Effect of sitting position on equal-dose spinal anaesthetic for caesarean section and post-partum tubal ligation.

BACKGROUND: We studied the hypothesis that an equal spinal anaesthetic dose administered in the sitting position to patients undergoing post-partum tubal ligation (PPTL) and caesarean section (CS) would yield similar sensory block characteristics and analgesic efficacy. METHODS: This prospective, non-randomised trial recruited 20 women undergoing PPTL within 48 h of vaginal delivery and 20 undergoing CS. Spinal anaesthesia comprising intrathecal hyperbaric bupivacaine 12 mg and morphine 100 µg was administered at L3/4 with patients sitting. Our primary end point was the maximal dermatomal sensory block (to cold). RESULTS: Baseline demographics were comparable, but PPTL patients had greater parity, with mean ± standard deviation 17.54 ± 11.2 h from delivery to spinal anaesthesia, and shorter duration of surgery, 17.54 ± 11.2 vs. 40.3 ± 15.5 min. Similar maximal sensory blocks (to cold) were achieved in group PPTL vs. CS, T4 (T1-T5) vs. T3 (T1-T5), P = 0.104, in comparable times, 8.6 ± 2.6 vs. 7.6 ± 3.0 min, P = 0.267. PPTL patients had significantly faster two-segment block regression (70.7 ± 23.5 vs. 97.6 ± 23.9 min, P = 0.001) and to T10 (120.8 ± 35.6 vs. 145.1 ± 24.3 min, P = 0.016), with less hypotension (25% vs. 65%, P = 0.025) and phenylephrine (20.0 ± 60.6 µg vs. 120.0 ± 119.6 µg, P = 0.005). CONCLUSION: The same dose of hyperbaric bupivacaine 12 mg and morphine 100 µg administered in the sitting position to both PPTL and CS parturients yielded similar maximal sensory blocks, but PPTL exhibited faster block regression and less hypotension/vasopressor requirement.

Contributors to surgical in-patient satisfaction--development and reliability of a targeted instrument.

OBJECTIVE: Patient satisfaction is an important indicator of healthcare system performance. High patient satisfaction is associated with greater trust in caregivers, improved compliance with treatment recommendations and a better quality of life (QOL). There are few validated instruments to measure surgical patients satisfaction. The aim of this study was to develop a culturally-specific patient satisfaction instrument, for use as an outcome measure in evaluating surgical services. DESIGN: Patient focus groups were convened to explore dimensions of the peri operative hospital experience. Forums uncovered pertinent domains of interest and identified terminology understood by patients. A preliminary set of items reflecting patient satisfaction was developed. Test-retest reliability of a new surgical patient satisfaction instrument was assessed in 42 subjects at hospital discharge. RESULTS: Domains that emerged included; admission processes and hospital environment, information provision, nursing care, doctor and nurse interaction, and ancillary staff services. Staff attitudes and human qualities were highly valued, as was prompt attention to requests for assistance. Clarity or quality of medical information did not appear to influence in-patient satisfaction. A new measure of surgical patient satisfaction, Hong Kong Index of Inpatient Happiness (HK2Happ), was developed from focus group consultation. Test-retest generated an Intra Class Correlation of 0.868-0.935, indicating a highly stable tool. CONCLUSION: The initial version of HK2Happ was reliable in assessing surgical patient satisfaction. The measure is now undergoing validity testing across different surgical patient populations for generalization and generation of a short form of discriminant items.

Laparoscopic radical prostatectomy: single centre experience after 5 years.

