Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 31
Filter
1.
Cell Death Dis ; 6: e2019, 2015 Dec 17.
Article in English | MEDLINE | ID: mdl-26673664

ABSTRACT

Hair cell damage is a side effect of cisplatin and aminoglycoside use. The inhibition or attenuation of this process is a target of many investigations. There is growing evidence that STAT1 deficiency decreases cisplatin-mediated ototoxicity; however, the role of STAT function and the molecules that act in gentamicin-mediated toxicity have not been fully elucidated. We used mice lacking STAT1 to investigate the effect of STAT1 ablation in cultured organs treated with cisplatin and gentamicin. Here we show that ablation of STAT1 decreased cisplatin toxicity and attenuated gentamicin-mediated hair cell damage. More TUNEL-positive hair cells were observed in explants of wild-type mice than that of STAT1(-/-) mice. Although cisplatin increased serine phosphorylation of STAT1 in wild-type mice and diminished STAT3 expression in wild-type and STAT1(-/-) mice, gentamicin increased tyrosine phosphorylation of STAT3 in STAT1(-/-) mice. The early inflammatory response was manifested in the upregulation of TNF-α and IL-6 in cisplatin-treated explants of wild-type and STAT1(-/-) mice. Expression of the anti-inflammatory cytokine IL-10 was altered in cisplatin-treated explants, upregulated in wild-type explants, and downregulated in STAT1(-/-) explants. Cisplatin and gentamicin triggered the activation of c-Jun. Activation of Akt was observed in gentamicin-treated explants from STAT1(-/-) mice. Increased levels of the autophagy proteins Beclin-1 and LC3-II were observed in STAT1(-/-) explants. These data suggest that STAT1 is a central player in mediating ototoxicity. Gentamicin and cisplatin activate different downstream factors to trigger ototoxicity. Although cisplatin and gentamicin triggered inflammation and activated apoptotic factors, the absence of STAT1 allowed the cells to overcome the effects of these drugs.


Subject(s)
Hair Cells, Auditory/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-jun/metabolism , STAT1 Transcription Factor/deficiency , STAT3 Transcription Factor/metabolism , Animals , Autophagy , Genes, jun , Hair Cells, Auditory/cytology , Mice , Mice, Inbred C57BL , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism
2.
Neuroscience ; 181: 28-39, 2011 May 05.
Article in English | MEDLINE | ID: mdl-21354273

ABSTRACT

The matrix metalloproteinases (MMPs) are a family of proteins involved in the remodelling and homeostasis of the extracellular matrix. These proteases have been well studied in the retina and the brain, marking their importance in neuronal cell survival and death [Chintala (2006) Exp Eye Res 82:5-12; Candelario-Jalil et al. (2009) Neuroscience 158:983-994]. The neuroepithelia of the eye and the inner ear share common characteristics. Therefore, we hypothesized that MMPs could play a similar role in the cochlea as described in the retina. We focused on the localization and function of MMP-2 and MMP-9 in the cochlea, by determining their expression and activity under normal conditions and after cochlear damage via aminoglycoside exposition. We examined their expression in 5-day-old Wistar rat cochleas by RT-PCR, real-time PCR, and Western blot. We used immunohistochemistry to investigate their location in the cochleas of adult C57BL/6 mice. We also determined whether or not the exposure of the organs of Corti to aminoglycosides would change MMP-2 and MMP-9 expression patterns. Western blotting identified MMP-2 and MMP-9 in neonatal spiral ganglion, stria vascularis, and to a lesser extent the organ of Corti. Neonatal mRNA expression of MMP-2 was approximately equivalent in all three tissues, while MMP-9 mRNA was highest in spiral ganglion. Immunohistochemistry showed MMP-2 primarily in adult spiral ganglion neurons and inner hair cells, while MMP-9 was found mainly in spiral ganglion neurons, inner hair cells and supporting cells. Organs of Corti treated with gentamicin for 24 h showed an upregulation of MMP-2 and MMP-9 proteins, but did not show a significant upregulation of mRNA expression 3, 6, 12, 24, and 36 h after gentamicin exposure. Inhibition of MMP activity in organs of Corti incubated with an MMP inhibitor in organotypic cultures resulted in hair cell death-suggesting that a basal level of MMP activity is required for hair cell survival.


