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We studied bone mineral content (BMC), bone mineral density (BMD), cortical thickness/total width (CT/TW) ratio and cortical area/total area (CA/TA) ratio in boys with constitutional delay of puberty and the effect of short-term testosterone treatment on bone mass. Seventeen boys (age 13.1-15.8 years) who met the family history and the clinical criteria of constitutional delay of puberty were selected and enrolled in the study. All subjects were eating a diet assuring an adequate intake of calories and calcium. A subset of 8 boys (group A) was treated with testosterone depot (100 mg/month x 6 months) while 9 boys (group B) were not. At inclusion, BMC and BMD were reduced in the patients according to their chronological age (BMC -4.04 +/- 1.34 standard deviation scores [SDS]; BMD -2.95 +/- 0.56 SDS), statural age (BMC -1.75 +/- 0.79 SDS; BMD -1.69 +/- 0.78 SDS), and bone age (BMC -1.80 +/- 0.65 SDS; BMD -1.86 +/- 0.68 SDS). No significant differences between the groups were found (group A: BMC 0.480 +/- 0.57 g/cm, BMD 0.488 +/- 0.037 g/cm2, CT/TW ratio 0.43 +/- 0.4, CA/TA ratio 0.68 +/- 0.04; group B: BMC 0.476 +/- 0.060, p = NS vs. group A; BMD 0.491 +/- 0.036 g/cm2, p = NS vs. group A). At 12 months of follow-up, BMC, BMD, CT/TW ratio, and CA/TA ratio significantly increased in group A (BMC 0.70 +/- 0.13 g/cm, delta +41.1 +/- 28.8%, p < 0.003 vs. 0 month; BMD 0.617 +/- 0.082 g/cm2, delta +26.2 +/- 13.6%, p < 0.005 vs. 0 month; CT/TW ratio 0.52 +/- 0.05, delta +20.59 +/- 10.65%, p < 0.001 vs. 0 month; CA/TA ratio 0.77 +/- 0.05 vs. 0 month; CT/TW ratio 13.60 +/- 6.65%, p < 0.004 vs 0 month), but not in group B (BMC: 0.48 +/- 0.05 g/cm; delta +5.1 7.8%, p = NS vs. 00 month; BMD: 0.492 +/- 0.037 g/cm2; delta +0.54 +/- 8.7%, p = NS vs. 0 month; CT/TW ratio 0.44 +/- 0.04, delta +4.04 +/- 6.75%, p = NS vs. 0 month; CA/TA ratio 0.68 +/- 0.05, delta +2.39 +/- 5.90%, p = NS vs. 0 month). We conclude that boys with constitutional delay of puberty have reduced BMC and BMD. The delay in statural and bone ages did not totally account for the decreased bone mass. Testosterone treatment for 6 months significantly increased BMC, BMD, CT/TW ratio, and CA/TA ratio in these patients, but definitive conclusions on the efficacy of the treatment in improving adult bone mass can be drawn only when our patients reach early childhood.

Growth hormone-insulin-like growth factor-I status and response to growth hormone therapy (0.6 IU/kg/week sc, six times a week for 12 months) were evaluated in 12 girls (chronological age 9.4 +/- 1.6 years) suffering from central precocious puberty with growth velocity less than 4 cm/year and no substantial increase or decrease in predicted adult height during gonadotropin releasing hormone Bn-RH) analogue treatment (D-Trp6-LH-RH, 60 micrograms/kg im/28 days). At baseline, large variations were observed in nocturnal growth hormone (GH) means (pathological values stimulated levodopa GH peaks (pathological values (< 10.0 micrograms/l) 28.6%) and serum insulin-like growth factor-I (IGF-I) levels. Neither GH-nor IGF-I levels were correlated with growth velocity. During recombinant GH therapy, growth velocity increased significantly (baseline 3.0 +/- 0.9 cm/year; 6 months 6.4 +/- 1.9 cm/year, p < 0.001 versus baseline; 12 months 6.0 +/- 1.3 cm/year, p < 0.0001 versus baseline). There was a significant increase in height SDS for bone age (baseline -1.6 +/- 0.5 SDS; 12 months -1.04 +/- 0.6 SDS; p < 0.002) and in predicted adult height (baseline 152.0 +/- 3.6 cm; 12 months 155.9 +/- 3.4 cm; p < 0.002). Our results suggest that combined therapy with Gn-RH analogues and recombinant GH can improve growth velocity and predicted adult height in girls with central precocious puberty and impaired height prognosis during Gn-RH analogue treatment.