OBJECTIVE: To summarise our experience of laparoscopic radical prostatectomy in a single centre in Hong Kong over 5 years. DESIGN: Retrospective study. SETTING: Urology Division, Department of Surgery, Tuen Mun Hospital, Hong Kong. PATIENTS: A total of 87 patients who underwent laparoscopic radical prostatectomy from March 2002 to May 2007. MAIN OUTCOME MEASURES: Peri-operative data and follow-up information. RESULTS: The operative procedure used entailed Montsouris technique and its modifications, including the latest method involving the extraperitoneal descending technique. In all, 87 patients underwent the operation; in two, the procedure was converted to open surgery. Peri-operative parameters which showed improvement included: operating time, blood loss, resort to blood transfusions, and the complication rate. There was no operation-related mortality. In organ-confined disease, a clear surgical margin was achieved in 93% of the patients, but in those whose disease was not organ-confined, the positive margin rate was 87%. Among patients with organ-confined disease, 13% had evidence of biochemical recurrence. Hormonal therapy was started in five patients, none of whom died during the follow-up period (mean, 24 months). Continence recovered in 69% of the patients by 6 months and in 92% by 12 months post-surgery. Assessment of erectile function before and after the surgery was problematic and estimated to be 20% among patients having the nerve-sparing procedure performed. CONCLUSION: Although Hong Kong has a relatively low incidence for prostate cancer, it was possible to develop laparoscopic radical prostatectomy with acceptable early results. Further follow-up is warranted before formulating definitive conclusions about this procedure.

Molecular cloning, expression, localization, and gene organization of PTX1, a human nuclear protein that is downregulated in prostate cancer.

A cDNA, designated PTX1, has been isolated by subtractive hybridization on the basis that it is expressed in normal prostate but not in prostate carcinoma. The full-length cDNA was subsequently established by 5' and 3' RACE. Nucleotide sequence analysis of the 5'- and 3'-RACE clones yielded a composite cDNA of 1327 bp, which predicted a protein of 377 amino acid residues with a putative nuclear import signal (RRLNRKK) at its N terminus. The PTX1 gene was localized to human chromosome 12 and was found to be ubiquitously expressed. A segment of the cDNA was expressed in E. coli to produce a fragment of the PTX1 protein for the generation of specific antibodies. The resulting antibodies detected a 73-kDa protein in both nuclear and cytoplasmic extracts of prostate, although the level in the cytoplasmic extract was much lower. Using immunohistochemical analysis, the PTX1 protein was localized mainly in the nuclei of glandular epithelia of normal prostate. The nuclear staining was greatly reduced in prostate carcinoma. The gene organization of PTX1 was established by comparing the cDNA sequence with the published human genomic sequence.

Structural cassette mutagenesis in a de novo designed protein: proof of a novel concept for examining protein folding and stability.

The solution to the protein folding problem lies in defining the relative energetic contributions of short-range and long-range interactions. In other words, the tendency of a stretch of amino acids to adopt a final secondary structural fold is context dependent. Our approach to this problem is to address whether an amino acid sequence, a "cassette," with a defined secondary structure in the three-dimensional structure of a native protein, can adopt a different conformation when placed into a different protein environment. Thus, we designed de novo a disulfide-bridged two-stranded alpha-helical parallel coiled coil, where each polypeptide chain consisted of 39 residues, as a "cassette holder." The 11-residue cassette would be inserted into the center of each polypeptide chain between the two nucleating alpha-helices to replace the control sequence. This Structural Cassette Mutagenesis model permits the analysis of short-range interactions within the inserted cassette as well as long-range interactions between the nucleating helices and the cassette region. The cassette holder, with a control sequence as the cassette, had a GdnHCl transition midpoint during denaturation of 5.6M. To demonstrate the feasibility of our model, an 11-residue beta-strand cassette from an immunoglobulin fold was inserted. The cassette was fully induced into the alpha-helical conformation with a [GdnHCl]1/2 value of 3.2M. To demonstrate the importance of short-range interactions (beta-sheet/alpha-helical propensities of amino acid side chains) in modulating structure and stability, a series of 1-5 threonine residues (highest beta-sheet propensity) were substituted into the solvent-exposed portions of the cassette in the alpha-helical conformation. Each successive substitution systematically decreased the stability of the coiled coil with peptide T4b (4 Thr residues) having a [GdnHCl]1/2 value of 2.2M. The single substitution of Ile in the hydrophobic core of the cassette with Ala or Thr had the most dramatic effect on protein stability (peptide 120T, [GdnHCl]1/2 value of 1.4M). Though these substitutions were able to modulate stability, they were not able to disrupt the alpha-helical conformation of the cassette, showing the importance of the nucleating alpha-helices on either side of the cassette in controlling conformation of the cassette. We have demonstrated the feasibility of our model protein to accept a beta-strand cassette. The effect of cassettes containing other beta-strands, beta-turns, loops, regions of undefined structure, and helical segments on conformation and stability of our model protein will also be determined.

