U1 small nuclear RNA genes are subject to dosage compensation in mouse cells.

Multiple copies of a gene that encodes human U1 small nuclear RNA were introduced into mouse C127 cells with bovine papilloma virus as the vector. For some recombinant constructions, the human U1 gene copies were maintained extrachromosomally on the viral episome in an unrearranged fashion. The relative abundance of human and mouse U1 small nuclear RNA varied from one cell line to another, but in some lines human U1 RNA accounted for as much as one-third of the total U1. Regardless of the level of human U1 expression, the total amount of U1 RNA (both mouse and human) in each cell line was nearly the same relative to endogenous mouse 5S or U2 RNA. This result was obtained whether measurements were made of total cellular U1 or of only the U1 in small nuclear ribonucleoprotein particles that could be precipitated with antibody directed against the Sm antigen. The data suggest that the multigene families encoding mammalian U1 RNA are subject to some form of dosage compensation.

[Term-breech presentation: predictive factors of cesarean section for vaginal-birth failure]. / Présentation du siège à terme: facteurs prédictifs de césarienne en cours de travail.

OBJECTIVES: In our maternity ward, vaginal-breech birth is favoured when the protocol for patient's eligibility is respected. But in our practice, 20% patients have a cesarean section during labor for vaginal-birth failure, with a higher rate of neonatal and maternal morbidities. This study tried to consider if some obstetrical elements, not usually used, could help select the candidates in a more efficient way. PATIENTS AND METHODS: This retrospective study included all the singleton-breech deliveries with a gestational age over 37 weeks in our maternity ward from 1994 to 2004. An analysis of obstetrical elements, available before and during labour, has been carried out. RESULTS: Three hundred and seventy-six deliveries were indexed for a vaginal-birth trial. We carried out a cesarean section in 21.3% cases. We have found five obstetrical elements, not used in the vaginal birth protocol, which were significant risk factors for a cesarean section: nulliparity (OR=0.25 [0.11-0.57]), maternal height (OR=0.56 [0.30-1.03]), ultrasound estimated fetal weight (OR=7.76 [2.29-26.28]), fundal height (OR=3.9 [2.41-8.62]) and dystocia in first stage of labour (OR=4.97 [2.67-9.25]). DISCUSSION: A cesarean section during labor is responsible for a high-morbidity rate. We have to reduce this obstetrical event, especially in breech presentation, by a better selection of patients. From this study, we have created a statistical model to screen patients, but we have to lead a prospective study to validate it.

Synthetic human parathyroid hormone-like protein stimulates bone resorption and causes hypercalcemia in rats.

Parathyroid hormone-like adenylate cyclase-stimulating proteins (hACSPs) have been implicated as one of the calcemic, bone-resorbing agents in patients with humoral hypercalcemia of malignancy. We report the synthesis of an amino-terminal hACSP fragment, Tyr36 hACSP (1-36) amide. The synthetic hACSP is a potent agonist of renal membrane adenylate cyclase (Km, 1.7 X 10(-10)) and of bone cell adenylate cyclase (Km 1 X 10(-9)M). It is a potent bone-resorbing agent in vitro, stimulating 45Ca release from fetal rat long bones at a concentration of 10(-9) M. When infused via osmotic minipumps into rats, it is also a potent calcemic factor in vivo, inducing a rise in serum calcium from (mean +/- SD) 10.6 +/- 0.6 to 19.7 +/- 3.2 mg/dl when infused at 1.4 micrograms/h and from 9.9 +/- 0.7 to 11.4 +/- 1.2 mg/dl when infused at 0.14 micrograms/h. These findings indicate that biologically active hACSP fragments can be synthesized. One such synthetic peptide possesses the in vitro and in vivo bioactivities demonstrated in native, tumor-derived hACSPs. It is also a potent calcemic, bone-resorbing agent.

Identification of transcripts encoding a parathyroid hormone-like peptide in messenger RNAs from a variety of human and animal tumors associated with humoral hypercalcemia of malignancy.

