Overfeeding and Substrate Availability, But Not Age or BMI, Alter Human Satellite Cell Function.

Satellite cells (SC) aid skeletal muscle growth and regeneration. SC-mediated skeletal muscle repair can both be influenced by and exacerbate several diseases linked to a fatty diet, obesity, and aging. The purpose of this study was to evaluate the effects of different lifestyle factors on SC function, including body mass index (BMI), age, and high-fat overfeeding. For this study, SCs were isolated from the vastus lateralis of sedentary young (18-30 years) and sedentary older (60-80 years) men with varying BMIs (18-32 kg/m2), as well as young sedentary men before and after four weeks of overfeeding (OVF) (55% fat/ + 1000 kcal, n = 4). The isolated SCs were then treated in vitro with a control (5 mM glucose, 10% fetal bovine serum (FBS)) or a high substrate growth media (HSM) (10% FBS, 25 mM glucose, and 400 µM 2:1 oleate-palmitate). Cells were assessed on their ability to proliferate, differentiate, and fuel substrate oxidation after differentiation. The effect of HSM was measured as the percentage difference between SCs exposed to HSM compared to control media. In vitro SC function was not affected by donor age. OVF reduced SC proliferation rates (-19% p < 0.05) but did not influence differentiation. Cellular proliferation in response to HSM was correlated to the donor's body mass index (BMI) (r2 = 0.6121, p < 0.01). When exposed to HSM, SCs from normal weight (BMI 18-25 kg/m2) participants exhibited reduced proliferation and fusion rates with increased fatty-acid oxidation (p < 0.05), while SCs from participants with higher BMIs (BMI 25-32 kg/m2) demonstrated enhanced proliferation in HSM. HSM reduced proliferation and fusion (p < 0.05) in SCs isolated from subjects before OVF, whereas HSM exposure accelerated proliferation and fusion in SCs collected following OVF. These results indicated that diet has a greater influence on SC function than age and BMI. Though age and BMI do not influence in vitro SC function when grown in controlled conditions, both factors influenced the response of SCs to substrate challenges, indicating age and BMI may mediate responses to diet.

Residue Distribution and Depletion of Ractopamine in Goat Tissues After Exposure to Growth-Promoting Dose.

The objectives of the present study was to investigated the ractopamine (RAC) distribution and depletion process in various tissues of goat including liver, kidney, spleen, lung, heart, fat, bile, brain and the eyes. The experiment was carried out on 21 goats (18 treated and 3 controls). Treated goats were orally administered RAC in a dose of 1 mg/kg body mass per day for last 28 days and randomly sacrificed on withdrawal days of 0.25, 1, 3, 7, 14 and 21. RAC in all matrices were determined by ultra-high performance liquid chromatography-quadrupole orbitrap high resolution mass spectrometry. After 21 days treatment discontinuation, the levels of RAC in bile reached at 13.48 ± 3.36 mg/L, which was significantly higher than that in the other tissues. The concentrations of RAC were followed by kidney, the excretory organ and liver, the major metabolic organ (4.49 ± 0.16 mg/kg for kidney and 1.81 ± 0.11 mg/kg for liver, respectively). The residual concentration of the drug in the eyes of goat was less than that in bile, kidney, liver, lung and spleen on withdrawal days 0.25. RAC residues was higher than the limits of detection = 0.15 µg/mL in liver on Day 21. These findings demonstrated that liver can serve as an alimentary matrix and as a matrix for the control of RAC abuse hypothetically except for urine.

First-in-man study with a novel PEGylated recombinant human insulin-like growth factor-I.