Subject(s)
Aminoglycosides/toxicity , Cochlea/enzymology , Cochlea/growth & development , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Neurotoxins/toxicity , Animals , Animals, Newborn , Cochlea/drug effects , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase Inhibitors , Mice , Mice, Inbred C57BL , Organ Culture Techniques , Rats , Rats, Wistar
3.
Eur J Cancer ; 47(9): 1407-13, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21273057

ABSTRACT

Families at high risk for Lynch syndrome can effectively be recognised by microsatellite instability (MSI) testing. The aim of the present study is to compare the effectiveness of a MSI test for the identification of Lynch syndrome in patients selected by a pathologist mainly based on young age at diagnosis (MSI-testing-indicated-by-a-Pathologist; MIPA), with that of patients selected by a clinical geneticist mainly based on family history (MSI-testing-indicated-by-Family-History; MIFH). Patients with a Lynch syndrome associated tumour were selected using MIPA (n=362) or MIFH (n=887). Germline DNA mutation testing was performed in 171 out of 215 patients (80%) with a MSI positive tumour. MSI was tested positive in 20% of the MIPA-group group compared to 16% in the MIFH-group (P=0.291). In 91 of 171 patients with MSI positive tumours tested for germline mutations were identified as Lynch syndrome patients: 42% in the MIPA-group and 56% in the MIFH-group (P=0.066). Colorectal cancer (CRC) or endometrial cancer (EC) presenting at an age below 50 years would have led to the diagnosis of Lynch syndrome in 89% of these families (CRC below 50 years: 88% and EC below 50 years: 12%). Families detected by MIPA were characterised more often by extracolonic Lynch syndrome associated malignancies, especially EC (P<0.001). Our results indicate that recognition of Lynch syndrome by CRC or EC below 50 years is as effective as a positive family history. Families from patients selected by individual criteria more often harbour extracolonic Lynch syndrome associated malignancies.


Subject(s)
Colorectal Neoplasms, Hereditary Nonpolyposis/diagnosis , Colorectal Neoplasms/diagnosis , Adaptor Proteins, Signal Transducing/genetics , Adult , Age Factors , Aged , Cohort Studies , Colorectal Neoplasms/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , DNA Methylation , Family Health , Female , Germ-Line Mutation , Humans , Male , Microsatellite Instability , Middle Aged , Models, Genetic , MutL Protein Homolog 1 , Nuclear Proteins/genetics , Risk
4.
J Med Genet ; 47(7): 464-70, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20591884

ABSTRACT

BACKGROUND: Colorectal, endometrial and upper urinary tract tumours are characteristic for Lynch syndrome (hereditary non-polyposis colon carcinoma, HNPCC). The aim of the present study was to establish whether carriers of mutations in mismatch repair genes MLH1, MSH2 or MSH6 are at increased risk of urinary bladder cancer. METHODS: Carriers and first degree relatives of 95 families with a germline mutation in the MLH1 (n=26), MSH2 (n=43), or MSH6 (n=26) gene were systematically questioned about the occurrence of carcinoma. The cumulative risk of cancer occurring before the age of 70 years (CR70) was compared to the CR70 of the general Dutch population. Microsatellite instability (MSI) testing and/or immunohistochemistry (IHC) for mismatch repair proteins was performed on bladder tumour tissue. RESULTS: Bladder cancer was diagnosed in 21 patients (90% men) from 19 Lynch syndrome families (2 MLH1, 15 MSH2, and 4 MSH6). CR70 for bladder cancer was 7.5% (95% CI 3.1% to 11.9%) for men and 1.0% (95% CI 0% to 2.4%) for women, resulting in relative risks for mutation carriers and first degree relatives of 4.2 (95% CI 2.2 to 7.2) for men and 2.2 (95% CI 0.3 to 8.0) for women. Men carrying an MSH2 mutation and their first degree relatives were at highest risks: CR70 for bladder and upper urinary tract cancer being 12.3% (95% CI 4.3% to 20.3%) and 5.9% (95% CI 0.7% to 11.1%). Bladder cancer tissue was MSI positive in 6/7 tumours and loss of IHC staining was found in 14/17 tumours, indicating Lynch syndrome aetiology. CONCLUSION: Patients with Lynch syndrome carrying an MSH2 mutation are at increased risk of urinary tract cancer including bladder cancer. In these cases surveillance should be considered.


