Effects of MMP inhibitors incorporated within dental adhesives.

Matrix metalloproteinase (MMP) inhibition has been shown to reduce adhesive bond degradation when applied as a pre-conditioner, adding to clinical steps in the placement of adhesives, but their incorporation within dental adhesives has not been fully explored. This study examined the effect of including 2 MMP inhibitors (BB94 and GM6001) within the primers of 3 commercially available adhesives. Fluorometric assay and zymography showed that adhesives with MMP inhibitors had high affinity toward both synthetic fluorogenic FRET peptides (95%) and dentin powder substrates, respectively. The immediate microtensile bond strength was enhanced for 2 types of adhesives following the addition of both inhibitors. However, no changes were detected between the control and the inhibitor groups following 3-month storage. The modified two-step etch-and-rinse and single-step systems showed less Rhodamine B penetration to the "hybrid layer" and to the "adhesive", respectively. The incorporation of BB94 and GM6001 within the primers resulted in the inhibition of dentin MMPs with improved initial bond strength and enhanced sealing ability.

Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts.

Tissue engineering of skeletal muscle from cultured cells has been attempted using a variety of synthetic and natural macromolecular scaffolds. Our study describes the application of artificial scaffolds (collagen sponges, CS) consisting of collagen-I with parallel pores (width 20-50 microm) using the permanent myogenic cell line C(2)C(12). CS were infiltrated with a high-density cell suspension, incubated in medium for proliferation of myoblasts prior to further culture in fusion medium to induce differentiation and formation of multinucleated myotubes. This resulted in a parallel arrangement of myotubes within the pore structures. CS with either proliferating cells or with myotubes were grafted into the beds of excised anterior tibial muscles of immunodeficient host mice. The recipient mice were transgenic for enhanced green fluorescent protein (eGFP) to determine a host contribution to the regenerated muscle tissue. Histological analysis 14-50 days after surgery showed that donor muscle fibres had formed in situ with host contributions in the outer portions of the regenerates. The function of the regenerates was assessed by direct electrical stimulation which resulted in the generation of mechanical force. Our study demonstrated that biodegradable CS with parallel pores support the formation of oriented muscle fibres and are compatible with force generation in regenerated muscle.

Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells.

Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state. To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter. An optimized promoter, P(TF), was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked P(TF) to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens. Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (P(NSE)) leads to barnase expression and cell death in neuronal cells after tc withdrawal. Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed.

The genomic basis of K(V)3.4 potassium channel mRNA diversity in mice.

K(V)3.4 belongs to the shaw subfamily of shaker-type potassium channels. It conducts fast inactivating, high threshold currents in the central nervous system and in fast-twitch skeletal muscle fibers. The corresponding mouse gene, Kcnc4, consists of five exons spanning a region of 20 kb. Approximately 700 bp of regulatory sequence were delineated. It is GC-rich and lacks typical TATA and CAAT motifs. Instead, seven Sp-1 and three E-box elements define putative regulatory sequences. The mouse K(V)3.4 mRNA has a size of 3639 bp, 1120 bp of which are 3' untranslated region. A transcript initiated from an alternative 5'-exon was identified by RACE and verified by genomic analysis. This isoform, designated K(V)3.4d, is predominantly expressed in skeletal muscle and probably results from alternative promoter usage. It encodes a channel protein with a novel N-terminal cytoplasmic domain. It lacks the conserved sequence motifs encoding the shaw-type tetramerization domain and the 'ball' peptide, which confers fast inactivation properties. Another splice variant, K(V)3.4c, is derived by exon skipping in the C-terminal region and is expressed at similar levels in brain and muscle. These data demonstrate that differential splicing and alternative transcription start sites are utilised to generate a set of K(V)3.4 variants in skeletal muscle and brain, presumably involved in the regulation of excitability.

Tumor necrosis factor alpha induces a metalloprotease-disintegrin, ADAM8 (CD 156): implications for neuron-glia interactions during neurodegeneration.

ADAM proteases, defined by extracellular disintegrin and metalloprotease domains, are involved in protein processing and cell-cell interactions. Using wobbler (WR) mutant mice, we investigated the role of ADAMs in neurodegeneration and reactive glia activation in the CNS. We found that ADAM8 (CD 156), a suspected leukocyte adhesion molecule, is expressed in the CNS and highly induced in affected CNS areas of WR mice, in brainstem and spinal cord. ADAM8 mRNA and protein are found at low levels throughout the normal mouse CNS, in neurons and oligodendrocytes. In the WR CNS regions in which neurodegeneration occurs, ADAM8 is induced in neurons, reactive astrocytes, and activated microglia. Similarly, the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) is upregulated and shows the same cellular distribution. In primary astrocytes from wild-type and WR mice, in primary cerebellar neurons, and in mouse motoneuron-like NSC19 cells, ADAM8 expression was induced up to 15-fold by mouse TNF-alpha, in a dose-dependent manner. In both cell types, ADAM8 was also induced by human TNF-alpha, indicating that TNF receptor type I (p55) is involved. Induction of ADAM8 mRNA was suppressed by treatment with an interferon-regulating factor 1 (IRF-1) antisense oligonucleotide. We conclude that IRF-1-mediated induction of ADAM8 by TNF-alpha is a signaling pathway relevant for neurodegenerative disorders with glia activation, proposing a role for ADAM8 in cell adhesion during neurodegeneration.