Rcho-1 trophoblast cell placental lactogens: complementary deoxyribonucleic acids, heterologous expression, and biological activities.

In this report, we have investigated placental lactogens (placental lactogen-I, PL-I; PL-I variant, PL-Iv; PL-II) expressed by differentiated Rcho-1 trophoblast cells. A complementary DNA (cDNA) library to differentiated Rcho-1 trophoblast cells was constructed and screened with probes to detect PL-I and PL-II. Sequence analysis of three independent Rcho-1 PL-I cDNAs indicated that they significantly differed from the previously reported PL-I sequence but more closely resembled a related cDNA referred to as PL-I mosaic (PL-Im). Upon further analysis, Rcho-1 PL-I/PL-Im transcripts could be detected in Rcho-1 trophoblast cells and normal developing placental tissue; however, the previously reported PL-I transcript could not be identified from the same sources. Given these results, we examined the original PL-I cDNA by PCR and nucleotide sequence analyses. The sequence differed from the original report and was found to be identical to the Rcho-1 PL-I and PL-Im cDNA clones. Thus, PL-I, Rcho-1 PL-I, and PL-Im are equivalent and should be referred to as PL-I. The PL-I gene was localized to chromosome 17 of the rat genome, similar to other PRL family members. Rcho-1 PL-II cDNAs were identical to the published PL-II sequence. PL-Iv cDNAs were isolated from differentiated Rcho-1 cells via an RT-PCR strategy and found to be identical to previously isolated PL-Iv cDNAs. Rcho-1 PL-I and PL-II cDNAs were subcloned into the pcDNA3 expression vector and recombinant protein produced in HRP-1 cells. Both recombinant Rcho-1 PL-I and PL-II proteins significantly stimulated the proliferation of lactogen-dependent rat Nb2 lymphoma cells and mouse mammary epithelial cells. In summary, we show that the Rcho-1 PL-I corresponds to PL-Im and Rcho-1 PL-Iv and PL-II are identical to their previously described placental counterparts. Additionally, both recombinant Rcho-1 PL-I and PL-II proteins are biologically active.

Placental lactogen-I variant utilizes the prolactin receptor signaling pathway.

Placenta lactogen-I variant (PL-Iv) is a member of a family of proteins expressed by the rat placenta with characteristics similar to prolactin (PRL). In this report, we present the molecular cloning, chromosomal localization, and heterologous expression of PL-Iv. Nucleotide sequence analysis of the PL-Iv cDNA clone predicted a precursor protein of 223 amino acids, including a 28-amino acid signal sequence. The PL-Iv gene was localized to chromosome 17 of the rat genome, which also carries other members of the PRL gene family. PL-Iv heterologously expressed in Chinese Hamster ovary (CHO) cells exhibited similar immunoreactive and electrophoretic characteristics with PL-Iv produced by the rat placenta. N-terminal sequencing verified the identity and purity of the recombinant PL-Iv species and the site of cleavage of the signal peptide from the mature secreted PL-Iv species. Recombinant PL-Iv was shown to bind to ovarian and liver PRL receptors, stimulate the proliferation of Nb2 lymphoma cells, and activate Jak2. Each of these actions is consistent with PL-Iv utilizing the PRL receptor signal transduction pathway.

Molecular cloning and sequence analysis of the cDNA encoding porcine acrosin inhibitor.

A full-length cDNA encoding the porcine acrosin inhibitor has been isolated from a boar seminal vesicle cDNA library. Nucleotide sequence analysis of the 667-bp cDNA predicts a precursor protein of 97 amino acid residues, which includes a 26-residue signal peptide and a 71-residue secreted protein. The predicted amino acid sequence of the mature protein agrees completely with that of the sperm-associated acrosin inhibitor determined by conventional amino acid sequence analysis. However, the asparagine/aspartic acid and glutamine/glutamic acid substitutions, as reported in the seminal plasma counterpart, have not been observed. Southern blot analysis shows only a single hybridizing band with three different restriction endonucleases, suggesting the presence of a single copy of the acrosin inhibitor gene in the porcine genome.

Transcriptional activation of cytochrome P450 side chain cleavage enzyme expression during trophoblast cell differentiation.