OBJECTIVE: This study is a first time assessment of safety and tolerability, pharmacokinetics, and pharmacodynamics of RO5046013 in human, in comparison with unmodified rhIGF-I. DESIGN: The study was conducted as a single-center, randomized, double-blinded, placebo-controlled, single ascending dose, parallel group study in a clinical research unit in France. A total of 62 healthy volunteers participated in this clinical trial. RO5046013 was given as single subcutaneous injection, or as intravenous infusion over 48h, at ascending dose levels. The active comparator rhIGF-I was administered at 50µg/kg subcutaneously twice daily for 4days. Safety and tolerability, pharmacokinetics, and pharmacodynamics of RO5046013 were evaluated. RESULTS: PEGylation resulted in long exposure to RO5046013 with a half-life of 140-200h. Exposure to RO5046013 increased approximately dose proportionally. RO5046013 was safe and well tolerated at all doses, injection site erythema after SC administration was the most frequent observed AE. No hypoglycemia occurred. Growth hormone (GH) secretion was almost completely suppressed with rhIGF-I administration, whereas RO5046013 caused only a modest decrease in GH at the highest dose given IV. CONCLUSIONS: PEGylation of IGF-I strongly enhances half-life, reduces the negative GH feedback and hypoglycemia potential, and therefore offers a valuable alternative to rhIGF-I in treatment of relevant diseases.

Temporal-controlled Dexamethasone Releasing Chitosan Nanoparticle System Enhances Odontogenic Differentiation of Stem Cells from Apical Papilla.

INTRODUCTION: The spatial and temporal control of stem cell differentiation into odontoblast-like cells remains one of the major challenges in regenerative endodontic procedures. The current study aims to synthesize and compare the effect of dexamethasone (Dex) release from 2 variants of Dex-loaded chitosan nanoparticles (CSnp) on the odontogenic differentiation of stem cells from apical papilla (SCAP). METHODS: Two variants of Dex-loaded CSnp were synthesized by encapsulation (Dex-CSnpI) and adsorption (Dex-CSnpII) methods. The physicochemical characterization of Dex-CSnpI and Dex-CSnpII was assessed by transmission electron microscopy, Zetasizer, and Fourier transform infrared spectroscopy, whereas the Dex release kinetics was assessed by spectrophotometry. A previously characterized SCAP cell line was cultured onto CSnp, Dex-CSnpI, or Dex-CSnpII. The biomineralization potential was determined by alizarin red staining. Alkaline phosphatase, dentin sialophosphoprotein, and dentin matrix protein-1 gene expressions were analyzed by real-time reverse-transcription polymerase chain reaction. RESULTS: Dex-CSnpI resulted in slower release of Dex compared with Dex-CSnpII, but both demonstrated sustained release of Dex for 4 weeks. Biomineralization of SCAP was significantly higher (P < .05) in presence of Dex-CSnpII compared with that in Dex-CSnpI at 3 weeks. Alkaline phosphatase gene expression was significantly higher in the presence of Dex-CSnpII compared with Dex-CSnpI, with peak expression seen at 2 weeks (P < .05). The expression of odontogenic specific marker dentin matrix protein-1 was significantly higher in presence of Dex-CSnpII compared with Dex-CSnpI at 3 weeks (P < .05). CONCLUSIONS: Collectively, these data suggest that sustained release of Dex results in enhanced odontogenic differentiation of SCAP. These findings highlight the potential of temporal-controlled delivery of bioactive molecules to direct the spatial- and temporal-controlled odontogenic differentiation of dental stem cells.

Sustained delivery of BMP-2 enhanced osteoblastic differentiation of BMSCs based on surface hydroxyapatite nanostructure in chitosan-HAp scaffold.

