Developmental changes in skin collagen biosynthesis pathway in posthatch male and female chickens.

The developmental changes in skin collagen biosynthesis pathway in male and female chickens were evaluated. Concentration of collagen, levels of mRNA for collagen type I subunits and for lysyl hydroxylase, and the level of three lysyl oxidase-derived cross-links: dehydro-dihydroxylysinonorleucine (DHLNL), dehydro-hydroxylysinonorleucine (HLNL), and dehydro-histidinohydroxymerodesmosine (HHMD) were determined during 4 wk posthatching. Skin collagen content increased with age and was higher in males than in females. In both sexes, the expression of the genes coding for alpha 1 and alpha 2 of collagen type I decreased with age: alpha 1(I) gene expression decreased from Day 3 onwards, whereas the reduction in alpha 2(I) gene expression started 1 wk later. At all ages examined, the expression of both genes was higher in male than in female skin. Males and females lysyl hydroxylase gene expression remained low until Day 16, after which an increase in the enzyme gene expression was observed. An increase in skin HLNL content was observed from Day 3 in both sexes reaching a peak in males at Day 9 and in females 1 wk later. The DHLNL content, which was higher in males than in females at all ages tested, dramatically decreased in both male and female skin from 3 d of age, reaching its lowest level at Day 16, and remained at that low level thereafter. The skin content of HHMD in males and females followed an oscillatory behavior with higher peaks in the male skin. The results suggest that the higher tensile strength of male skin than female skin may be due to the elevated skin collagen content that resulted from increased expression in collagen type I genes on the one hand, and from the higher amounts of various collagen cross-links on the other.

Synthesis and phosphorylation of osteopontin by avian epiphyseal growth-plate chondrocytes as affected by differentiation.

The regulation of synthesis and phosphorylation of osteopontin in relation to avian epiphyseal growth-plate chondrocyte differentiation was studied in situ and in culture. Osteopontin gene expression was evaluated in the tibia growth-plate of 3-week-old chickens by in situ hybridization. The gene was expressed mainly at the lower hypertrophic zone where cartilage matrix is calcified and endochondral bone formation is initiated. Within the hypertrophic region, a poorly labeled area separated the layer of osteopontin-positive hypertrophic chondrocytes from those associated with endochondral bone formation. In culture, proliferative chondrocytes show no alkaline phosphatase activity in contrast to ascorbic acid-treated chondrocytes which display the enzyme activity. Chondrocytes not treated with ascorbic acid, exhibited lower levels of osteopontin mRNA than the treated cells. The phorbol ester TPA--an activator of protein kinase C--and to a lesser extent FGF but not EGF, stimulated osteopontin gene expression. Chondrocytes secreted low levels of phosphorylated osteopontin to the medium. EGF treatment resulted in the appearance of phosphorylated osteopontin in the medium, without affecting the synthesis of other proteins. FGF and TGF beta, but not IGF-I or IGF-II, also caused phosphorylation of osteopontin. Ascorbic acid-treated chondrocytes secreted higher levels of phosphorylated osteopontin than the non-treated cells, but addition of FGF or TPA did not stimulate osteopontin phosphorylation any further. Parathyroid hormone caused a dose-dependent attenuation of osteopontin phosphorylation and inhibited the EGF-dependent osteopontin phosphorylation. The results suggest that osteopontin gene expression and phosphorylation in chondrocytes are regulated by separate mechanisms. The response to the various controlling agents varies with the state of differentiation. Both processes--the synthesis and phosphorylation of osteopontin--are under the control of local growth factors which are involved in bone growth and calcification.

Cell damage by excess CuZnSOD and Down's syndrome.