The syndrome of humoral hypercalcemia of malignancy (HHM) appears to be mediated in many instances by a parathyroid hormone-like peptide, which has recently been purified, sequenced, and cloned. Using a probe representing the coding region of the human PTH-like peptide, we examined by Northern analysis poly (A)+ RNA from a variety of human and animal tumors associated with HHM. Hybridizing transcripts were identified in mRNA from each of 12 human and each of four animal HHM-associated tumors, with a complex hybridization pattern observed in the human mRNAs and a relatively simple pattern observed in the animal mRNAs. Poly (A)+ RNA prepared from tumors of similar histological types unassociated with HHM failed to hybridize with the probe. Messenger RNA-dependent biological activity from the animal tumors was entirely eliminated in a hybridization-arrest experiment using a complementary oligonucleotide spanning the region of homology between human PTH and the PTH-like peptide. These findings indicate that the PTH-like peptide is associated with the syndrome of HHM in a wide spectrum of tumor types from a variety of mammalian species and that the PTH-like sequence in the proximal amino terminus of the peptide is highly conserved.

Orientation-dependent transcriptional activator upstream of a human U2 snRNA gene.

We examined the structure of the promoter for the human U2 snRNA gene, a strong RNA polymerase II transcription unit without an obvious TATA box. A set of 5' deletions was constructed and assayed for the ability to direct initiation of U2 snRNA after microinjection into Xenopus oocytes. Sequences between positions -295 and -218 contain an activator element which stimulates accurate initiation by 20- to 50-fold, although as few as 62 base pairs of 5' flanking sequence are sufficient to direct the accurate initiation of U2 RNA. When the activator was recloned in the proper orientation at either of two different upstream locations, the use of the normal U2 start site was stimulated. Inversion of the element destroyed the stimulation of accurate U2 initiation, but initiation at aberrant upstream start sites was enhanced by the element in both orientations. A 4-base-pair deletion that destroyed the activity of the element lies within a sequence (region III) which is highly conserved among U2 genes from different organisms. Mutations in the activator also affected the ability of the U2 template to compete with a wild-type U1 gene in coinjection experiments. We propose that the element enhances the efficiency of transcription in part by facilitating the association of a limiting factor with transcription complexes. Human U1 snRNA genes possess a region homologous to U2 region III, and we suggest that upstream activator elements may be a general feature of snRNA promoters.

Osteosynthesis of periprosthetic type A and B femoral fractures using an unlocked plate with integrated cerclage cable and trochanteric hook: A multicenter retrospective study of 45 patients with mean follow-up of 20 months.

INTRODUCTION: Femoral periprosthetic fracture (FPF) is a frequent complication in dependent elderly persons, with a limited life expectancy. Their management is difficult and the choice between osteosynthesis and prosthesis is still matter of discussion. To date, there is no study on unlocked plate with integrated cerclage cable and trochanteric hook for this indication. The objectives of this study were to analyze fracture healing, complication rate and functional outcome. Our hypothesis is that this technique allows a high rate of consolidation and a return to the previous state in terms of autonomy and place of residence. MATERIALS AND METHODS: We conducted a retrospective multicenter study between 2010 and 2015. The inclusion criteria were: patients with type A and B FPF according to the classification of Vancouver who received osteosynthesis hook plate. The evaluation focused on the consolidation period, complications and pre and postoperative Parker and Katz scores. Death, nonunion, dislocation, infection and failure of fixation were considered major complications. RESULTS: Forty-five patients met the inclusion criteria and were evaluated at mean 20 months (6-72). All fractures consolidated at a mean 7 weeks (6-10), except one that has not undergone further surgery in the absence of functional impairment. Parker score decreased from 6.4 to 4.9 (p=0.03) and Katz score from 4.8 to 4.3 (p=0.045). Five patients died within the year of the operation. Five patients living at home preoperatively were admitted to an institution, the others returned to their retirement home or nursing home. CONCLUSION: This plate allows for a quick and effective management of patients with FPF. The low rate of complications and the very good consolidation rate lead us to use the same plate even for class B2 or B3 fractures in some patients with precarious health condition who cannot tolerate major revision surgery: Elderly, ASA score >3, loss of autonomy, Katz score <4.