Elevated expression of membrane type 1 metalloproteinase (MT1-MMP) in reactive astrocytes following neurodegeneration in mouse central nervous system.

Reactive astrocytes occurring in response to neurodegeneration are thought to play an important role in neuronal regeneration by upregulating the expression of extracellular matrix (ECM) components as well as the ECM degrading metalloproteinases (MMPs). We examined the mRNA levels and cellular distribution of membrane type matrix metalloproteinase 1 (MT1-MMP) and tissue inhibitors 1-4 of MMPs (TIMPs) in brain stem and spinal cord of wobbler (WR) mutant mice affected by progressive neurodegeneration and astrogliosis. MT1-MMP, TIMP-1 and TIMP-3 mRNA levels were elevated, whereas TIMP-2 and TIMP-4 expression was not affected. MT1-MMP was expressed in reactive astrocytes of WR. In primary astrocyte cultures, MT1-MMP mRNA was upregulated by exogeneous tumor necrosis factor alpha. Increased plasma membrane and secreted MMP activities were found in primary WR astrocytes.

A functional knock-out of titin results in defective myofibril assembly.

Titin, also called connectin, is a giant muscle protein that spans the distance from the sarcomeric Z-disc to the M-band. Titin is thought to direct the assembly of sarcomeres and to maintain sarcomeric integrity by interacting with numerous sarcomeric proteins and providing a mechanical linkage. Since severe defects of such an important molecule are likely to result in embryonic lethality, a cell culture model should offer the best practicable tool to probe the cellular functions of titin. The myofibroblast cell line BHK-21/C13 was described to assemble myofibrils in culture. We have now characterized the sub-line BHK-21-Bi, which bears a small deletion within the titin gene. RNA analysis revealed that in this mutant cell line only a small internal portion of the titin mRNA is deleted. However, western blots, immunofluorescence microscopy and immunoprecipitation experiments showed that only the N-terminal, approx. 100 kDa central Z-disc portion of the 3 MDa titin protein is expressed, due to the homozygous deletion in the gene. Most importantly, in BHK-21-Bi cells the formation of thick myosin filaments and the assembly of myofibrils are impaired, although sarcomeric proteins are expressed. Lack of thick filament formation and of ordered actin-myosin arrays was confirmed by electron microscopy. Myogenisation induced by transfection with MyoD yielded myofibrils only in myotubes formed from wild type and not from mutant cells, ruling out that a principal failure in myogenic commitment of the BHK-21-Bi cells might cause the observed effects. These experiments provide the first direct evidence for the crucial role of titin in both thick filament formation as a molecular ruler and in the coordination of myofibrillogenesis.

Steroid RU 486 inducible myogenesis by 10T1/2 fibroblastic mouse cells.

For reconstruction or repair of damaged tissues, an artificially regulated switch from proliferation to differentiation would be of great advantage. To achieve conditional myogenesis, we expressed MyoD in mouse C3H 10T1/2 fibroblastic cells, using a gene regulation system based on the synthetic steroid RU 486. By stable co-transfection of a plasmid construct with the RU 486 dependent activator and an integrating inducible MyoD construct, a cell clone, designated 10T-RM, was obtained in which MyoD expression was stringently controlled by RU 486. 12 h after addition of 10 nM RU 486 to 10T-RM cells, saturation levels of MyoD mRNA were observed and >/=2 days later, mRNA for embryonal myosin heavy chain (MyHC(emb)) was abundant and mononucleated cells fused into myotubes.

A role for polyproline motifs in the spinal muscular atrophy protein SMN. Profilins bind to and colocalize with smn in nuclear gems.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the loss of alpha-motoneurons in the spinal cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have been identified on human chromosome 5q. The corresponding SMN protein is expressed ubiquitously. It is coded by seven exons and contains conspicuous proline-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to bind to profilins (PFNs), small proteins engaged in the control of actin dynamics. We tested whether profilins interact with SMN via its polyproline stretches. Using the yeast two-hybrid system we show that profilins bind to SMN and that this binding depends on its proline-rich motifs. These results were confirmed by coimmunoprecipitation and by in vitro binding studies. Two PFN isoforms, I and II, are known, of which II is characteristic for central nervous system tissue. We show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons. In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN in the cytoplasm of the cell body and in nuclear gems. Likewise, SMN and PFN I colocalize in gems of HeLa cells. Although SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the SMN genes are expressed ubiquitously, our findings suggest that the interaction of PFN II with SMN may be involved in neuron-specific effects of SMN mutations.

