Xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome: a complex genotype-phenotype relationship.

Patients with the rare genetic disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS) have defects in DNA nucleotide excision repair (NER). The NER pathway involves at least 28 genes. Three NER genes are also part of the basal transcription factor, TFIIH. Mutations in 11 NER genes have been associated with clinical diseases with at least eight overlapping phenotypes. The clinical features of these patients have some similarities but also have marked differences. NER is involved in protection against sunlight-induced DNA damage. While XP patients have 1000-fold increase in susceptibility to skin cancer, TTD and CS patients have normal skin cancer risk. Several of the genes involved in NER also affect somatic growth and development. Some patients have short stature and immature sexual development. TTD patients have sulfur deficient brittle hair. Progressive sensorineural deafness is an early feature of XP and CS. Many of these clinical diseases are associated with developmental delay and progressive neurological degeneration. The main neuropathology of XP is a primary neuronal degeneration. In contrast, CS and TTD patients have reduced myelination of the brain. These complex neurological abnormalities are not related to sunlight exposure but may be caused by developmental defects as well as faulty repair of DNA damage to neuronal cells induced by oxidative metabolism or other endogenous processes.

Genotypic heterogeneity and clinical phenotype in triple A syndrome: a review of the NIH experience 2000-2005.

Triple A syndrome (AAAS, OMIM#231550) is an autosomal recessive condition characterized by adrenal insufficiency, achalasia, alacrima, neurodegeneration and autonomic dysfunction. Mutations in the AAAS gene on chromosome 12q13 have been reported in several subjects with AAAS. Over the last 5 years, we have evaluated six subjects with the clinical diagnosis of AAAS. Three subjects had mutations in the AAAS gene-- including one novel mutation (IVS8+1 G>A)-- and a broad spectrum of clinical presentations. However, three subjects with classic AAAS did not have mutations in the AAAS gene on both alleles. This finding supports the notion of genetic heterogeneity for this disorder, although other genetic mechanisms cannot be excluded.

Bone morphogenetic protein but not transforming growth factor-beta enhances bone formation in canine diaphyseal nonunions implanted with a biodegradable composite polymer.

BACKGROUND: The purpose of the present study was to create an effective bone-graft substitute for the treatment of a diaphyseal nonunion. METHODS: A standardized nonunion was established in the midportion of the radial diaphysis in thirty mongrel dogs by creating a three-millimeter segmental bone defect (at least 2 percent of the total length of the bone). The nonunion was treated with implantation of a carrier comprised of poly(DL-lactic acid) and polyglycolic acid copolymer (50:50 polylactic acid-polyglycolic acid [PLG50]) containing canine purified bone morphogenetic protein (BMP) or recombinant human transforming growth factor-beta (TGF-beta1), or both, or the carrier without BMP or TGF-beta1. Five groups, consisting of six dogs each, were treated with implantation of the carrier alone, implantation of the carrier with fifteen milligrams of BMP, implantation of the carrier with 1.5 milligrams of BMP, implantation of the carrier with fifteen milligrams of BMP and ten nanograms of TGF-beta1, or implantation of the carrier with ten nanograms of TGF-beta1. At twelve weeks after implantation, the radii were examined radiographically and the sites of nonunion were examined histomorphometrically. RESULTS: We found that implantation of the polylactic acid-polyglycolic acid carrier alone or in combination with ten nanograms of TGF-beta1 failed to induce significant radiographic or histomorphometric evidence of healing at the site of the nonunion. The radii treated with the carrier enriched with either 1.5 or fifteen milligrams of BMP showed significantly increased periosteal and endosteal bone formation on histomorphometric (p < 0.05) and radiographic (p < 0.02) analysis. CONCLUSIONS: Bone formation in a persistent osseous defect that is similar to an ununited diaphyseal fracture is increased when species-specific BMP incorporated into a polylactic acid-polyglycolic acid carrier is implanted at the site of the nonunion. TGF-beta1 at a dose of ten nanograms per implant did not induce a similar degree of bone formation or potentiate the effect of BMP in this model. CLINICAL RELEVANCE: The biodegradable implant containing BMP that was used in the present study to treat diaphyseal nonunion is an effective bone-graft substitute.

A cell culture model for androgen effects in motor neurons.