Is amputation a viable treatment option in lower extremity trauma?

INTRODUCTION: There is currently no consensus on how to treat patients with lower extremity trauma. Should amputation be performed early on to avoid complications or should the limb be saved at any price? The goal of this study was to show that early amputation is a viable treatment option in lower extremity trauma cases. MATERIAL AND METHODS: Twenty patients who underwent early amputation and 16 patients who underwent limb-salvage were included with a minimum follow-up of 1year. The main endpoints were the Mangled Extremity Severity Score (MESS) used to predict amputation, complications, sequelae, bone union and functional outcomes. RESULTS: The amputees had a higher MESS score than those treated conservatively (7.8 vs. 4.9, P<0.00001), had a shorter hospital stay (P<0.022) and had fewer postoperative complications (P<0.003), especially infection-related (P<0.001). The prevalence of infection in limb-salvage patients was 61%. There was no significant difference between the two groups in terms of quality of life. DISCUSSION: In cases of lower extremity trauma, early amputation and limb-sparing treatment each have their advantages and disadvantages. Early amputation seems to be better in cases of complications, despite similar quality of life in the two groups in the long-term. It is a viable treatment option in cases of lower extremity trauma. Amputation must not be considered as a failure, but a deliberate choice due to the functional impact of complications that occur after limb-salvage. LEVEL OF EVIDENCE: Level IV study.

The concept of a waiting period for preoperative patient consent: Prospective study of 51 shoulder arthroscopy cases.

INTRODUCTION: The French Code of Public Health (CSP) does not explicitly require that patients should be given a certain amount of time to think about a procedure, except for cosmetic surgery, where 15 days is required (Art. L 6322-2 CSP). We hypothesized that patients require a waiting period during their decision-making process for scheduled shoulder arthroscopy procedure. MATERIALS AND METHODS: This prospective observational study of 51 patients analysed the concept of a waiting period based on a 10-item questionnaire. A comparative statistical approach was used and the P values were calculated using a paired Wilcoxon rank-sum test. RESULTS: Of the 51 patients, 42 (82%) rejected the concept of a waiting period before the procedure and 37 patients (73%) did not want a mandatory waiting period imposed by law. DISCUSSION: This study looked at the decision-making process during scheduled orthopaedic surgery and differentiated between the conscious and unconscious approach corresponding to an active and passive waiting period. A waiting period does not allow patients to make a conceptually deliberative decision that conforms to the criteria defined by the French Health Authority. This study rejects the need for a mandatory waiting period imposed on surgeons and patients as it does not integrate itself into the informative model of ethical decision-making for scheduled shoulder arthroscopy. TYPE OF STUDY: Prospective, observational; level of evidence IV.

Identification of an up-stream promoter of the human parathyroid hormone-related peptide gene.

Previous evidence has suggested that the human PTH-related peptide (PTHRP) gene uses two promoters, one a short down-stream element lying immediately between two 5' exons (1 and 2) and a second lying in an unknown up-stream location. We approached identification of the up-stream element in three steps. First, Northern analysis carried out using progressively 5' fragments of the gene as probes identified a candidate region some 2.5 kilobases up-stream of exon 1. Second, a battery of overlapping 5' cRNA probes was used in RNase protection experiments to identify two previously unrecognized exons, 212 and 93 basepairs in length (termed exons 1A and 1B to distinguish them from the previously designated exon 1, which was termed exon 1C). Third, primer extension experiments were performed with oligonucleotides complementary to each of the 5' exonic sequences. These experiments identified a transcription start site up-stream of exon 1A and also demonstrated that the 5' exons of the PTHRP gene could be spliced together in several combinations. The up-stream promoter element contains a TATA box, but does not otherwise resemble the down-stream PTHRP gene promoter or the PTH gene promoter. We conclude that the human PTHRP gene contains eight exons spanning more than 15 kilobases of genomic DNA, with promoter elements lying immediately up-stream of exons 1A and 2. The identification of these elements will permit functional analysis of their roles in mediating tissue- and tumor-specific PTHRP gene expression.

