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Objective: This study aimed to evaluate the antibacterial efficacy of different concentrations of GH12 on a simulated multispecies biofilm comprising Enterococcus faecalis, Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis., Methods: Single rooted teeth were decoronated, cut into 1.5 mm sections to obtain dentine discs which were randomly allocated into five groups: (n=12 each), Group 1: Phosphate Buffered Solution (PBS) - negative control, Group II: 5% Sodium hypochlorite (NaOCl) - positive control, Group III: Minimum Inhibitory Concentration (MIC) of GH12, Group IV: 2x MIC of GH12, Group V: 4x MIC of GH12. Colony forming units, Crystal violet assay and scanning electron microscopy examinations were performed. One-way ANOVA and Turkey's test were applied for statistical analysis using the SPSS software version 22.0., Results: Group II (NaOCl) showed maximum reduction in bacterial load followed by Group V (GH12 16mg/mL) with no statistically significant difference (p=1.000). On comparing the mean CFU reduction, the maximum reduction was identified for S. mutans and the least was for P. gingivalis. There was marked erosion observed in the NaOCl group whereas the GH12 group showed no erosive changes in the morphology and no bacterial colonies was identified., Conclusion: The findings revealed that GH12 at higher concentrations inhibits and disrupts the growth of multispecies endodontic biofilm comparable to NaOCl but without erosive effects to the dentine, further highlighting its potential to be used as an antimicrobial solution. (EEJ-2023-10-147).

Introduction: Root resorption poses a significant challenge in dental practice, with external cervical resorption (ECR) being a common manifestation. ECR is often asymptomatic until advanced stages, complicating its diagnosis and management. Various factors contribute to its etiology, ranging from trauma to orthodontic treatment. The classification system proposed by Patel et al. (2018) offers a comprehensive framework for characterizing ECR lesions based on location and extent. Treatment strategies for ECR involve a combination of endodontic intervention and restorative techniques, with bioactive materials like mineral trioxide aggregate (MTA) and Biodentine emerging as promising options. However, the biomechanical behavior of teeth restored with these materials in the context of ECR remains underexplored., Materials and Methods: This study utilized finite element analysis (FEA) to assess stress distribution in teeth with simulated ECR lesions of varying sizes and locations, restored with MTA or Biodentine. Three-dimensional models of maxillary central incisors were generated based on CBCT scans, incorporating periodontal ligament and surrounding bone structures. Eight experimental models representing different ECR configurations were created and subjected to FEA using Optistruct software based on dimensional classification given by Patel et al., in 2018, A70 M & A70B: 1Ap, A130 M & A130B: 1Bp, B70 M & B70B: 2Ap, B130 M & B130B: 2Bp. All the models were tested for stress distribution by restoring the lesions with either M: MTA or B: Biodentine. Oblique load of 100 N was applied at 45°angle to the long axis 2 mm lingual to incisal edge. vonMises Stress distribution in enamel, dentine, restoration and at all the interfaces were observed., Results: The analysis revealed that both MTA and Biodentine restorations exhibited uniform stress distribution around ECR lesions, with no significant differences based on lesion location or size. Maximum stress concentrations were observed around the restorations, particularly in subcrestal lesions. However, overall stress levels were comparable between MTA and Biodentine restorations, indicating similar biomechanical performance., Conclusion: Finite element analysis provides valuable insights into the biomechanical behavior of teeth with ECR lesions restored with MTA and Biodentine. Both materials exhibit similar stress distribution patterns and offer adequate reinforcement against mechanical forces. Clinicians can confidently utilize MTA or Biodentine in the management of ECR, considering their favorable biomechanical properties and clinical outcomes. Further research is necessary to validate these findings and optimize treatment protocols for ECR., (© 2024 Published by Elsevier B.V. on behalf of Craniofacial Research Foundation.)

Aim: The aim of this study was to compare the effect of two calcium silicate-based and an epoxy resin-based root canal sealers on postoperative pain and analgesic intake following single-visit root canal treatment., Materials and Method: Ninety patients with at least one first or second molar tooth diagnosed as symptomatic irreversible pulpitis and symptomatic apical periodontitis were selected and allocated into three groups (n=30) according to the sealer used. Root canals were prepared using Protaper Gold instruments (Dentsply Sirona) in a crown down technique and irrigated with 2.5% NaOCl (Calyx, India) and saline solution. Root canal filling was then accomplished with a single cone obturation technique and treated in a single visit by the same endodontist. Patients were told to use a Visual Analog Scale (VAS) to rate their postoperative pain severity as none, minimal, moderate, or severe after 6 h, 24 h, 48 h, 5 days and 7 days following obturation using the appropriate sealers. The need for analgesic intake was also recorded. The data were statistically analyzed., Results: Results showed a significant difference among the studied groups. Bio-C Sealer Ion+ reported the least pain score followed by Nishika Canal Sealer BG and AH plus sealer at all the time intervals recorded. The intergroup analysis, revealed was a significant difference in postoperative pain at 6 h (p=0.000) and 24 h (p = 0.028), but not at 48 h, 5 day or 7 days (P > 0.05). VAS ratings for all the three groups decreased over time. Also, there were significant differences between the means of analgesic intake among 3 groups (p=0.022). Analgesic intake in group BIO-C Sealer Ion+ is significantly lesser than AH Plus and Nishika Canal Sealer BG group., Conclusion: Calcium silicate-based sealer (Nishika Canal Sealer BG and Bio-C Sealer Ion+) resulted in significantly lower levels of pain as compared to epoxy resin-based sealer (AH Plus) at 6h and 24-h interval, there was no significant difference in postoperative pain occurrence at 48-h, 5 day and 7-day period. The analgesic intake in Bio-C Sealer Ion+ group is significantly lesser than Nishika Canal Sealer BG and AH Plus group., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Journal of Conservative Dentistry and Endodontics.)

