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Langevin mode theory and the coarse-grained elastic network model (ENM) for proteins are combined to yield the Langevin network model (LNM). Hydrodynamic radii of 6 A were assigned to each alpha-carbon on the basis of matching experimental translational and rotational diffusion constants of lysozyme, myoglobin, and hemoglobin with those calculated using a rigid body bead model with hydrodynamic interactions described by the Rotne-Prager tensor. LNM analysis of myosin II indicates that all ENM-like modes are overdamped at water viscosities. The low-frequency LNM modes in the pre-power stroke structure (PDB code: 1VOM) are substantially less mixed than the corresponding modes of the post-power stroke structure (1Q5G). Results from a four-bead model of the myosin "lever arm" indicate that coupling between modes increases as the array departs from linearity and are consistent with the results for 1VOM and 1Q5G. The decay times for all overdamped Langevin modes are shorter than the calculated rotational tumbling times found for lysozyme and myosin.

Cave organisms occupy a special place in evolutionary biology because convergent morphologies of many species demonstrate repeatability in evolution even as they obscure phylogenetic relationships. The origin of specialized cave-dwelling species also raises the issue of the relative importance of isolation vs. natural selection in speciation. Two alternative hypotheses describe the origin of subterranean species. The 'climate-relict' model proposes allopatric speciation after populations of cold-adapted species become stranded in caves due to climate change. The 'adaptive-shift' model proposes parapatric speciation driven by divergent selection between subterranean and surface habitats. Our study of the Tennessee cave salamander complex shows that the three nominal forms (Gyrinophilus palleucus palleucus, G. p. necturoides, and G. gulolineatus) arose recently and are genealogically nested within the epigean (surface-dwelling) species, G. porphyriticus. Short branch lengths and discordant gene trees were consistent with a complex history involving gene flow between diverging forms. Results of coalescent-based analysis of the distribution of haplotypes among groups reject the allopatric speciation model and support continuous or recurrent genetic exchange during divergence. These results strongly favour the hypothesis that Tennessee cave salamanders originated from spring salamanders via divergence with gene flow.

Neuroimaging and neurophysiology evidence suggests that component operations in working memory (WM) emerge from the coordinated interaction of posterior perceptual cortices with heteromodal regions in the prefrontal and parietal cortices. Still, little is known about bottom-up and top-down signaling during the formation and retrieval of WM representations. In the current set of experiments, we combine complementary fMRI and EEG measures to obtain high-resolution spatial and temporal measures of neural activity during WM encoding and retrieval processes. Across both experiments, participants performed a face delayed recognition WM task in which the nature of sensory input across stages was held constant. In experiment 1, we utilized a latency-resolved fMRI approach to assess temporal parameters of the BOLD response during stage-specific encoding and retrieval waveforms. Relative to the latency at encoding, the PFC exhibited an earlier peak of fMRI activity at retrieval showing stage-specific differences in the temporal dynamics of PFC engagement across WM operations. In experiment 2, we analyzed the first 200 ms of the ERP response during this WM task providing a more sensitive temporal measure of these differences. Divergence of the ERP pattern during encoding and retrieval began as early as 60 ms post-stimulus. The parallel fMRI and ERP results during memory-guided decisions support a key role of the PFC in top-down biasing of perceptual processing and reveal rapid differences across WM component operations in the presence of identical bottom-up sensory input.

Refreshing is a basic reflective component process that can serve to prolong activation of task-relevant information. Neuroimaging work has shown that left middle frontal gyrus (MFG) and supramarginal gyrus (SMG) are selectively engaged during refreshing. Functional MRI (fMRI), however, is not able to determine if these regions are necessary for refreshing. In this experiment, we utilize repetitive transcranial magnetic stimulation (rTMS) to assess the behavioral effect of functionally deactivating these regions. We report a selective slowing of response times (RTs) to refresh words following MFG stimulation, consistent with a role of lateral prefrontal cortex (PFC) in top-down control mechanisms necessary for refreshing. In contrast, SMG stimulation slowed participants in both refreshing and repeating words, indicating a more general role of SMG in verbal processing.

Certain head and neck surgical cases require the patient to be positioned prone. Such positioning carries with it an attendant subset of risks and complications not otherwise encountered in more traditional supine positioning. Gaining awareness of these risks and complications, and developing proactive positioning strategies, will enable the surgical team to position the patient optimally for the procedure and provide for every consideration of patient safety. This article consists of a specific literature review of those issues directly related to the anatomical and physiological concerns arising from prone positioning. Particular attention is paid to the cardiopulmonary, renal, ophthalmologic, and neurological vulnerabilities unique to this position. Proper planning by the surgical team and utilization of the correct equipment are a necessity. A tailored approach to the needs of the individual patient and an intimate awareness of the potential pitfalls will contribute to better outcomes when using the prone position.

Adjuvant therapies are often used to enhance endometrial thickness during IVF. This retrospective cohort analysis investigated if women undergoing oocyte donation cycles with sonographic evidence of endometrial insufficiency benefit from adjuvant medicaltherapy. Infertile patients received 503 mock cycles followed by 503 anonymous oocyte donation cycles. One hundred and twenty-three patients received adjuvant therapy during a donor oocyte cycle. Patients who had a mock endometrial thickness > or = 8 mm experienced a significant decrease in endometrial thickness with the donor oocyte cycle regardless of the use of adjuvant therapy (P < 0.001). In contrast, those patients with a mock endometrial thickness < 8 mm experienced a significant increase in donor oocyte cycle endometrial thickness regardless of adjuvant therapy use (P < 0.001). The patients with a mock endometrial thickness < 8 mm experienced a significant improvement in pregnancy rates when taking adjuvant therapy (87.7 versus 73.5%, P < 0.05). Adjuvant therapy significantly improved both pregnancy (87.8 versus 76.8%, P < 0.01) and live birth rates (74.8 versus 63.7%, P < 0.05) in the entire patient population. While the use of adjuvant therapy did not significantly improve ultrasonographic endometrial thickness, it did improve outcome rates.

Background: Black thyroid pigmentation has been considered pathognomonic for chronic minocycline ingestion for more than 30 years. Although never conclusively linked to overt disease, evidence clearly exists that minocycline is a competitive inhibitor of thyroid peroxidase in metabolically active thyroid tissue. This offers a potential mechanism of pigment accumulation, which can account for the occasional finding of hypopigmentation in thyroid carcinomas. To our knowledge, an association with tetracycline derivatives other than minocycline has not been documented., Methods: Herein is a case report of a patient with gross black thyroid tissue containing a hypopigmented papillary thyroid carcinoma. Twelve days before surgery, the patient was placed on doxycycline, a tetracycline derivative, to optimize an in vitro fertilization regimen., Results: The gross specimen was diffusely black in color with a 1.5-cm hypopigmented focus of papillary thyroid cancer. Hematoxylin-eosin staining, as well as electron micrographs, was consistent with findings associated with minocycline ingestion., Conclusions: Black thyroid is rare and only previously associated with chronic minocycline ingestion. This report documents a black thyroid in a patient after short-term exposure to doxycycline. Pigment accumulation in normal tissue is thought to occur by inhibition of thyroid peroxidase. Minocycline is a competitive inhibitor of this enzyme in its native configuration. Thyroid carcinomas are known to have abnormal thyroid peroxidase, which could account for reports of hypopigmented tumors within grossly darkened thyroid tissue. Hypopigmented foci within such "black thyroid" deserve through pathologic examination., (Copyright 2005 Wiley Periodicals, Inc.)