Down's Syndrome (DS), the phenotypic expression of human trisomy 21, is presumed to result from overexpression of certain genes residing on chromosome 21 at the segment 21q22-the Down locus. The "housekeeping" enzyme CuZn-superoxide dismutase (CuZnSOD) is encoded by a gene from that region and its activity is elevated in DS patients. Moreover, the recent discovery that familial ALS is associated with mutations in the gene encoding CuZnSOD, focused attention on the entanglement of oxygen-free radicals in cell death and neuronal disorders. To investigate the involvement of CuZnSOD gene dosage in the etiology of the syndrome we have developed both cellular and animal models which enabled us to investigate the physiological consequences resulting from overexpression of the CuZnSOD gene. Rat PC12 cells expressing elevated levels of transfected human CuZnSOD gene were generated. These transformants (designated PC12-hSOD) closely resembled the parental cells in their morphology, growth rate, and response to nerve growth factor, but showed impaired neurotransmitter uptake. The lesion was localized to the chromaffin granule transport mechanism. These results show that elevation of CuZnSOD activity interferes with the transport of biogenic amines into chromaffin granules. Since neurotransmitter uptake plays an important role in many processes of the central nervous system, CuZnSOD gene-dosage may contribute to the neurobiological abnormalities of Down's Syndrome. As an approach to the development of an animal model for Down's Syndrome, several strains of transgenic mice which carry the human CuZnSOD gene have been prepared. These animals express the transgene as an active enzyme with increased activity from 1.6 to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. To investigate the contribution of CuZnSOD gene dosage in the neuropathological symptoms of Down's Syndrome, we analyzed the tongue muscle of the transgenic-CuZnSOD mice. The tongue neuromuscular junctions (NMJ) in the transgenic animals exhibited significant pathological changes; withdrawal and destruction of some terminal axons and the development of multiple small terminals. The ratio of terminal axon area to postsynaptic membranes decreased, and secondary folds were often complex and hyperplastic. The morphological changes in the transgenic NMJ were similar to those previously seen in the transgenic NMJ and were similar to those previously seen in muscles of aging mice and rats as well as in tongue muscles of patients with Down's Syndrome. The findings suggest that CuZnSOD gene dosage is involved in the pathological abnormalities of tongue NMJ observed in Down's Syndrome patients.(ABSTRACT TRUNCATED AT 400 WORDS)

cis-acting DNA elements involved in oocyte-specific expression of mouse sperm receptor gene mZP3 are located close to the gene's transcription start site.

We report that cis-acting DNA elements involved in oocyte-specific expression of the mouse sperm receptor gene (mZP3) are located close to the gene's transcription start site. Mice bearing a transgene that consists of only 153 nt of mZP3 5'-flanking region fused to the firefly luciferase gene (153-ZP3/LUC) expressed the reporter gene in ovary not in a wide variety of tissues; although two of three lines carrying 153-ZP3/LUC also expressed the transgene in forebrain and hypothalamus. Within the ovaries of transgenic mice, luciferase activity was restricted to growing oocytes. However, levels of luciferase activity in these oocytes were lower than those in oocytes from mice bearing transgenes that contain a larger segment of mZP3 5'-flanking region (470-6,500 nt) fused to the firefly luciferase gene. Mice bearing a transgene that consists of 470 nt of mZP3 5'-flanking region and mZP3 intragenic sequences (ZDT) were also analyzed. The presence of mZP3 intragenic sequences did not result in significantly increased levels of firefly luciferase activity in oocytes of mice carrying the ZDT transgene. Overall, these results suggest that as little as 153 nt of mZP3 5'-flanking region is sufficient to target expression of the firefly luciferase gene to mouse oocytes and that the mZP3 intragenic sequences probably do not contain enhancer elements. Rather, enhancer elements are probably present between-153 and -470 nt of the mZP3 5'-flanking region.

Growth hormone receptors in avian epiphyseal growth-plate chondrocytes.

Growth hormone receptor (GH-R) gene expression was evaluated in avian growth-plate cartilage by Northern blot and hybridization using the avian GH-R probe. A single transcript of approximately 5.2 kb was demonstrated in cultured growth-plate chondrocytes as well as in growth-plate extracts. GH receptor gene expression was inhibited by chicken GH (cGH) in a dose- and time-dependent manner. Chicken GH was more potent in down-regulating the GH-R gene expression than hGH, but on the other hand cGH exhibited a lower affinity to avian chondrocytes receptor than did the human hormone. Addition of ascorbic acid to the culture media caused cell differentiation: induction of alkaline phosphatase activity and attenuation of collagen type II gene expression. No differences in the GH-R gene expression were observed in the nondifferentiated cells compared with the differentiated cells. Chicken GH did not form any complex with the purified hGH binding protein (hGHBP), did not bind to human lymphocytes GH receptor, and did not affect Nb2 cell proliferation. These systems represent somatogenic and lactogenic types of GH receptors, respectively. In summary, avian growth-plate chondrocytes in situ and in culture exhibit GH-R and these receptors are capable of binding GH. Thus, the failure of GH to affect avian chondrocytes' proliferation was not due to either the absence of receptors on the cell membrane or to a lack in its binding activity, but rather may be due to events farther downstream.

