Wolfgang Quitschke

BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.

Multiple Nucleosome Positioning Sites Regulate the CTCF-Mediated Insulator Function of the H19 Imprinting Control Region†

ABSTRACT The 5′ region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.

58,000 dalton intermediate filament proteins of neuronal and nonneuronal origin in the goldfish visual pathway

Abstract: A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two‐dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal‐enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one‐dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one‐dimensional gel electrophoresis.

Survey of Intermediate Filament Proteins in Optic Nerve and Spinal Cord: Evidence for Differential Expression

Abstract: The distribution of intermediate filament proteins in optic nerve and spinal cord from rat, hamster, goldfish, frog, and newt were analyzed by two‐dimensional gel electrophoresis. General as well as specific monoclonal and polyclonal antibodies were reacted against putative intermediate filament proteins. In vitro incubations of excised optic nerve in the presence of [35S]methionine distinguished between neuronal and nonneuronal intermediate filament proteins. The proteins of the intermediate filament complex in the two tissues for rat and hamster were similar. The typical neurofilament triplet and glial fibrillary acidic protein (GFAP) were observed. Vimentin was more concentrated in the optic nerve than in the spinal cord. The goldfish, newt, and frog contained neurofilament proteins in the 145–150K range and in the 70–85K range. In addition, predominant neurofilament proteins in the 58–62K molecular‐weight range were found in all three species. In contrast to mammalian species, the goldfish, newt, and frog displayed extensive heterogeneity between optic nerve and spinal cord in the expression of both neuronal and nonneuronal intermediate filament proteins. The distinctive presence of low‐molecular‐weight intermediate filament proteins and their high concentration in the optic nerve and spinal cord of these nonmammalian vertebrates is discussed in terms of neuronal development and regeneration.

In Vitro Protein Synthesis in the Goldfish Retinotectal Pathway During Regeneration: Evidence for Specific Axonal Proteins of Retinal Origin in the Optic Nerve

Abstract: Four proteins with molecular weights of 58,000 can be separated as a linear array by two‐dimensional gel electrophoresis. They are highly concentrated in the goldfish optic nerve and are designated as ON1, ON2, ON3, and ON4. Proteins ON1 and ON2 are undetectable in the optic nerve after disconnection and their concentration is gradually restored during regeneration. In vitro incubations of retinas, optic nerves, or tecta in the presence of [35S]methionine indicate that proteins ON1 and ON2 are of retinal origin. The labeling rate of these proteins in the retina increases fourfold after optic nerve crush whereas the overall labeling rate in the retina remains largely constant. Their synthesis cannot be detected in tissues de void of retinal ganglion cells. This is consistent with the view that ON1 and ON2 are synthesized by retinal ganglion cells and are consequently of neuronal origin in the optic nerve. In contrast, similar experiments indicate that ON3 and ON4 are of nonneuronal origin. They are synthesized in the optic nerve in the absence of retinal ganglion cells.

Specific optic nerve proteins during regeneration of the goldfish retinotectal pathway.

A cluster of proteins originally detected in the goldfish optic tectum is found in high concentration in the optic nerve. When the proteins are solubilized in sodium dodecylsulfate and analyzed by two-dimensional gel electrophoresis, a well resolved linear array is observed. The optic nerve proteins are designated here as ON1, ON2, ON3 and ON4. Proteins ON1 and ON2 can be further resolved into components (ON1a + ON1b) and (ON2a + ON2b), respectively. ON1a and ON2a are specific to the goldfish retinotectal pathway in that they are not found in brain areas which do not receive retinal inputs. The effect of optic nerve disconnection on the concentration of the ON proteins was investigated. A large decrease in the levels of ON1 and ON2 is observed within 5 days of optic nerve crush and their concentration is restored to normal levels during the regeneration process. In contrast, the concentration of ON3 and ON4 is stable following optic nerve disconnection. It is suggested that ON1 and ON2 originate from the retina and that they are located in the axons of the optic nerve while ON3 and ON4 are of non-neuronal origin. These proteins which are prominent in the optic nerve are compared to previously reported tectal proteins. Their cellular origin and the possibility that they may be associated with the neurofilament complex is discussed.

Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

BackgroundCommercially available curcumin preparations contain a mixture of related polyphenols, collectively referred to as curcuminoids. These encompass the primary component curcumin along with its co-purified derivatives demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids have numerous biological activities, including inhibition of cancer related cell proliferation and reduction of amyloid plaque formation associated with Alzheimer disease. Unfortunately, the solubility of curcuminoids in aqueous solutions is exceedingly low. This restricts their systemic availability in orally administered formulations and limits their therapeutic potential.ResultsMethods are described that achieve high concentrations of soluble curcuminoids in serum. Solid curcuminoids were either mixed directly with serum, or they were predissolved in dimethyl sulfoxide and added as aliquots to serum. Both methods resulted in high levels of curcuminoid-solubility in mammalian sera from different species. However, adding aliquots of dimethyl sulfoxide-dissolved curcuminoids to serum proved to be more efficient, producing soluble curcuminoid concentrations of at least 3 mM in human serum. The methods also resulted in the differential solubility of individual curcuminoids in serum. The addition of dimethyl sulfoxide-dissolved curcuminoids to serum preferentially solubilized curcumin, whereas adding solid curcuminoids predominantly solubilized bisdemethoxycurcumin. Either method of solubilization was equally effective in inhibiting dose-dependent HeLa cell proliferation in culture. The maximum concentration of curcuminoids achieved in serum was at least 100-fold higher than that required for inhibiting cell proliferation in culture and 1000-fold higher than the concentration that has been reported to prevent amyloid plaque formation associated with Alzheimer disease. Curcuminoids were also highly soluble in solutions of purified albumin, a major component of serum.ConclusionThese results suggest the possibility of alternative therapeutic approaches by injection or infusion of relatively small amounts of curcuminoid-enriched serum. They also provide tools to reproducibly solubilize curcuminoids for analysis in cell culture applications. The differential solubility of curcuminoids achieved by different methods of solubilization offers convenient alternatives to assess the diverse biological effects contributed by curcumin and its derivatives.

A noncomputerized scanning method for determining relative protein quantities and synthesis rates on two-dimensional electrophoretic gels

Abstract A densitometric method that permits the determination of relative amounts and synthesis rates of proteins separated by two-dimensional gel electrophoresis is presented. The method is applicable to proteins that have been stained by Coomassie blue or proteins that have been radiolabeled. The analysis is achieved by scanning selected spots with an ordinary densitometer in the horizontal and vertical direction and relating the response to the volume of an ellipsoid. A linear dependence is observed between the amount of protein or incorporated radioactivity and the measured optical density. The advantage of this method is that specialized scanning instruments and computer analysis are not required. The method is most useful for the analysis of a few specific proteins which change in their relative amount or specific activity due to experimental manipulations. Difficulties in the analysis of protein spots derived from the twodimensional gel electrophoresis technique are discussed and compared to an analysis of bands from the one-dimensional electrophoresis technique.

Homology and diversity between intermediate filament proteins of neuronal and nonneuronal origin in goldfish optic nerve

Abstract: The predominant intermediate filament proteins of the goldfish optic nerve have molecular weights of 58K. They can be separated into a series of four major isoelectric variants of neuronal (ON1 and ON2) and nonneuronal (ON3 and ON4) origin. The extent of homology between the goldfish 58K intermediate filament proteins themselves and to rat optic nerve vimentin and glial fibrillary acidic protein (GFAP) was investigated. Unlabeled and [32P]orthophosphate‐labeled proteins were subjected to partial hydrolysis by V8 protease, chymotrypsin, and CNBr. The results show that the goldfish intermediate filament proteins share with vimentin and GFAP a 40K chymotrypsin‐resistant core fragment. Phosphorylated moieties appear to be located outside the core region since they are preferentially cleaved off by chymotrypsin and not found associated with the 40K core. In addition, the goldfish ON proteins contain the antigenic site within the core that is common to most intermediate filaments. V8 or CNBr digestion indicates that many fragments that are common to ON1 and ON2 are clearly distinct from fragments that are common to ON3 and ON4. In addition, structural variability is observed between the goldfish intermediate filament proteins and vimentin and GFAP. The results are discussed in terms of intermediate filament structure and their possible role in nerve growth.

Plasma Hyaluronan-binding Protein Is a Serine Protease

CTCF is an essential factor for optimal transcription from the amyloid β-protein precursor promoter. A proteolytic activity detected in bovine, rabbit, horse, and human serum cleaves CTCF at three major sites, resulting in a modified mobility shift pattern of the fragments that retain DNA binding ability. The protease was purified to electrophoretic homogeneity, partially sequenced, and identified as the plasma hyaluronan-binding protein. The proteolytic activity was selectively abolished by various serine protease inhibitors, including the Kunitz-type protease inhibitor domain of amyloid β-protein precursor. Reduction with β-mercaptoethanol showed that the 70-kDa protein consists of two polypeptides with apparent molecular masses of 44 and 30 kDa. The serine protease domain was localized to the 30-kDa polypeptide as determined by [3H]diisopropylfluorophosphate binding.