Objective: Increasing evidence suggests that hyperinsulinemia plays an important role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the timing for the onset of hyperinsulinemia is not clear. The objective of this study was to examine the effect of peripubertal hyperinsulinemia on the maturing female reproductive axis., Methods: Hyperinsulinemia was induced in 28-day-old peripubertal female rats by infusing insulin (0.04 IU/d) via subcutaneously implanted Alzet minipumps (Model #2004; Durect Corp, Cupertino, CA; constant flow rate 0.25 muL/h) for 4 weeks. Control animals were administered normal saline. Estrus cyclicity was monitored regularly. Upon termination of the experimental period, the animals were killed, trunk blood and pituitaries were collected for hormone assays, and ovaries were collected for histological and immunocytochemical studies., Results: In contrast to the control animals, hyperinsulinemic animals had (1) erratic estrus cycles, with prolonged (2 to 3 days) metestrus-diestrus or diestrus-proestrus stages; (2) significantly (P <.05) decreased levels of serum progesterone, and significantly (P <.05) increased levels of serum testosterone and dehydroepiandrostene sulfate; (3) prematurely luteinized ovarian follicles with prominent thecal and interfollicular stromal proliferation; and (4) markedly reduced expression of growth differentiation factor-9 (GDF-9) and activin receptors (ActR) I and IB in the ovaries., Conclusion: Peripubertal hyperinsulinemia in rats causes hormonal and ovarian changes similar to those in women with PCOS. Based on these novel findings, we speculate that peripubertal hyperinsulinemia may be a risk factor for the development of PCOS later in life.

Although numerous theoretical models implicate the prefrontal cortex (PFC) as a source of top-down control signals, empirical support is limited. In this review, we highlight direct evidence for this view of PFC function and discuss several lines of other supportive findings. Most evidence to-date is only suggestive due to methodological challenges in tracking the spatiotemporal dynamics of cortical networks. However, we discuss several promising approaches that could further our understanding of the role of the PFC in goal-directed behavior.

Objective: To examine the effects of chronic methylphenidate use on the reproductive axis of adolescent female rats., Design: Controlled prospective study., Setting: University research laboratory., Animal(s): Twenty prepubertal female Sprague Dawley rats., Intervention(s): Subcutaneous implantation of drug-filled Alzet minipumps (Durect Corporation, Cupertino, CA) for infusing methylphenidate (450 microg/d, treated) or physiological saline (control) for 4 weeks. Estrous cyclicity was checked from 3 weeks of pump implantation till the termination of the experiments. Animals were killed after 4 weeks of treatment., Main Outcome Measure(s): Estrous cyclicity, pituitary and peripheral FSH and LH, serum estrogen and progesterone, ovarian histology, and immunocytochemistry for localizing growth differentiation factor-9 and activin receptors-I., Result(s): Compared with the control group, the treated animals exhibited the following: [1] poor vaginal opening and erratic estrous cyclicity; [2] undeveloped, disrupted, or prematurely luteinized ovarian follicles; [3] absence of growth differentiation factor-9 and of activin receptors I and IB in the oocyte; and [4] high levels of LH in the pituitary., Conclusion(s): Chronic methylphenidate administration during adolescence perturbs pubertal onset, adversely affects maturation of the female reproductive axis by retarding pituitary LH release, and adversely affects ovarian folliculogenesis. These novel findings may have significant clinical implications in evaluating the effects of methylphenidate abuse on adolescent health.

Objective: To determine the predictive value and to define prognostic threshold measurements for mean ovarian volume (MOV) in patients undergoing IVF., Design: Prospective cohort analysis., Setting: Tertiary care center., Patient(s): Two hundred sixty-seven patients., Intervention(s): Transvaginal ultrasound before starting gonadotropins., Main Outcome Measure(s): Number of oocytes retrieved, basal hormone levels, and cycle outcomes., Result(s): The MOV for the population was 4.78 +/- 2.6 cm(3) (range 0.9-21.1 cm(3)). The MOV significantly correlated with the majority of prestimulation and poststimulation IVF parameters. Threshold analysis demonstrated a lower pregnancy rate associated with a MOV of <2 cm(3) (31.6% vs. 55.6%). Threshold analysis revealed a trend toward higher cancellation rate associated with a MOV of <2 cm(3) (21.1% vs. 7.3%)., Conclusion(s): Although MOV correlated with IVF stimulation parameters, its use as an adjunct in counseling patients during IVF appears to be of limited value. A MOV <2 cm(3) was associated clinically with a higher cancellation rate (21.1%) and a lower pregnancy rate (31.6%) in those cycles not cancelled. However, these values do not deviate far from the mean national IVF outcome rates. There was no absolute MOV that was predictive of pregnancy outcome or cycle cancellation.

Objective: To determine the predictive value and define threshold values for basal antral follicle count in patients undergoing IVF., Design: Prospective cohort analysis. Tertiary care center. Two hundred eighty-nine patients. Transvaginal ultrasonography before starting gonadotropin administration., Main Outcome Measures: Number of oocytes retrieved, basal hormone levels, and cycle outcomes., Results: Pregnant patients had significantly more antral follicles (13.8 +/- 7.5 vs. 12.4 +/- 10.0). Patients in whom cycles were canceled had significantly fewer antral follicles (7.6 +/- 4.8 vs. 13.7 +/- 8.8). Antral follicle count significantly correlated with most prestimulation and poststimulation IVF variables. Threshold analysis demonstrated a lower pregnancy rate (23.5% vs. 57.6%) and a higher cancellation rate (41% vs. 6.4%) associated with having four or fewer antral follicles., Conclusion(s): The basal antral follicle count identified patients who responded poorly to IVF stimulation. Having four or fewer antral follicles was associated with a high cancellation rate (41%) and, in patients without a cancelled cycle, a low pregnancy rate (23%). However, no antral follicle count absolutely predicted pregnancy or cycle cancellation.

