[Impact of hyperoxia on the phenotype of pulmonary artery smooth muscle cells].

Objective: To investigate the influence of varied oxygen (O2) concentration environments on the phenotypic transformation of pulmonary artery smooth muscle cells (PASMC) and the mechanism of pulmonary hypertension. Methods: Primary rat PASMC were isolated and cultured through the process of enzymatic digestion. Following identification, the stable passaged PASMC were subjected to a 6-hour incubation in sealed containers with normal O2 content (group C) and relative O2 content comprising 55% (group H55), 75% (group H75), and 95% (group H95). mRNA and protein expression of α-Actin (α-SMA), smooth muscle 22α (SM22α), osteopontin (OPN), and matrix metalloproteinase-2 (MMP-2) were measured using real-time quantitative PCR and western blot analysis. Results: The H55 group displayed no significant difference from the C group in terms of mRNA and relative protein expression levels for α-SMA, SM22α, OPN, and MMP-2 (all P>0.05). On the other hand, groups H75 and H95 exhibited a reduction in mRNA and relative protein expression of α-SMA and SM22α, along with an increase in mRNA and relative protein expression of OPN and MMP-2 when compared with both the C and H55 groups (all P<0.05). The H95 group showed a higher relative mRNA expression of MMP-2 as compared to the H75 group (P<0.05). Conclusions: Oxygen concentration environments of 75% or higher can serve as the foundation for the pathogenesis of pulmonary hypertension, essentially by inducing a phenotypic transformation in PASMC towards adopting a robust secretory function. This induction is contingent upon the concentration of oxygen present.

[Preoperative dynamic contrast-enhanced MRI can reduce the rate of tumor-positive resection margins after breast conserving surgery in patients with early non-mass breast carcinoma].

Objective: To investigate the value of preoperative dynamic contrast-enhanced MRI in reducing the rate of tumor-positive resection margins after breast conserving surgery in patients with early non-mass breast carcinoma. Methods: Seventy-two patients with early non-mass breast carcinoma received ultrasonographic and mammographic examination and subsequently underwent dynamic contrast-enhanced MRI examination before breast conserving surgery. The control group consisted of 74 patients who had early non-mass breast carcinoma. They only received ultrasonographic and mammographic examination and didn't undergo contrast-enhanced MRI examination. The comparison of the rate of tumor-positive resection margins between two groups was performed. The MRI findings that had the significant influence on the rate of tumor-positive resection margins were analyzed using Logistic regression model. Results: In 28 patients (28/72, 38.9%), dynamic contrast-enhanced MRI could correct or supplement the ultrasonographic and mammographic findings and resulted in the reasonable change of surgical program. The preoperative MRI examination group (n=30) had lower rate of tumor-positive resection margins than control group for invasive ductal carcinoma (23.3% vs 40.0%, P=0.02), but there was no significant difference (21.4% vs 26.9%, P=0.10) between two groups for ductal carcinoma in situ (n=28). The preoperative MRI examination group (n=14) had lower rate of tumor-positive resection margins than control group for the other pathologic types of breast carcinoma (14.3% vs 38.9%, P=0.02). The statistical analysis on the basis of Logistic regression model showed that some main MRI findings, including change surrounding the tumor, distance between tumor and nipple and tumor size, had the significant influence on the rate of tumor-positive resection margins. Conclusion: Preoperative dynamic contrast-enhanced MRI significantly increased the accuracy of resection margins evaluation, and greatly reduced the rate of tumor-positive resection margins after breast conserving surgery in patients with early non-mass breast carcinoma.

Urinary incontinence after radical retropubic prostatectomy is not related to patient body mass index.

To determine if a relationship exists between patient body habitus and urinary incontinence after radical retropubic prostatectomy (RRP) for clinically localized prostate cancer. A questionnaire developed by combining parts of lower urinary tract symptom questionnaires concerning voiding symptoms after RRP was mailed to 268 consecutive patients who underwent RRP over a 2-year period. The interval between surgery and questionnaire administration was greater than 24 months for each patient. No interval was greater than 54 months. The questionnaire attempted to overcome the subjectivity of patient documented urinary incontinence by probing different aspects of each patient's voiding symptoms. Body mass index (BMI), obtained from preoperative anesthesia records, was used as the measurement for body habitus. Pearson correlations were used to determine relationships between BMI and responses and the independent t-test was used to determine differences between grouped responses and BMI. One hundred and eighty-two of 268 (68%) questionnaires were returned. No relationship was detected between BMI and patient estimates of urinary control, QOL relating to urinary symptoms, severity of stress incontinence, or use of protection (pad use). As well, no statistically significant relationship was found between BMI and a patient's willingness to undergo RRP again, based on his voiding symptoms, if given the choice. In conclusion, although patient body habitus may be related to other clinical outcomes following RRP, there does not appear to be a relationship of BMI to post-RRP urinary incontinence.