Two distinct tumor-derived, parathyroid hormone-like peptides result from alternative ribonucleic acid splicing.

A novel PTH-like peptide has recently been isolated and cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The deduced product of the initial clones to be reported is a 177 amino-acid protein, consisting of a 36 amino-acid precursor sequence followed by a 141 amino-acid mature peptide. Southern analysis of genomic DNA is compatible with a single-copy gene, but Northern analysis of mRNAs from both tumors and normal tissues consistently reveals multiple hybridizing transcripts, suggesting the possibility of alternative RNA splicing. We provide here direct evidence of alternative RNA splicing by the identification of a second cDNA clone in a human renal carcinoma cDNA library. As compared to the initial clone, this cDNA contains a foreshortened 5'-untranslated region but is otherwise identical until the distal portion of the coding region, at which point it diverges completely to encode a 166 amino-acid mature peptide with 27 amino acids of unique C-terminal sequence. The relative lengths of the primary translation products encoded by these two cDNAs were confirmed by transcription and translation in vitro, and both products were shown to be processed by added microsomes. The unique 3'-ends of these two cDNAs, as well as that of a third cDNA isolated by another laboratory, were used to identify one or more hybridizing transcripts corresponding to each cDNA in mRNA from a human renal carcinoma as well as in mRNA from normal human keratinocytes. We conclude that alternative RNA splicing results in mRNAs which encode multiple PTH-like peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid regulation of parathyroid hormone-related peptide gene transcription in a human neuroendocrine cell line.

A PTH-related peptide (PTHRP) has been identified and its cDNA cloned from tumors associated with the syndrome of humoral hypercalcemia of malignancy. The PTHRP and PTH genes appear to represent members of a gene family. Whereas the PTH gene is expressed exclusively in the parathyroids, the PTHRP gene appears to be widely expressed, but little is known concerning the regulation of its expression in any site. We studied the regulation of PTHRP gene expression in a human carcinoid cell line (NCI-H727) which has neuroendocrine features and also produces calcitonin, calcitonin gene-related peptide, and chromogranin-A. We found that the synthetic glucocorticoid triamcinolone produced time- and dose-dependent decreases in steady state PTHRP and calcitonin mRNA levels in NCI-H727 cells. This effect was blocked by the competitive glucocorticoid inhibitor RU-486. Messenger RNA stability and transcription run-off experiments revealed that triamcinolone decreased PTHRP and calcitonin expression by repressing the transcription rates of both genes.

Expression of messenger ribonucleic acids encoding a parathyroid hormone-like peptide in normal human and animal tissues with abnormal expression in human parathyroid adenomas.

A novel PTH-like peptide has recently been purified and cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. We surveyed the expression of mRNAs encoding this peptide in normal tissues by Northern analysis. One or more low abundance hybridizing transcripts was identified in poly(A)+ RNA prepared from human keratinocytes, thyroid, bone marrow, and fibroblasts, from bovine hypothalamus, pituitary, parathyroid, adrenal cortex, and adrenal medulla, and from rat brain, stomach mucosa, and fetal but not adult liver. One or more hybridizing transcripts was also identified in poly(A)+ RNA prepared from a number of established lines, including rat pituitary (GH4), rat pheochromocytoma (PC 12), human osteosarcoma (TE-85), and human medullary carcinoma (TT) cells. Northern analysis of mRNAs from abnormal human parathyroid tissue revealed an overexpression of transcripts for the PTH-like peptide which appeared to be specific for adenomatous or autonomous glands. These findings suggest that the PTH-like peptide is expressed in a number of endocrine and nonendocrine tissues, that it is developmentally expressed in at least one tissue (fetal liver), and that the regulation of its expression is abnormal in human parathyroid adenomas.

[Obesity and primiparity: Risky delivery?]. / Obésité et primiparité: accouchement à risque ?