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG(1-80)) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1-1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK(1) and CCK(2) receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK(1)-R and CCK(2)-R on IEC cells. High-affinity (K(d) = 0.5-1.0 nM) binding sites for (125)I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG >or= COOH-terminally extended G17 >or= G-Gly > G17 > *CCK-8 (* significant difference; P < 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Cell-penetrating peptides derived from hydrophilic regions of the homeoprotein Antennapedia (Antp) or the transcription-regulating factor Tat have been used to transport several peptide and oligonucleotide cargoes into the interior of cells. Such vector peptides penetrate cells, in part, because they contain multiple lysine and arginine residues. Using radiolabeled peptide cargoes covalently linked to Antp- or Tat-related vectors, or to D-Arg heptamers, we found that a significant amount of the label remained tightly bound to plastic and glass surfaces. Binding of the labeled conjugates was due entirely to the cationic vector moieties. Under certain conditions, such non-specific binding could be mistaken for cellular penetration., (Copyright 2002 Elsevier Science Inc.)

Purpose: Recently ovarian volume has been touted as a means to evaluate ovarian reserve in assisted reproductive technology cycles. In this study, a novel method of determining ovarian size was evaluated and compared to the standard three-dimensional ovarian volume measurement during in vitro fertilization (IVF)., Methods: This prospective observational study consisted of 60 consecutive patients undergoing baseline transvaginal ultrasonography for IVF from July to August, 1999. The main outcome measures were mean ovarian size and mean ovarian volume., Results: The patients' ages ranged from 23 to 43 years with a mean age of 33.86 +/- 4.5 years. The mean ovarian size was 2.19 +/- 0.4 cm (range 1.40-3.40). The mean ovarian volume was 5.02 +/- 2.7 cm3 (range 1.71-16.5 cm3). By linear regression there was a 90% correlation between the two methods of ovarian measurement (r = 0.90, p < 0.01)., Conclusions: These results demonstrated a strong correlation between these two methods of determining ovarian size. Mean ovarian diameter measured in the largest sagittal plane is a good estimation of ovarian volume and may be used to quickly assess ovarian status prior to undergoing IVF.

Background: Endometrial cavity fluid (ECF) is occasionally observed during assisted reproductive technology (ART) cycles. However, few reports have described its prevalence or significance., Methods and Results: We examined the relationships between ECF, clinical pregnancy rate (CPR), tubal factor infertility and ultrasound-visible (USV) hydrosalpinges. In 843 ART cycles involving 721 patients, ECF was observed during stimulation in 57 cycles and after human chorionic gonadotrophin (HCG) administration in 12 cycles, with an overall incidence of 8.2% (69/843). When ECF was observed during stimulation, the cancellation rate due to poor ovarian response was significantly higher (29.8 versus 16.9%, P <0.05) and the CPR per started cycle was significantly lower (26.3 versus 42.4%, P <0.05) than cycles without ECF. When ECF developed after HCG administration, the CPR was similar compared with that of the group for which ECF was not observed. In the 327 cycles involving tubal factor infertility patients, USV hydrosalpinges were noted in 71 cycles (71/327; 21.7%), and ECF developed in five of those cycles (5/71; 7.0%). A total of 27 cycles during which ECF developed (27/57, 47.4%) involved non-tubal factor patients., Conclusions: ECF during stimulation was associated with increased cancellation rates and lower CPRs per started cycle, and was not associated with USV hydrosalpinges. Furthermore, ECF observed after HCG administration did not impact CPR and may represent a different clinical entity.

Objective: To determine the predictive value(s) of beta-hCG serum levels for pregnancy outcome following blastocyst transfer., Design: Retrospective review., Setting: University-based assisted reproductive technology (ART) program., Patients: All ART patients enrolled from January 1998 to December 1999., Intervention(s): None., Main Outcome Measure(s): Beta-hCG serum levels and pregnancy outcomes., Result(s): Of the 836 ART cycles initiated, 608 embryo transfers met study criteria and were assigned to one of two groups: 248 day 5 blastocyst transfers or 360 day 3 embryo transfers. In the day 5 blastocyst group, 147 pregnancies occurred (59.2%), and day 3 transfers resulted in 165 pregnancies (45.8%). For day 3 and day 5 transfers, mean values of beta-hCG on day 16 post-retrieval of spontaneous abortions were lower than ongoing pregnancies (P< .05). A beta-hCG value on day 16 of >300 mIU/mL predicted an ongoing pregnancy for day 5 transfer group in 97% of pregnancies compared with 92% for day 3 embryo transfers. A multiple gestation was observed in 70% of pregnancies with a beta-hCG level >400 mIU/mL in the day 5 group compared with 63% for the day 3 group. The incidence of higher-order multiple gestations was significantly lower in the day 5 blastocyst group (P< .05)., Conclusion(s): Beta-hCG serum levels on day 16 post-retrieval were highly predictive of pregnancy outcome after a blastocyst transfer.

Objective: To determine whether exposure of developing endometrium to supraphysiologic E2 levels during controlled ovarian hyperstimulation (COH) in IVF cycles inhibits endometrial receptivity., Design: Retrospective analysis of IVF-ET and ovum donation data., Setting: Tertiary-care teaching hospital., Patient(s): Four hundred ten patients <33 years of age undergoing IVF-ET and 181 anonymous ovum donors (<33 years of age) and their associated ovum recipients., Main Outcome Measure(s): Implantation, pregnancy, and delivery rates., Result(s): Ovarian response to COH (duration of stimulation, peak E2 level, area under the curve for E2 exposure, and number of oocytes retrieved) was similar for IVF-ET patients and ovum donors. Donors were younger than IVF-ET patients (mean age, 27.5 +/- 0.2 years vs. 30.4 +/- 0.1 years). A similar number of embryos with similar number of blastomeres were transferred in IVF-ET patients and ovum recipients. The fragmentation rate at time of transfer differed slightly between groups (5.2 +/- 0.2% vs. 4.3 +/- 0.3%). Implantation, pregnancy, and delivery rates did not differ between IVF-ET patients and recipients of donor oocytes., Conclusion(s): Exposure of the developing endometrium to controlled ovarian hyperstimulation during IVF cycles does not inhibit embryo implantation or affect pregnancy and delivery rates.

Objective: To compare reproductive outcome between women with normal ovarian reserve and women with abnormal ovarian reserve., Design: Retrospective., Setting: Tertiary care center., Patient(s): Nine thousand eight hundred and two patients who had basal follicle-stimulating hormone (FSH) concentrations measured as part of an infertility evaluation., Intervention(s): Monitoring of early pregnancy., Main Outcome Measure(s): Pregnancy loss rates, live birth rates., Result(s): Of 1,034 patients with diminished ovarian reserve (DOR) (FSH > or =14.2 IU/L), 28 (2.7%) conceived. Twenty of these pregnancies (20/28; 71.4%) were lost in the first trimester. Pregnancy loss rates in women with DOR were 57.1% in women <35 years old, 63.5% in women 35-40 years old, and 90.0% in women >40 years old. These rates of pregnancy loss were significantly higher compared to age-matched patients with normal ovarian reserve., Conclusions(s): Women with DOR have exceedingly high rates of pregnancy loss, regardless of age. Women with diminished ovarian reserve should be counseled that, in addition to a low probability of conception, live birth rates are poor.