The surface characteristics of biomaterials, especially regarding the sustained delivery of bone morphogenetic protein-2 (BMP-2), can possibly provide a novel and effective drug delivery system that can enhance osteogenesis. In this study, we evaluated the BMP-2 adsorption and release ability of the surface biomimetic hydroxyapatite (HAp) nanostructure on a new HAp-coated genipin-chitosan conjugation scaffold (HGCCS), and the resulting osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. HGCCS exhibited a loading efficiency of 65% (1.30 µg), which is significantly higher than 28% (0.56 µg, p < 0.01) for the genipin cross-linked chitosan framework, as quantified by an enzyme-linked immunosorbent assay. More importantly, we found that the release of BMP-2 from HGGCS sustained for at least 14 days in simulated body fluid in vitro, which is much better than the burst release within 3 days for CGF. Moreover, the BMP-2 release from HGCCS induced an increase in alkaline phosphatase activity as an indicator of osteogenic differentiation of seeded BMSCs for 14 days in vitro. HGCCS also stimulated a high mRNA expression of osteogenic differentiation makers, runt-related transcription factor 2 for 14 days, osteopontin for 3 days, and osteocalcin for 14 days. The results of this study suggest that the surface biomimetic HAp nanostructure of HGCCS used as a delivery system for BMP-2 is capable of promoting osteogenic differentiation in vitro. These findings demonstrated that HAp nanostructure assembled on organic porous scaffold could work as both calcium source and absorption/release platform, which opened a new research avenue for cell growth factor release, and provided a promising strategy for design and preparation of bioactive scaffold for bone tissue engineering.

Excretion profile of corticosteroids in bovine urine compared with tissue residues after therapeutic and growth-promoting administration of dexamethasone.

The illicit use of dexamethasone as growth-promoting agent in animal breeding is still practiced within the EU constituting a health risk for meat consumers. An experimental study was developed to assess dexamethasone urinary excretion and tissue distribution (liver, kidney, and muscle) in male calves after therapeutic and growth-promoting administration. Urine and tissue samples collected from treated and untreated bovines were also investigated for the presence of other natural and synthetic corticosteroids (prednisolone, prednisone, hydrocortisone, and cortisone), in order to study a possible correlation with dexamethasone administration and to clarify prednisolone origin. Analyses were performed by a multi-residue LC-MS/MS method developed and validated according to the Commission Decision 2002/657/EC. The results confirm the rapid rate of dexamethasone urinary excretion, irrespective of the dosage, the duration and the route of administration, and the disappearance of cortisone and hydrocortisone during the treatment. Dexamethasone was distributed to the tissues where the elimination rate proceeded relatively slower as suggested by the presence of residues one month after the withdrawal of the therapeutic treatment. An increase in the number of positive findings for prednisolone, in association with higher levels of cortisone and hydrocortisone, was observed in urine samples collected from slaughterhouse rather than those collected at the farm. Prednisone residues were found only in one urine sample that showed the highest levels of prednisolone, hydrocortisone, and cortisone. The occurrence of prednisolone residues in urine and even in tissue samples confirms the endogenous nature of this molecule.

Absorption and metabolism of the mycotoxin zearalenone and the growth promotor zeranol in Caco-2 cells in vitro.

SCOPE: Zearalenone (ZEN) and α-zearalanol (α-ZAL, zeranol) were studied in differentiated Caco-2 cells and in the Caco-2 Millicell® system in vitro to simulate their in vivo intestinal absorption and metabolism in humans. METHODS AND RESULTS: In addition to metabolic reduction/oxidation, extensive conjugation with glucuronic acid and sulfate of the parent compounds and their phase I metabolites was observed. The positional isomers of the glucuronides and sulfates were unambiguously identified: Sulfonation occurred specifically at the 14-hydroxyl group, whereas glucuronidation was less specific and, in addition to the preferred 14-hydroxyl group, involved the 16- and 7-hydroxyl groups. Using the Caco-2 Millicell® system, an efficient transfer of the glucuronides and sulfates of ZEN and α-ZAL and their phase I metabolites into both the basolateral and the apical compartment was observed after apical administration. The apparent permeability coefficients (P(app) values) of ZEN, α-ZAL and the ZEN metabolite α-zearalenol were determined, using an initial apical concentration of 20 µM and a permeation time of 1 h. CONCLUSION: According to the P(app) values, the three compounds are expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycones and as glucuronide and sulfate conjugates in humans.

The metabolism of olaquindox in rats, chickens and pigs.