OBJECTIVES: To evaluate the risk of materno-foetal complications in obese primiparous women (Body Mass Index (BMI)≥30) MATERIALS AND METHODS: A retrospective study was conducted in our tertiary referral labour ward from 1st January 2009 to 31st December 2010, including primiparous women delivering living cephalic singleton pregnancies after 37 weeks of amenorrhea. Two groups were compared: obese patients (BMI≥30) and non-obese (BMI<30). Obstetrical and neonatal data were collected. RESULTS: Among 1636 primiparous women, 132 (8%) had a BMI≥30. Induction of labor, prolonged pregnancy and post-partum hemorrhage were significatively more frequent in obese group (P<0.001). Those patients have an increased risk of cesarean section (P<0.001). Mean birth weight of newborns from obese mothers is significatively higher (3493g vs 3265g, P<0.001), as is the frequency of macrosomia (16.7% vs 5.2%, P<0.001). CONCLUSION: Obesity is associated with obstetrical complications in primiparous obese women, who are to be considered risky patients at their arrival in the labour ward, and specific obstetrical management must be planned to improve maternal and neonatal outcomes.

[Active management of the second twin for vaginal delivery: ruptured versus intact membranes. Apropos a series of 182 patients]. / Accouchement manœuvrier du deuxième jumeau: membranes rompues versus membranes intactes. À propos d'une série de 182 patientes.

AIM: The vaginal management of the second twin (T2) differs throughout our country. In 2009, the French National College of Gynecologists and Obstetricians released practice guidelines, with low-level evidence, encouraging active management of the second twin, using maneuvers with intact membrane. PATIENTS AND METHODS: In our level III labour ward, these maneuvers are systematically performed when the second twin is in a breech or transverse presentation and after ruptured membranes in most cases. We studied 182 twin pregnancies with active management of the T2 at more than 28 weeks of gestation, from 1st January 1996 to 31st December 2010, by comparing the membrane status during delivery of T2. RESULTS: The results did not show any significant differences between the two groups of patients concerning the neonatal or maternal results. The technique is feasible, even by residents. Our results show that total breech extraction of the second twin with ruptured membranes is feasible, which allows for reappraisal of the national recommendations. CONCLUSION: In our practice, it is possible to actively deliver the T2 with ruptured membranes without altering maternal and fetal prognosis and thus simplifying learning for young doctors.

Expression of transcripts encoding a parathyroid hormone-related peptide in abnormal human parathyroid tissues.

A PTH-related peptide (PTHRP) has been identified and its cDNA cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The human PTHRP gene has been recently isolated and found to be a complex transcriptional unit using multiple promoters and containing alternatively spliced 3' exons which result in three mRNA classes, each class encoding a PTHRP with a unique carboxy-terminus. The PTHRP gene appears to be expressed in a number of normal tissues, and PTHRP transcripts have been previously reported to be overexpressed in a small sample of human parathyroid adenomas. In the present study we surveyed RNA prepared from a total of 60 abnormal human parathyroid glands for PTHRP gene expression using a combination of Northern blotting and RNase protection techniques. Apparent overexpression of PTHRP mRNA was observed in two thirds of parathyroid adenomas, whereas no overexpression was found in 7 examples of sporadic primary hyperplasia, 5 examples of secondary hyperplasia, and 3 examples of parathyroid carcinoma. Apparent overexpression was also observed in 1 of 4 cases of multiple endocrine neoplasia type 1, 1 of 2 examples of multiple endocrine neoplasia type 2, and 1 gland considered to represent tertiary hyperparathyroidism. Northern analysis of poly(A)+ RNA prepared from three representative adenomas using region-specific probes indicated that two putative promoters are used and revealed a pattern of preferential splicing of transcripts to include the most distal 3' exon. These findings suggest that the PTHRP gene is commonly overexpressed in adenomatous parathyroid glands, but not in sporadic primary hyperplasia, that this overexpression does not seem to be dependent on the use of a single specific promoter, and that adenomatous parathyroid cells appear to preferentially use one of several alternative splicing pathways. It is presently not known whether PTHRP is secreted by abnormal parathyroid tissues and, if so, in what form.

Synthetic and partially-purified adenylate cyclase-stimulating proteins from tumors associated with humoral hypercalcemia of malignancy inhibit phosphate transport in a PTH-responsive renal cell line.

