-∞) were 301.12 (280.78-380.82) and 304.07 (286.60-394.21), respectively. No differences were detected between the formulations. The T/R ratios (90% CI) for C(max), AUC(0-)(t) and AUC(0-∞) were 100.17% (82.35-121.84), 99.99% (87.58-114.16) and 98.77% (87.02-112.11) for mosapride, and 100.99% (85.14-119.77), 99.31% (84.74-116.38) and 99.03% (85.07-115.28) for rabeprazole. No subject complained of adverse events. CONCLUSIONS: In this single-dose study, the mosapride/rabeprazole tablets (test formulation) met the criterion for bioequivalence with the reference formulations. Study limitations include single-dose, open-label design, and a small sample of healthy volunteers.

A recombinant human enzyme for enhanced interstitial transport of therapeutics.

Subcutaneously injected therapeutics must pass through the interstitial matrix of the skin in order to reach their intended targets. This complex, three-dimensional structure limits the type and quantity of drugs that can be administered by local injection. Here we found that depolymerization of the viscoelastic component of the interstitial matrix in animal models with a highly purified recombinant human hyaluronidase enzyme (rHuPH20) increased the dispersion of locally injected drugs, across a broad range of molecular weights without tissue distortion. rHuPH20 increased infusion rates and the pattern and extent of appearance of locally injected drugs in systemic blood. In particular, rHuPH20 changed the pharmacokinetic profiles and significantly augmented the absolute bioavailability of locally injected large protein therapeutics. Importantly, within 24 h of injection, the interstitial viscoelastic barriers were restored without histologic alterations or signs of inflammation. rHuPH20 may function as an interstitial delivery enhancing agent capable of increasing the dispersion and bioavailability of coinjected drugs that may enable subcutaneous administration of therapeutics and replace intravenous delivery.

Purification of brain peroxisomes and localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well-defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of subcellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40-fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG-CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.

Identification of an angiogenic mitogen selective for endocrine gland endothelium.

The known endothelial mitogens stimulate growth of vascular endothelial cells without regard to their tissue of origin. Here we report a growth factor that is expressed largely in one type of tissue and acts selectively on one type of endothelium. This molecule, called endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), induced proliferation, migration and fenestration (the formation of membrane discontinuities) in capillary endothelial cells derived from endocrine glands. However, EG-VEGF had little or no effect on a variety of other endothelial and non-endothelial cell types tested. Similar to VEGF, EG-VEGF possesses a HIF-1 binding site, and its expression is induced by hypoxia. Both EG-VEGF and VEGF resulted in extensive angiogenesis and cyst formation when delivered in the ovary. However, unlike VEGF, EG-VEGF failed to promote angiogenesis in the cornea or skeletal muscle. Expression of human EG-VEGF messenger RNA is restricted to the steroidogenic glands, ovary, testis, adrenal and placenta and is often complementary to the expression of VEGF, suggesting that these molecules function in a coordinated manner. EG-VEGF is an example of a class of highly specific mitogens that act to regulate proliferation and differentiation of the vascular endothelium in a tissue-specific manner.

Identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes. AA-CoA thiolase, hmg-coa synthase, MPPD, and FPP synthase.

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. The peroxisomal targeting signals for phosphomevalonate kinase and isopentenyl diphosphate isomerase have been identified. In the current study we identify the peroxisomal targeting signals required for four other enzymes of the cholesterol biosynthetic pathway: acetoacetyl-CoA (AA-CoA) thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, mevalonate diphosphate decarboxylase (MPPD), and farnesyl diphosphate (FPP) synthase. Data are presented that demonstrate that mitochondrial AA-CoA thiolase contains both a mitochondrial targeting signal at the amino terminus and a peroxisomal targeting signal (PTS-1) at the carboxy terminus. We also analyze a new variation of PTS-2 sequences required to target HMG-CoA synthase and MPPD to peroxisomes. In addition, we show that FPP synthase import into peroxisomes is dependent on the PTS-2 receptor and identify at the amino terminus of the protein a 20-amino acid region that is required for the peroxisomal localization of the enzyme. These data provide further support for the conclusion that peroxisomes play a critical role in cholesterol biosynthesis.

Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections.

We have adapted existing microwave irradiation (MWI) protocols and applied them to the processing and immunoelectron microscopy of both plastic-embedded and frozen sections. Rat livers were fixed by rapid MW irradiation in a mild fixation solution. Fixed liver tissue was either cryosectioned or dehydrated and embedded in Spurr's, Unicryl, or LR White resin. Frozen sections and sections of acrylic-embedded tissue were immunolabeled in the MW oven with an anti-catalase antibody, followed by gold labeling. Controls were processed conventionally at room temperature (RT). The use of MWI greatly shortened the fixation, processing, and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The higher immunogold labeling intensity was achieved after a 15-min incubation of primary antibody and gold markers under discontinued MWI at 37C. Quantification of the immunolabeling for catalase indicated a density increase of up to fourfold in the sections immunolabeled in the MW oven over that of samples immunolabeled at RT. These studies define the general conditions of fixation and immunolabeling for both acrylic resin-embedded material and frozen sections.

Pex17p is required for import of both peroxisome membrane and lumenal proteins and interacts with Pex19p and the peroxisome targeting signal-receptor docking complex in Pichia pastoris.

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Delta mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Delta mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal-receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.

Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane.

We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A Deltapex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25-amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

Pex19p interacts with Pex3p and Pex10p and is essential for peroxisome biogenesis in Pichia pastoris.

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Delta cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.

VEGF is required for growth and survival in neonatal mice.

We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP- mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development.

Peroxisome degradation by microautophagy in Pichia pastoris: identification of specific steps and morphological intermediates.

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl ) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor-product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate.

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins.

Effect of insulin-like growth factor binding proteins on the response of proximal tubular cells to insulin-like growth factor-I.

The insulin-like growth factor binding proteins (IGFBP) are major modulators of insulin-like growth factor-I (IGF-I) action, but relatively little is known about their production by kidney tubular cells or about their modulating effects on the action of IGF-I on these cells. In this study we demonstrated that rabbit proximal tubular cells express the genes for IGFBP-2, -4 and -5 and secrete 24 and 32 kDa size binding proteins. The rate of IGFBP production by these cells was regulated by several growth factors including hydrocortisone, which was potently stimulatory, and EGF, which was inhibitory. The overall effect of these kidney cell-secreted IGFBPs was to inhibit the mitogenic activity of IGF-I. Similarly, recombinant IGFBP-3, the major circulating IGFBP that in kidney is produced close to the proximal tubules, also inhibited IGF-I stimulated DNA synthesis in cultured rabbit proximal tubular cells and in cultured opossum kidney (OK) cells. IGFBP-3 also inhibited basal DNA synthesis in OK cells in the absence of added IGF-I, suggesting that this IGFBP may have an IGF-I independent action. These findings highlight the important effect that IGFBPs have on the action of IGF-I on kidney cells and support the notion that the changes in IGFBPs observed in various renal diseases may contribute to the pathophysiology of these diseases.

Normal platelets and megakaryocytes are produced in vivo in the absence of thrombopoietin.