The object of this study was to determine whether increased circulating levels of parathyroid hormone-related protein (PTH-rp) may explain the increased parathyroid hormone (PTH) bioactivity in pregnancy. In 41 healthy pregnant women (age 19-41 years), PTH-rp and corrected calcium levels were measured and compared with those of nonpregnant control women (n = 18, age 20-39 years). PTH-rp and corrected calcium levels were significantly higher in pregnant women (PTH-rp 21.9 +/- 7.9 pg/ml, P < 0.001; corrected calcium 2.38 +/- 0.07 mmol/liter, P = 0.001) than in nonpregnant women (PTH-rp 10.3 +/- 7.8 pg/ml; corrected calcium 2.30 +/- 0.10 mmol/liter). Our data indicate that circulating PTH-rp levels may significantly increase in pregnancy, suggesting a possible role of this peptide in the modification of calcium homeostasis in pregnant women.

In this report we confirm that the putative vitamin D response element (VDRE), located between -320 and -306 in the chicken calbindin-D28K gene, is not a binding site for the vitamin D3 receptor (VDR). In examining the ability of chicken intestinal nuclear extracts (CINE) to bind known VDREs, we observed a specific VDRE-binding activity, which is distinct from VDR. In fact, VDR-depleted CINE retains the ability to bind the rat osteocalcin VDRE. The VDRE-binding activity binds DNA with high affinity and contacts it at the same guanine residues as VDR. Its specificity in binding structural variants of the AGGTCA repeat is broader than that of VDR, as direct repeats spaced by 3, 4, and 5 base pairs are almost equally effective competitors when added to the probe in molar excess. Palindromic arrangements of the same motif are lower affinity competitors. The retinoid-X receptor is involved in the binding complex, as incubation of CINE with antibody to retinoid-X receptor results in a quantitative supershift. Antibodies to retinoic acid receptors (RAR alpha and -beta), T3 receptor, or chicken ovalbumin up-stream promoter-transcription factor had no apparent effect. These data suggest that species specificity is a relevant aspect of VDR/VDRE recognition, and that a novel factor(s), different from VDR, might be involved in the effect of vitamin D on gene expression.

Gonadal steroids drive the significant bone mineral increase that occurs at puberty. Oestrogen deprivation in women results in bone loss. We investigated bone mineralization by single photon absorptiometry in girls with central precocious puberty (n = 13, age 3.8-8.5 years) before and during 1 year of treatment with gonadotropin releasing hormone analogue (GnRH-a = long-acting D-Trp6-GnRH, 60 micrograms i.m. every 28 days). Before GnRH-a therapy, bone mineral density (BMD) was significantly higher in patients than in ten control girls matched for chronological age (patients 0.575 +/- 0.097 g/cm2, controls 0.433 +/- 0.049 g/cm2, P < 0.001). Patient BMD was not significantly different from that of ten control girls matched according to patient bone, age (0.550 +/- 0.046 g/cm2, P = NS). During GnRH-a treatment, pituitary-gonadal axis was suppressed and patient BMD significantly decreased (6 months: -6.0%, P < 0.002 vs baseline; 12 months: -8.0%, P < 0.001 vs baseline). We conclude that in girls with precocious puberty the activation of gonadal steroid secretion induces an increase in bone mineralization and that oestrogen deprivation by GnRH-a treatment caused a significant decrease in BMD.

We studied, in vivo, the effects of a calcitriol-containing ointment (3 micrograms of calcitriol per gram of petrolatum) topically applied on skin lesions in children affected by psoriasis vulgaris. Each patient was instructed to apply the ointment, about 1 g once a day at bedtime followed by occlusive dressing with plastic wrap, on one-side lesion (treated) and petrolatum alone on the equivalent controlateral site (placebo) (total weekly dose of calcitriol: approximately 21 micrograms/patient). After 4 weeks of topical treatment all children showed a complete clearing of their skin lesions on the treated side, without appreciable changes on the placebo side. Serum ionised calcium, calcium and calcitriol levels or urinary calcium excretion did not vary significantly throughout the period of therapy. These findings suggest that topical calcitriol may be an effective and safe alternative therapy for psoriasis in children.

Growth velocity and serum aminoterminal propeptide of type III procollagen (P-III-NP) were evaluated in 11 girls (age 3.8-8.5 years) with central precocious puberty during luteinizing hormone releasing hormone (LH-RH) analogue treatment (D-Trp6-LH-RH, 60 micrograms/kg im for 28 days, n = 7; D-ser(TBU)6-LH-RH, 1600 micrograms/day intranasally, n = 4). Before treatment, growth velocity (10.2 +/- 1.9 cm/year) and P-III-NP concentrations (12.8 +/- 4.3 micrograms/l) were in the pubertal range. During therapy, growth velocity significantly decreased to the prepubertal levels. P-III-NP concentrations decreased significantly after six months of therapy (7.9 +/- 3.7 micrograms/l, p < 0.001). Three girls with low growth velocity ( < 4 cm/year), stimulated growth hormone peak < 10 micrograms/l, and altered 12-h nocturnal growth hormone secretion at 12 and/or 18 months of treatment, had a more marked decrease in P-III-NP concentrations (patient 3: -65.9%; patient 5: -58.7%; patient 10: -61.0%) after 6 months of therapy. Our results suggest that LH-RH analogue treatment in central precocious puberty may impair growth. In these cases, measurement of serum P-III-NP levels may be an additional marker to monitor growth.