Androgens are known to alter the morphology, survival, and axonal regeneration of lower motor neurons in vivo. To understand better the molecular mechanisms of androgen action in neurons, we created a model system by stably expressing the human androgen receptor (AR) in motor neuron hybrid cells. Motor neuron hybrid cells express markers consistent with anterior horn cells and can be differentiated into a neuronal phenotype. When differentiated in the presence of androgen, AR-expressing cells, but not control cells, exhibit a dose-dependent change in morphology: androgen-treated cells develop larger cell bodies and broader neuritic processes while continuing to express neuronal markers. In addition, androgen promotes the survival of AR-expressing cells, but not control cells, under low-serum conditions. Our results demonstrate a direct trophic effect of androgens on lower motor neurons, mediated through the AR expressed in this population of neurons.

Characterization of an expanded glutamine repeat androgen receptor in a neuronal cell culture system.

Spinal and bulbar muscular atrophy (SBMA) is an inherited form of lower motor neuron degeneration caused by expansion of a CAG repeat in the androgen receptor (AR) gene. To study the mechanism by which this mutation causes neuronal pathology, we stably transfected a motor neuron hybrid cell line with human AR cDNAs containing either 24 or 65 repeats (AR24 and AR65, respectively). Both forms of receptor were able to bind ligand and activate transcription of a reporter construct equally well. Likewise, the subcellular localizations of AR24 and AR65 were similar, in both the presence and the absence of ligand. AR24- and AR65-expressing clones were phenotypically indistinguishable. They survived equally well after differentiation and were equally susceptible to damage by oxidative stress. Our studies thus demonstrate that, in a neuronal system, the expanded repeat AR functions like the normal repeat AR in several important ways. Because levels of AR65 expression were consistently lower than levels of AR24 expression, we propose that the loss of function of AR seen in SBMA may be due to decreased levels of receptor expression rather than to a difference in intrinsic properties. The postulated gain of function responsible for neuronal degeneration remains to be determined.

Spinal and bulbar muscular atrophy: a trinucleotide-repeat expansion neurodegenerative disease.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset motor neuronopathy that is caused by expansion of a trinucleotide (CAG) repeat in the androgen-receptor gene. The length of this repeat varies as it is passed down through SBMA families, and correlates inversely with the age of onset of the disease. The motor-neuron degeneration that occurs in this disease is probably caused by a toxic gain of function in the androgen-receptor protein. Subsequent to the identification of the mutation in SBMA, other inherited neurodegenerative diseases have been found to be caused by the expansion of CAG repeats in the coding regions of other genes. Because these diseases probably share a common pathogenesis, investigation of SBMA might help to determine a general mechanism of neuronal degeneration.

Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes.

Vitamin D3 metabolites affect the proliferation and differentiation of cartilage cells. Previous reports have shown that rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], whereas those from the resting zone (RC) respond to 24,25-(OH)2D3. The aim of the present study was to determine whether 24,25-(OH)2D3 induces differentiation of RC cells into a 1,25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24,25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24,25-(OH)2D3, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1,25-(OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)2D3 caused these cells to lose their response to 1,25-(OH)2D3. These results indicate that 24,25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)2D3. 24,25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)2D3 responsiveness. These observations support the hypothesis that 24,25-(OH)2D3 plays a significant role in cartilage development.

Dexamethasone promotes von kossa-positive nodule formation and increased alkaline phosphatase activity in costochondral chondrocyte cultures.

This study examined the effect of dexamethasone on von Kossa-positive nodule formation and alkaline phosphate specific activity of costochondral chondrocytes at two distinct stages of maturation. The nodules formed by the more mature growth zone chondrocyte cultures contained von Kossa-positive deposits in the extracellular matrix that had a punctate morphology. The nodules formed by the less mature resting zone cells also contained von Kossa-positive deposits, but differentiation was delayed by three-to-five days compared to the growth zone cell cultures. Dexamethasone stimulated the number of nodules formed and shortened the length of time required for von Kossa-positive nodule formation in both types of cultures. During the first 48 h of exposure to dexamethasone, alkaline phosphatase specific activity in the cell layer of both resting zone and growth zone cultures was increased in a dose-dependent manner. At 12 days post-confluence and thereafter, enzyme activity was inhibited in the dexamethasone-treated cultures. Changes in matrix vesicle alkaline phosphatase specific activity reflected those changes seen in the cell layer after dexamethasone treatment, but with higher magnitude, suggesting that one effect of dexamethasone might be to regulate matrix vesicle function. With the exception of one culture, the chondrocytes did not synthesize type X collagen under any of the experimental conditions used. Fourier transform infrared spectroscopy (FTIR) failed to detect the presence of calcium phosphates in any of the cultures exposed to dexamethasone except one. These results demonstrate that dexamethasone promotes early differentiation events, including nodule formation and increased alkaline phosphatase activity, in costochondral chondrocyte cultures. The failure to detect type X collagen synthesis and mineralization in both dexamamethasone-treated and control cultures suggests that these cultures lack the factors necessary for terminal differentiation and mineralization.