Objective: The escalating prevalence of noncarious tooth wear stands as a critical concern in the backdrop of evolving lifestyles and dietary patterns. Dental erosion, a progressive condition induced by both endogenous and exogenous acidic influences, directly impacts enamel integrity, resulting in surface loss. The contemporary surge in carbonated beverage consumption further exacerbates this erosive milieu, underscoring the urgency for dental practitioners to adopt meticulous treatment strategies. Existing literature underscores a noteworthy 94% reduction in tooth erosion risk for individuals abstaining from sweetened soft beverages, emphasizing the imperative for a well-devised remineralization protocol to counter demineralized surfaces., Methodology: Seventy-three enamel specimens were taken. Forty samples were subjected to pre-operative hardness testing, and five samples were subjected to baseline EDX evaluation followed by grouping of samples (Group 1 = control Group; Group 2 = casein phosphopeptide-amorphous calcium phosphate fluoride [CPP-ACPF] Group; Group 3 = Biomin F Group; and Group 4 = self-assembling peptide [SAP] P-114 Group). A demineralization-remineralization cycle was carried out for 5 days followed by testing through Vickers Microhardness Tester, EDX Evaluation, and Scanning Electron Microscopy (SEM) Imaging. Statistical analysis was performed using one-way analysis of variance followed by intergroup analysis using Tukey's post hoc test with SPSS software 25.0 version., Results: The mean percentage change in microhardness values was 30.05% in Group 1, 24.21% in Group 2, 18.85% in Group 3, and 12.08% in Group 4. The mean Ca/P ratio of samples tested through EDAX was 2.20 at baseline, 1.40 in Group 1 (Control Group), 1.62 in Group 2 (CPP-ACPF), 1.82 in Group 3 (Biomin F), and 2.01 in Group 4 (SAP-P114). Postintervention values were statistically significant from baseline values in both parameters., Conclusion: Curodont Protect exhibits superior efficacy, offering valuable insights for future in vivo studies and clinical applications. The multifaceted evaluation, encompassing microhardness testing, SEM analysis, and EDXS assessment, contributes to a nuanced interpretation of the agents' impact, paving the way for informed decisions in clinical practice and future research endeavors., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Journal of Conservative Dentistry and Endodontics.)

Objective: The study aimed to compare and evaluate the effect of biodentine (BD) alone, BD along with Lyophilised freeze dried platelet rich concentrate (LPC + BD), and BD along with low-level laser therapy (BD + LLLT) after pulpotomy in mature permanent molars with irreversible pulpitis., Materials and Methods: The study was designed as a randomized, pragmatic, parallel, double-blinded clinical trial registered under the Clinical Trial Registry-India (CTRI/2020/02/023245). 120 permanent molars fulfilling the inclusion and exclusion criteria with symptoms of irreversible pulpitis were randomized after performing pulpotomy into three pulp capping groups: Group 1, BD; Group 2, lyophilized freeze-dried platelet-rich concentrate + BD (LPC + BD); and Group 3, Low level laser therapy + BD Group 3, LLLT + BD. The intergroup comparison was done using one-way analysis of variance followed by the Bonferroni test. The level of significance and confidence interval were 5% and 95%, respectively. Interobserver reliability was measured using Cohen's kappa analysis., Results: At 1 week, there was a significant difference (P < 0.005) observed in the mean postoperative pain levels between the three groups with Group 1 (BD) exhibiting the highest postoperative pain followed by Group 2 (LPC + BD) and least pain was exhibited by Group 3 (LLLT + BD). A similar pattern was observed regarding the analgesic intake with maximum frequency in Group 1 (BD) and least with Group 3 (LLLT + BD). No significant difference in success rates was reported among the groups., Conclusion: Pulpotomy as a treatment option for mandibular molars with irreversible pulpitis has an acceptable clinical success rate; however, long-term overall success rate remains questionable. The outcomes of incorporating adjunctive modalities with BD are remarkable and show tremendous potential for continued development and research., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Journal of Conservative Dentistry and Endodontics.)

Objective: To investigate the concentration of Matrix metalloproteinases-9 (MMP-9) and Tumor necroses fac- tor-alpha (TNF- α) in pulpal blood at various stages of pulpal inflammation in diabetics and to establish the relationship between these two biomarkers., Methods: 77 patients, each having a tooth with pulpal exposure due to caries presenting with distinct stages of pulpitis were grouped into 2 main study groups as based on the HbA1c Levels-Group 1: Non-Diabetics (Control Group) (HbA1c < 5.6%) and Group 2: Type 2 Diabetics (Experimental Group) (HbA1c>6.5%; Random Plasma Glucose > 200) and diabetes mellitus with less than 10-year history. Depending on the radiological and clinical diagnosis, these two groups were again sub-divided into 2 subgroups: Sub-group A: Tooth with Symptomatic Irreversible Pulpitis. Sub Group B: Tooth with Reversible Pulpitis. Thus, for comparison purposes, a total of 4 sub-divisions were formed: Sub-group 1A- Non-Diabetic, Symtomatic Irreversible Pulpitis, Sub- group 1B: Non-Diabetic, Reversible Pulpitis, Sub-group 2A: Diabetic, Symptomatic Irreversible Pulpitis, Sub- group 2B: Diabetic, Reversible Pulpitis. Blood sample was collected from pulp chamber after partial pulpo- tomy was done. The total levels of MMP-9 and TNF-α were assessed by enzyme linked immunosorbent assays (ELISA). Inter-group comparison in levels of MMP-9 and TNF- α were conducted using the Kruskal Wallis test and pairwise comparison was done Mann-Whitney U test., Results: The inter-group comparison in levels of MMP-9 and TNF- α were conducted using the Kruskal Wallis test and pairwise comparison was done using Mann-Whitney U test. Pearson correlations were conducted in order to investigate correlations between the paired TNF-α and MMP-9 values and also their correlation with the blood sugar levels within the pulp diagnosis groups. MMP-9 and TNF-α levels were significantly higher (p<0.005) in irreversible pulpits than reversible pulpits and also in Type-2 diabetics than non-diabetics. High- est level of MMP-9 and TNF-α was found in Group 2A (Diabetic, symptomaticirreversible pulpitis) and lowest in Group 1B (Non-Diabetic, reversible pulpitis). There exists a very high significant positive correlation between MMP-9 and TNF-α (p<0.005)., Conclusion: These findings show that the inflammatory mediators MMP-9 and TNF-α are significantly in- creased in pulpal blood samples of diabetic patients. Also, in diabetic patients diagnosed with reversible pul- pitis, higher levels of inflammatory pulpal biomarkers were reported that could compromise the success of Vital Pulp Therapy (VPT) and may necessitate endodontic intervention. MMP-9 and TNF- α were reported to have a positive correlation. (EEJ-2023-01-04).