Spatiotemporal progression of neurodegeneration and glia activation in the wobbler neuropathy of the mouse.

The wobbler mouse (phenotype WR; genotype wr/wr) has been investigated as a model for neurodegenerative diseases like SMA and ALS. A new diagnostic marker based on a polymorphism in the closely linked chaperonine gene Cct4 enabled us to diagnose the allelic status at the wr locus within the original background strain C57BL/6. Using this marker, we investigated the spatiotemporal progression of neuropathology in WR mice from postnatal day (d.p.n.) 10 to 60. Neurodegeneration starts at 13 d.p.n. in the thalamus (N. ventralis), in deep cerebellar nuclei, brain stem (N. vestibularis) and spinal cord interneurons. The motor nuclei of spinal nerves and motoneurons degenerate from 15 d.p.n. onward. Reactive astrocytes are observed around 17 d.p.n. in the white and grey matter of the spinal cord. Microgliosis occurs only from 23 d.p.n. onward. Our data demonstrate that in the WR disease, neurodegeneration in thalamus, cerebellum, and brain stem precedes motoneuron degeneration, astrogliosis and microgliosis.

The protein kinase N (PKN) gene PRKCL1/Prkcl1 maps to human chromosome 19p12-p13.1 and mouse chromosome 8 with close linkage to the myodystrophy (myd) mutation.

Protein kinase N (PKN) is a fatty acid- and Rho-activated serine/threonine protein kinase involved in the regulation of cell motility by association with cytoskeletal components such as neurofilament and alpha-actinin. We determined the chromosomal location of the human PKN gene PRKCL1 by fluorescence in situ hybridization and by radiation hybrid mapping. The corresponding mouse gene Prkcl1 was mapped by segregation analysis. We found by FISH that PRKCL1 is localized to chromosome 19p12-p13.1 and, more precisely, by radiation hybrid mapping, about 11 cR from EST WI-6344 in subband 19p12. Prkcl1 maps to mouse chromosome 8 between D8Mit6 and junb. This region of mouse Chr 8 shows a scrambled syntenic conservation to human chromosomes 4q, 8p, and 19p. As the mouse mutation myodystrophy myd has been mapped to the same region, Prkcl1 is a candidate gene for myd.

Expression of the potassium channel KV3.4 in mouse skeletal muscle parallels fiber type maturation and depends on excitation pattern.

We report the detailed expression pattern of the voltage-dependent potassium channel KV3.4 (rat homologue, Raw3) in mouse skeletal muscle. Using semi-quantitative RT-PCR, we show that its expression is detectable at embryonic day 17 and rises to adult levels within 2 weeks after birth. Expression is fiber type-dependent, with mRNA levels being 5-6-fold lower in the mixed slow/fast soleus muscle than in the fast tibialis anterior and extensor digitorum longus muscles. Fast muscles from myotonic mice exhibit low KV3.4 mRNA levels similar to those of wild-type soleus. In denervated extensor digitorum longus, KV3.4 expression declines to perinatal levels. We conclude that KV3.4 expression in mouse skeletal muscle is regulated by the pattern of excitation.

The mouse Clc1/myotonia gene: ETn insertion, a variable AATC repeat, and PCR diagnosis of alleles.

Myotonias are muscle diseases in which the function of the muscular chloride channel ClC-1 is impaired. Null alleles of the corresponding Clc1 gene on mouse chromosome (Chr) 6 provide animal models for human myotonias. It was shown that the allele adr (Clc1adr) is due to an insertion of an ETn type transposon that is transcribed and leads to multiple splicing events; the allele mto (Clc1adr-mto) involves a stop codon near the N-terminus. We have determined the genomic organization of the mouse Clc1 gene and the sequence requirements for the transposon insertion in the Clc1adr allele. The mouse Clc1 gene is composed of 23 exons, ranging from 39 to 372 bp, and spans approximately 23 kb of genomic DNA. The exon/intron organization is highly homologous to that of the human CLCN1 gene; the homology of the coding sequence is 97% to rat and 89% to human. In the adr allele the ETn transposon is inserted into intron 12, the largest intron. Whereas the 5' and 3' LTR sequences of the ETn transposon are homologous to those reported for other insertional mutations of the mouse, no consensus motif for an insertion target site could be defined. On the basis of flanking sequences, we provide duplex PCR diagnoses for the adr, adr-mto, and wild-type alleles of Clc1. Close to the 3' end of intron 12, a tetranucleotide repeat (AATC)n was found that is polymorphic between mouse species Mus musculus, M. molossinus, M. castaneus, and M. spretus, and can thus be used for chromosomal mapping studies.