Culture surfaces coated with various implant materials affect chondrocyte growth and metabolism.

The effect on chondrocyte metabolism of culture surfaces sputter-coated with various materials used for orthopaedic implants was studied and correlated with the stage of cartilage cell maturation. Confluent, fourth-passage chondrocytes from the costochondral resting zone and growth zone of rats were cultured for 6 or 9 days on 24-well plates sputter-coated with ultrathin films of titanium, titanium dioxide, aluminum oxide, zirconium oxide, and calcium phosphate (1.67:1). Corona-discharged tissue culture plastic served as the control. The effect of surface material was examined with regard to cell morphology; cell proliferation (cell number) and DNA synthesis ([3H]thymidine incorporation); RNA synthesis ([3H]uridine incorporation); collagenase-digestible protein, noncollagenase-digestible protein, and percentage of collagen production; and alkaline phosphatase-specific activity, both in the cell layer and in trypsinized chondrocytes. Cell morphology was dependent on surface material; only cells cultured on titanium had an appearance similar to that of cells cultured on plastic. While titanium or titanium dioxide surfaces had no effect on cell number or [3H]thymidine incorporation, aluminum oxide, calcium phosphate, and zirconium oxide surfaces inhibited both parameters. Cells cultured on aluminum oxide, calcium phosphate, zirconium oxide, and titanium dioxide exhibited decreased collagenase-digestible protein, noncollagenase-digestible protein, and percentage of collagen production, but [3H]uridine incorporation was decreased only in those chondrocytes cultured on aluminum oxide, calcium phosphate, or zirconium oxide. Chondrocytes cultured on titanium had greater alkaline phosphatase-specific activity than did cells cultured on plastic, but the incorporation of [3H]uridine and production of collagenase-digestible protein, noncollagenase-digestible protein, and percentage of collagen was comparable. The response of chondrocytes from the growth zone and resting zone to culture surface was comparable, differing primarily in magnitude. Cell maturation-dependent effects were evident when enzyme activity in trypsinized and scraped cells was compared. These results indicate that different surface materials affect chondrocyte metabolism and phenotypic expression in vitro and suggest that implant materials may modulate the phenotypic expression of cells in vivo.

Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of beta-glycerophosphate and ascorbic acid.

Matrix vesicles, media vesicles, and plasma membranes from three well-characterized, osteoblast-like cells (ROS 17/2.8, MG-63, and MC-3T3-E1) were evaluated for their content of enzymes capable of processing the extracellular matrix. Matrix vesicles were enriched in alkaline phosphatase specific activity over the plasma membrane and contained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collagenase, lysozyme, hyaluronidase, and tissue inhibitor of metalloproteinases (TIMP) were not found in any of the membrane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 micrograms/ml), beta-glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcification. Ascorbate or beta-glycerophosphate alone had no effect, but in combination produced significant increases in both active and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix vesicles being the most dramatic. This correlated with an increase in the formation of von Kossa-positive nodules. The results of the present study indicate that osteoblast-like cells produce matrix vesicles enriched in proteoglycan-degrading metalloproteinases. In addition, the observation that matrix vesicles contain significantly increased metalloproteinases under conditions favorable for mineralization in vitro lends support to the hypothesis that matrix vesicles play an important role in extracellular matrix processing and calcification in bone.

Gender-specific, maturation-dependent effects of testosterone on chondrocytes in culture.