Current practice and future directions in pathology and laboratory evaluation of the sentinel node.

We developed the techniques of lymphatic mapping and sentinel node (SN) biopsy to improve the management of patients with high-risk (thick and deep) primary melanoma. The SN is the first lymph node on the direct lymphatic drainage path from the primary tumor. This node is uniquely immune-modulated by the primary tumor and is the node most likely to contain the earliest stages of metastases. Accurate assessment of the SN requires careful evaluation of multiple sections removed from the areas of the node most likely to contain tumor. These sections are stained with hematoxylin and eosin and by immunohistochemistry with antibodies directed to tumor-associated markers (S-100, HMB-45, and Melan-A/MART-1) in the case of melanoma and to cytokeratins for breast cancer. Studies are in progress to determine whether molecular biology techniques will detect additional nodes that contain truly occult tumor deposits.

Pressure measurements in closed aneurysmal sac during abdominal aortic aneurysm resection.

PURPOSE: This study determined the relationship between closed aneurysmal sac pressure (ASP) and mean blood pressure (BP) during open abdominal aortic aneurysm (AAA) resection and evaluated the contribution of inferior mesenteric and lumbar artery blood flow to ASP after proximal and distal clamping. METHODS: We measured ASP after proximal and distal clamping by placing an 18-gauge needle connected to a BP transducer into the excluded aneurysmal sac in 25 consecutive patients from April 1999 to August 2000. Simultaneous measurement of the mean systemic BP was also recorded. The ratio of ASP to mean BP in relation to the number of actively bleeding lumbar arteries (N-LA), diameter of the AAA (D-Cm), and volume of the thrombus in the AAA (Vol-TA) were recorded. RESULTS: The mean ASP was 43.32 +/- 15.19 mm Hg, with an ASP to mean BP ratio of 0.47 +/- 0.15. The N-LA in the closed aneurysmal sac ranged from 0 to 6 (mean, 3.4 +/- 1.78). The D-Cm as determined by means of computed tomography (CT) scan of the aorta ranged from 5 to 8 cm in its largest anteroposterior/transverse diameter. The average Vol-TA was 6.15 +/- 4.49 mL. Inferior mesenteric artery blood flow contributed to ASP in three patients (12%). There was no correlation between ASP to mean BP ratios and the N-LA (P =.127), D-Cm (P =.882), or Vol-TA (P =.252). CONCLUSION: Closed ASP and ASP ratios are highly variable and do not correlate with N-LA, D-Cm, or the Vol-TA.

Accelerated treatment of breast cancer.

PURPOSE: Radiation therapy (RT) restricted to the tumor bed, by means of an interstitial implant, and lasting 4 to 5 days after lumpectomy was prospectively evaluated in early-stage breast cancer patients treated with breast-conserving therapy (BCT). The goals of the study were to determine whether treatment time can be reduced and whether elective treatment of the entire breast is necessary. MATERIALS AND METHODS: Between January 1993 and January 2000, 174 cases of early-stage breast cancer were managed with lumpectomy followed by RT restricted to the tumor bed using an interstitial implant. Each brachytherapy patient was matched with one external-beam RT (ERT) patient derived from a reference group of 1,388 patients treated with standard BCT. Patients were matched for age, tumor size, histology, margins of excision, absence of an extensive intraductal component, nodal status, estrogen receptor status, and tamoxifen use. Median follow-up for both the ERT and brachytherapy groups was 36 months. RESULTS: No statistically significant differences were noted in the 5-year actuarial rates of ipsilateral breast treatment failure or locoregional failure between ERT and brachytherapy patients (1% v 0%, P =.31 and 2% v 1%, P =.63, respectively). In addition, there were no statistically significant differences noted in rates of distant metastasis (6% v 3%, P =.24), disease-free survival (87% v 91%, P =.55), overall survival (90% v 93%, P =.66), or cause-specific survival (97% v 99%, P =.28). CONCLUSION: Accelerated treatment of breast cancer using an interstitial implant to deliver radiation to the tumor bed alone over 4 to 5 days seems to produce 5-year results equivalent to those achieved with conventional ERT. Extended follow-up will be required to determine the long-term efficacy of this treatment approach.