Intestinal cells from chicken embryos were grown in chemically defined, serum-free medium. The majority of cultured cells exhibits an epithelial-like morphology. As demonstrated by indirect immunofluorescence, the epithelial cells, and not the contaminating fibroblasts, express Calbindin-D28K only after 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, is added to the culture medium. The highly sensitive reverse transcriptase-polymerase chain reaction shows that both Calbindin-D28K mRNA and the corresponding primary unprocessed transcripts (pre-mRNA) are dramatically increased in cultured intestinal cells treated with 1,25-dihydroxyvitamin D3, thus indicating that Calbindin-D28K is induced by the increased rate of transcription of the corresponding gene.

Northern blot hybridization experiments showed that Apolipoprotein A-I (Apo A-I) mRNA is present at high concentration in chicken myotubes cultured in vitro, while it is virtually absent in fibroblasts and myoblasts. Myotubes are also capable of translating and secreting in the culture medium a protein which is specifically immunoprecipitated by anti-Apo A-I antibodies and has the same electrophoretic mobility as Apo A-I purified from circulating high-density lipoproteins. The appearance of Apo A-I mRNA in myotubes depends on the transcriptional activation of the corresponding gene, as it was shown by hybridizing 32P-labeled RNA synthesized in isolated nuclei to Apo A-I cDNA. The activation of the Apo A-I gene is regulated by the muscle cell coordinately with muscle-specific genes. In fact, treatment with TPA, a powerful inhibitor of differentiation, efficiently prevents myoblasts from producing Apo A-I mRNA, as well as muscle actin mRNA, and causes myotubes to quickly cease Apo A-I mRNA synthesis. The existence of a strict relationship between Apo A-I mRNA concentration and myogenic cell differentiation was also confirmed by experiments with quail myoblasts transformed with a temperature-sensitive mutant of the Rous Sarcoma Virus. Cells raised at the permissive temperature (undifferentiated phenotype) do not contain Apo A-I as well as alpha-actin mRNAs, while shifting to the nonpermissive temperature (differentiated phenotype) causes a rapid increase in Apo A-I and alpha-actin mRNA concentration.

A transcribed RNA sequence encompassing the junction between the first intron and the second exon of the chicken calbindin D28K gene was copied in a cDNA fragment and subsequently amplified by polymerase chain reaction. When intestinal RNA is used as template, the appearance of the 161 bp amplified fragment is strictly dependent on the vitamin D status of the animal. In fact no amplified fragment is obtained when the RNA is extracted from the intestine of vitamin D-deficient chickens, while it is easily detected when the RNA is extracted only 30 min after injection with 1,25-dihydroxycholecalciferol. Conversely, the amplified fragment is obtained, irrespectively of the vitamin D status of the animal, when the RNA template is extracted from the brain. The appearance of unspliced RNA sequences upon vitamin D induction is followed, after a 30 min lag, by the appearance of the corresponding mature mRNA sequences.

The gene for vimentin, an intermediate-filament protein, is growth regulated. We used Southern blot analysis and in situ chromosome hybridization to determine the location of the human vimentin gene. Our results show that there is only one copy of the vimentin gene and that it is located on the short arm of chromosome 10 (10pter-10q23) close to the interleukin-2 receptor gene, which is also growth regulated. In situ hybridization studies suggest that the most likely location of the vimentin gene is 10p13. Sequence similarities and homologies of human vimentin to other genes are presented.

The present work describes the construction of a cDNA library in pBR322 plasmid from an mRNA population enriched for the intestinal calcium-binding protein (CaBP) mRNA of the chick. We report the isolation of one recombinant clone containing a vitamin D-regulated sequence, which is complementary to part of the CaBP mRNA. Northern blot hybridization experiments allowed us to identify a 1900 nucleotide RNA species as the CaBP mRNA.