Olaquindox is a growth-promoting feed additive for food-producing animals. Its toxicities were reported to be closely related to the metabolism. To provide the interpretation of toxicities in animals, this study explored the metabolism of olaquindox in rats, chickens and pigs of different genders by qualitative metabolite profiling. Animals were fed olaquindox in an oral dose, and then their urine, plasma, feces, liver, kidney and muscle were collected. Liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry was used for structural investigation and identification of metabolites. The structures of metabolites were elucidated based on the accurate MS² spectra and comparison of their changes in accurate molecular masses and fragment ions with those of parent drug or metabolite. A total of 18, 18 and 16 metabolites of rats, chickens and pigs were identified, respectively. Among the identified metabolites, 8 known metabolites were confirmed as an early study had stated, and 15 metabolites were found for the first time in vivo. The major metabolic pathways of olaquindox were proposed to be N-O reduction and oxidation of hydroxyl to carboxylic acid followed by N-O reduction. The qualitative species difference on the metabolite profiles of olaquindox among the three species was observed. However, metabolite profiles of olaquindox appeared to be qualitatively similar between female and male for the same species. The proposed metabolic pathways of olaquindox in animals will provide comprehensive data to clarify the metabolism of olaquindox among different species, and will give scientific explanation for toxicities and residues of olaquindox.

Residue depletion and tissue-plasma correlation of methyl-3-quinoxaline-2-carboxylic acid after dietary administration of olaquindox in pigs.

A residue depletion study was performed to investigate the tissue kinetics and tissue-plasma correlation of methyl-3-quinoxaline-2-carboxylic acid (MQCA), the marker residue of olaquindox (OLA), in pigs. Twenty-five pigs were randomly divided into a test and a control group. The former group was treated with 100 mg/kg OLA in its feed for 30 consecutive days, and the latter was given blank feed for the same period. One control and four treated animals were slaughtered at 0.5, 3, 10, 17, and 28 days post-medication. Muscle, liver, kidney, fat, and plasma samples were collected and analyzed using the validated high-performance liquid chromatography method (HPLC). Results showed that the tissue concentration of MQCA in the liver > kidney > fat > muscle, at almost all time points. The half-lives of MQCA in the muscle, liver, kidney, fat, and plasma were 12, 8, 15, 8, and 6 days, respectively. A withdrawal period of 38 days was calculated using the statistical method recommended by the European Medical Evaluation Agency (EMEA). Good correlations between tissue and plasma MQCA levels were found in the present study with correlation coefficients of more than 0.92. These correlations would be helpful in the routine monitoring of OLA in porcine tissues, without sacrificing the animals.

Insulin-like growth factor-I deficiency in children with growth hormone insensitivity: current and future treatment options.

Growth hormone (GH) acts directly at the growth plate and through the production of insulin-like growth factor (IGF)-I. At least 50% of the hormone circulates bound to GH binding protein, and its secretion is controlled by growth hormone-releasing hormone and somatostatin. Once GH binds to two GH receptors, the janus activated kinase/signal transducers and activators of transcription (JAK/STAT) protein pathway is activated, resulting in the production of IGF-I. Serum IGF-I is produced predominantly in the liver and circulates in a 140 kDa complex, along with its binding protein, IGF binding protein 3, and acid-labile protein. Recombinant human (rh) IGF-I (mecasermin) is approved by the US FDA and the European Medicines Agency for the treatment of patients with severe primary IGF deficiency or for patients with GH1 gene deletion who have developed neutralizing antibodies to GH. It has been shown to increase growth velocity in children with either condition. In the past, there have been adverse events, particularly hypoglycemia, reported with the administration of mecasermin. However, a recent report of long-term therapy with mescasermin in children with severe IGF-I deficiency has concluded that although adverse events are common, they are rarely severe enough to interrupt or modify treatment. The serum half-life of mecasermin is shorter in patients with GH insensitivity syndrome and low serum levels of its binding protein, the insulin-like growth factor binding protein (IGFBP)-3 and acid-labile subunit, compared with the serum half-life in normal volunteers or in patients with an IGF1 gene deletion who have normal levels of IGFBP-3. Mecasermin rinfabate, a complex of rhIGF-I and rhIGFBP-3, appears to prolong the serum half-life and might counteract acute adverse events, particularly hypoglycemia, associated with the administration of mescasermin. Mecasermin rinfabate, however, is no longer available in the USA or Europe for treating conditions involving short stature, because of a legal requirement. Mecasermin has been shown to be effective in increasing height velocity and adult height in patients with severe GH resistance and in IGF1 gene deletion. There has been some interest in using mecasermin to treat patients with partial GH resistance or idiopathic short stature. At the present time, the data are insufficient to make this recommendation.