Background: Embryo transfer represents one of the most critical procedures in the practice of assisted reproduction. The objective of this study was to identify retrospectively the minimum number of embryo transfers required to train providers properly in this skill., Methods and Results: The study group consisted of 204 patients who received embryo transfers between January 1996 and March 2000 in a university-based programme of assisted reproduction. The main outcome measure was clinical pregnancies per embryo transfer. Five Fellow trainees performed a total of 204 embryo transfers for an overall pregnancy rate of 45.5% per embryo transfer (93/204). In comparison, the programme pregnancy rate per transfer for experienced providers was 47.3% (560/1179). A chronological graph of each individual trainee's experience for the first 50 embryo transfers performed suggested a lower initial pregnancy rate for three of the five trainees. To determine whether a learning curve might exist, results of the first 25 transfers were compared as a subgroup with the second 25 transfers. Pregnancy rates were lower for the 1-25 transfer subgroup than in the 26-50 subgroup for three of the five Fellow trainees, although the difference was not statistically significant., Conclusion: Clinical pregnancy rates of Fellows-in-training were indistinguishable statistically from those of experienced staff by 50 transfers.

Recent literature advocates carotid endarterectomy on duplex alone. The authors hypothesized that carotid angiography adds information that alters clinical management in a substantial number of patients compared to the use of carotid duplex examination alone. The records of 182 consecutive patients who underwent carotid artery duplex and subsequent carotid/cerebral angiography for suspected carotid artery stenosis between January 1998 and April 1999 were reviewed retrospectively. Carotid artery duplex examinations were stratified based on stenosis: < or =39%, 40% to 59%, 60% to 79% (moderate), 80% to 99% (severe), 100%. Carotid stenosis on angiograms was determined by NASCET criteria. New information found at angiography included vertebral, subclavian, or arch atherosclerosis, intracranial pathosis, or a change in duplex stenosis category to a degree of stenosis not requiring surgery. Clinical importance was attributed to angiograms that altered the patients' management plan. Angiography provided additional information in 53% (97/182) of patients. Vertebral disease was found in 25.1%, subclavian disease in 16.4%, intracranial disease in 15.3%, aortic arch disease in 3.3%. Patient treatment was altered in 30% (55/182). Angiographic findings downgraded the stenosis to medical therapy in 20.9% (38/182). The surgical plan was influenced in 5.5% (10/182). Nine intracranial aneurysms were discovered. Carotid angiography was essential for vascular bypass surgery planning in 3.3% (6/182). Angioplasty was performed in 2.2% (4/182). The accurate determination of stenosis is critical in determining optimal treatment of patients with carotid artery stenosis. Routine carotid angiography remains valuable in the clinical treatment of these patients.

Objective: To determine the predictive value and define threshold levels for basal antral follicle number and mean ovarian diameter in patients undergoing ART cycles., Design: Retrospective., Setting: Tertiary care center., Patients: Two hundred seventy-eight patients who had ovarian measurements performed on cycle day 3 before beginning treatment with gonadotropins., Intervention: Pretreatment ovarian ultrasound measurements., Main Outcome Measure: Number of oocytes retrieved, hormone levels, and cycle outcomes., Results: A direct linear correlation was observed between mean ovarian diameter and basal follicle number. Both measures demonstrated a positive linear correlation with recovered oocytes, basal E(2), and peak E(2). Both demonstrated a negative linear correlation with ampules of gonadotropins administered, days of stimulation, patient age, cycle day 3 FSH, and FSH:LH ratio. An antral follicle count of

Objective: Our purpose was to compare outcomes of women with triplet gestations conceived via assisted reproductive technology who chose expectant management or multifetal pregnancy reduction., Study Design: We performed a retrospective review of all women who initiated assisted reproductive technology cycles from August 1995 through July 1997 with ultrasonographic documentation of triplets exhibiting fetal heart tones at 9 weeks of gestation (N = 127). Patients were then uniformly referred to a maternal-fetal medicine specialist and to 3 centers offering multifetal pregnancy reduction., Results: Thirty-six percent of patients (46/127) chose multifetal pregnancy reduction with 95% undergoing reduction to twins. In the expectant management group, 13.6% of pregnancies were reduced spontaneously after 9 weeks of gestation. The "take home" infant per delivery rates for the multifetal pregnancy reduction and expectant management groups were 87% and 90.1%, respectively (P =.66). The mean gestational ages at delivery (+/-SE) for the multifetal pregnancy reduction and expectant management groups were 33.25 +/- 1. 03 weeks and 32.04 +/- 0.58 weeks (P =.23), and the mean birth weights of infants delivered at >24 weeks of gestation were 2226 +/- 79 and 1796 +/- 44, respectively (P <.0001). There were no significant differences in perinatal mortality, gestational age at delivery, or "take home" infant per delivery rates between these groups., Conclusions: These data suggest that multifetal pregnancy reduction does not have a significant impact on the probability of live birth or on gestational age at delivery for women with triplets conceived with assisted reproductive technology.

Objective: To evaluate the effect of individual providers on pregnancy outcome after embryo transfer., Design: Retrospective data analysis., Setting: University-based tertiary-care assisted reproductive technology program with 10 physician-providers., Patient(s): Six hundred and seventeen women who underwent 854 fresh embryo transfers between January 1996 and January 1999., Intervention(s): Pregnancies after embryo transfer were recorded for each provider., Main Outcome Measure(s): Establishment of a clinical pregnancy., Result(s): Three hundred ninety-three clinical pregnancies resulted from 854 embryo transfers, for an overall clinical pregnancy rate of 46.0% per embryo transfer. Three hundred forty-seven (40.6%) pregnancies were ongoing. The clinical pregnancy rate varied significantly between providers: for example, 17.0% (47 transfers) vs. 54.3% (57 transfers) (P<.05). Similarly, the ratio of high-grade embryos required to produce a gestational sac differed between providers. The number or quality of embryos transferred did not differ significantly., Conclusion(s): Significant differences were observed in pregnancy rates after embryo transfer done by different providers, suggesting that embryo transfer technique may influence pregnancy outcome in assisted reproductive technology.

