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Biomedical subjects

K Giese

Publications and source records attributed to K Giese.

At least 19 recordsLinked to original sources
PubMed2006

A novel siRNA-lipoplex technology for RNA interference in the mouse vascular endothelium.

For the application of RNA interference (RNAi) in vivo the functional delivery of short interfering RNAs (siRNAs) is still the major obstacle. Therefore, delivery technologies need to be established for the systemic application of RNAi in vivo. Here we report uptake, biodistribution and in vivo efficacy of siRNA molecules formulated into siRNA-lipoplexes. The applied formulation is based on complex formation of positively charged liposomes, a mixture of cationic and fusogenic lipids complexed with the negatively charged siRNA. We determined by fluorescence microscopy the temporal and spatial distribution of fluorescently labeled siRNA-lipoplexes, the body clearance and endothelial cell type specific uptake after single intravenous injection. Furthermore, by using siRNA molecules for targeting endothelia-specifically expressed genes, such as CD31 and Tie2, we were able to demonstrate downregulation of the corresponding mRNA and protein in vivo. Taken together, we show the applicability of this non-viral delivery technology for inducing RNAi in the vasculature of mice after systemic application.

Animals↗
PubMed2006

RNA interference in the mouse vascular endothelium by systemic administration of siRNA-lipoplexes for cancer therapy.

RNA interference (RNAi) entails the potential for novel therapeutic strategies through the silencing of disease-causing genes in vivo. However, recent studies have raised an issue regarding applicable routes of administration for small interfering RNA (siRNA) molecules as therapeutics. In this study, we demonstrate that liposomally formulated siRNA molecules, the so-called siRNA-lipoplexes, but not naked siRNAs, are delivered to the tumor endothelial cells in vivo by microscopy. In addition, functional intracellular delivery of formulated siRNA targeting the tumor suppressor PTEN is shown in endothelial cells of the liver and tumor. Finally, the therapeutic potential of systemically administered siRNA(CD31)-lipoplexes is established by inhibition of tumor growth in two different xenograft mouse models. Our findings corroborate the applicability of this liposomal siRNA delivery technology for inducing RNAi to modulate gene expression levels in angiogenesis-dependent processes. In addition, our results advocate CD31 as a promising therapeutic target for antiangiogenic intervention. Therefore, our study provides a basis for the development of antiangiogenic cancer therapies based on RNAi.

3T3 Cells↗
PubMed2005

Dynamical hydrogen atom tunneling in dichlorotropolone: a combined quantum, semiclassical, and classical study.

Based on the Cartesian Reaction Surface framework we construct a four-dimensional potential for the tropolone derivative 3,7-dichlorotropolone, a molecule with an intramolecular O-H...O hydrogen bond. The reduced configuration space involves the in-plane hydrogen atom coordinates, a symmetric O-O vibrational mode, and an antisymmetric mode related to deformations of the seven-membered ring. The system is characterized in terms of quantum mechanical computations of the low-lying eigenstates as well as a classical and semiclassical analysis of spectra obtained via Fourier transforming autocorrelation functions. For the semiclassical analysis we utilize the amplitude-free correlation function method [K. Hotta and K. Takatsuka, J. Phys. A 36, 4785 (2003)]. Our results demonstrate substantial anharmonic couplings leading to highly correlated wave functions even at moderate energies. Furthermore, the importance of dynamical tunneling in tropolone is suggested since many low-lying states--including the ground state--lie above the classical saddle point but nevertheless appear as split pairs.

Journal Article↗
PubMed2004

Identification of novel effectors of invasive cell growth downstream of phosphoinositide 3-kinase.

Conventional approaches to identifying cancer targets are complicated by the chromosomal instability of tumour cells, and typically result in a large number of differentially expressed candidate genes with uncertain disease relevance. Here we present a novel approach which aims to elucidate the molecular changes that are induced after loss of tumour suppressor function. Using gene silencing tools, we mimic the loss of tumour suppressor function to identify key regulators of tumour initiation and progression. Loss of function of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) correlates with increased invasive cell growth due to the resulting chronic activation of the PI 3-kinase (phosphoinositide 3-kinase) pathway. Induced activation of PI 3-kinase either by inhibiting PTEN expression or by using p110*, a constitutively active PI 3-kinase, increased signalling and the invasive growth potential of cells. Using this unbiased approach we have identified novel downstream effectors of PI 3-kinase/PTEN signalling that mediate the behaviour of cells with a hyperactive PI 3-kinase pathway. These molecules represent candidate targets for therapeutic intervention in patients with PTEN-deficient tumours.