A full-length chicken apolipoprotein A-I (apoAI) cDNA has been cloned into an expression vector, pRSVapoAI. This plasmid was transfected into a monkey kidney (COS-1) cell line in order to study apolipoprotein-lipid assembly. Chicken apoAI is the major apolipoprotein of chicken high-density lipoprotein (HDL), which is less complex in apolipoprotein content than the HDL of human plasma. The transient transfected COS-1 cells synthesized and secreted authentic plasma apoAI. Under serum-free medium conditions, COS cells secreted only proapoAI. A small portion (15%) of the secreted apoAI floated at a density 1.07-1.20 g/ml. Upon incubation with fetal bovine serum at 10 degrees C, a majority of the apoAI was recovered in the HDL density (1.06-1.20 g/ml) region. Secreted apoAI was labeled when transfected COS cells were incubated with [U-14C]palmitate, but the incorporation of radioactivity was not the result of fatty acid acylation through ester bond formation. These results indicate that heterologous COS-1 cells are capable of synthesizing and secreting apoAI, and that intracellular association of apoAI with lipids is not necessary for secretion.

We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.

The cytosol fraction of rat heart contains an initiation factor-like protein component that behaves like the eukaryotic factor (eIF-2) in binding [35S]-Met-tRNAf in the presence of GTP. The ternary initiator complex thus formed is able to bind to heart ribosomes. In the left ventricle of rat heart undergoing hypertrophic growth upon constriction of the descending aorta, the [35S]-Met-tRNAf binding activity of the cytosol protein(s) gradually increases after the operation from 40%, at 48 h, to 90%, at 10 days; slightly lower activation is seen in the [35S]-Met-tRNAf binding to ribosomes. Polysomal RNA is extracted from sham operated and hypertrophic rat hearts and translated in a reticulocyte cell free system. The translation products are analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis: no clearcut qualitative difference is observed in the pattern obtained from sham operated and hypertrophic animals.

We have identified in a human cDNA library a clone (hp2F1) whose cognate RNA is growth-regulated. The insert has been sequenced and the nucleotide sequence shows a strong homology to the nucleotide sequences of the ADP/ATP carrier cDNA and gene, respectively, isolated from Neurospora crassa and Saccharomyces cerevisiae. The putative amino acid sequence of hp2F1 shows an 87% homology to the amino acid sequence of the ADP/ATP carrier from beef heart mitochondria. We conclude that the insert of hp2F1 contains the full coding sequence of a human ADP/ATP carrier. The steady-state RNA levels of the ADP/ATP carrier are growth-regulated. They increase when quiescent cells are stimulated by serum, platelet-derived growth factor, or epidermal growth factor, but not by platelet-poor plasma or insulin. RNA levels of the ADP/ATP carrier decrease instead when growing HL-60 cells are induced to differentiate by either phorbol esters or retinoic acid.

A BamHI-HindIII restriction fragment containing the 5'-terminal portion of the gene encoding chicken Calbindin D28K was sequenced and used as a probe in Northern-blot hybridizations to RNA extracted from the brain and intestine of chickens at various stages of development. In both tissues Calbindin D28K mRNA consists of a family of three species, which differ by size. In the intestine Calbindin D28K mRNAs appear at hatching and reach a peak at day 7. In the brain the same RNA species are easily detected at least 7 days before hatching, show a moderate increase at hatching and remain essentially constant during the first 10 days of adult life. The concentration of Calbindin D28K mRNAs in the intestine is strictly dependent on Vitamin D, while it is not in the cerebellum.

When WI-38 human diploid fibroblasts become confluent, they stop synthesizing DNA and dividing. Addition of serum causes the quiescent cell to reenter the cell cycle. Prolonged quiescence after confluence decreases and delays the response to serum. For a few days after reaching confluence, WI-38 cells also respond to platelet-poor plasma. During this period, although not cycling, WI-38 cells still express c-myc and other growth-regulated genes, as measured by steady-state RNA levels. If the quiescence is prolonged further, c-myc expression (and that of two other growth-regulated genes) is no longer detectable, and its disappearance coincides with a loss of response to platelet-poor plasma. These results suggest that, also under physiological conditions, the expression of c-myc and other growth-regulated genes can cooperate with platelet-poor plasma in inducing cellular DNA synthesis in human diploid fibroblasts.

Calcyclin was originally defined as a cDNA clone (2A9) whose cognate RNA is growth-regulated and whose sequence shows strong similarities to the sequences of the S-100 protein, a calcium-binding protein, as well as to a subunit of the major cellular substrate for tyrosine kinase. Using the full-length cDNA, we have now isolated from a human genomic library several phages containing calcyclin sequences. One of the phages, ch. 28-10, contains the entire calcyclin gene, plus extensive flanking sequences. The calcyclin gene is a unique copy gene and has 3 exons. The 5' flanking sequence has been characterized, both structurally and functionally. Besides a TATA box, it contains, in the region proximate to the cap site, GC boxes and a sequence with a strong homology to the enhancer core of the SV40 promoter. Other enhancer-like elements are found scattered in both the 5' and 3' flanking regions. The proximate 5' flanking region is very active in driving the transient expression of linked reporters in transfection experiments. Finally, the calcyclin gene has been localized to the long arm of human chromosome 1, near the ski oncogene.

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.