Purpose: To examine the effect of intracytoplasmic sperm injection (ICSI) on embryo fragmentation and implantation rates in those embryos chosen for transfer compared to conventional in vitro fertilization (IVF)., Methods: We compared 253 infertility patients (71 ICSI and 182 IVF) with respect to age, semen analysis, number of embryos transferred, embryo fragmentation, implantation rate, and pregnancy rate. Embryo fragmentation was determined by one observer at the same laboratory over the entire study period., Results: A statistically significant difference was observed in mean embryo grade between IVF (2.2 +/- 0.84) and ICSI (2.5 +/- 0.77), P = 0.01. Additionally, the IVF patients had significantly more nonfragmented (grade I) embryos compared to the ICSI group, P < 0.01., Conclusions: These data suggest that ICSI, irrespective of semen parameters, may increase embryo fragmentation and produce fewer nonfragmented grade I embryos while maintaining implantation and pregnancy rates similar to conventional IVF.

Objective: We previously identified a protein, Brx, that interacted with estrogen receptor alpha. Sequence analysis determined that Brx is a novel member of the Dbl family of oncoproteins involved in signaling pathways that regulate cell growth. Because the Brx protein was found to be highly expressed in hormoneresponsive breast epithelium, the objective of this study was to determine whether Brx was expressed in both normal and neoplastic ovarian tissues., Study Design: A polyclonal antiserum directed against the Brx protein was used to perform immunolocalization on sections from 5 normal ovaries and 20 ovarian neoplasms. Chromosomal localization of the brx gene was accomplished by means of fluorescence in situ hybridization. Northern and Western blot analyses were performed on extracts prepared from human ovaries., Results: Brx protein was localized to the cytoplasm of granulosa cells from mature graafian follicles, the corpus luteum, and islands of hilar cells in normal ovaries. In tumors with low malignant potential and ovarian carcinomas the neoplastic epithelium stained strongly for Brx protein. Northern and Western blot analyses, respectively, confirmed expression of Brx messenger ribonucleic acid and protein in normal ovary. Finally, the brx gene was localized to 15q25., Conclusion: The proto-oncogene brx is expressed in specific normal human ovarian tissues and is also present in ovarian epithelial neoplasms.

Objective: To compare an ovarian stimulation protocol using microdose follicular phase GnRH agonist (GnRH-a) and oral contraceptive (OC) pills to a luteal phase GnRH-a protocol., Design: Retrospective analysis., Setting: University affiliated IVF program., Patient(s): One hundred seventy patients who underwent IVF and ET in 1996., Intervention(s): Patients were assigned to either a midluteal start of leuprolide acetate (LA) 1 mg/d, reduced to 0.5 mg/d after addition of gonadotropins (LUT), or OC pills until cycle day 0 followed by 20 microg of LA every 12 hours on cycle day 3 with addition of gonadotropins on cycle day 5 (MICRO)., Main Outcome Measure(s): Number of FSH ampules, days of stimulation, peak E2, and number of oocytes retrieved., Result(s): There were no statistically significant differences in the main outcome measures between the two groups using an age-matched ANOVA. Clinical pregnancy rate per cycle start was not statistically different (LUT = 54%, and MICRO = 37%). The cancellation rate was significantly higher in the MICRO group (22.5% vs. 8.2%)., Conclusion(s): Given the higher cancellation rate in the microdose group, a randomized clinical trial is required to determine the possible benefit of a lower dose of GnRH-a in patients with normal ovarian function.

Collection of cervical secretions for local immunological assessment requires that the secretions be collected prior to the Pap smear to avoid contamination with blood. The objective of the present study was to determine whether gentle collection of cervical secretions prior to a Pap smear collection influences the quality of the Pap smear. A total of 266 women were recruited. Half of the participants were assigned to collection of cervical secretions prior to Pap smear collection with Weck-cel sponges. The remaining half had only the Pap smear collection performed. Pap smear slides were reviewed and evaluated for quality by the Bethesda System adequacy criteria without knowledge of randomization. The proportions of limited or inadequate slides in the two study groups were compared by using the Pearson chi-square test. No significant differences were observed between the two study groups when overall Pap smear quality was evaluated (P = 0.29). Comparison of the two study groups with respect to individual adequacy criteria, including presence of air drying artifact, presence of obscuring blood, absence of metaplastic or endocervical cells from the transformation zone, scant cellularity, and presence of obscuring inflammatory cells, also revealed no significant differences between the two study groups. Results from the present study suggest that the collection of cervical secretions with Weck-cel sponges does not adversely impact the quality of subsequently obtained Pap smears.

Numerous studies of rat pituitaries have reported that inhibin suppresses the synthesis and release of FSH and decreases the release of LH. The latter effect seems to be related to the down-regulation of receptors for GnRH. The studies reported here identified cellular changes behind the inhibitory effects of inhibin on gonadotropes to learn whether its effects are mediated by changes in subtypes of gonadotropes. Cell populations from diestrous day 2 and proestrous (morning) rats were collected, dispersed to single cell populations, and plated in medium containing either recombinant 32-kDa inhibin or porcine follicular fluid for 24 h. GnRH binding was detected by exposing the cells to a biotinylated analog (Bio-GnRH) for 10 min before fixation, followed by avidin-peroxidase labeling protocols to detect the biotin on the analog. In parallel fields, the cells were further identified by immunolabeling for LH or FSH beta-subunits or for GH with a different colored reaction product. The most striking changes were seen in cells from proestrous rats. Inhibin reduced the percentages of Bio-GnRH target cells in the population by 60% and the area and density of Bio-GnRH label on the remaining cells. Inhibin reduced the percentages of FSH cells by 30% and caused nearly a 60% reduction in the binding of Bio-GnRH by this cell type (from 83% of FSH cells to 32% of FSH cells). Inhibin also reduced the area of FSH cells and the density of FSH stores. Inhibin's effects on LH cells were limited to a reduction in the area of the cells and the density of LH stores, but not the number of LH cells. In addition, it reduced the percentages of LH cells with Bio-GnRH receptors from 84% to 40%. When cells with GH were analyzed, inhibin had no effect on their percentages, areas, or GH stores. In populations from proestrous rats, inhibin reduced the percentages of GH cells with Bio-GnRH binding from 38% to 21%. These data suggest that inhibin's target cell is the abundant multihormonal gonadotrope that contains LH, FSH, and GH and predominates during proestrus. Inhibin's effects are most severe on FSH cells, which suggests that it may either selectively affect FSH synthesis and stores in bihormonal gonadotropes and/or affect monohormonal FSH cells. Thus, mechanisms behind its inhibitory effects include 1) a reduction in the percentage of Bio-GnRH target cells, 2) a reduction in the area of Bio-GnRH-binding sites on individual cells, and 3) a reduction in the stores of FSH and the percentages of FSH cells. These last effects are consistent with known reductions in FSH synthesis. The effects of inhibin on LH secretion may be secondary to the effects on Bio-GnRH receptors in bihormonal gonadotropes.