This study examined the effects of testosterone on chondrocytes in vitro in order to determine whether the effects of testosterone were dependent on the stage of chondrocyte maturation and gender specific. Cells derived from male or female rat costochondral growth zone and resting zone cartilage were used as the cell culture model. [3H]Thymidine incorporation, cell number, alkaline phosphatase specific activity, and percent collagen production were used as indicators. Alkaline phosphatase specific activity in matrix vesicles and plasma membranes isolated from male and female chondrocyte cultures was measured to determine which membrane fraction was targeted by the hormone. The role of fetal bovine serum in the culture medium was also addressed. The results demonstrated that testosterone decreases cell number and [3H]thymidine incorporation in male chondrocytes, suggesting that it may promote differentiation of these cells. Alkaline phosphatase specific activity is stimulated in growth zone cells, with no effect on resting zone cells. The increase in enzyme activity is targeted to the matrix vesicles. Cells cultured in serum-free medium exhibit a dose-dependent inhibition of alkaline phosphatase activity when cultured with testosterone, even in the presence of phenol red. Testosterone-dependent stimulation of enzyme activity is seen only in the presence of serum, suggesting that serum factors are also necessary. Testosterone increased the percent collagen production in male cells only, regardless of the cartilage zone of origin. The results of this study indicate that the effects of testosterone are dependent on the time of exposure, presence of serum, and sex and stage of maturation of the chondrocytes. Testosterone-dependent stimulation of alkaline phosphatase specific activity is targeted to matrix vesicles.

Evidence for receptors specific for 17 beta-estradiol and testosterone in chondrocyte cultures.

Recently, sex hormones were shown to stimulate chondrocyte differentiation and matrix protein synthesis in vitro in a sex-specific and maturation-dependent manner. The aim of the present study was to determine whether cytosolic receptors in these cells would specifically bind 17 beta-estradiol and testosterone, and if so, whether binding was gender- and maturation-dependent. Confluent, fourth passage cultures of cells derived from male or female rat costochondral growth zone and resting zone cartilage were homogenized and specific binding of 17 beta-estradiol or testosterone measured in the cytosolic fraction. Scatchard analysis indicated the presence of a high-affinity 17 beta-estradiol receptor (Kd = 4.5 to 8.7 x 10(-11) M), with low binding capacity (3.9 to 11.2 fmol/mg protein). Chondrocytes from female rats were found to have a significantly greater binding capacity for 17 beta-estradiol than chondrocytes from male rats. However, cells from both sexes had binding capacities that were independent of cell maturation. A high-affinity testosterone receptor (Kd = 4.3 to 6.3 x 10(-11) M) with low binding capacity (4.1 to 5.9 fmol/mg protein) was found in both males and females, but no difference in binding capacity was noted, either as a function of gender or stage of cell maturation. Immunohistochemistry using antibodies against 17 beta-estradiol and testosterone and the 17 beta-estradiol nuclear receptor (D-75) confirmed that 17 beta-estradiol and testosterone receptors were present in chondrocytes from both male and female rats. These data demonstrate that chondrocytes from growth zone and resting zone cartilage are capable of binding both 17 beta-estradiol and testosterone. This suggests that these hormones mediate their direct effects on chondrocytes via receptors specific for their appropriate ligand. The sex-specific effects of 17 beta-estradiol may be due to differences in receptor number between chondrocytes derived from female and male rats. In contrast, the sex-specific effects of testosterone may be regulated at the post receptor level since no differences in binding capacity were found between males and females.

Nongenomic regulation of chondrocyte membrane fluidity by 1,25-(OH)2D3 and 24,25-(OH)2D3 is dependent on cell maturation.

1,25-(OH)2D3 and 24,25-(OH)2D3 regulate rat costochondral chondrocyte cultures in a metabolite-specific manner; 1,25-(OH)2D3 targets primarily growth zone cells (GC) and 24,25-(OH)2D3 targets primarily resting zone cells (RC). Some of the effects are nongenomic, since incubation of isolated membrane fractions with the metabolites results in regulation of enzyme activities comparable to that seen in culture. This study examined whether changes in membrane fluidity might be one mechanism involved in the nongenomic regulatory pathway. Chondrocyte cultures were incubated with the vitamin D metabolites and changes in plasma membrane fluidity monitored using the fluorophore, TMA-DPH, which is specific for membranes exposed to external fluids. Isolated matrix vesicles were also incubated directly with the metabolites and anisotropy of the membrane, as well as alkaline phosphatase-specific activity, determined. 1,25-(OH)2D3 caused a rapid and constant increase in alkaline phosphatase-specific activity in GC matrix vesicles; 24,25-(OH)2D3 caused an increase in RC matrix vesicle enzyme activity that was both dose- and time-dependent. Matrix vesicles produced by GC had a lower degree of fluidity than their parent plasma membranes or RC plasma membranes and matrix vesicles. Fluidity of the GC membrane fractions was increased by 1,25-(OH)2D3 in a dose- and time-dependent manner. 1,25-(OH)2D3 had no effect on the fluidity of the RC membranes. 24,25-(OH)2D3 caused a decrease in fluidity in GC at later time points. This metabolite caused an increase in fluidity of RC plasma membranes that returned to normal levels by 6 h; however, the increase induced in the matrix vesicles remained elevated throughout the 24-h experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)

Iodinated tomoxetine derivatives as selective ligands for serotonin and norepinephrine uptake sites.