Zonal release of proteins within tissue engineering scaffolds.

The manufacture of a scaffold for tissue engineering applications that can control the location and timing of growth factor release is described. The scaffold is formed by the sintering of poly(DL-lactic acid) (P(DL)LA) microparticles, plasticized with poly(ethylene glycol) (PEG), although the method can be used for many other polymer types. The microparticles were loaded with model proteins, trypsin and horseradish peroxidase (HRP), or recombinant human bone morphogenetic protein-2 (rhBMP-2). Entrapment efficiencies above 75% were achieved using a solid-in-oil-in-water system. Controlled release of active protein was achieved for at least 30 days. Microparticles were built into protein-loaded or protein-free layers and release of the protein was restricted to zones within the scaffold. Cell response to rhBMP-2 was tuneable by changing the dose of the rhBMP-2 released by varying the ratio of protein-loaded and protein-free microparticles within scaffolds. Zonal activity of rhBMP-2 on C2C12 cells was demonstrated. The scaffolds may find utility in applications where gradients of growth factors within 3D templates are required or where zonation of tissue growth is required.

[Research advances in the controlled release of growth factor related to blood vessel tissue engineering].

Growth factors play an important role in cell adhesion and proliferation as well as in tissue regeneration. By incorporating growth factors into polymer scaffolds, controlled release of them can be performed. The release mechanism is varied with the incorporation methods. In this paper, the latest advances in the controlled release of growth factors by blending, hydrogel, microsphere embedding and chemical bonding are reviewed. The potential application of ultrafine fibric embedding in growth factor delivery is described as well.

Manipulation of hydrogel assembly and growth factor delivery via the use of peptide-polysaccharide interactions.

The design of materials in which assembly, mechanical response, and biological properties are controlled by protein-polysaccharide interactions could provide materials that mimic the biological environment and find use in the delivery of growth factors. In the investigations reported here, a heparin-binding, coiled-coil peptide, PF4 ZIP, was employed to mediate the assembly of heparinized polymers. The heparin-binding affinity of this peptide was compared with that of other heparin-binding peptides (HBP) via heparin-sepharose chromatography and surface plasmon resonance (SPR) experiments. Results from these experiments indicate that PF4 ZIP demonstrates a higher heparin-binding affinity and heparin association rate when compared to the heparin-binding domains of antithrombin III (ATIII) and heparin-interacting protein (HIP). Viscoelastic hydrogels were formed upon the association of PF4 ZIP-functionalized star poly(ethylene glycol) (PEG-PF4 ZIP) with low-molecular-weight heparin-functionalized star PEG (PEG-LMWH). The viscoelastic properties of the hydrogels can be altered via variations in the ratio of LMWH to PF4 ZIP. bFGF release from these gels have also been investigated. Comparison of the bFGF release profiles with the hydrogel erosion profiles indicates that bFGF delivery from this class of hydrogels is mainly an erosion-controlled process and the rates of bFGF release can be modulated via choice of HBP or via variations in the mechanical properties of the hydrogels. Manipulation of hydrogel physical properties and erosion profiles will provide novel materials for controlled growth factor delivery and other biomedical applications.

Pharmacodynamic models for agents that alter production of natural cells with various distributions of lifespans.