Objective: To evaluate and compare the effect of diode laser assisted bleaching, ultrasonic scaling and powered tooth brushing on surface roughness and bacterial adherence on class V cavities restored with composites., Materials and Methods: A total of one hundred and twenty samples (40 samples each of Brilliant Everglow, Beautifil II and Heytec-N) were prepared in standardized stainless steel molds. The samples were further subdivided into four subgroups i.e. one control group (without any intervention) and three experimental groups - diode laser assisted bleaching, ultrasonic scaling and powered tooth brushing consisting of 10 sample each. Surface roughness was measured quantitatively with the help of 3D Optical Profilometer. For bacterial adherence analysis S. mutans strain (ATCC 25175) was cultured in BHI medium and samples were evaluated for the presence of viable bacteria using the Colony Forming Unit (CFU) count. Results obtained were then tabulated and subjected to statistical analysis., Results: Diode laser bleaching caused a significant increase in surface roughness and bacterial adherence with lowest mean change exhibited by Heytec-N followed by Beautifil II and highest by Brilliant Everglow group. Similarly, Ultrasonic scaling increased the surface roughness of all the three tested samples with significant difference between the groups. Powered tooth brushing had no effect on the surface roughness and bacterial adherence of the tested composites., Conclusion: Diode assisted laser bleaching and ultrasonic caused significantly higher surface roughness and bacterial adherence values for all the tested composites. It may therefore be recommended to do finishing and polishing of restorations after such procedures., (© 2023 Published by Elsevier B.V. on behalf of Craniofacial Research Foundation.)

Objective: The present study was conducted to evaluate the presence of aerobic bacteria, anaerobic bacteria, E. faecalis, F. nucleatum, Propionibacteria sp., Actinomyces sp., and their reduction at various stages of endodontic retreatment with the use of conventional protocol (5.25 % Sodium Hypochlorite (NaOCl) as the irrigant along with Calcium Hydroxide (Ca (OH)2) as intracanal medicament and advocated protocol (SmearOFF as the irrigant along with 2% Chlorhexidine (CHX) gel as intracanal medicament)., Methods: Twenty eight patients fulfilling the eligibility criteria were selected for root canal retreatment and randomly allocated into two groups. Group 1: Final irrigant as SmearOFF+Chlorhexidine 2% gelas intracanal medicament (n=14). Group 2: Final irrigant as 5.25% NaOCl+Ca(OH)2 as intracanal medicament (n=14). With aseptic environment, access opening was performed followed by Gutta Percha (GP) removal and sample S1 was collected for bacterial analysis. The biomechanical preparation was done by using Reciproc system with additional finishing with XP-Endo Finisher R. Sample S2 was then collected for bacterial analysis after the final irrigation protocol in the respective groups. Intracanal medicaments were placed for one week and sample S3 was collected. All the samples were subjected to qualitative analysis using PCR and quantification was done by Colony Forming Unit (CFU) analysis., Results: Aerobic [28/28], Anaerobic [28/28], Propionibacterium sp. [20/28] and F. nucleatum [24/28] were the most frequently isolated in S1 sample followed by Actinomyces sp. [16/28] and E. faecalis sp. [19/28]. Chemico-mechanical preparation followed by irrigation (S2 sample) resulted in significant reduction of all types of bacteria in both groups. Group-1 (SmearOFF as the final irrigant) had significantly superior efficacy against aerobic bacteria, E. faecalis and F. nucleatum (P<0.05) as compared to Group-2 (NaOCl). After medicament placement, significant differences between the groups were noted only for the E. Faecalis group. For the S3 samples, the mean bacterial reduction was significant in Aerobic and F. nucleatum in S3 samples for Group 1 and Group 2., Conclusion: Chemico-mechanical preparation followed by irrigation resulted in significant reduction in bacterial load irrespective of the final irrigant. SmearOFF was significantly better than NaOCl in minimizing bacterial load of E. faecalis and F. nucleatum. 2% Chlorhexidine gel has superior antimicrobial efficacy against E. faecalis and may be recommended in secondary endodontic treatment.

Background: Journal of Clinical and Experimental Dentistry (J Clin Exp Dent; JCED) is an English language journal published by the Spanish Society of Oral Surgery, and has been online since 2009. It is indexed in PubMed Central and Scopus since 2012, with monthly publications since 2016. The purpose of this article was to review and analyse the publications in this journal since its inception, over a period of 11 years (2009-2019)., Material and Methods: This paper assessed the number, type and subjects of the articles published in the journal over 11 years. The institutions of the first authors, number of PubMed citations and the Hirsch (h5) index was assessed and analysed., Results: The manuscripts published in JCED have gradually increased over the years, with Original research articles accounting for the bulk of contributions. The journal publishes articles mainly from the subjects of Oral Pathology and Operative Dentistry and Endodontics. Articles published in JCED are indexed in PubMed Central (since 2012), Scopus, DOI system, and Google Scholar. A country-wise mapping of the (first) author's institutions revealed significant contributions from researchers from all over the world. With an h5 index of 26, the journal was ranked among the top six multispeciality journals. The most cited articles were the literature reviews on common oral lesions (recurrent apthous stomatitis and candidiasis)., Conclusions: The journal has contributed to the growth of scientific literature pertaining to subjects from all the fields of dentistry. Over the past 11 years, JCED has served as a platform for large number of manuscripts in all the disciples of dentistry, from researches all over the world. Key words: Publication trends, Journal of Clinical and Experimental Dentistry, Bibliometrics., Competing Interests: Conflicts of interest None declared., (Copyright: © 2020 Medicina Oral S.L.)