Subject(s)
Colorectal Neoplasms, Hereditary Nonpolyposis/complications , Genetic Predisposition to Disease , MutS Homolog 2 Protein/genetics , Urinary Bladder Neoplasms/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adult , Aged , Carcinoma/complications , Carcinoma/genetics , Female , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Male , Middle Aged , MutL Protein Homolog 1 , MutS Homolog 2 Protein/metabolism , Mutation , Nuclear Proteins/metabolism , Pedigree , Risk Factors , Urinary Bladder Neoplasms/complications , Urothelium
5.
HNO ; 55(11): 862-70, 2007 Nov.
Article in German | MEDLINE | ID: mdl-17487462

ABSTRACT

BACKGROUND: Stem cell therapy is especially interesting for inner ear related diseases, since the hair cells are very sensitive and do not regenerate. Hair cell loss is therefore irreversible and is accompanied by hearing loss. In the last few years, different research groups have transplanted stem cells into the inner ear with promising results. In the presented study, our aim was to gain insight into how neuronal stem cells behave when they are transplanted, both in vitro and in vivo, into a damaged inner ear. METHODS: Neuronal stem cells from E9.5 day old mouse embryos were collected and infected with an adenoviral vector encoding green fluorescent protein (GFP). GFP+ cells were then transplanted into a damaged organ of Corti in vitro or into a damaged mouse inner ear in vivo. RESULTS: We were able to detect GFP+ cells close to the organ of Corti in vitro and in the organ of Corti in vivo. The GFP+ cells do not seem to be randomly distributed in either the in vitro or in vivo situation. Most interestingly, GFP+ cells could be detected close to places where hair cells had been lost in vivo. CONCLUSION: Neuronal stem cells are interesting candidates to replace lost hair cells. However, a great deal of research is still needed before they can enter clinical trials.


Subject(s)
Cochlea/pathology , Cochlear Diseases/pathology , Cochlear Diseases/surgery , Nerve Regeneration , Neurons/pathology , Neurons/transplantation , Stem Cell Transplantation/methods , Animals , Cells, Cultured , Mice , Mice, Inbred C57BL , Treatment Outcome
6.
Br J Cancer ; 96(10): 1605-12, 2007 May 21.
Article in English | MEDLINE | ID: mdl-17453009

ABSTRACT

The cancer risk is unknown for those families in which a microsatellite instable tumour is neither explained by MLH1 promoter methylation nor by a germline mutation in a mismatch repair (MMR) gene. Such information is essential for genetic counselling. Families suspected of Lynch syndrome (n = 614) were analysed for microsatellite instability, MLH1 promoter methylation and/or germline mutations in MLH1, MSH2, MSH6, and PMS2. Characteristics of the 76 families with a germline mutation (24 MLH1, 2 PMS2, 32 MSH2, and 18 MSH6) were compared with those of 18 families with an unexplained microsatellite instable tumour. The mean age at diagnosis of the index patients in both groups was comparable at 44 years. Immunohistochemistry confirmed the loss of an MMR protein. Together this suggests germline inactivation of a known gene. The Amsterdam II criteria were fulfilled in 50/75 families (66%) that carried a germline mutation in an MMR gene and in only 2/18 families (11%) with an unexplained microsatellite instable tumour (P<0.0001). Current diagnostic strategies can detect almost all highly penetrant MMR gene mutations. Patients with an as yet unexplained microsatellite instable tumour likely carry a different type of mutation that confers a lower risk of cancer for relatives.