In order to develop selective radioactive ligands for the study of presynaptic monoamine uptake sites, iodinated derivatives of tomoxetine were synthesized and evaluated in radioligand binding assays. Iodotomoxetine derivatives showed high affinity for serotonin (5-HT) uptake sites using a rat cortical membrane preparation. Compound 1R,(R)-(-)-N-methyl-3-(4-iodo-2-methylphenoxy)-3-phenylpropanamine , was the most potent and showed high stereoselectivity for 5-HT uptake sites (Ki, R isomer = 0.65 nM, S isomer = 13.9 nM). Changing the position of the methyl group or eliminating the methyl group at the phenoxy ring resulted in a loss of stereoselectivity. Substitution of the methyl group of tomoxetine with iodine gave the R and S isomers of N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine 4R and 4S. These compounds displayed stereoselectivity for the norepinephrine (NE) (Ki values = 0.24 and 9.35 nM for R and S isomers, respectively). The in vitro binding data suggest that 1R and 4R are potential radioiodinated ligands for pharmacological studies of 5-HT and NE uptake sites, respectively.

Stimulus-induced coordinate changes in mRNA abundance in single postsynaptic hippocampal CA1 neurons.

The molecular effects of use-dependent changes in synaptic transmission were studied in individual CA1 pyramidal neurons from rat hippocampal slices. Potentiation of excitatory postsynaptic currents was associated with coordinate changes in the relative abundance of several mRNAs 30 min to 3 hr after stimulation. There was a 300% increase in calcium/calmodulin-dependent protein kinase II mRNA levels concordant with a 50% decrease in protein kinase C beta 1 isoform mRNA. A 2-fold increase in zif-268 mRNA was seen, while increases in c-fos and c-jun mRNA levels were inconsistent, gamma-Aminobutyric acid A receptor beta 1 subunit mRNA levels increased 3-fold. Potentiation-induced changes were prevented by N-methyl-D-aspartate receptor blockade. Changes in mRNA abundance in individual cells, with synaptic and glial interactions intact, combine to produce a molecular fingerprint of a potentiated CA1 neuron.

[3H]opipramol labels a novel binding site and sigma receptors in rat brain membranes.

Opipramol (OP), a clinically effective antidepressant with a tricyclic structure, is inactive as an inhibitor of biogenic amine uptake. [3H]Opipramol binds saturably to rat brain membranes (apparent KD = 4 nM, Bmax = 3 pmol/mg of protein). [3H]Opipramol binding can be differentiated into haloperidol-sensitive and -resistant components, with Ki values for haloperidol of 1 nM (Bmax = 1 pmol/mg of protein) and 350 nM (Bmax = 1.9 pmol/mg of protein), respectively. The drug specificity of the haloperidol-sensitive component is the same as that of sigma receptors labeled with (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperdine. The haloperidol-resistant component does not correspond to any known neurotransmitter receptor or uptake recognition site. It displays high affinity for phenothiazines and related structures such as perphenazine, clopenthixol, and flupenthixol, whose potencies are comparable to that of opipramol. Because certain of these drugs are more potent at the haloperidol-resistant opipramol site than in exerting any other action, it is possible that this opipramol-selective site may mediate their therapeutic effects.

Pharmacologic profile of MDMA (3,4-methylenedioxymethamphetamine) at various brain recognition sites.

We report here an in vitro pharmacologic profile for MDMA (3,4-methylenedioxymethamphetamine) at various brain recognition sites. The rank order of affinities of MDMA at various brain receptors and uptake sites are as follows: 5-HT uptake greater than alpha 2-adrenoceptors = 5-HT2 serotonin = M-1 muscarinic = H-1 histamine greater than norepinephrine uptake = M-2 muscarinic = alpha 1-adrenoceptors = beta-adrenoceptors greater than or equal to dopamine uptake = 5-HT1 serotonin much greater than D-2 dopamine greater than D-1 dopamine. MDMA exhibited negligible affinities (greater than 500 microM) at opioid (mu, delta and kappa), central-type benzodiazepine, and corticotropin-releasing factor receptors, and at choline uptake sites and calcium channels.