Post-traumatic stress disorder (PTSD) is a well-established psychological disorder after severe traumatic injury but remains poorly recognized. Recent changes in the "Resources for Optimal Care of the Injured Trauma Patient 2014" stress the need for comprehensive screening and referral for PTSD and depression after injury. Our purpose was to review the current PTSD literature and perform a retrospective chart review to evaluate screening at our institution. We hypothesized a lack of documentation and thus referral of these patients to mental health professionals. We performed a literature review of 43 publications of risk factors for PTSD in the civilian adult population followed by a retrospective review. Records were analyzed for basic demographics, risk factors found in the literature, and referrals to mental health providers. Risk factors included amputation, dissociative symptoms, female gender, history of mental health disorder, and peri-traumatic emotionality. Traumatic amputation status and gender were recorded in all patients. History of mental health disorder was present in 11.5 per cent patients, absent in 80.75 per cent, and not recorded in 7.75 per cent with an overall documentation of 91.75 per cent. Dissociative symptoms and peri-traumatic emotionality were recorded in 0.5 per cent and 1.0 per cent of patients, respectively. Only 13 patients of 400 (3.25%) were referred to mental health professionals. Despite extensive evidence and literature supporting risk factors for the development of PTSD, identification and treatment at our level 1 trauma center is lacking. There is a need for consistent screening among trauma centers to identify PTSD risk factors and protocols for risk reduction and referrals for patients at risk.

With the limitations posed by increasing work hour restrictions, surgical residency programs are focusing more on maximizing the educational benefit of their conferences. The Morbidity and Mortality (M&M) conference serves as a forum to discuss adverse events and patient care improvement using evidence-based medicine. The matrix format (MF) is an enhancement to the traditional format (TF), focusing on the case selection process and a postconference newsletter reiterating the relevant literature review and discussion points. Our institution adopted the MF to evaluate both its short- and long-term educational values. Surveys were distributed to residents and faculty within the Department of General Surgery to assess their interest and satisfaction, perception of educational value, and efficiency with the MF compared with the TF. Responses were obtained from 22/22 (100%) residents for the TF and 11/23 (48%) for the MF. Faculty responses were 19/19 (100%) and 9/16 (56%), respectively. Reasons for an overall decreased response with the MF were not investigated further, as participation was strictly voluntary. Our results confirmed an overall approval of the MF by both residents and faculty. Faculty reported an improved efficiency of the conference (P < 0.039), encompassing improved content and presentation quality. Residents reported an improved overall interest and satisfaction with the MF (P < 0.001) as well as an improvement in the educational value (P < 0.007). Residents spent less time preparing presentations and reported learning greater educational benefit to the conference when preparing their own presentations (P < 0.001). In conclusion, the MF resulted in a greater overall satisfaction for residents and attending surgeons with an increased investment by the audience and overall improvement in perceived educational benefit.

A cutaneous ciliated cyst is a rare entity found predominantly in the lower extremities and perineal region of young females. Although initially described by Hess in 1890, the present day term, "cutaneous ciliated cyst," was proposed by Farmer in 1978 and includes a wide array of cyst types. Despite their typical female predominance and location, many have described cutaneous ciliated cysts in males and atypical locations. In addition, Mullerian cysts in the posterior mediastinum and the retroperitoneum have been reported. To date, only 40 cases have been reported in the literature of a Mullerian-type, cutaneous ciliated cyst. Here, we report a case of 13-year-old female with one in the gluteal cleft, initially presenting as a pilonidal cyst. We also discuss the differential diagnosis of pediatric sacrococcygeal lesions and pathogenesis of a Mullerian-type, cutaneous ciliated cyst.

Background: Recent efforts by the Accreditation Council for Graduate Medical Education to standardize resident education and demonstrate objective clinical proficiency have led toward more accurate documentation of resident competencies. Particularly with regard to bedside procedures, hospitals are now requiring certification of competency before allowing a provider to perform them independently. The current system at our institution uses a time-consuming, online verification system. This study provided an alternative method through an identification card with a list of bedside procedures. Our aim was an easier verification method for nurses, allowing fewer delays of bedside procedures and more time for nursing to patient care., Methods: We performed a prospective, controlled study, using general surgical residents and surgical intensive care nurses. Subjects performed an initial survey of their experience with the current online system in place to identify resident bedside procedure competency. Phase I involved educating the subjects about this current system followed by another survey. Phase II involved introducing our proficiency card. After 3 months, we conducted a final survey to evaluate opinions on the proficiency card, comparing it with the online verification method., Results: Nursing postintervention responses indicated that significantly less time was required to validate a resident's proficiency (P = .04). Prior to the introduction of the proficiency card, only 15% of nurses reported a verification time of 5 minutes or less, compared with 64% postintervention. In addition, nurses rated the card validation as an easier, more efficient method of verification (P = .02)., Conclusions: We believe that its continued use will not only improve the adherence to a mandatory hospital policy but also result in a less-cumbersome verification process, allowing more time for physician and nurse-to-patient care.