Subject(s)
Carcinoma/genetics , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , Colorectal Neoplasms/genetics , Genetic Predisposition to Disease , Microsatellite Instability , Adaptor Proteins, Signal Transducing/genetics , Adult , Aged , Algorithms , DNA Methylation , DNA Mismatch Repair , DNA-Binding Proteins/genetics , Family , Germ-Line Mutation , Humans , Middle Aged , MutL Protein Homolog 1 , MutS Homolog 2 Protein/genetics , Nuclear Proteins/genetics , Promoter Regions, Genetic , Risk Factors
7.
Br J Cancer ; 95(6): 757-62, 2006 Sep 18.
Article in English | MEDLINE | ID: mdl-16909138

ABSTRACT

To establish an efficient, reliable and easy to apply risk assessment tool to select families with breast and/or ovarian cancer patients for BRCA mutation testing, using available probability models. In a retrospective study of 263 families with breast and/or ovarian cancer patients, the utility of the Frank (Myriad), Gilpin (family history assessment tool) and Evans (Manchester) model was analysed, to select 49 BRCA mutation-positive families. For various cutoff levels and combinations, the sensitivity and specificity were calculated and compared. The best combinations were subsequently validated in additional sets of families. Comparable sensitivity and specificity were obtained with the Gilpin and Evans models. They appeared to be complementary to the Frank model. To obtain an optimal sensitivity, five 'additional criteria' were introduced that are specific for the selection of small or uninformative families. The optimal selection is made by the combination 'Frank >or=16% or Evans2 >or=12 or one of five additional criteria'. The efficiency of the selection of families for mutation testing of BRCA1 and BRCA2 can be optimised by using a combination of available easy to apply risk assessment models.


Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/diagnosis , Models, Statistical , Ovarian Neoplasms/diagnosis , Patient Selection , Adult , Breast Neoplasms/genetics , Cohort Studies , Female , Genetic Predisposition to Disease , Humans , Middle Aged , Mutation , Ovarian Neoplasms/genetics , Pedigree , Predictive Value of Tests , Probability , Retrospective Studies , Risk Assessment , Sensitivity and Specificity
8.
Neuroscience ; 137(3): 971-80, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16338090

ABSTRACT

Sensorineural hearing loss is often associated with damage of cochlear hair cells and/or of the neurons of the auditory pathway. This damage can result from a variety of causes, e.g. genetic disorders, aging, exposure to certain drugs such as aminoglycosides, infectious disease and intense sound overexposure. Intracellular events that mediate aspects of aminoglycoside-mediated damage to hair cells have been partially unraveled. Several independent research groups have demonstrated a crucial role of mitogen-activated protein kinase signaling in aminoglycoside-induced ototoxicity. Mitogen-activated protein kinases are important mediators of signal transduction from the cell surface to the nucleus. Jun N-terminal kinases, members of the mitogen-activated protein kinase family, are strongly activated in cell culture conditions by stress inducing stimuli, including ultraviolet light, heat shock and tumor necrosis factor; therefore they are also referred to as stress-activated protein kinases. In hair cells aminoglycoside treatment was shown to activate the Jun N-terminal kinase signaling pathway. Activation of Jun N-terminal kinase leads to phosphorylation and thereby activation of transcription factors and consequently to altered gene expression. There are many nuclear Jun N-terminal kinase substrates including c-Jun, ATF-2, and Elk-1 proteins. One of the downstream targets of Jun N-terminal kinase is the transcription factor activating protein-1. Activating protein-1 is a dimeric complex composed of members of the Fos and Jun proteins. A variety of different stimuli is known to induce activating protein-1 activity. Induction of activating protein-1 is thought to play a central role in reprogramming gene expression in response to external stimuli. In this study we have analyzed the effect of gentamicin treatment on the downstream targets of Jun N-terminal kinase. Our results demonstrate that gentamicin treatment of explants of organ of Corti results in increased activating protein-1 binding activity. The main component of these activating protein-1 complexes is the c-Fos protein. Moreover, we show that the activating protein-1 induction is transient and occurs exclusively in hair cells of rat organ of Corti explants.