Animals↗
PubMed2003

Implementation of a practical model for light and heat distribution using laser-induced thermotherapy near to a large vessel.

This paper introduces a simulation model for light and heat transport in tissues including perfusion effects. The model enables an efficient simulation of the damaged zone induced with an optical fibre for laser interstitial thermotherapy (LITT). It is designed specially for, but not limited to, tissue ablation in the neck region near to vessels. We describe in detail the effects of the rise in temperature caused by the absorption of light in tissue, using the heat equation and including the cooling effects of flow in vessels and of microperfusion in tissue in order to determine the extent of thermal damage. The extent of the necrosis zone is calculated with a damage function at each point of a finite element method (FEM) mesh. The FEM mesh is implemented with FEMLAB 2.3 as an add-on for finite element modelling for Matlab 6.5. LITT for tumour ablation in liver and some other anatomical regions is a well-known and established method (Bundesärztekammer und Kassenärztliche Bundesvereinigung 2002 Assessment der Bundesärztekammer und der Kassenärztlichen Bundesvereinigung, Köln). Investigations of treatments using LITT in the neck region are still in progress. We propose a refined model to validate the LITT method in the future in another anatomic region, e.g., in the highly sensitive region of the neck. Our calculations show that in order to induce a lesion with a maximum diameter of about 1 cm near to a large vessel, an application time between 3 and 4 min is needed using a laser power of about 10 W with a Nd:YAG 1064 nm radiation wavelength.

Body Temperature↗
PubMed2001

Dental erosion in children and adolescents--a cross-sectional and longitudinal investigation using study models.

OBJECTIVE: To investigate the prevalence and incidence of dental erosion in children and adolescents. METHODS: Lesions were registered for all tooth surfaces of primary and permanent teeth using pre-orthodontic study models. A total of 1,000 individuals (mean age 11.4+/-3.3 years) were included; 265 of them were followed over a 5-year period using their final orthodontic casts. RESULTS: In the primary teeth, 26.4% of the individuals had no erosive lesions, 70.6% had at least one tooth with grade 1 erosion and 26.4% had grade 2 erosion. Grade 1 erosion was found in 44% of the occlusal surfaces of molars (36% of the incisal surfaces of the canines) and grade 2 erosion in 11% (9%). Lesions affecting oral or vestibular surfaces were negligible. In the permanent teeth, 11.6% of individuals had at least one tooth with grade 1 erosion but only 0.2% had at least one tooth with grade 2 erosion. The most affected teeth were the mandibular first molars (7% with grade 1 lesions). Lesions affecting oral or vestibular surfaces were negligible. Mandibular first molars were identified as possible marker teeth for the onset of erosive lesions. Within the last two decades, the percentage of subjects with at least one tooth with a grade 1 or 2 erosive lesion significantly increased for primary and with grade 1 for permanent teeth (P< or =0.001). The longitudinal observation revealed that subjects with erosive lesions in their primary dentition had a significantly increased risk for erosion in their permanent teeth (P< or =0.001). CONCLUSION: In Germany, dental erosion seems to be a significant, but not a serious, problem for dental health in adolescents.

Adolescent↗
PubMed2000

The I kappa B kinase (IKK) complex is tripartite and contains IKK gamma but not IKAP as a regular component.

A critical step in the activation of NF-kappa B is the phosphorylation of I kappa Bs by the I kappa B kinase (IKK) complex. IKK alpha and IKK beta are the two catalytic subunits of the IKK complex and two additional molecules, IKK gamma/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-kappa B signaling. IKAP and IKK gamma belong to distinct cellular complexes. Quantitative association of IKK gamma was observed with IKK alpha and IKK beta. In contrast IKAP was complexed with several distinct polypeptides. Overexpression of either IKK gamma or IKAP blocked tumor necrosis factor alpha induction of an NF-kappa B-dependent reporter construct, but IKAP in addition affected several NF-kappa B-independent promoters. Whereas specific down-regulation of IKK gamma protein levels by antisense oligonucleotides significantly reduced cytokine-mediated activation of the IKK complex and subsequent NF-kappa B activation, a similar reduction of IKAP protein levels had no effect on NF-kappa B signaling. Using solely IKK alpha, IKK beta, and IKK gamma, we could reconstitute a complex whose apparent molecular weight is comparable to that of the endogenous IKK complex. We conclude that while IKK gamma is a stoichiometric component of the IKK complex, obligatory for NF-kappa B signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-kappa B activation.