Species-dependent pharmacological properties of the melanocortin-5 receptor.

The genes encoding the melanocortin-3 receptor and melanocortin-5 receptor have been cloned from rhesus monkey. Heterologous expression in CHO cells indicated species dependent in vitro pharmacological properties for the human and rhesus melanocortin-5 receptors. Several peptides including NDP-alpha-MSH, alpha-MSH, MT-II and ACTH1-24 are more potent at the rhesus melanocortin-5 receptor than the human melanocortin-5 receptor by more than 10-fold. In contrast, we found no species difference in pharmacological properties between the human and rhesus melanocortin-3 receptors. Such a species-dependent pharmacological difference for melanocortin-5 receptor appears to be an exception compared to other G protein-coupled receptors from human and rhesus monkey.

Structure-function studies on the cyclic peptide MT-II, lactam derivative of alpha-melanotropin.

The alanine-substituted and the retro, enantio, and retro-enantio analogs of MT-II, a potent agonist at melanocortin (MC) receptors, were prepared by solid-phase synthesis and evaluated for their ability to bind and activate human MC3, MC4, and MC5 receptors. Replacement of His with Ala resulted in [Ala6]-MT-II with affinity and agonist potency at human MC3, MC4, and MC5 receptors similar to MT-II. Substitution of Arg with Ala gave compound 100-fold less potent than MT-II, but replacement of Phe or Trp with Ala led to inactive compounds (at the micromolar concentrations). The significant drop of potency of the retro, enantio, and retro-enantio analogs of MT-II, demonstrated a crucial role of side-chain topology, and to a lesser degree, of peptide backbone in interactions of MT-II with the melanocortin receptors. The nuclear magnetic resonance analysis of MT-II suggested involvement of Phe and Arg residues in H-bonds stabilizing the bent conformations of the peptide backbone.

Detection of tyrosinase mRNA in formalin-fixed, paraffin-embedded archival sections of melanoma, using the reverse transcriptase in situ polymerase chain reaction.

Most studies of the reverse transcriptase in situ polymerase chain reaction technique have reported results from assessments of cultured cells, frozen sections, and cytospin preparations. For application to routine diagnosis, it will be necessary to adapt the technique for use with formalin-fixed, paraffin-embedded tissues, the materials that are generally available. We have evaluated the feasibility of such an approach, using surgical pathology archival material from 25 UCLA patients: 15 tissues from primary and metastatic melanoma, 7 from nonmelanocytic tumors, including cancer of the lung, colon, kidney and skin and a thyroid adenoma, and 3 nontumorous tissues. Seven of 15 melanoma tissues gave a strong positive signal, 5 gave a weak signal, and 3 were negative. None of the 10 nonmelanoma tissues gave a positive signal. The specific reaction product was mainly located in the cytoplasm. None of the nonmelanocytic tumors or normal tissues demonstrated this pattern of cytoplasmic staining. Some nonspecific nuclear staining was observed in melanocytic and nonmelanocytic tumors and must not be overread as a true positive result. It is possible to detect tyrosinase mRNA in formalin-fixed, paraffin-embedded tissue sections of melanoma, but the technique remains too demanding for routine application.

Localization of leptin binding domain in the leptin receptor.

The leptin receptor is a member of the class I cytokine receptor family and is involved in the control of appetite and body weight. The predicted amino acid sequence of the extracellular region of the cloned leptin receptor differs from that of many other cytokine receptors in that it contains two homologous segments representing potential ligand binding sites. After the analysis of various deletion and substitution mutants of the leptin receptor, we found that the first potential binding motif is not required for leptin binding and receptor activation, whereas modification of the second potential binding motif can lead to inactive receptor mutants. Further deletion analysis generated a minimal binding domain that retains high affinity leptin binding. The leptin binding domain thus has been localized to residues 323-640, which contain the second segment of cytokine receptor domain/fibronectin type 3 domain (residues 428-635). Coexpression of the active isoform of leptin receptor (OB-Rb) with an inactive mutant lacking high affinity leptin binding site led to suppression of the activity mediated by OB-Rb, suggesting that the leptin receptor may exist as a multimeric complex in the absence of leptin.