Subject(s)
DNA/metabolism , Gentamicins/toxicity , Hair Cells, Auditory/pathology , Nerve Degeneration/pathology , Protein Synthesis Inhibitors/toxicity , Transcription Factor AP-1/metabolism , Actins/biosynthesis , Actins/genetics , Animals , Binding, Competitive/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cells, Cultured , Electrophoretic Mobility Shift Assay , Genes, fos/genetics , Hair Cells, Auditory/drug effects , Hair Cells, Auditory/metabolism , Immunohistochemistry , MAP Kinase Kinase 4/physiology , Nerve Degeneration/metabolism , Organ Culture Techniques , Protein Binding , Rats , Rats, Sprague-Dawley , Signal Transduction/physiology , Transcription Factor AP-1/genetics
9.
J Microencapsul ; 21(2): 137-49, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15198426

ABSTRACT

The prolonged delivery of hydrophilic drug salts from hydrophobic polymer carriers at high drug loading is an ambitious goal. Pamidronate disodium salt (APD) containing implants prepared from spray-dried microparticles were investigated using a laboratory ram extruder. An APD-containing polymer matrix consisting of an APD-chitosan implant embedded in the biodegradable polymer D,L-poly(lactide-co-glycolide acid-glucose) (PLG-GLU) was compared with a matrix system with the micronized drug distributed in the PLG-GLU. The APD-chitosan matrix system showed a triphasic release behaviour at loading levels of 6.86 and 15.54% (w/w) over 36 days under in-vitro conditions. At higher loading (31.92%), a drug burst was observed within 6 days due to the formation of pores and channels in the polymeric matrix. In contrast, implants containing the micronized drug showed a more continuous release profile over 48 days up to a loading of 31.78% (w/w). At a drug loading of 46.17% (w/w), a drug burst was observed. Using micronized drug salts and reducing the surface area available for diffusion, parenteral delivery systems for highly water-soluble drug candidates were shown to be technically feasible at high drug loadings.


Subject(s)
Chitin/analogs & derivatives , Diphosphonates , Drug Compounding/methods , Drug Implants , Adjuvants, Pharmaceutic , Anti-Inflammatory Agents , Biocompatible Materials , Biodegradation, Environmental , Chitosan , Feasibility Studies , Lactic Acid , Microscopy, Electron, Scanning , Microspheres , Pamidronate , Polyesters , Polyglycolic Acid , Sucrose , Surface Properties
10.
Neuroscience ; 122(4): 1025-35, 2003.
Article in English | MEDLINE | ID: mdl-14643769

ABSTRACT

To identify possible intracellular mediators of hair cell (HC) death due to ototoxins, we treated basal-turn, neonatal, rat HCs in vitro with several intracellular signaling inhibitors, prior to and during gentamicin exposure. The general guanine nucleotide-binding protein (G-protein) inhibitor, GDP-betaS (1 mM), provided potent HC protection, suggesting involvement of G-proteins in the intracellular pathway linking gentamicin exposure to HC death. ADP-betaS had minimal effect, indicating that the protection is specific to guanosine diphosphate (GDP)-binding, rather than a general reaction to nucleotides. Azido-GTP(32) photolabeling and gel electrophoresis indicated activation of an approximately 21 kDa G-protein in HCs after exposure to gentamicin. Spectroscopic analysis of peptide fragments from this band matched its sequence with H-Ras. The Ras inhibitors B581 (50 microM) and FTI-277 (10 microM) provided potent protection against damage and reduced c-Jun activation in HC nuclei, suggesting that activation of Ras is functionally involved in damage to these cells due to gentamicin.


Subject(s)
Gentamicins/toxicity , Hair Cells, Auditory/drug effects , ras Proteins/metabolism , Animals , Cochlea/drug effects , Cochlea/metabolism , Cochlea/pathology , Enzyme Inhibitors/pharmacology , Growth Inhibitors/pharmacology , Hair Cells, Auditory/metabolism , Hair Cells, Auditory/pathology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology , ras Proteins/antagonists & inhibitors
11.
J Microencapsul ; 20(4): 509-24, 2003.
Article in English | MEDLINE | ID: mdl-12851051