Indirect pharmacodynamic response (IDR) models were developed for agents which alter the generation of cell populations with arbitrary lifespan distributions. These models extend lifespan based IDR models introduced previously [J. Pharmacokinet. Biopharm. 27: 467, 1999] for cell populations with the same lifespan ("delta" distribution). Considered are cell populations exhibiting time-invariant lifespan distributions described by the probability density function l(tau). It is assumed that cell response (R) is produced at a zero-order rate (kin(t)) and is eliminated from the population when the cell lifespan expires. The cell loss rate is calculated as kin*l(t), where '*' denotes the convolution operator. Therapeutic agents can stimulate or inhibit production rates according to the Hill function: 1 +/- H(C(t)) where H(C(t)) contains the capacity (Smax) and potency (SC50) parameters and C(t) is a pharmacokinetic function. The production rate is kin(t) = kin.[ 1+/-H(C(t))]. The operational model is dR/dt = kin(t)-kin*l(t) with the baseline condition R0 = kin.TR, where TR is the mean lifespan. Single populations as well as populations with precursors were examined by simulation to establish the role of lifespan distribution parameters (mean and standard deviation) in controlling the response vs. time profile. Estimability of parameters was assessed. Numerical techniques of solving differential equations with the convolution integral were proposed. In addition, the models were applied to literature data to describe the stimulatory effects of single doses of recombinant human erythropoietin on reticulocytes in blood. The estimates of Smax and SC50 for these agents were obtained along with means and standard deviations for reticulocyte lifespan distributions. The proposed models can be used to analyze the pharmacodynamics of agents which alter natural cell production yielding parameters describing their efficacy and potency as well as means and standard deviations for cell lifespan distributions.

Histopathological effects of boldenone in cattle.

Histopathology of male cattle previously found positive for béta-boldenone in urine in the Netherlands and in Italy was studied. The animals were derived from practice and several weeks had passed after the finding of béta-boldenone before the animals were examined. The animals consisted of 34 male veal calves and one finishing bull. In the prostate gland hypersecretion, cyst formation (45%) and hyperplasia of the urethral epithelium was observed, in the bulbo-urethral gland similar alterations were present. The testis showed reduced development and degeneration of the germinal epithelium (70%), leading to debris and syncytial cell formation in the lumina. Stromal proliferation was evident. In some animals the liver was sampled and showed periportal fibrosis, bile duct proliferation and sometimes necrosis. The bull also showed degeneration of the germinal epithelium of the testis and absence of sperm production, the prostate gland showed some secretion and had an atrophic appearance. It is concluded that béta-boldenone may lead to degeneration of the germinal epithelium of the testis and hypersecretion and cyst formation in the prostate and bulbo-urethral gland, which alterations may heal in time.

Effect of acute ethanol exposure on hepatic stimulator substance (HSS) levels during liver regeneration: protective function of HSS.

Ethanol administration in rats induces liver damage and suppression of liver regeneration. To further understand the underlying mechanism, we investigated the effects of ethanol on hepatic stimulator substance (HSS) levels during liver regeneration caused by partial hepatectomy. The hepatotrophic action of HSS to ethanol-treated partially hepatectomized rats was also examined. Rats received repetitive ethanol or saline doses beginning 1 hr prior to 70% partial hepatectomy (PH), and the animals were killed at 16, 24, 32, 40, 48, and 60 hr after PH. Our results showed that ethanol inhibited hepatic regenerative capacity and prolonged liver regenerative process. HSS biological activity in ethanol-administered rats peaked at 48 hr after PH, in contrast to saline-treated ones where activity peaked at 24 hr. Additionally, exogenous HSS administration to ethanol-treated partially hepatectomized rats increased liver proliferating capacity and suppressed the elevation of serum ALT activity. These results showed that ethanol modifies the time course of HSS biological activity during the regenerating process. The observed suppression of HSS activity at 24 hr after PH was in relation with a reduction of DNA synthesis. Exogenous administration of HSS to ethanol-treated partially hepatectomized rats restored DNA synthesis and ameliorated serum AST levels, indicating that HSS could be used in the treatment of ethanol-induced hepatic failures.

Growth factor releasing porous poly (epsilon-caprolactone)-chitosan matrices for enhanced bone regenerative therapy.