Carrier Proteins↗
PubMed2000

Identification of a novel aspartic-like protease differentially expressed in human breast cancer cell lines.

Four different human breast cancer cell lines were examined to search for genes associated with tumor growth and metastasis. Each of these cell lines, MDA-MB-453, MCF-7, MDA-MB-231 and MDA-MB-435, displays different phenotypic characteristics ranging from poorly to highly tumorigenic and metastatic. The differences in gene expression profiles of these cell lines generated by differential display technique should allow one to identify candidates as putative oncogenes or tumor/metastasis suppressor genes. A novel cDNA expressed in the highly tumorigenic and metastatic cell line, MDA-MB-435, was identified and isolated by this approach. The function for this gene, designated ALP56 (aspartic-like protease 56 kDa), in tumor progression is suggested by the homology of the encoded protein to aspartic proteases, such as cathepsin D. The amino acid residues in two catalytic domains of this family are highly conserved in those domains of ALP56. Northern hybridization indicated that the expression of ALP56 is associated with growth and metastasis of MDA-MB-435 tumors in immunodeficient mice. In situ hybridization of biopsies from breast cancer and colon cancer patients indicated that ALP56 is upregulated in human primary tumors and liver metastasis. These results suggest that this novel gene correlates with human tumor progression.

Amino Acid Sequence↗
PubMed1997

Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin.

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes mellitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2-5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity.

Animals↗
PubMed1997

Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1.

LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) alpha enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCR alpha enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCR alpha enhancer.

DNA, Superhelical↗
PubMed1996

Positive selection system to screen for inhibitors of human immunodeficiency virus-1 transcription.

We describe a transcription-based assay system to screen for antiviral drugs in vivo. The system consists of two transcription units, a cytomegalovirus promoter driving a reporter gene physically linked to an HIV-1 promoter oriented in the opposite direction. Based on the arrangement of the transcription units, enhanced HIV-1 promoter activity in the presence of the viral transactivating Tat protein downregulates reporter gene expression initiated from the CMV promoter. Inhibitors of HIV-1 transcription relieve the suppression and are identified by an increase in reporter gene expression. This positive selection system allows discrimination between drugs that nonspecifically block cellular functions by cytotoxicity and molecules that specifically repress HIV-1 promoter activity.

Alkaline Phosphatase↗
PubMed1996

Reduction of food intake and weight gain by the ob protein requires a specific secondary structure and is reversible.

BACKGROUND: Obesity, the condition of excessive accumulation of fat is a poorly understood disorder and is a risk factor for type II diabetes, hypertension, and hyperlipidaemia. Recently, a putative mouse obese gene was cloned and its product, termed ob protein, was shown to be involved in the regulation of body weight. MATERIALS AND METHODS: Bacterial and insect cells were used for expression of recombinant mouse ob protein. Amino-terminal sequence analysis and site-directed mutagenesis were used to identify and characterize the mature form of ob protein. Genetically obese mice and wild-type rats were used to determine the biological activity of ob protein. RESULTS: Mouse ob protein is synthesized as a precursor molecule, the mature form of which was found in mouse serum. Biochemical analysis identified the processing site in the ob precursor molecule and an intramolecular disulfide bond in the mature form that is necessary for activity. Reduction of food intake and weight gain after administration of ob protein to genetically obese mice and wild-type rats is reversible. DISCUSSION: This study demonstrates that ob protein is a secreted satiety factor which regulates body weight and reduces food intake even in animals with no genetic body weight abnormalities. The failure of ob protein to effect these parameters in db/db mice supports the hypothesis that these mice are deficient in a signaling molecule that normally responds to the ob protein.

Amino Acid Sequence↗
PubMed1995

Structural basis for DNA bending by the architectural transcription factor LEF-1.

Lymphoid enhancer-binding factor (LEF-1) and the closely related T-cell factor 1 (TCF-1) are sequence-specific and cell-type-specific DNA-binding proteins that play important regulatory roles in organogenesis and thymocyte differentiation. LEF-1 participates in regulation of the enhancer associated with the T cell receptor (TCR)-alpha gene by inducing a sharp bend in the DNA and facilitating interactions between Ets-1, PEBP2-alpha, and ATF/CREB, transcription factors bound at sites flanking the LEF-1 site. It seems that LEF-1 plays an architectural role in the assembly and function of this regulatory nucleoprotein complex. LEF-1 recognizes a specific nucleotide sequence through a high-mobility-group (HMG) domain. Proteins containing HMG domains bind DNA in the minor groove, bend the double helix, and recognize four-way junctions and other irregular DNA structures. Here we report the solution structure of a complex of the LEF-1 HMG domain and adjacent basic region with its cognate DNA. The structure reveals the HMG domain bound in the widened minor groove of a markedly distorted and bent double helix. The basic region binds across the narrowed major groove and contributes to DNA recognition.