Hypophosphatemia and hyperphosphaturia characteristically occur in patients with humoral hypercalcemia of malignancy (HHM). To determine if a tumor product causes these abnormalities in phosphate metabolism, rather than, for example, hypercalcemia, we investigated the effect of partially-purified adenylate cyclase-stimulating activity (ACSA) from human and animal HHM-associated tumors on sodium-dependent phosphate transport (Na PiT) in a PTH-responsive renal epithelial cell line. Thirty minute exposure to 7 X 10(-10) MbPTH (1-34) equivalents of ACSA from the human and animal tumors, reduced NaPiT by 20% and 14%, respectively. We also recently isolated an adenylate cyclase-stimulating protein (hACSP) from two human tumors associated with HHM and identified a cDNA clone for this protein which encodes a 141 amino-acid peptide. Based on the deduced amino-acid sequence, we synthesized tyr36 (1-36) hACSP. This synthetic peptide induced a 22% decrease in the initial rate of NaPiT by the epithelial monolayer. Its inhibitory activity was roughly equipotent to that of bPTH (1-34). We conclude that the ACSP derived from HHM-associated tumors decreases phosphate transport in renal epithelial cells. This peptide appears to play a key role in mediating the changes in phosphate metabolism in this syndrome.

The orir to ori+ mutation in spontaneous yeast petites is accompanied by a drastic change in mitochondrial genome replication.

The orir petite mutants of Saccharomyces cerevisiae show a very low level of suppressivity (5-12%; suppressivity is the percentage of diploid petites issued from a cross of the parental haploid petite with a wild-type cell), indicating a poor replication efficiency of their mitochondrial genome. The latter is made up of repeat units containing two inverted ori sequences and arranged as tandem pairs in inverted orientation relative to their nearest neighbors. After subcloning orir petites or crossing with wild-type cells a large number of ori+ petites are found in the progeny. In contrast to the orir petites, from which they are derived, these ori+ petites are characterized by high suppressivity levels (approx. 90%) and contain mitochondrial genomes made up of tandem repeat units containing single ori sequences. The structural changes underlying the orir to ori+ mutation are therefore accompanied by a dramatic increase in suppressivity, indicating that the elimination of inverted ori sequences causes a drastic change from very poor to very good replicative efficiency in the mitochondrial genome. Finally, crosses of ori0 petites with wild-type cells were also studied; the results obtained have clarified the reasons for the high frequency of petites having genomes similar to those of orir petites after mutagenesis with ethidium bromide.

Structure of the mouse gene encoding parathyroid hormone-related peptide.

The parathyroid hormone-related peptide (PTHRP) was initially isolated from tumors associated with the syndrome of humoral hypercalcemia of malignancy. The human PTHRP gene is a complex transcriptional unit which uses multiple promoters and contains alternatively spliced 3' exons that result in mRNAs encoding three different deduced products. We report here the structure of the mouse PTHRP gene. The mouse gene has a considerably simpler organization than its human counterpart. This organization includes a single 3' exon and an apparent single 3' splicing pathway, leading to an mRNA encoding a 139-amino acid mature PTHRP. In addition, the mouse gene appears to be predominantly under the control of a short proximal promoter element. By RNase protection analysis, we identified PTHRP mRNA in specimens prepared from a variety of normal rodent tissues, including a number of tissues not previously recognized as sites of PTHRP gene expression.

The mitochondrial genomes of spontaneous orir petite mutants of yeast have rearranged repeat units organized as inverted tandem dimers.

We have investigated the structure and organization of the mitochondrial genomes of two related orir (ori-rearranged) spontaneous petite mutants of Saccharomyces cerevisiae. In these mutant genomes every repeat unit contains an inverted terminal duplication harboring a second (inverted) ori sequence, and tandem pairs of repeat units alternate with tandem pairs in inverted orientation. We have shown that orir genomes are organized as the genomes with inverted repeat units of ethidium bromide (EtBr)-induced petites, and we have clarified the mechanism by which such mutant mitochondrial genomes arise.