N-hydroxysuccinimide (NHS) esters of biotin are reported to react specifically with amino groups of peptides and proteins. However, we have found that these reagents can readily acylate other functional groups in specific peptide sequences under relatively mild conditions. We have extended our inquiry of sequence-dependent acylation by evaluating the reactivity of a variety of commonly employed biotinylation reagents typically used for amino group modification. These included the p-nitrophenyl ester of biotin, NHS-esters of biotin containing aminohexanoic acid spacer arms, and a sulfonated NHS-biotin ester that contained a disulfide bond within its spacer. The decapeptide [D-Lys6]gonadotropin releasing hormone was employed as a model peptide. Reaction products were characterized by high-performance liquid chromatography, amino acid compositional analysis, reaction with hydroxylamine, and mass spectrometry. In addition to the O-acylation of Ser4 and Tyr5 in this peptide, we have also identified a novel biotinylation of the Arg8 side chain.

Malignant hyperthermia is a complication of general anesthesia that is especially problematic if it occurs during renal transplantation because myoglobinemia, shock, and ischemia play a role in injuring the transplanted kidney. In this report, we describe a case of malignant hyperthermia, its clinical course, and the measures taken to successfully treat it and preserve the function of a kidney allograft.

The Hynobiidae is generally regarded as the most phylogenetically basal and least derived extant family of terrestrial salamanders. As in the other families of terrestrial salamanders, prey capture in the Hynobiidae is accomplished by lingual prehension. In Hynobius, the prey capture system appears to be a mosaic of derived and primitive features. This, in conjunction with previous studies, suggests that the hyolingual systems of all families of terrestrial salamanders have evolved various degrees of specialization since the appearance of the common ancestral condition. We propose that the generalized feeding system for the extant terrestrial salamanders includes a hyolingual skeleton comprised of one basibranchial, one pair of radial or radial-like structures, two pairs of ceratobranchials, two pairs of epibranchials, one pair of ceratohyals, and one urohyal arranged in a configuration similar to that of Hynobius; a simple, sac-like secondary tongue pad; a lift and thrust system of tongue projection; a four-part gape cycle; and a forward head and body surge. Modifications to this general plan, previously described for the disparate families, include various changes in the size, shape, and definition of the tongue pad, changes in the specific types of structures and configurations in the anterior hyolingual skeleton, secondary ossification in the posterior hyolingual skeleton, the appearance of various protrusion, projection, and flipping systems for tongue protraction, simplification of the kinematic gape profile, and loss of the forward head and body surge. The evolutionary trends in these modifications have provided a rich data set from which much phylogenetic information has been inferred. © 1996 Wiley-Liss, Inc., (Copyright © 1996 Wiley-Liss, Inc.)

Reaction of the decapeptide gonadotropin releasing hormone (GnRH) with the Bolton-Hunter reagent produced a single major derivative. Mass spectrometric analysis of this derivative at M-Scan Corporation revealed that O-acylation of the Ser4 hydroxyl had occurred. Formation of the O-acylated Ser4 derivative was dependent on the presence of the His2 residue in GnRH. Similar experiments with several unrelated peptides revealed that the Bolton-Hunter reagent will readily acylate hydroxyl groups on serine, tyrosine, and threonine side chains located two positions from a histidine residue (e.g., His-X-Ser). Such O-acylated peptides can be formed under mild reaction conditions and appear to be relatively stable. Recognition of this sequence-specific O-acylation can be critical when labeling peptides with the Bolton-Hunter reagent and when interpreting experiments in which such modified peptides are used.

Recent findings have indicated that certain hydroxyl groups of peptides have enhanced intrinsic chemical reactivity and can be O-acylated by N-hydroxysuccinimide esters of biotin. Analytical procedures are described for the identification of O-acylated derivatives of seryl, threonyl, and tyrosyl residues after reaction with the N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid. The identification and chemical characterization of O-biotinylated residues included amino acid analysis, peptide sequence determination, and mass spectrometric analysis. The application of these analytical procedures provided unequivocal identification of O-biotinylated residues for a number of peptides investigated. In a separate study, we also show that O-biotinylated amino-terminal seryl residues occurring after proteolysis of peptides or proteins can rapidly undergo an O-->N acyl shift resulting in blockage of sequence analysis by the Edman degradation procedure.

Pituitary cells with GnRH receptors increase over 2-fold during diestrus to reach a peak during the morning of proestrus. This is followed by a rapid fall during the afternoon of proestrus to reach a nadir by estrus. The objective of this study was to learn the identity of the new target cells added during diestrus. This was particularly important in view of recent evidence showing that gonadotropes with LH beta and FSH beta mRNA have GH antigens. Pituitary cells from diestrous and proestrous rats were exposed to biotinylated GnRH (Bio-GnRH) for 10 min. Bio-GnRH was detected by avidin peroxidase, and then the cells were immunolabeled for pituitary hormones. The percentages of cells labeled for Bio-GnRH rose during diestrus from 6.6 +/- 0.8% in the morning to 11.9 +/- 0.7% by evening (mean +/- SD). By the morning of proestrus, the percentages of Bio-GnRH target cells increased further to 16 +/- 0.7%. The percentages of pituitary cells dual labeled for LH beta antigens and Bio-GnRH rose from 4.3 +/- 0.6% to 9% +/- 1% during diestrus and averaged 13 +/- 0.7% by the morning of proestrus. At this time, 90% of cells with LH antigens bound Bio-GnRH. When percentages of pituitary cells with FSH beta antigens and Bio-GnRH-binding sites were analyzed, there was an increase during diestrus from 4 +/- 0.4% to 9.7 +/- 0.7%; a peak level of 14 +/- 0.9% was reached by the morning of proestrus. Bio-GnRH binding was expressed by 86% of FSH cells during this peak. Finally, GH antigens were also detected in GnRH target cells. The percentage of cells dual labeled for Bio-GnRH and GH increased from 4 +/- 0.8% to 8 +/- 1% during diestrus and the morning of proestrus. During the diestrous and proestrous peak periods of expression, Bio-GnRH binding was seen in 32% of GH cells. None of the other pituitary cell types showed significant GnRH binding. These studies showed that most of the new GnRH-receptive cells stem from maturing gonadotropes. Half of the GnRH-receptive cells also contain GH antigens, which correlated with results from previous studies that showed GH antigens in cells with gonadotropin mRNAs. This might reflect expression of gonadotrope functions by a subset of GH cells. Alternatively, the GH antigens may be bound to GH receptors in gonadotropes. This latter possibility may signify a paracrine regulation of gonadotrope function by GH.