Amino Acid Sequence↗
PubMed1995

Assembly and function of a TCR alpha enhancer complex is dependent on LEF-1-induced DNA bending and multiple protein-protein interactions.

In this study we examine the molecular basis for the synergistic regulation of the minimal TCR alpha enhancer by multiple proteins. We find that reconstitution of TCR alpha enhancer function in nonlymphoid cells requires expression of the lymphoid-specific proteins LEF-1, Ets-1 and PEBP2 alpha (CBF alpha), and a specific arrangement of their binding sites in the enhancer. We show that Ets-1 cooperates with PEBP2 alpha to bind adjacent sites at one end of the enhancer, forming a ternary complex that is unstable by itself. Stable occupancy of the Ets-1- and PEBP2 alpha-binding sites in a DNase I protection assay was found to depend on both a specific helical phasing relationship with a nonadjacent ATF/CREB-binding site at the other end of the enhancer and on LEF-1. The HMG domain of LEF-1 was found previously to bend the DNA helix in the center of the TCR alpha enhancer. We now show that the HMG domain of the distantly related SRY protein, which also bends DNA, can partially replace LEF-1 in stimulating enhancer function in transfection assays. Taken together with the observation that Ets-1 and members of the ATF/CREB family have the potential to associate in vitro, these data suggest that LEF-1 can coordinate the assembly of a specific higher-order enhancer complex by facilitating interactions between proteins bound at nonadjacent sites.

Activating Transcription Factors↗
PubMed1994

Distinct DNA-binding properties of the high mobility group domain of murine and human SRY sex-determining factors.

The mammalian sex-determining gene SRY (sex-determining region on Y chromosome) encodes a member of the high mobility group (HMG) family of regulatory proteins. The HMG domain of the SRY protein represents a DNA binding motif that displays rather unusually weak evolutionary conservation of amino acids between human and mouse sequences. Together with the previous finding that the human (h) SRY gene is unable to induce a male phenotype in genetically female transgenic mice, these observations raise questions concerning the DNA binding properties of SRY proteins. Here, we present data that indicate that the DNA binding and bending properties of the HMG domains of murine (m) SRY and hSRY differ from each other. In comparison, mSRY shows more-extensive major-groove contacts with DNA and a higher specificity of sequence recognition than hSRY. Moreover, the extent of protein-induced DNA bending differs from the HMG domains of hSRY and mSRY. These differences in DNA binding by hSRY and mSRY may, in part, account for the functional differences observed with these gene products.

Animals↗
PubMed1994

HMG domain proteins: architectural elements in the assembly of nucleoprotein structures.

The high-mobility group (HMG) domain is a DNA-binding motif that is shared abundant non-histone components of chromatin and by specific regulators of transcription and cell differentiation. The HMG family of proteins comprises members with multiple HMG domains that bind DNA with low sequence specificity, and members with single HMG domains that recognize specific nucleotide sequences. Common properties of HMG domain proteins include interaction with the minor groove of the DNA helix, binding to irregular DNA structures, and the capacity to modulate DNA structure by bending. DNA bending induced by the HMG domain can facilitate the formation of higher-order nucleoprotein complexes, suggesting that HMG domain proteins may have an architectural role in assembling such complexes.

Amino Acid Sequence↗
PubMed1994

[In vivo fluorescence spectroscopy of human skin: principles and application to penetration measurements].

The fluorescence properties of untreated human skin and the penetration of topically applied drugs were measured in vivo using a commercially available fluorescence spectrometer which was adapted to in vivo-measurements on skin by means of optical fibres and a remote viewing head. The intensity and characteristics of the intrinsic fluorescences of human skin and their dependences upon several parameters like pigmentation, water content of the skin or depth of the horny layer are shown. The principles of the penetration measurements based on the fluorescence of the skin or the fluorescence of the drug topically applied to the skin are described and measurements of the penetration of some drugs are reported.

Fiber Optic Technology↗