Thrombopoietin (TPO) has been established as the major regulator of megakaryocyte and platelet production. In vitro and in vivo studies have demonstrated that TPO affects both megakaryocyte proliferation and maturation. In vitro, TPO has been reported to be essential for full development of megakaryocytes and platelets. These studies are in contrast to results observed in vivo in mice deficient in the TPO or c-mpl gene (TPO-/- and c-mpl-/-). Both TPO-/- and c-mpl-/- mice exhibit a 90% reduction in megakaryocyte and platelet levels. But even with this small number of circulating platelets, these mice do not have any excessive bleeding. Ultrastructural analysis indicates that platelets and megakaryocytes present in the knockout mice are morphologically normal. Characterization of platelet function shows that platelets from knockout mice are functionally identical to the wild-type platelets as measured by upregulation of 125I-fibrinogen binding to platelets in response to adenosine diphosphate (ADP) stimulation and by platelet attachment to the immobilized extracellular matrix proteins, collagen and von Willebrand factor (vWF). These results demonstrate that in vivo, TPO is required for the control of megakaryocyte and platelet number but not for their maturation. Other factors with megakaryocytopoietic activity may be able to compensate for the maturational role of TPO and lead to the formation of normal megakaryocytes and platelets in TPO-/- and c-mpl-/- mice.

Regulation of the serum concentration of thrombopoietin in thrombocytopenic NF-E2 knockout mice.

The mechanisms that regulate circulating levels of thrombopoietin (Tpo) are incompletely understood. According to one favored model, the rate of Tpo synthesis is constant, whereas the serum concentration of free Tpo is modulated through binding to c-Mpl receptor expressed on blood platelets. Additionally, a role for c-Mpl expressed on megakaryocytes is suggested, particularly by the observation that serum Tpo levels are not elevated in human immune thrombocytopenic purpura. Whereas direct binding of Tpo to platelets has been demonstrated in vitro and in vivo, the role of megakaryocytes in modulating serum Tpo levels has not been addressed experimentally. The profoundly thrombocytopenic mice lacking transcription factor p45 NF-E2 do not show the predicted increase in serum Tpo concentration. To evaluate the fate of the ligand in these animals, we injected 125I-Tpo intravenously into mutant and control mice. In contrast to normal littermates, NF-E2 knockout mice show negligible association of radioactivity with blood cellular components, consistent with an absence of platelets. There is no corresponding increase in plasma-associated radioactivity to suggest persistence in the circulation. However, a greater fraction of the radioligand is bound to hematopoietic tissues. In the bone marrow this is detected virtually exclusively in association with megakaryocytes, whereas in the spleen it is associated with megakaryocytes and small, abnormal, platelet-like particles or megakaryocyte fragments that are found within or in close contact with macrophages. These findings implicate the combination of megakaryocytes and the latter particles as a sink for circulating Tpo in NF-E2 knockout mice, and provide an explanation for the lack of elevated serum Tpo levels in this unique animal model of thrombocytopenia.

Characterization of UT2 cells. The induction of peroxisomal 3-hydroxy-3-methylglutaryl-coenzyme a reductase.

In the liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum but also in the peroxisomes. However, to date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and that is localized exclusively to peroxisomes. This cell line was obtained by growing UT2 cells (which lack the endoplasmic reticulum HMG-CoA reductase) in the absence of mevalonate. The cells exhibited a marked increase in a 90-kDa HMG-CoA reductase that was localized exclusively to peroxisomes. The wild type Chinese hamster ovary cells contain two HMG-CoA reductase proteins, the well characterized 97-kDa protein, localized in the endoplasmic reticulum, and a 90-kDa protein localized in peroxisomes. The UT2 cells grown in the absence of mevalonate containing the up-regulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study.

Insulin-degrading enzyme does not require peroxisomal localization for insulin degradation.