ABSTRACT

The preparation of microparticles (MP) with a high loading of hydrophilic, low molecular weight drugs is an ambitious goal. This study investigated the microencapsulation of a bisphosphonate salt (BP) into a biodegradable star branched terpolymer Poly(D,L-lactide-co-glycolide-D-glucose) (PLG-GLU). Two aqueous solvent evaporation microencapsulation-techniques were studied, namely the water-in-oil-in-water-technique (WOW) and the solid-in-oil-in-water-technique (SOW) as well as a non-aqueous microencapsulation method based on suspension of the drug in organic solvents (SOO). The aqueous microencapsulation techniques showed several disadvantages, which rendered it difficult to prepare MP with high drug loading (approximately 30% w/w). A modified SOO-technique allowed the preparation of highly loaded MP up to 28% (w/w). A micronized drug substance and a polymer solvent system consisting of equal volumes of acetonitrile (ACN) and dichloromethane (DCM) were essential features of the SOO-process. A morphologic examination of the internal structure by confocal laser scanning microscopy demonstrated that these MP contain many vacuoles and pores, leading to an unfavourable initial burst release of APD. This process needs further optimization with respect to drug release and may then be of general interest for the preparation of highly-loaded MP with other drug salts and hydrophilic macromolecules.


Subject(s)
Anti-Inflammatory Agents/administration & dosage , Diphosphonates/administration & dosage , Drug Compounding/methods , Polyesters/chemistry , Drug Carriers/chemistry , Drug Delivery Systems , Feasibility Studies , Microscopy, Electron, Scanning , Microspheres , Molecular Weight , Pamidronate , Solvents
12.
Genet Couns ; 14(2): 149-54, 2003.
Article in English | MEDLINE | ID: mdl-12872808

ABSTRACT

Renal cell carcinomas (RCC) occur in both sporadic and familial forms. The best known example of a familial RCC syndrome is the Von Hippel Lindau cancer syndrome. In addition, RCC families segregating constitutional chromosome 3 translocations have been reported. The list of these latter families is rapidly expanding. We have initiated a survey of all Dutch families known to segregate chromosome 3 translocations for (i) the ocurrence of RCCs and (ii) the establishment of refined risk estimates. This information will be critical for genetic counseling and clinical patient management. Within the families 'at risk' that we have identified so far, this approach has already led to early RCC detection and surgical intervention.


Subject(s)
Carcinoma, Renal Cell/genetics , Chromosomes, Human, Pair 3/genetics , Kidney Neoplasms/genetics , Translocation, Genetic/genetics , Tumor Suppressor Proteins , Ubiquitin-Protein Ligases , Female , Humans , Ligases/genetics , Male , Middle Aged , Point Mutation/genetics , Risk Factors , Von Hippel-Lindau Tumor Suppressor Protein
13.
Laryngorhinootologie ; 81(12): 853-6, 2002 Dec.
Article in German | MEDLINE | ID: mdl-12486621

ABSTRACT

The hair cells (HCs) are the most vulnerable elements in the cochlea and damage to them is the most common cause of sensorineural hearing loss (SNHL). Understanding the intracellular events that lead to the death of HCs is a key to developing protective strategies. Recently, it has been shown that the c-Jun-N-terminal kinase (JNK) pathway is activated in HCs in response to aminoglycosides and CEP-1347, an inhibitor of the JNK signaling pathway protected HCs from ototoxicity. We have studied another inhibitor (CEP-11 004) of this signaling pathway in its ability to protect HCs from aminoglycoside ototoxicity in vitro. Organ of Corti explants from p5 rat basal turns were maintained in tissue culture and treated with CEP-11 004 for 12 hours. They were then treated with CEP-11 004 plus gentamicin for 72 hours. Significantly less HC death was observed compared to gentamicin alone. CEP-11 004 alone had no effect on HCs. We conclude that the JNK signaling pathway plays a role in aminoglycoside ototoxicity signaling.


Subject(s)
Carbazoles/pharmacology , Gentamicins/toxicity , Hair Cells, Auditory/drug effects , Indoles/pharmacology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Animals , Cell Count , Culture Techniques , JNK Mitogen-Activated Protein Kinases , Microscopy, Fluorescence , Rats , Rats, Sprague-Dawley
14.
J Control Release ; 76(3): 209-25, 2001 Oct 19.
Article in English | MEDLINE | ID: mdl-11578737