Elevated levels of homocysteine produce detrimental effects in humans but its role in preterm birth is not known. Here we used a mouse model of hyperhomocysteinemia to examine the relevance of homocysteine to preterm birth. The mouse carries a heterozygous deletion of cystathionine β-synthase (Cbs(+/-)). Gestational period was monitored in wild type and Cbs(+/-) female mice. Mouse uterine and placental tissues, human primary trophoblast cells, and human myometrial and placental cell lines were used to determine the influence of homocysteine on expression of specific genes in vitro. The activity of BKCa channel in the myometrial cell line was monitored using the patch-clamp technique. We found that hyperhomocysteinemia had detrimental effects on pregnancy and induced preterm birth in mice. Homocysteine increased the expression of oxytocin receptor and Cox-2 as well as PGE2 production in uterus and placenta, and initiated premature uterine contraction. A Cox-2 inhibitor reversed these effects. Gpr109a, a receptor for niacin, induced Cox-2 in uterus. Homocysteine upregulated GPR109A and suppressed BKCa channel activity in human myometrial cells. Deletion of Gpr109a in Cbs(+/-) mice reversed premature birth. We conclude that hyperhomocysteinemia causes preterm birth in mice through upregulation of the Gpr109a/Cox-2/PGE2 axis and that pharmacological blockade of Gpr109a may have potential in prevention of preterm birth., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The serial transverse enteroplasty procedure (STEP) was introduced as a bowel-lengthening procedure to reduce complications related to short bowel syndrome (SBS). Although some have described it as a useful adjunct to the Bianci procedure, others have acknowledged it as a primary procedure. We present a case of jejunal atresia in which two STEP procedures were performed 7 months apart to increase small bowel length. A 1-day-old, term girl presented with a known bowel obstruction diagnosed in utero. A laparotomy revealed a Type IIIb jejunal atresia with no remaining small bowel or cecum. A STEP procedure with an end jejunostomy and ascending colon mucous fistula lengthened the small bowel from 35 to 50 cm. A repeat procedure 7 months later lengthened it to 89 cm. The STEP procedure results in slower intestinal transit time and increases enterocytes contact with oral intake. We performed it during our initial exploration to increase small bowel size by 30 per cent. A repeat procedure 7 months later increased length to 89 cm. The use of multiple, staged STEP procedures avoided the need for bowel transplantation and long-term total parenteral nutrition dependence, demonstrating its effectiveness as a primary procedure for the surgical management of SBS.

Bizarre parosteal osteochondromatous proliferation (BPOP), or Nora's lesion, was first described by Nora et al. in 1983 as a rare, tumor-like lesion involving the bones of the hands and feet. Popliteal artery pseudoaneursyms in the pediatric population are also unusual. Here, we present a case of a young male with a popliteal artery pseudoaneurysm and distal femur lesion originally thought to be an osteochondroma. A 10-year old, Caucasian male was referred to our facility following an MRI concerning for a popliteal artery pseudoaneurysm. On physical exam, there was a palpable 5 × 5-cm pulsatile mass in the upper popliteal fossa with a normal pulse exam bilaterally. A computed tomographic angiogram demonstrated a 4.5-cm by 1.8-cm by 3.6-cm pseudoaneurysm adherent to a 3.5-cm thick, exostotic lesion of the posterior right femur. He was taken to the operating room for repair of the popliteal pseudoaneurysm and resection of his bone lesion. The final pathology was consistent with a popliteal pseudoaneurysm, osteochondroma, and bizarre parosteal osteochondromatous proliferation (BPOP), otherwise known as Nora's lesion. The location of the lesion and the age of our patient were both atypical for BPOP and to our knowledge, this represents the first report of a resulting popliteal artery pseudoaneurysm., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: interventions to prevent hospital readmission depend on the identification of patients at risk. The LACE index predicts readmission (and death) and is in clinical use internationally. The LACE index was investigated in an older UK population., Methods: randomly selected alive-discharge episodes were reviewed. A LACE score was calculated for each patient and assessed using receiver operator characteristic (ROC) curves. A logistic regression model was constructed, compared with the LACE and validated in a separate population., Results: a total of 507 patients were included with a mean (SD) age of 85 (6.5) years; 17.8% were readmitted and 4.5% died within 30 days. The median LACE score of those readmitted compared with those who were not was 12.5 versus 12 (P = 0.13). The Lace index was only a fair predictor of both 30-day readmission and death with c-statistics of 0.55 and 0.70, respectively. Only the emergency department visit was an independent predictor of readmission, with a c-statistic of 0.61 for readmission. In a validation cohort of 507 cases, the c-statistic of the regression model was 0.57., Conclusion: the LACE index is a poor tool for predicting 30-day readmission in older UK inpatients. The absence of a simple predictive model may limit the benefit of readmission avoidance strategies.

Hypertrophic pyloric stenosis (HPS) is a common cause of nonbilious vomiting in the neonatal period with an incidence of approximately 1 to 3 per 1000 live births. The Ramstedt pyloromyotomy has been the standard treatment since 1912. In 1991, Alain et al. reported a novel approach to HPS using laparoscopy. Since this original description, the laparoscopic pyloromyotomy has become progressively more popular and, in many institutions, has replaced the open approach. Similarly, malrotation is a condition affecting the neonatal population, resulting from incomplete intestinal rotation about the superior mesenteric artery during weeks 10 through 11 of development. If left untreated, it can lead to abnormal mesenteric attachments and a narrowed mesenteric base, placing the patient at risk for midgut volvulus. The standard surgical treatment has been the open Ladd procedure first described in 1932. In 1996, Gross described a minimally invasive procedure to address malrotation. The association of concurrent pyloric stenosis and malrotation has rarely been reported in the pediatric literature. This is the first published report of a laparoscopic treatment of HPS and malrotation simultaneously.

By using four different cell isolation procedures, we previously identified two morphologically and biochemically distinct Leydig cell populations in rat testis. The light cells were vacuolated and bound 125I-labeled human choriogonadotropin (hCG) with high affinity but upon hCG stimulation in vitro, cAMP and testosterone production by these cells were minimal. On the other hand, the heavier cells displayed typical Leydig cell morphology and bound very little hCG but vigorously produced cAMP and testosterone (Browne, E.S., Bhalla, V.K., 1991, J. Androl. 12:132-139). This study examines the distribution of LH/hCG receptor mRNAs in the two cell types. The light cell fraction contains larger transcripts of LH/hCG receptor but the heavier Leydig cells contain shorter transcripts. The observations raises the intriguing possibility that shorter rather than larger LH/hCG receptor transcripts are responsible for the induction of a biologically functional, G-protein coupled, LH/hCG receptor in Leydig cells.