Chemical modification studies of peptide hormones and random peptides have revealed that seryl hydroxyl groups had enhanced reactivity toward acylating reagents when they occurred in the linear triads His-Xaa-Ser or Ser-Xaa-His (Xaa = any amino acid). O-acylation of serine within these triads was achieved by reaction with N-hydroxysuccinimide esters of biotin (NHS-biotin) and succinic anhydride. Seryl residues not occurring in His-Xaa-Ser/Ser-Xaa-His triads showed no reactivity towards NHS-biotin under reaction conditions described. Results of histidine replacement studies and studies of the pH dependence of O-biotinylation indicated that the increased nucleophilicity of the seryl hydroxyl group was due to intramolecular interaction between the seryl and histidyl residues. Our findings provide strong evidence that such triads represent novel consensus motifs in peptides.

A procedure employing C18 reversed-phase high-performance liquid chromatography (HPLC) is described for evaluating the kinetics of biotinylation of specific residues of peptides after reaction with N-hydroxysuccinimide esters of biotin. Utilizing this HPLC method, we determined the observed pseudo-first-order reaction rate constant (k'1) of biotinylation of lysyl residues in two model peptides, [biotinyl-Ser108]ProA-egg laying hormone (108-121) and pGlu-Lys-Trp-Ala-Pro, to be 1.22.10(-2)s-1 and 1.08.10(-2)s-1, respectively, in 0.05 M sodium phosphate buffer, pH 8.2, at 25 degrees C. The respective reaction half-lives of the two peptides were 57 s and 64 s. In addition, HPLC analytical methods were established for determining the time-course of hydrolysis of biotinylating reagents at acidic and alkaline pH and for evaluating biotin reagent homogeneity.

Mouse anterior pituitary cells cultured for 2 days were stimulated with one of three biotinylated-GnRH probes ([biotinyl-Lys6]-[D-Lys6]GnRH, [biotinyl-Ser4]-[D-Lys6]GnRH, [biotinyl-Ser4]-[D-Trp6, des-Gly10]GnRH) in the cold (4 degrees C) for 1 hr. These cells were subsequently fixed and an avidin-gold complex was conjugated to the bound GnRH. After a second fixation, the gold label was silver-enhanced for viewing with a scanning electron microscope. Gonadotropes were identified as a result of the labeling procedure, measured for size, and the number of GnRH receptor sites counted. Gonadotropes ranged from 3 to 13 microns in diameter and contained from 23.2 +/- 3.3 to 338.4 +/- 25.2 sites per cell depending upon the size of the cell and the ligand employed. The methods described should be applicable for studying the topographical distribution of a variety of cellular receptors.

Gonadotropin-releasing hormone (GnRH) and two of its analogs ([D-Lys6]GnRH and des-Gly10-[D-Trp6]-GnRH) were reacted with sulfonated N-hydroxysuccinimide esters of biotin that have been reported to react specifically with primary amino groups. Fractionation by reversed-phase high performance liquid chromatography demonstrated the occurrence of multiple biotinylated derivatives for each reacted peptide. These results were unexpected since GnRH and des-Gly10-[D-Trp6]GnRH contained no reactive amino groups and [D-Lys6]GnRH had only one. Reaction of the biotinylated derivatives with hydroxylamine indicated that significant O-biotinylation had occurred. Mass spectrometric analyses established the stoichiometry of biotinylation and confirmed that substantial O-biotinylation of residue Ser4, and to a minor extent Tyr5, of GnRH and the two analogs had occurred. In contrast, the biotinylation of selected peptides unrelated to GnRH under identical reaction conditions indicated no significant evidence of O-acylation of seryl residues. Strikingly, biotinylation of GnRH under denaturing conditions largely abolished O-acylation, indicating that the observed O-biotinylation was dependent on peptide conformation. All the O-biotinylated derivatives displayed significantly reduced bioactivity. Taken together, these results give strong evidence that the Ser4 hydroxyl of GnRH has a significantly elevated intrinsic reactivity, which raises new questions concerning its putative role in the conformation and mode of action of the hormone. These results also demonstrate for the first time that the N-hydroxysuccinimide-biotin esters are capable of significant O-acylation and may be generally useful reagents for detecting highly reactive hydroxyamino acid residues.

Chemical modification of the egg-laying hormone (ELH) of Aplysia by reaction with the N-hydroxysuccinimide ester of biotin, which contained 6-aminohexanoic acid as spacer, yielded seven distinct derivatives that were readily separated by reversed-phase high performance liquid chromatography. The derivatives were chemically characterized by amino acid compositional analysis, sequence analysis, and mass spectrometry. The seven derivatives resulted from combinations of differential modification of the three amino groups in the ELH molecule located at Ile1 (alpha-NH2), Lys8, and Lys36. Of the seven derivatives formed, only one, monobiotinyl Lys36-ELH, was biologically active in eliciting egg-laying activity and altering the electrophysiological activity of the abdominal ganglion neuron R15 and LB and LC cluster neurons. In addition, evaluation of the time course of biotinylation of ELH revealed that the relative rate of amino group reactivity was epsilon-NH2-Lys36 greater than epsilon-NH2-Lys8 much greater than alpha-NH2-Ile1. The slow rate of reaction of the terminal alpha-amino group suggested that it was relatively inaccessible to biotinylation, possibly due to conformational factors or to ion-pair formation with an unidentified carboxyl group. Loss of bioactivity of ELH monobiotinylated on the alpha-amino group, coupled with the unusually low reactivity of the alpha-amino group, provided strong evidence for the importance of the alpha-amino group in ELH function. Furthermore, the development and availability of a bioactive ELH probe should greatly facilitate the isolation, characterization, and localization of the ELH receptor.

A method is described for the determination of the stoichiometry of biotinylation of peptides and proteins after reaction with an N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid (NHS-epsilon Ahx-biotin). The method of analysis, based on the quantification of phenylthiocarbamyl derivatives of 6-aminohexanoic acid, is able to measure low picomolar amounts of biotinyl derivative. Analyses were performed using an automated on-line hydrolyzer-derivatizer followed by high-performance liquid chromatography. Compositional analyses determined for known peptides were in excellent agreement with analyses obtained by mass spectrometry. Procedures are also described for the production of biotinylated protein probes that can be labeled reproducibly to contain specific amounts of biotin. The analytical advantage and steric freedom provided by the 6-aminohexanoic acid spacer arm argue strongly for the NHS-epsilon Ahx-biotin reagent to be a reagent of choice for the biotinylation of peptides and proteins.

The identification and characterization of biotinylated lysyl residues in a polypeptide chain by automated sequence analysis is described. An in depth analytical study was conducted for the delineation of N epsilon modification of lysyl residues with N-hydroxysuccinimide esters of biotin and 6-aminohexanoic biotin. Confirmation of the structure of the phenylthiohydantoin derivatives of N epsilon biotinylated lysine was achieved by mass spectrometry. The analytical study focused on the identification of biotinylated lysine-4 of neuropeptide Y which served as a model peptide for the analytical procedures detailed.