Molecular cloning of the dog beta 1 and beta 2 adrenergic receptors.

We report the isolation of the genes encoding the beta 1 and beta 2 adrenergic receptors from dog genomic DNA. Sequence analysis of both genes revealed intronless open reading frames of 473 and 415 amino acid residues, receptively. Heterologous expression of both receptors in CHO cells indicated that both receptors are functionally similar to the human homologs. Comparing the dog beta 1 and beta 2 adrenergic receptors, the beta 1 receptor appears to bind to G proteins more tightly than the beta 2 receptor. Heterologously expressed receptors provide a convenient system for evaluating novel receptor agonists and antagonists.

Relative contribution of polar interactions and conformational compatibility to the binding of neurokinin-1 receptor antagonists.

Based on single residue substitutions, previous studies suggested that Gln165, His197, and His265 of the neurokinin-1 receptor interact directly with many nonpeptide antagonists, including CP-96,345. To further test this model, all three residues have been substituted simultaneously with alanine. The Q165A-H197A-H265A triple mutant bound CP-96,345 and eight analogs with similar affinity (2-20 microM), even though the same series of compounds bound to the wild-type receptor with affinities over a range of 1000-fold. These observations correspond exactly to the prediction of the binding site model. The micromolar binding affinity of all tested CP-96,345 analogs for the triple mutant seems to reflect solely van der Waals interactions, which suggests a significant contribution of conformational compatibility (or shape complementarity) to binding affinity. The primary role of conformational compatibility in ligand binding was consistent with the observation that simply transferring the residues involved in polar interactions with beta2-agonists into the neurokinin-1 receptor did not lead to increased binding affinity for the beta2-agonists. Taken together, these results support a general principle of ligand-receptor binding in which specific polar interactions can take place only if the overall ligand conformation is compatible with the stereochemistry of the binding pocket. In addition, double-residue and triple-residue substitutions, in combination with single-residue substitutions, can provide an alternative route to reveal multiple interactions that may not be detectable by single-residue substitutions and represent a novel approach to examine ligand-receptor interactions in the absence of high-resolution structural data.

Conformational compatibility as a basis of differential affinities of tachykinins for the neurokinin-1 receptor.

The neurokinin-1 receptor is characterized by sub-nanomolar affinity for substance P and 30-100 nM affinity for other substance P-related peptides, including neurokinin B and septide. We have characterized a neurokinin-1 receptor mutant containing a Y216A substitution in the fifth transmembrane segment. This receptor mutant binds substances P with sub-nanomolar affinity but loses much of its peptide discrimination capability, exhibiting 1-2 nM binding affinity for other tachykinins. Kinetic measurements of ligand binding indicate that the increased binding affinity of neurokinin B and septide for the Y216A mutant compared to the wild-type receptor is due to a 100-fold increase in the association rate constant without appreciable change in the dissociation rate constant. The substantially increased association rate constant for the Y216A mutant suggests that the mutant receptor is probably more flexible in accommodating the approaching peptide molecule. It is proposed that a major determinant of peptide specificity for the wild-type neurokinin-1 receptor is the overall conformational compatibility between the receptor and the ligand, rather than residue-specific interactions with the divergent N-terminal residues of different peptides. Furthermore, the highly conserved nature of Tyr-216 in the G protein coupled receptor family suggests that this residue may also play an important role in the receptor activation process in general.

Identification of residues involved in ligand binding to the neurokinin-2 receptor.

Several residues of the human neurokinin-2 receptor have been identified to be critical for the binding of peptide agonists and non-peptide antagonists. Amino acid substitutions in the first and second extracellular segments and the second transmembrane segment led to substantial reduction in peptide affinity without affecting the affinity of antagonist SR48968. These effects are identical to those observed for homologous residues in the neurokinin-1 receptor, suggesting that these three regions are involved in high-affinity peptide binding to both receptor subtypes. On the other hand, some conserved residues in the fourth to seventh transmembrane segments are required for peptide binding to only one receptor subtype but not both. The conserved nature and location of these receptor residues suggest that the distance between bound peptide and helices 4-7 varies depending on the receptor subtype. It is likely that the conformational compatibility between a ligand and a given receptor determines the magnitude of binding affinity, and thus receptor subtype selectivity. While many single-residue substitutions did not affect the binding affinity of the antagonist SR48968, two double mutants in the sixth and seventh transmembrane segments were found to reduce its affinity substantially. Therefore, receptor residues participate cooperatively in the binding of SR48968. These results demonstrate the usefulness of combining single-residue substitutions in studying and confirming the role of receptor residues in ligand binding. Finally, the overlapping nature of agonist and antagonist binding sites is consistent with the observation that substitutions of some residues modify the binding affinities of both peptide agonists and non-peptide antagonists.