The JAR human placental choriocarcinoma cell line transports serotonin, accumulating the monoamine inside the cell against a concentration gradient. The transport is energized by an NaCl gradient. Tricyclic (imipramine and desipramine) and non-tricyclic (paroxetine and fluoxetine) antidepressants inhibit the transporter markedly, but reserpine and 5-hydroxytryptophan do not. Ouabain, gramicidin, and nigericin, which reduce or abolish the transmembrane Na+ gradient, and phloridzin, which interferes with glucose transport into the cells, inhibit the transport. Preincubation of the cells with glucose-free medium also causes similar inhibition. The activity of the serotonin transporter in this cell line is stimulated in response to overnight (16-h) incubation with increasing concentrations of cholera toxin (0.1-1,000 ng/ml). Under these conditions the stimulation is maximal at 10 ng/ml cholera toxin (3.1 +/- 0.2-fold). Cholera toxin increases the cAMP content of these cells by several hundredfold within 2 h. Isobutylmethylxanthine (100 microM), dibutyryl cAMP (100 microM), and forskolin (100 microM) mimic the action of cholera toxin, eliciting a 1.6-2.5-fold stimulation of the serotonin transporter activity. The stimulatory effect of cholera toxin is antagonized significantly by simultaneous incubation of the cells with 50 microM N-(2-aminoethyl)-5-isoquinolinesulfonamide, a protein kinase inhibitor. The effect of cholera toxin on serotonin transport is specific because, under similar conditions, cholera toxin inhibits 3-O-methyl-D-glucose transport and does not influence taurine transport in this cell line. There is also no significant change in the protein content of the cells after cholera toxin treatment. Kinetic analysis reveals that cholera toxin causes an increase in the maximal velocity (7.89 +/- 0.67 to 17.55 +/- 1.06 pmol/mg of protein/5 min) and a decrease in the Michaelis-Menten constant (0.52 +/- 0.09 to 0.29 +/- 0.04 microM). These data show that the JAR human placental choriocarcinoma cell line expresses a high affinity serotonin transporter that is sensitive to inhibition by antidepressants and that the activity of the transporter is under cAMP-dependent regulation.

The dose-response relationship between luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated biological response and 125I-labeled hCG binding was studied in purified Leydig cells from adult rat testes. The concentration of hCG needed for one-half maximal stimulation of cyclic adenosine monophosphate (cAMP) and testosterone production (ED50) was 2.16 x 10(-11)mol/L and 5.6 x 10(-13)mol/L, respectively. This suggests that extremely low levels of hormone in the range of 10(-13)mol/L hCG are sufficient to generate enough cAMP (5.66 pmol; 2.83 x 10(-9)mol/L) for steroidogenesis, thereby preserving the catalytic potential of the receptor-cyclase system. Most of the cAMP formed at 10(-13)mol/L hCG was released into the medium, and the intracellular cAMP was much less and barely detectable (0.98 x 10(-9)mol/L; 1.96 pmol/2 x 10(6) cells). The specific binding of 125I-labeled hCG to purified Leydig cells at a correspondingly higher hCG concentration (3 x 10(-10)mol/L) was extremely low and did not display a dose-dependent increase in binding. Assuming the specific binding to represent 100% occupancy of high affinity receptors (14.2 fmol/2 x 10(6) cells per 2 ml), each mole of bound hCG generated 15,423 mol cAMP and 12,817 mol testosterone. The results show that the hormone interacts with cellular receptors as a catalyst to generate the biological response. Moreover, the true affinity of hormone-receptor interaction responsible for the physiologic action is possibly much greater than previously reported for this system. This information should prove useful for reconstitution studies using the hormone receptor/G-protein/adenylate cyclase system in vitro in soluble form.

Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10(-10) M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable 125I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the 125I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

Two human chorionic gonadotropin (hCG) responsive cells from rat testicular interstitium were previously isolated on a discontinuous gradient of Percoll. The light cells were non-steroidogenic and bound 125I-labeled hCG with high affinity (Kd 3.0 x 10(-10) mol/L), whereas the steroidogenic heavier cells (Leydig cells) produced cyclic adenosine monophosphate (cAMP) and testosterone in response to hCG stimulation with very little hCG binding. In that study, the heavier cell fraction was contaminated with germ cells, red blood cells, and other cells. These cells have now been further purified on a continuous gradient of Percoll (20 to 60%, v/v), and have resolved into three visible bands. The cells in subfraction I, predominantly damaged Leydig cells, germ cells, and/or residual light cells, bind 125I-labeled hCG with high affinity (Kd 4.09 x 10(-10) mol/L) without producing cAMP and testosterone in response to hCG. Subfraction III consists mainly of red blood cells. The cells in subfraction II, identified as typical Leydig cells by electron microscopy, produce cAMP and testosterone in response to hCG but, again, bind only a small amount of hCG (4.5 +/- 0.3 fmol/2 x 10(6) cells/250 microliters/per hour at 37 degrees C). Thus, further purification of the heavier cell fraction from a discontinuous gradient of Percoll on a continuous gradient of Percoll yields Leydig cells, free of contaminating germ cells and red blood cells, which actively produce cAMP and testosterone with a very low level of hCG binding, the affinity of which is undetectable by current binding techniques.