The purpose of this study was to investigate P450scc expression during trophoblast differentiation. Biochemical characteristics of P450scc protein and mRNA identified in rat trophoblast tissues were similar to those identified in the rat adrenal gland. Furthermore, P450scc was localized to trophoblast giant cells. This observation prompted an examination of progesterone biosynthesis and P450scc expression in Rcho-1 cells. Rcho-1 cells were derived from a transplantable rat choriocarcinoma, their differentiation can be regulated, and they have the capacity to express the trophoblast giant cell phenotype. Progesterone was produced by Rcho-1 cells and increased approximately 100-fold as the cells progressed from proliferation to differentiation. P450scc protein and mRNA accumulation also increased during trophoblast differentiation. P450scc expression within the Rcho-1 cell line was restricted to trophoblast giant cells. To further investigate the regulation of P450scc expression during trophoblast differentiation, we examined a plasmid construct, containing 894 base pairs of DNA 5' upstream from the P450scc transcriptional start site linked to a human growth hormone reporter gene, following stable transfection into Rcho-1 cells. The transfected P450scc regulatory DNA permitted the expression of human growth hormone which paralleled expression of the endogenous P450scc gene. In conclusion, transcriptional activation of the P450scc gene accompanies trophoblast giant cell differentiation.

Heterologous expression and characterization of prolactin-like protein-A. Identification of serum binding proteins.

In this report, we describe the heterologous expression of prolactin-like protein-A (PLP-A) in Chinese hamster ovary (CHO) cells, the characterization of recombinant PLP-A, and the identification of serum PLP-A-binding proteins. CHO cell and native placental PLP-A showed similar immunoreactive characteristics and electrophoretic mobilities. N-terminal sequencing verified the identity and purity of the recombinant PLP-A species and the site of cleavage of the signal peptide from the mature secreted PLP-A species. Recombinant PLP-A lacked activity in standardized prolactin and growth hormone in vitro bioassays. Antibodies generated to recombinant PLP-A facilitated the cellular localization of PLP-A and the identification of high molecular weight PLP-A complexes. Cross-linking analyses of radioiodinated PLP-A with serum harvested from late gestation rats indicated the presence of two major cross-linked complexes migrating under reducing conditions at 130 and 250 kDa and two minor cross-linked complexes migrating at 70 and 110 kDa. Binding of PLP-A to serum proteins was specific for PLP-A and not effectively competed by other members of the prolactin/growth hormone family. The PLP-A binding species were also found in serum from non-pregnant female and male rats.

Decidual prolactin-related protein. Identification, molecular cloning, and characterization.

In this report, we describe the isolation, molecular cloning, and characterization of a new member of the prolactin (PRL)-growth hormone (GH) family expressed in rat decidual tissue. A 29-kDa protein was isolated from medium conditioned by decidual explants. The protein possessed an affinity for concanavalin A and cross-reactivity with antibodies to two rat placental proteins, PRL-like protein-B (PLP-B) and PLP-C and with antibodies to human PRL. NH2-terminal sequencing of the isolated decidual protein indicated that it shared significant sequence identity with the NH2 terminus of PLP-C. The decidual protein was termed decidual prolactin-related protein (dPRP). A PLP-C cDNA was used to identify dPRP cDNAs from a rat decidual cDNA library. Nucleotide sequence analyses of the dPRP cDNAs predicted a mature protein of 239 amino acids, including a 28-amino acid signal sequence. The predicted dPRP amino acid sequence contains two putative N-linked glycosylation sites and 6 cysteine residues. The 6 cysteines are located in positions homologous to the cysteines of PLP-C and PRL. Additional sequence similarities with members of the PRL-GH family are evident. The dPRP gene was localized to rat chromosome 17, which also carries other members of the PRL gene family. Northern blot analysis showed that the dPRP cDNA clone specifically hybridized to a 1.0-kilobase mRNA. The relationship of dPRP with other members of the PRL-GH family and its putative role(s) in the physiology of pregnancy are discussed.

Recombinant porcine prorelaxin produced in Chinese hamster ovary cells is biologically active.

Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studied so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay.

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.

Molecular cloning and characterization of prolactin-like protein C complementary deoxyribonucleic acid.