Although considerable evidence implicates insulin-degrading enzyme (IDE) in the cellular metabolism of insulin in many cell types, its mechanism and site of action are not clear. In this study, we have examined the relationship between insulin-degrading enzyme's peroxisomal location and its ability to degrade insulin by mutation of its peroxisomal targeting signal (PTS), the carboxy terminal A/S-K-L tripeptide. Site-directed mutagenesis was used to destroy the peroxisomal targeting signal of human insulin-degrading enzyme by changing alanine to leucine (AL.pts), leucine to valine (LV.pts), or by deleting the entire tripeptide (DEL.pts). The alanine or leucine mutants, when expressed in COS cells, were indistinguishable from wild-type insulin-degrading enzyme with respect to size (110 kDa), amount of immunoreactive material, ability to bind insulin, in vitro activity, and cellular degradation of insulin. In contrast, the deletion mutant was shorter in size (approximately 0 kDa) and unable to bind the hormone. Thus, although the tripeptide at insulin-degrading enzyme's carboxy terminus appeared to confer enzyme stability, the conserved sequence was not required for insulin degradation. Finally, an immunocytofluorescence study showed that, whereas a significant amount of the wild-type protein was localized in peroxisomes, none of the peroxisomal targeting mutants could be detected in these organelles. These findings indicate that insulin-degrading enzyme does not require peroxisomal localization for insulin degradation and suggest that this enzyme has multiple cellular functions.

Metabolism of thrombopoietin (TPO) in vivo: determination of the binding dynamics for TPO in mice.

Previous in vivo studies have established that plasma thrombopoietin (TPO) levels are regulated by binding to c-Mpl on platelets and that, in vitro, platelets bind and degrade TPO. To determine if the in vivo metabolism of TPO was specific and saturable, we injected normal CD-1 mice IV with trace amounts of 125I-rmTPO with or without a saturating concentration of rmTPO. The amount of radioactivity present in the spleen, blood cell fraction, platelet fraction, tibia/fibula, and femur was significantly greater in the mice receiving 125I-rmTPO alone. Conversely, the amount of radioactivity present in the plasma was significantly greater in the mice receiving both 125I-rmTPO and rmTPO, thus suggesting the uptake of rmTPO by the spleen, platelets, and bone marrow in vivo was saturable. Platelet and spleen homogenates from animals receiving 125I-rmTPO alone showed a degradation pattern of 125I-rmTPO similar to that observed in vitro using mouse platelet rich plasma. To determine the in vivo binding dynamics for rmTPO, mice were injected with 125I-rmTPO alone or with increasing concentrations of rmTPO; spleen and blood cell-associated radioactivity was determined at 2 hours postinjection. A 4-parameter curve fit of the data indicated that the "in vivo binding affinity" for rmTPO was approximately 6.4 microg/kg. These data indicate that after a dose of approximately 6.4 microg/kg, 50% of all c-Mpl receptors will be saturated with rmTPO. Electron microscopy indicated that radioactivity was present bound to and within megakaryocytes and platelets in both sternum and spleen and platelets in circulation. Together these data demonstrate that in vivo, 125I-rmTPO is mainly metabolized by platelets and to a small extent by cells of the megakaryocyte lineage, via a specific and saturable mechanism.

Management for quality: continuous quality improvement to increase access to outpatient mental health services.

OBJECTIVE: A health maintenance organization (HMO) examined whether continuous quality improvement could be used to address the problem of long waiting times for outpatient mental health services from a closed panel of providers during peak periods of demand. METHODS: A task force at a staff model HMO in Burlington, Vermont, used continuous quality improvement methods to identify and solve specific service access problems in five categories: quality, protocol, and standards; systems and processes; management and administration; clinical practice management; and public relations and marketing. RESULTS: Over a two-year period, the task force identified 13 specific problems, for which solutions were implemented. For example, two new support positions were created to meet clinicians' needs. Triage categories were defined, and acceptable waiting times for appointments, along with goals for percent compliance, were established. A weekly training program in brief psychotherapy and an extensive group psychotherapy program were implemented. A network of community providers was formed to complement the HMO's fixed provider panel during periods of high demand. The average waiting time was reduced from 22 days to six days, and patients' satisfaction increased markedly. CONCLUSIONS: Use of continuous quality improvement can guide clinical leaders in their central role of reinstating clinical quality as the goal of management. The author suggests that continuous quality improvement with balanced clinical and administrative leadership is the means to forge the needed synthesis of quality and cost capable of improving mental health.