Rat testicular interstitial cells have been separated by discontinuous/continuous gradient of Percoll, yielding four cell fractions. The light cells in fraction I bound luteinizing hormone/human chorionic gonadotropin (LH/hCG) with high affinity but were not steroidogenic in response to hormone. Fraction II consisted mainly of germ cells. Although fraction III contained Leydig cells, this fraction was contaminated with germ cells and was less responsive to hormone as compared to the Leydig cells in fraction IV. The Leydig cells in fraction IV produced cAMP and testosterone in response to hormone action in a manner which was critically dependent upon cell concentration. The production of cyclic adenosine monophosphate (cAMP) in the presence of saturating concentrations of hCG (2.4 X 10(-10) M) was linear as a function of cell concentration up to 7.0 X 10(6) cells/1.25 ml and thereafter, a slight inhibition (26%) was seen at 10 X 10(6) cells/1.25 ml. The average value for cAMP production by hCG was 133.8 +/- 8.5 pmol cAMP/2 X 10(6) cells. The production of testosterone was biphasic, increasing linearly up to 5 X 10(6) cells/1.25 ml and decreasing thereafter. Two million cells, in the presence of 2.4 X 10(-10) M hCG, produced an average of 24.2 +/- 1.7 ng of testosterone in reaction volumes ranging from 1 to 2 ml whereas the same number of cells only produced 5.1 +/- 0.6 ng of testosterone in 250 microliters. The binding of 125I-labeled hCG to the same batch of cells increased with increasing cell concentrations as expected but under the conditions of maximal steroidogenesis at low cell concentrations (1.25, 2.0, and 2.5 X 10(6) cells/1.25 ml), it was barely detectable. Thus, we conclude that there is an inverse relationship between the parameters of binding and biological response in purified Leydig cells.

In order to determine the significance of carbohydrate residues of human chorionic gonadotropin (hCG) in receptor interaction and signal transduction leading to steroidogenesis, the effect of deglycosylated hCG (DG-hCG) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fraction I light cells, previously found to bind 125I-labeled hCG with high affinity without producing testosterone, also bound 125I-labeled DG-hCG with high affinity (Kd 7.2.10(-10) M) without stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studies in which DG-hCG was shown to be an antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as result of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological response in the same cell; and (d) DG-hCG partially antagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.

Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal. These studies reveal that: 1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH. The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4 degrees, 27 degrees, and 37 degrees. 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors. 3. The equilibrium dissociation constant (Kd) of 125I-hFSH binding to the soluble factor was 6.0 +/- 0.58 X 10(-10) M, consistent with the values obtained from the membrane binding studies. Similarly, the Kd value for 125I-hLH to the soluble factor(s) was 3.33 +/- 0.3 X 10(-9) M, comparable to the values obtained from the membrane binding studies. Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH. Furthermore, two classes of binding sites for 125I-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 +/- 0.5 X 10(-11) M and 1.35 +/- 1.2 X 10(-9) M. The apparent Hill coefficient of interaction of 125I-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites. 4. The binding of 125I-hLH and 125I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher Kd for hLH (294 +/- 13.8 mug/ml) compared to hFSH (66.6 +/- 4 mug/ml). These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 X 10(-9) M to 6.0 X 10(-10) M versus the ratio of 294 mug/ml to 66.6 mug/ml. 5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin. The binding of FSH at low factor concentrations was found to be 5- to 10-fold greater than prolactin, growth hormone, and albumin. 6. The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood. 7. The effect of ethanol upon solubilization of the factor(s) has been investigated. The factor(s) can be extracted with buffer or water alone. However, 10 to 25% of ethanol (v/v) facilitates the process of solubilization. The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes. The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent. 8. Finally the effect of soluble factors on classical membrane binding was investigated...

We and others have recently reported an involvement of calcium (Ca2+)-mediated intracellular pathways in the release of antral gastrin in response to bombesin (BBS), while cyclic adenosine 3'5'-monophosphate (cAMP) potentiated the gastrin response to BBS. In this study we examined the effect of cyclic nucleotides on BBS-induced gastrin release from isolated perfused rat stomachs. Dibutyryl cyclic AMP (dbcAMP, 1 mM), and Rolipram (a phosphodiesterase inhibitor, 0.5 microM), stimulated basal gastrin secretion and potentiated BBS-induced gastrin release. The stimulation of gastrin release by BBS was not altered by Wiptide (a cAMP dependent protein kinase inhibitor, 1.0 microM), but was surprisingly inhibited by dbcGMP (1 mM). The cAMP content in antral mucosa or in the perfusates was not changed after infusion of BBS. These findings coupled with previous results suggest that BBS-provoked gastrin release is principally coupled to a Ca2+-mediated intracellular pathway, and that an activation of the adenylate cyclase mediated pathway is not involved. Intracellular cGMP, however, may participate in the negative regulation of gastrin release induced by BBS.

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (Kd) and number of hormone binding sites (Bmax) are highly sensitive to changes in hormone and/or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher Kd as well as Bmax values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436--453).