A mouse oocyte-specific protein that binds to a region of mZP3 promoter responsible for oocyte-specific mZP3 gene expression.

The gene encoding mZP3, the mouse sperm receptor, is expressed exclusively in growing oocytes during oogenesis. To investigate the molecular basis of oocyte-specific mZP3 gene expression, we generated several lines of mice harboring a transgene that contains 470 bp of mZP3 gene 5'-flanking sequence (nucleotides -470 to +10) fused to the firefly luciferase gene coding region. Three of four expressing transgenic lines exhibited luciferase activity only in growing oocytes, suggesting that the 470-bp fragment is sufficient to direct Iocyte-specific expression of the luciferase gene. Results of DNase I footprinting and gel mobility shift assays suggested the presence of an ovary-specific protein that binds to a small region (nucleotides-99 to -86) within the 470-bp fragment of the mZP3 promoter, with 5'-G(G/A)T(G/A)A-3' representing the minimal sequence required for binding. Southwestern (DNA-protein) gel blots revealed the presence of an oocyte-specific, approximately 60,000-Mr protein, called OSP-1, that binds to the minimal sequence. Changes in levels of OSP-1 during oogenesis and early cleavage are consistent with the pattern of mZP3 gene expression during these developmental stages in mice. Therefore, OSP-1 may be a mammalian oocyte-specific transcription factor involved in regulating oocyte-specific mZP3 gene expression.

Down syndrome clinical symptoms are manifested in transfected cells and transgenic mice overexpressing the human Cu/Zn-superoxide dismutase gene.

Down syndrome (DS), the phenotypic expression of human trisomy 21, is presumed to result from overexpression of certain genes residing on chromosome 21 at the segment 21q22-the Down locus. The "housekeeping" enzyme CuZn-superoxide dismutase (CuZnSOD) is encoded by a gene from that region and its activity is elevated in DS patients. To investigate the possible involvement of CuZnSOD gene dosage in the etiology of the syndrome we have developed both cellular and animal models which enabled us to investigate the physiological consequences resulting from overexpression of the CuZnSOD gene. 1. Rat PC12 cells expressing elevated levels of transfected human CuZnSOD gene were generated. These transformants (designated PC12-hSOD) closely resembled the parental cells in their morphology, growth rate, and response to nerve growth factor, but showed impaired neurotransmitter uptake. The lesion was localized to the chromaffin granule transport mechanism. We found that the pH gradient (delta pH) across the membrane, which is the main driving force for amine transport, was diminished in PC12-hSOD granules. These results show that elevation of CuZnSOD activity interferes with the transport of biogenic amines into chromaffin granules. Since neurotransmitter uptake plays an important role in many processes of the central nervous system, CuZnSOD gene-dosage may contribute to the neurobiological abnormalities of Down's syndrome. 2. As an approach to the development of an animal model for Down syndrome, several strains of transgenic mice that carry the human CuZnSOD gene have been prepared. These animals express the transgene in a manner similar to that of humans, with 0.9 and 0.7-kilobase transcripts in a 1:4 ratio, and synthesize the human enzyme in an active form capable of forming human-mouse enzyme heterodimers. CuZnSOD activity is increased from 1.6 to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. 3. To investigate the possible involvement of CuZnSOD gene dosage in the neuropathological symptoms of Down's syndrome, we analyzed the tongue muscle of the transgenic mice that express elevated levels of human CuZnSOD. The tongue neuromuscular junctions (NMJ) in the transgenic animals exhibited significant pathological changes, namely, withdrawal and destruction of some terminal axons and the development of multiple small terminals. The ratio of terminal axon area to postsynaptic membrane decreased, and secondary folds were often complex and hyperplastic. The morphological changes in the transgenic NMJ were similar to those previously seen in muscles of aging mice and rats as well as in tongue muscle of patients with Down's syndrome.(ABSTRACT TRUNCATED AT 400 WORDS)

Diminished serotonin uptake in platelets of transgenic mice with increased Cu/Zn-superoxide dismutase activity.