Drug releasing porous poly(epsilon-caprolactone) (PCL)-chitosan matrices were fabricated for bone regenerative therapy. Porous matrices made of biodegradable polymers have been playing a crucial role as bone substitutes and as tissue-engineered scaffolds in bone regenerative therapy. The matrices provided mechanical support for the developing tissue and enhanced tissue formation by releasing active agent in controlled manner. Chitosan was employed to enhance hydrophilicity and biocompatibility of the PCL matrices. PDGF-BB was incorporated into PCL-chitosan matrices to induce enhanced bone regeneration efficacy. PCL-chitosan matrices retained a porous structure with a 100-200 microm pore diameter that was suitable for cellular migration and osteoid ingrowth. NaHCO3 as a porogen was incorporated 5% ratio to polymer weight to form highly porous scaffolds. PDGF-BB was released from PCL-chitosan matrices maintaining therapeutic concentration for 4 week. High osteoblasts attachment level and proliferation was observed from PCL-chitosan matrices. Scanning electron microscopic examination indicated that cultured osteoblasts showed round form and spread pseudopods after 1 day and showed broad cytoplasmic extension after 14 days. PCL-chitosan matrices promoted bone regeneration and PDGF-BB loaded matrices obtained enhanced bone formation in rat calvarial defect. These results suggested that the PDGF-BB releasing PCL-chitosan porous matrices may be potentially used as tissue engineering scaffolds or bone substitutes with high bone regenerative efficacy.

Transport of exogenous growth factors and cytokines to the cytosol and to the nucleus.

In recent years a number of growth factors, cytokines, protein hormones, and other proteins have been found in the nucleus after having been added externally to cells. This review evaluates the evidence that translocation takes place and discusses possible mechanisms. As a demonstration of the principle that extracellular proteins can penetrate cellular membranes and reach the cytosol, a brief overview of the penetration mechanism of protein toxins with intracellular sites of action is given. Then problems and pitfalls in attempts to demonstrate the presence of proteins in the cytosol and in the nucleus as opposed to intracellular vesicular compartments are discussed, and some new approaches to study this are described. A detailed overview of the evidence for translocation of fibroblast growth factor, HIV-Tat, interferon-gamma, and other proteins where there is evidence for intracellular action is given, and translocation mechanisms are discussed. It is concluded that although there are many pitfalls, the bulk of the experiments indicate that certain proteins are indeed able to enter the cytosol and nucleus. Possible roles of the internalized proteins are discussed.

Ectopic expression of Delta4 impairs hematopoietic development and leads to lymphoproliferative disease.

Notch signaling plays a critical role in cell fate determination in many developmental systems, including the hematopoietic system. We and others have recently cloned a novel Notch ligand called Delta4. In this study, we show the effect of retrovirus-mediated ectopic expression of Delta4 in hematopoietic cells. Lethally irradiated mice transplanted with bone marrow cells expressing Delta4 initially suffered from leukopenia and thrombocytopenia. Although all lineages were affected, the deficit in B cells and platelets was the most durable and profound. A rapid expansion of CD4(+)CD8(+) cells occurred shortly after transplantation. CD4(+)CD8(+) cells progressively invaded all tissues analyzed except the thymus, which surprisingly was atrophic. CD4(+)CD8(+) cells were mainly non-Delta4-transduced cells, strongly suggesting that the disease was not cell autonomous. Around 15 weeks after transplantation, mice died from this severe lymphoproliferative disorder, which was not transplantable in late-stage disease into secondary recipients. Mice transduced with a soluble form of Delta4 behaved like control mice. Characterization of early hematopoietic development revealed that Delta4 expression impaired formation of day-12 spleen colony-forming units (CFU-Ss) and, to a greater extent, pre-CFU-Ss. No effect was observed on myeloid colony-forming cells (CFU-Cs), indicating that Delta4 specifically acted on the earliest hematopoietic stem cell compartment. These results show that constitutive expression of Delta4 in hematopoietic cells impairs the development of B cells, platelets, and early stem cells and induces a lethal lymphoproliferative disease.