We have previously shown that incubation of rat pituitary cells in the presence of LHRH results in specific enhancement of nuclear estrogen receptor (ER) binding which cannot be accounted for by simple intracellular translocation of cytoplasmic receptor. In the present study, the role of cAMP in this response has been examined. Suspended pituitary cells from adult ovariectomized rats primed with estradiol were incubated with varying concentrations of LHRH or a highly active LHRH analog (LHRH-A) for 30 min at 37 degrees C, and levels of cAMP were determined. Total cAMP levels changed only in response to a concentration of 100 pmol/pituitary of either peptide; the stimulation by LHRH was twice that by LHRH-A. When the priming dose level of estradiol was reduced from 1.0 to 0.5 micrograms/day, stimulation of cells by 100 pmol of LHRH caused a much greater increase in cAMP levels. Separate incubation of subcellular fractions with a wide dose range of dibutyryl-cAMP (DBcAMP) resulted in a progressive loss of cytosol receptor binding capacity (which was also observed in whole cultured or suspended cells), but no significant concomitant change in nuclear receptor binding; however, whole cells in suspension or culture did show an increase in nuclear ER activity in the presence of 100 nM DBcAMP. This response was qualitatively, but not quantitatively, similar to that elicited by LHRH. When the effects of whole cell incubation with LHRH and LHRH-A on nuclear ER were compared with their effects on total cAMP levels, no correlation was observed; rising levels of cAMP did, however, coincide with falling levels of cytosol ER.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism by which luteinizing hormone (LH) promotes the production of testosterone in Leydig cells by binding to its high affinity sites was reinvestigated. Collagenase dispersed interstitial cells when purified by the application of a variety of techniques such as unit gravity sedimentation, gradient centrifugation, and a combination of the two procedures, were separated into two LH/hCG responsive cell fractions. The two types of interstitial cells displayed distinct biochemical and morphological characteristics. One cell type (the light cell) bound 125I-labeled human chorionic gonadotropin (125I-labeled hCG) with high affinity (Ka approximately equal to 3.33 x 10(9) M-1) but testosterone was not produced by this cell type as a result of hCG target cell receptor interaction. On the other hand, hCG stimulated the production of testosterone in another cell type (the dark/heavier cell). Steroidogenesis was maximally stimulated (700-800 percent over basal) by concentrations of hCG in the range of 3 x 10(-10) M, but high affinity binding sites for 125I-labeled hCG were not detectable. The residual binding that occurred did not obey saturation kinetics and was predominantly nonspecific. The stimulation of steroidogenesis by hCG in dark/heavier cells was dose and time dependent. Addition of dibutyryl or bromo cAMP (1 mM) to the cell suspension resulted in production of testosterone demonstrating the involvement of an hCG sensitive adenylate cyclase system in the transfer signaling process. These observations suggest the lack of a direct association between the occupancy of high affinity binding sites by hCG and testosterone production in rat Leydig cells. The stimulation of a biological response by a pathway independent of hCG occupancy of high affinity binding sites on Leydig cell is discussed and morphology of light and dark/heavier cells is presented. Autoradiographic evidence substantiates the conclusions.

The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.

Two testicular interstitial cell fractions, light and heavier, biochemically and morphologically distinct were obtained by a unit gravity sedimentation procedure. Binding sites for 125I-labeled human chorionic gonadotropin (hCG) were preferentially localized in the light cell fraction (apparent Kd = 2.02 X 10(-10) M; Bmax = 1.17 X 10(-5) nmol/2 X 10(6) cells). These cells did not synthesize testosterone in response to hCG, but the basal release of testosterone was higher than by cells in the heavier fraction (2.49 +/- 0.02 ng/2 X 10(6) cells in the light versus 0.22 +/- 0.00 ng/2 X 10(6) cells in the heavier fraction). The cells in the heavier fraction bound little or no hCG. The binding data from this fraction did not obey saturation kinetics, but testosterone levels were elevated 700-800% in the presence of hCG (i.e. basal value 0.22 +/- 0.00 ng/2 X 10(6) cells versus 1.81 +/- 0.04 ng/2 X 10(6) cells in hCG-stimulated cells). Electron microscopy revealed that heavier cells had features typical of Leydig cells such as large ovoid nucleus with peripherally located heterochromatin, numerous mitochondria with tubular cristae, some lipid droplets, extensively developed smooth endoplasmic reticulum, and well developed Golgi complex. The cells in the light fraction contained an ovoid nucleus with one or more deep infoldings, and their most notable cytoplasmic feature was the presence of numerous vacuoles of varying sizes and shapes. Based upon this and the investigation which follows (Bhalla, V.K., Flasch, M.V., Browne, E.S., Sohal, G.S., and Sharawy, M.M. (1987) J. Biol. Chem. 262, 5322-5332), we conclude that occupancy of high affinity hCG binding sites, generally assumed to be coupled to steroidogenesis, is not necessarily related to the elicitation of this biological response.

The effect of reaction volume upon the binding of gonadotropins by particulate receptors was studied. Two experimental approaches were used: one involved increasing the reaction volume of the binding assay (i.e. diluting the hormone and receptor concentrations and will be referred to as buffer coincubation studies) and the other involved incubating the testicular homogenate in various buffer volumes prior to the binding assay (buffer preincubation stidies). The results showed that the number of hormone binding sites inferred from Scatchard analysis was inversely related to the reaction volume in the coincubation as well as in the preincubation studies. Time-dependent dissociation of receptors from the intact testis was demonstrated by perifusion studies and the loss of receptors from intact testis correlated with the appearance of soluble factors (Bhalla, V.K., Haskell, J., Grier, H. and Mahesh, V.B. (1976) J. Biol. Chem. 251, 4947--4957) in the eluate obtained. The results obtained along with those presented in the preceding manuscript (Chen, C.J.H., Lindeman, J.G., Trowbridge, C.G. and Bhalla, V.K. (1979) Biochim. Biophys. Acta 284, 407--435) question the validity of the rapid equilibrium model which assumes reversible hormone occupancy of a fixed number of receptor sites. An alternate binding model is proposed herein and its implications are discussed.

By using pair-feeding technique, the number of gonadotropin binding sites per unit mass of testicular homogenate was measured 15, 30, 45 and 60 days after ethanol administration to 60-day-old male rats. The ingestion of ethanol (5%, v/v) as a part of a nutritionally adequate liquid diet resulted in 30, 35 and 40% reduction in gonadotropin receptors by 15, 30 and 45 days, respectively. The body weights in the alcohol-fed rats were comparable to pair-fed controls. These findings suggest that the presence of ethanol in the liquid diet depresses the level of gonadotropin receptors in the rat testis.

Two previous reports from this laboratory showed that the binding of 125I-labeled human choriogonadotropin and 125I-labeled human luteinizing hormone to rat testicular receptors is partially irreversible and the binding parameters obtained from Scatchard analysis of the data can be drastically altered simply by changing the reaction volume of the binding assays (Chen, C.J.H., Lindeman, J.G., Trowbridge, C.G. and Bhalla, V.K. (1979) Biochim, Biophys. Acta 584, 407--435; Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim, Biophys. Acta 584, 436--453). It is reported herein that the binding reaction between follicle-stimulating hormone and testicular receptors displays very similar characteristics. The results support the previous conclusion that receptor concentrations fluctuate in the membranes and that the extent of their loss from tissue membranes in vitro is dependent upon time and temperature of incubation, the volume of buffer present, and the quantity of hormone used.