ABSTRACT

The degradation and drug carrier properties of poly(ethylene carbonate) (PEC) were investigated in vitro and in rats and rabbits. PEC was found to be specifically degraded in vivo and in vitro by superoxide radical anions O2-*, which are, in vivo, mostly produced by inflammatory cells. No degradation of PEC was observed in the presence of hydrolases, serum or blood. PEC is biodegraded by surface erosion without significant change in the molecular weight of the residual polymer mass. The non-hydrolytic biodegradation by cells producing O2-* is unique among the polymers used as biodegradable drug carriers. The main degradation product of PEC in aqueous systems is ethylene glycol, formed presumably by hydrolysis of ethylene carbonate. The splitting off of a five-membered ring structure from the polymer chain indicates a chain reaction mechanism for the biodegradation. PEC is a suitable drug carrier, particularly for labile drugs. Using human interleukin-3 and octreotide as model drugs, surface erosion of the PEC formulations was indicated by a 1:1 correlation between drug release and polymer mass loss.


Subject(s)
Polyethylenes/chemistry , Animals , Chemistry, Pharmaceutical , Drug Carriers , Drug Implants , Fluorescent Antibody Technique , Humans , Interleukin-3/administration & dosage , Interleukin-3/pharmacokinetics , Male , Materials Testing , Microspheres , Molecular Weight , Pharmaceutical Solutions , Powders , Rabbits , Rats , Rats, Wistar , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacokinetics , Tablets
15.
EMBO Rep ; 2(5): 423-30, 2001 May.
Article in English | MEDLINE | ID: mdl-11375935

ABSTRACT

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytoplasm and degraded by the proteasome. Processing intermediates of N-linked oligosaccharides on incompletely folded glycoproteins have an important role in their folding/refolding, and also in their targeting to proteolytic degradation. In Saccharomyces cerevisiae, we have identified a gene coding for a non-essential protein that is homologous to mannosidase I (HTM1) and that is required for degradation of glycoproteins. Deletion of the HTM1 gene does not affect oligosaccharide trimming. However, deletion of HTM1 does reduce the rate of degradation of the mutant glycoproteins such as carboxypeptidase Y, ABC-transporter Pdr5-26p and oligosaccharyltransferase subunit Stt3-7p, but not of mutant Sec61-2p, a non-glycoprotein. Our results indicate that although Htm1p is not involved in processing of N-linked oligosaccharides, it is required for their proteolytic degradation. We propose that this mannosidase homolog is a lectin that recognizes Man8GlcNAc2 oligosaccharides that serve as signals in the degradation pathway.


Subject(s)
Fungal Proteins/metabolism , Glycoproteins/metabolism , Protein Folding , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Animals , Carbohydrate Conformation , Carboxypeptidases/genetics , Carboxypeptidases/metabolism , Cathepsin A , Cysteine Endopeptidases/metabolism , Fungal Proteins/genetics , Gene Deletion , Genes, Fungal , Glycoproteins/chemistry , Glycoproteins/genetics , Hexosyltransferases , Humans , Immunoblotting , Mannosidases/chemistry , Mannosidases/genetics , Mannosidases/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins , Molecular Sequence Data , Multienzyme Complexes/metabolism , Oligosaccharides/metabolism , Proteasome Endopeptidase Complex , SEC Translocation Channels , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Temperature , Ubiquitins/metabolism
16.
Genes Chromosomes Cancer ; 31(1): 23-32, 2001 May.
Article in English | MEDLINE | ID: mdl-11284032

ABSTRACT

We identified a novel familial case of clear-cell renal cancer and a t(3;6)(q12;q15). Subsequent cytogenetic and molecular analyses showed the presence of several abnormalities within tumour samples obtained from different patients. Loss of the der(3) chromosome was noted in some, but not all, of the samples. A concomitant VHL gene mutation was found in one of the samples. In addition, cytogenetic and molecular evidence for heterogeneity was obtained through analysis of several biopsy samples from one of the tumours. Based on these results and those reported in the literature, we conclude that loss of der(3) and subsequent VHL gene mutation may represent critical steps in the development of renal cell cancers in persons carrying the chromosome 3 translocation. Moreover, preliminary data suggest that other (epi)genetic changes may be related to tumour initiation.