Reduced levels of the neurotransmitter serotonin in blood platelets is a clinical symptom characteristic of individuals with Down's syndrome. To investigate the possible involvement of the Cu/Zn-superoxide dismutase (CuZnSOD) gene, which resides at the Down locus on chromosome no. 21, in the etiology of that symptom, we examined blood platelets of transgenic mice harboring the human CuZnSOD gene. It was found that platelets of transgenic CuZnSOD animals, which overexpress the transgene, contain lower levels of serotonin than nontransgenic littermate mice, due to a reduced rate of uptake of the neurotransmitter by the dense granules of the platelets. We found that the pH gradient (delta pH) across the dense granule membrane, which is the main driving force for serotonin transport, was diminished in dense granules of transgenic-CuZnSOD. Furthermore, a significantly lower than normal serotonin accumulation rate was also detected in dense granules isolated from blood platelets of Down's syndrome individuals. These findings suggest that CuZnSOD gene dosage is affecting the dense granule transport system and is thereby involved in the depressed level of blood serotonin found in patients born with Down's syndrome.

Premature aging changes in neuromuscular junctions of transgenic mice with an extra human CuZnSOD gene: a model for tongue pathology in Down's syndrome.

We examined the tongue muscles in several strains of transgenic mice carrying the human Zn-Cu superoxide dismutase (CuZnSOD) gene. The presence of the extra gene was confirmed in mated progeny and the gene product activity was measured in the tongue and found to be much higher than in normal littermate controls. Using electron microscopic morphometry, the neuromuscular junctions of the transgenic mice showed significant changes resembling excessive aging, with atrophy, degeneration, withdrawal, and sometimes destruction of the terminal axons, as well as the development of multiple small terminals. The myofibers showed little change except for slight hypertrophy and an increased variability in size. They also had more megamitochondria, fat droplets and lipofuscin bodies. Excess CuZnSOD generates H2O2 and hydroxyl radicals which affect both NMJ membranes and plasticity, and which may produce premature aging. The findings resemble those observed in tongues of patients with Down's syndrome, in whom an extra CuZnSOD gene is present as part of the trisomy of chromosome 21.

Down's syndrome: abnormal neuromuscular junction in tongue of transgenic mice with elevated levels of human Cu/Zn-superoxide dismutase.

To investigate the possible involvement of Cu/Zn-superoxide dismutase (CuZnSOD) gene dosage in the neuropathological symptoms of Down's syndrome, we analyzed the tongue muscle of transgenic mice that express elevated levels of human CuZnSOD. The tongue neuromuscular junctions (NMJ) in the transgenic animals exhibited significant pathological changes, namely, withdrawal and destruction of some terminal axons and the development of multiple small terminals. The ratio of terminal axon area to postsynaptic membrane decreased, and secondary folds were often complex and hyperplastic. The morphological changes in the transgenic NMJ were similar to those previously seen in muscles of aging mice and rats as well as in tongue muscle of patients with Down's syndrome. The findings suggest that CuZnSOD gene dosage is involved in the pathological abnormalities of tongue NMJ observed in Down's syndrome patients.

The immune system during the pre-cancer and the early cancer period. IL-2 production by PBL from post-menopausal women with and without endometrial carcinoma.

We report on alterations in IL-2 production and cell proliferation following PHA stimulation of peripheral blood lymphocytes (PBL) from stage-I endometrial carcinoma (EC) patients, and on mechanisms involved in these alterations. Our study includes 3 groups: EC patients, post-menopausal women at high risk of developing EC, and age-matched healthy women. IL-2 production was markedly lower in most EC patients than in healthy controls. Varying levels of IL-2 were produced by PBL from women in the high-risk group. The proliferative response of PBL to PHA appeared to correlate with levels of IL-2 production. Our results suggest that macrophages are involved, in part, in the modulation of T-cell functions of EC patients.