The effects of adolescent morphine exposure on the sexual maturation of male rats, their reproductive capacity and the development of their progeny were examined. Groups of prepubescent male rates (25-27 days of age) were implanted with morphine- or placebo-pellets (one on Day 1, then two pellets on Days 4, 7 and 10); the pellets were not removed to assure the sustained release of morphine for 3 to 4 weeks and to avoid the confounding effects of a precipitated withdrawal syndrome. Groups of animals were sacrificed at weekly intervals through adulthood for an assessment of reproductive endocrine function. A large group, however, was also bred with drug-naive primiparous females at 85 days of age (8 weeks after morphine or placebo pellet implantation), when the acute and chronic effects of morphine on reproductive endocrine parameters had dissipated; their fertility and the development of the male and female progeny was characterized. Our results indicated that morphine exposure during adolescence led to a pronounced inhibition of a number of indices of sexual maturation (e.g., serum testosterone and luteinizing hormone levels and reduced weights of the testes and seminal vesicles). Breeding morphine- and placebo-implanted male rats with drug-naive females resulted in smaller liters derived from morphine-treated fathers when compared to controls, but in all other respects the development of the offspring in the two groups were equivalent. However, upon reaching adulthood, a number of selective endocrine differences were detected in morphine-derived offspring when compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein synthetic activity of anterior hypothalamic and neocortical brain slices from androgenized neonatal female rats was studied in vitro in an attempt to clarify the mechanism of action of testosterone on the developing hypothalamus. The short-term effects of testosterone propionate injections of the animals on the protein synthetic activity of the slices were assayed by means of incorporation of 14C-leucine into acid-insoluble protein. Although a high dose (1 mg/animal) of testosterone propionate stimulated increased incorporation in neocortical and anterior hypothalamic slices, a lower dose and other approaches failed to demonstrate a stimulation regionally specific to the anterior hypothalamus. It is concluded that if testosterone has specific stimulatory effects on protein synthesis in the anterior hypothalamus of neonatally androgenized rats, these are not readily demonstrable by methods used with other steroid target tissues.

The effects of a single morphine pellet (75 mg) implanted in developing male rats at 27 days of age on reproductive endocrine parameters were compared to those found in adult (65-day-old) animals after the same treatment. The pellets were left in place to provide the release of morphine during critical phases of puberty and sexual maturation and to prevent an abrupt withdrawal syndrome upon pellet removal which would confound our results. Developing rats were sacrificed at representative intervals after pellet insertion to assess the development of key indices of reproductive endocrinology; adult rats were sacrificed at the same time intervals to permit an evaluation of age-related differences in the sensitivity to opiate-induced endocrine disturbances. Our results showed that morphine markedly influenced a number of endocrine parameters associated with the maturation of the hypothalamic-pituitary-gonadal axis in developing rats for prolonged periods of time, whereas the effects of the opiate in the adult rat were relatively modest and transient. In the developing rat, serum luteinizing hormone (LH), testosterone, the wet tissue weights of the seminal vesicles and testes and hypothalamic LH-releasing hormone (LHRH) levels were substantially depressed immediately after pellet implantation and these effects persisted for up to 4 weeks when compared to placebo-implanted, age-matched controls. In contrast to these results, adult rats showed only transient effects (less than 1 week) of morphine on certain reproductive endocrine parameters (e.g., serum LH, testosterone and the weights of the seminal vesicles) and no effects on others (e.g., testes weights and hypothalamic LHRH).(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbic acid is frequently used in in vitro studies of neurotransmitter-evoked release of luteinizing hormone-releasing hormone (LHRH) from hypothalamic fragments. Although it is assumed that ascorbate merely prevents the oxidative degradation of catecholamines, we have discovered that ascorbic acid itself produces significant increases in the release of LHRH. Our studies showed that ascorbic acid, at concentrations below 1 mM, produced a dose-dependent release of LHRH from incubated rat mediobasal hypothalamus (MBH). The magnitude of the ascorbate-induced release was in the range of 100-200% above controls; significant amounts of LHRH were released only if the MBH were incubated with ascorbate for time periods longer than 30 minutes. We also found that ascorbate-induced increases in LHRH were equivalent to those produced by another LHRH secretagogue, naloxone, and that the combined effects of the two substances were additive in nature. Although the mechanisms underlying this effect are not fully understood, nonspecific chemical reduction is probably not a factor since sodium metabisulfite did not induce the release of LHRH. It seems probable that ascorbate may enhance the activity of endogenous norepinephrine in the MBH and, thereby, lead to increased release of LHRH.

Relative potency estimates were performed by parallel line skin test assay (PLST) and ELISA inhibition methods for three polymerized allergen extracts (Bermuda grass, orchard grass, and a copolymer of short and giant ragweed) versus four unmodified RAST standardized reference extracts (Bermuda grass, orchard grass, and giant and short ragweed) in nine subjects. One subject experienced a systemic reaction, requiring treatment at the end of the PLST assay. Another subject had a systemic reaction during limited skin testing performed approximately 72 hours after completion of PLST. Relative potency values for the polymerized extracts obtained by PLST were much lower than those obtained by ELISA inhibition, but results were significantly (r = 0.95; p less than 0.01) correlated. Because polymerized allergen extracts are designed to be hypoallergenic, a skin test assay may underestimate their potency relative to an unmodified reference extract.

Naloxone produces large increases in serum luteinizing hormone (LH) levels in normal males and females, supporting a role for endogenous opioids (EOP) in the tonic inhibition of LH. Since the antagonist apparently exerts no important effects on the pituitary, the reasonable assumption has been made that it elevates gonadotropin levels by affecting the release of LH-releasing hormone (LHRH) from the hypothalamus. However, at present there is no direct in vivo evidence supporting this widely-held view. In an attempt to directly demonstrate that naloxone increases the secretion of LHRH, and thereby elevates serum LH levels, we examined whether a potent synthetic antagonist of LHRH ( [D-p Glu1, D-Phe2, D-Trp3,6]-LHRH, GPT-LHRH) blocked the effects of naloxone in male rats with a normal response to naloxone and in those with a markedly enhanced sensitivity to the drug induced by a brief period of morphine pellet implantation. Our results demonstrated that GT-LHRH antagonized equipotent doses of LHRH (100 ng/kg) and naloxone (0.5 mg/kg) over a similar time course with approximately the same AD50. Most importantly, however, we showed that the GPT-LHRH produced equivalent, parallel shifts to the right in the dose-response curves for LHRH and naloxone, indicative of competitive inhibition. We also found that GPT-LHRH completely abolished the enhanced response to naloxone's effects on LH which occurs in morphine-pretreated rats. Since we observed no competition between LHRH and naloxone for their binding sites in pituitary or brain, the only viable interpretation of our results is that naloxone increases LH by inducing the release of LHRH.