Subject(s)
Carcinoma, Renal Cell/genetics , Chromosomes, Human, Pair 3/genetics , Chromosomes, Human, Pair 6/genetics , Kidney Neoplasms/genetics , Translocation, Genetic/genetics , Adult , Female , Humans , In Situ Hybridization, Fluorescence , Karyotyping/methods , Loss of Heterozygosity/genetics , Male , Middle Aged , Nucleic Acid Hybridization/methods , Pedigree , Tumor Cells, Cultured
19.
Pharm Dev Technol ; 3(3): 325-36, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9742553

ABSTRACT

Formulations consisting of either sucrose or trehalose with glycine, lysine-HCl, or mannitol were studied to determine how the ratio of the excipients affects the design of the lyophilization program and the properties of the final cake. Glass transitions (Tg', Tg), crystallization temperatures, and eutectic melting temperatures were measured by differential scanning calorimetry, the physical state of the excipients was determined by x-ray powder diffraction, and residual moisture was measured by Karl Fischer titration. The addition of increasing amounts of glycine, lysine-HCl, or mannitol to a sucrose solution caused a progressive depression of the Tg', an effect that was more pronounced with the amino acids. In equivalent ratios with sucrose, the two amino acids induced a comparable Tg' shift due to their low Tg' values. For lysine-HCl, two apparent Tg' with midpoint temperatures of -69 and -56 degrees C were measured. For mannitol, the Tg' depression was unexpected because mannitol exhibits a higher Tg' than that of sucrose. During lyophilization, the ratio of the amorphous amino acids or mannitol to the sugar determined whether crystallization could be induced by an annealing step performed after freezing. Crystallization could be verified by a shift of the formerly depressed Tg' back to the value of the sugar and by the detection of the eutectic melting peak of the crystallized compound. The crystallized excipients served as excellent bulking agents. In the freeze-dried cake, amorphous glycine and even more amorphous mannitol lowered the Tg value. If the cake was stored above Tg, subsequent crystallization of mannitol occurred. The results emphasize that the qualitative and quantitative composition of a formulation has profound implications on the design of a lyophilization program and on the characteristics of the freeze-dried cake.


Subject(s)
Amino Acids/administration & dosage , Carbohydrates/administration & dosage , Freeze Drying , Mannitol/administration & dosage , Calorimetry, Differential Scanning , Freezing
20.
Pharm Dev Technol ; 3(3): 337-46, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9742554

ABSTRACT

This study assessed the impact of residual moisture, Tg, and excipient physical state of different formulations on the "in-process" and shelf-life stability of freeze-dried interleukin-6 (IL-6). The effect of an annealing procedure was also evaluated. Characterization of the lyophilizates was done by Karl Fischer titration, differential scanning calorimetry (DSC), and x-ray measurements. Analysis of protein stability was carried out by size exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and turbidity measurements. During freeze-drying, the most effective protection against aggregation was provided by completely amorphous formulations consisting of trehalose or sucrose either alone or in combination with glycine or mannitol. Other amorphous formulations like those of sucrose with lysine-HCl or dextran could not provide comparable stabilization. In lyophilizates containing a crystallized excipient such as glycine or mannitol, IL-6 suffered destabilization, which was less pronounced if an additional amorphous excipient was present. For the completely amorphous formulations, aggregation was prevented during a 9-month storage at 25 and 40 degrees C as long as the storage temperature did not exceed the Tg value of the lyophilizate, otherwise severe damage occurred. Formulations containing amorphous dextran or lysine-HCl could not effectively stabilize IL-6 even when stored below Tg. Annealing helped to improve cake robustness and appearance, but for lyophilizates containing an excipient crystallized by annealing an increase of IL-6 aggregation was observed despite a storage below Tg. Thus, the amorphous state of the excipients and a high Tg can be considered necessary conditions for preventing aggregation of freeze-dried IL-6. Whether the conditions are also sufficient depends on the choice of excipients. Destabilization can occur with some excipients despite their amorphous state as well as in the presence of crystallized excipients despite a storage below Tg. Compared to sucrose, trehalose is a more favorable excipient for protein lyophilization because it exhibits a higher Tg, possesses better stabilizing properties, and can reduce protein aggregation which may have been caused by annealing.


Subject(s)
Interleukin-6/chemistry , Dextrans/pharmacology , Drug Stability , Drug Storage , Freeze Drying , Glycine/pharmacology , Interleukin-6/administration & dosage , Mannitol/pharmacology , Trehalose/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...