Localization of the ligand binding site of the neurokinin-1 receptor: interpretation of chimeric mutations and single-residue substitutions.

Previous studies have indicated that substitution of the third or fourth extracellular segment of the human neurokinin-1 receptor with the equivalent segment from the neurokinin-3 receptor affects the binding affinities of peptide agonists and/or nonpeptide antagonists. To elucidate the roles of the divergent residues within these domains in ligand binding, single-residue substitutions in these regions were analyzed. Neurokinin B affinity was increased by one single-residue substitution (E172K) in the third extracellular segment and one double-residue replacement (L279R+K280W) in the fourth extracellular segment, and the affinity for the antagonist L-703,606 was reduced by one single-residue substitution (Y272A) in the fourth extracellular segment. The effect of these three specific substitutions is consistent with the prediction of chimeric mutations. However, the substantial reduction in ligand binding affinity observed upon multiple-residue substitutions in the third extracellular segment (residues 176-183 or 187-195) has not been reproduced by eliminating potential electrostatic interactions or substituting with the corresponding residues from the neurokinin-3 receptor, suggesting that the reduction in ligand binding affinity observed with some chimeric receptors is not due to the loss of direct electrostatic interactions. These data indicate that other factors such as conformational effects may complicate the interpretation of data obtained with chimeric receptors, and they demonstrate the need to evaluate chimeric receptors along with single-residue substitutions in the same region to localize specific residues involved in ligand binding. Furthermore, the available data suggest that one major determinant of peptide selectivity in the neurokinin-1 receptor may be the conformational compatibility between a peptide and the receptor.

Interaction of substance P with the second and seventh transmembrane domains of the neurokinin-1 receptor.

The neurokinin-1 receptor is a member of the G-protein-coupled receptor family and has the highest affinity for the endogenous peptide transmitter substance P. Previous studies have indicated that several residues in the first and second extracellular segments, and at least part of the transmembrane domain, of the human neurokinin-1 receptor are involved in substance P binding to the receptor. To further map the peptide binding site, single-residue substitutions in the transmembrane domains were analyzed. Asn-85, Asn-89, Tyr-92, and Asn-96 in the second transmembrane domain and Tyr-287 in the seventh transmembrane domain are required for the high-affinity binding of peptides, with Asn-85 possibly interacting with the C-terminus of substance P. In addition, Glu-78 in the second transmembrane domain and Tyr-205 in the fifth transmembrane domain appear to be involved in the receptor activation process. Some of the key residues for peptide binding are likely to be near those residues that are required for the binding of competitive antagonists (such as His-197, His-265, and Tyr-287). These data suggest that a volume exclusion effect can explain the competitive antagonism of substance P binding by non-peptide antagonists. Furthermore, the key residues identified thus far are required for the high-affinity binding of all three neurokinin peptides, consistent with a hypothesis that the conformational compatibility between the receptor and the peptide agonist may be a major determinant of peptide recognition.

Characterization of the binding of [125I-iodo-histidyl, methyl-Phe7] neurokinin B to the neurokinin-3 receptor.

We have characterized the binding of [125I-iodo-histidyl, methyl Phe7]neurokinin B (125I-NKB) to the human neurokinin-3 (NK3) receptor. 125I-NKB specifically binds to the NK3 receptor expressed in CHO cells with a Kd of 0.2 nM. The ligand displays little crossreactivity with the human NK1 and NK2 receptors. The binding of 125I-NKB to the human NK3 receptor and to rat cortex membranes is inhibited by neurokinin B with IC50 of 1.5 nM and 4 nM, respectively. In contrast, 350- to 500-fold higher concentrations of substance P and neurokinin A are required to inhibit binding to either receptor preparation. The data suggest that 125I-NKB is a high affinity, selective ligand for the human and rat NK3 receptor.