In this report, we describe the isolation and characterization of a full length cDNA clone for rat prolactin-like protein C (PLP-C) and describe the expression of PLP-C mRNA in the developing rat placenta. Nucleotide sequence analysis of the PLP-C cDNA clone predicted a mature protein of 238 amino acids, including a 30-amino acid signal sequence. The predicted PLP-C amino acid sequence contains seven cysteine residues, three tryptophan residues, and two putative N-linked glycosylation sites. Six of the cysteine residues in PLP-C are located in positions homologous to the cysteines of pituitary prolactin (PRL). Additional sequence similarities with pituitary PRL and other members of the rat placental PRL family are evident. The PLP-C gene was localized to rat chromosome 17. Northern blot analysis showed that the PLP-C cDNA clone specifically hybridized to a 1.0-kilobase mRNA. PLP-C mRNA was first detectable between days 13 and 14 of gestation, peaked by day 18 of gestation, and remained elevated until term. In situ hybridization analysis indicated that PLP-C mRNA was specifically expressed by spongiotrophoblast cells and some trophoblast giant cells in the junctional zone region of rat chorioallantoic placenta.

Identification and characterization of a new member of the prolactin family, placental lactogen-I variant.

This report describes the identification and characterization of a new member of the placental prolactin (PRL) family, termed placental lactogen-I variant (PL-Iv). PL-Iv was isolated from medium conditioned by late gestation placental explants. Rat PL-Iv was found to be closely related to rat PL-I. Amino-terminal sequence analysis indicated that PL-Iv shared approximately 88% sequence identity with the amino terminus of PL-I. PL-Iv proteins cross-reacted with antiserum to recombinant mouse PL-I and PL-Iv mRNA hybridized with a PL-I cDNA. Multiple PL-I and PL-Iv species were present in placental cytosol. Despite the structural similarities between PL-I and PL-Iv, distinct differences were also evident. Antibodies generated to the amino-terminal 19 amino acids of PL-Iv specifically recognized PL-Iv, while failing to recognize PL-I. Secreted PL-Iv had an affinity for concanavalin A, whereas secreted PL-I lacked affinity for the lectin. PL-I was predominantly secreted as a 36-40-kDa species and PL-Iv was predominantly secreted as a 33-kDa species. Furthermore, PL-I and PL-Iv were synthesized at different times during gestation and by different cell types. PL-I was synthesized by trophoblast giant cells during the first half of gestation, while PL-Iv was predominantly synthesized by spongiotrophoblast cells during the later stages of gestation. PL-Iv was shown to stimulate the proliferation of rat Nb2 lymphoma cells, an in vitro measure of lactogenic activity. In summary, PL-Iv shares structural similarities with PL-I; however, it shows other structural differences in addition to unique cell- and temporal-specific patterns of expression in the rat chorioallantoic placenta.

Transplantable rat choriocarcinoma cells express placental lactogen: identification of placental lactogen-I immunoreactive protein and messenger ribonucleic acid.

The purpose of this investigation was to evaluate the abilities of a transplantable rat choriocarcinoma (Rcho) to produce placental PRLs. The Rcho tumor was analyzed biochemically and histologically for the expression of placental PRLs. Expression of placental PRL mRNAs was determined by Northern blot and in situ hybridization analyses. Expression of placental PRL proteins was determined by Western blot and immunocytochemical analyses. Histologically, Rcho tumors were characterized by the appearance of giant cell surrounding hemorrhagic regions. Female rats bearing the Rcho tumor beneath their kidney capsule showed extensive mammary gland development. The Rcho tumors expressed placental lactogen-I (PL-I) mRNA and protein, but there was no evidence of placental lactogen-II (PL-II), PRL-like protein-A (PLP-A), or PRL-like protein-B (PLP-B). Rcho PL-I mRNA and proteins migrated as a 1-kilobase species and a 36- to 40-kDa species similar to those expressed by normal rat trophoblast tissues. The cell type responsible for Rcho PL-I production was the giant cell, similar to that observed in normal rat trophoblast tissues. In summary, we have demonstrated the production of PL-I by a transplantable rat choriocarcinoma (Rcho). The Rcho tumor resembles rat trophoblast tissue at early postimplantation stages (days 6-10 of gestation) and may be a useful tool for studying placental PRL expression during trophoblast differentiation.