Indocyanine green fluorescence angiography during low anterior resection for low rectal cancer: results of a comparative cohort study.

BACKGROUND: Anastomotic leak (AL) after low anterior resection (LAR) is associated with increased morbidity, mortality, cost and cancer recurrence rates. The aim of this study was to evaluate the impact of fluorescence angiography (FA) on AL following LAR for low rectal cancer. METHODS: This is a single surgeon retrospective cohort study with a historical, consecutively sampled case matched control group. The institution's prospectively maintained institutional review board (IRB)-approved database was queried for all patients who underwent a laparoscopic LAR for rectal neoplasia with a colorectal or coloanal anastomosis < 5 cm from the anal verge between 2013 and 2016. Patients were divided into two groups: patients in whom FA was employed (study group, 2015-2016) and those patients in whom it was not (control group, 2013-2015). All patients were diverted with a loop ileostomy. The primary outcome measured was the AL rate and the secondary outcome measured was change in surgical plan following FA. RESULTS: Sixty patients were included in the study: 30 patients in the FA group and 30 patients in the control group. Patients' demographics, the use of neoadjuvant chemoradiation, tumor stage, and mean height of anastomosis were comparable between the study groups. FA led to a change in surgical plan in four patients (13.3%) none of who suffered an AL. Two patients in the control group had a clinically and radiologically confirmed AL, whereas there were no leaks in the FA group (6.7% vs. 0%, p = 0.49). CONCLUSIONS: FA changed the surgical plan in 13.3% of LAR's, potentially reducing the incidence of AL in these high-risk patients.

Transanal total mesorectal excision for rectal cancer with indocyanine green fluorescence angiography.

BACKGROUND: The aim of this study was to evaluate the impact of fluorescence angiography (FA) on any change in proximal resection margin and/or anastomotic leak (AL) following transanal total mesorectal excision (TaTME) for rectal cancer (RC). METHODS: This retrospective cohort study was conducted at two centers by three senior surgeons. Both institutions' prospectively maintained Institutional Review Board-approved databases were retrospectively queried for all consecutive patients between July 2015 and May 2017 who had laparoscopic hybrid trans-abdominal total mesorectal excision (TME) and TaTME for RC with colorectal or coloanal anastomosis < 10 cm from the anal verge. All patients had intraoperative FA to assess colonic perfusion of the planned proximal resection margin before bowel transection and after construction of the anastomosis. Primary outcomes measured any changes in proximal resection margins and AL rates. RESULTS: Fifty-four patients (31 males; mean age 63 ± 12 years) were included; 30 (55%) of whom received neoadjuvant chemoradiation. The average anastomotic height was 3.6 cm from the anal verge and 8 (14.5%) patients required intersphincteric dissection. Forty-six patients (85%) had loop ileostomy. FA led to a change in the proximal resection margin in 10 patients (18.5%), one of whom had AL on postoperative day 3 requiring diagnostic laparoscopy and loop ileostomy. A second patient, without a change in the proximal resection margin, also had an AL. The overall AL rate was 3.7%. CONCLUSIONS: FA changed the planned proximal resection margin in 18.5% of patients, possibly accounting for the relatively low AL rate. FA is imperfect, and subjective but does have the potential to improve outcomes.

Magnetic resonance imaging following neoadjuvant chemoradiation and transanal endoscopic microsurgery for rectal cancer.

AIM: Full-thickness local excision after neoadjuvant chemoradiotherapy (CRT) for patients with rectal cancer and incomplete clinical response has been a treatment strategy for organ preservation. Follow-up of these patients is challenging since anatomic distortion and postoperative changes may be clinically indistinguishable from tumour recurrence. MRI may have a role in detecting recurrence. The aim of this study was to describe the MRI findings during follow-up in patients having local excision following CRT with and without local recurrence. METHOD: The data were collected retrospectively from a single centre. Fifty-three patients with rectal cancer who had full-thickness local excision after neoadjuvant CRT and near-complete response were eligible for the study. Patients with local recurrence were treated by radical salvage surgery. The main outcome was local MRI assessment findings during follow-up. RESULTS: Fifteen patients (five who developed local recurrence and 10 with no evidence of local recurrence) had MR images available for review and were included in the study. High signal intensity and thickening of the rectal wall were present in all patients with recurrent disease within the rectal wall. Overall, 80% of the patients with recurrence showed diffusion restriction. MRI mesorectal fascia status and circumferential resection margin showed agreement in all cases. A low signal intensity scar was seen in all patients without recurrent disease. CONCLUSION: MRI shows high signal intensity and thickening of the rectal wall in recurrent disease in comparison to a low signal intensity fibrotic scar in non-recurrent disease. These findings may be useful in surveillance of these patients.

Giant adrenal carcinoma associated with renal vein and inferior vena cava thrombus. Case report and literature review.

OBJECTIVE: To present a case of giant adrenal carcinoma associated with renal vein and inferior vena cava (IVC) thrombus. Up to now, there is no similar case reported in the national literature. METHODS: 75 year old woman with signs of virilization. CT-scan showed an 18 cm adrenal mass with venous thrombus and possible pulmonary metastases. The working diagnosis was primary suprarenal carcinoma. RESULT: Due to elderly age and advanced stage, including metastasis, we decided to not perform surgery, and initiate chemotherapy. CONCLUSIONS: Adrenal Cancer is an infrequent and very aggressive tumor. Surgery is the only curative treatment. In advanced stages chemotherapy is recommended, but with poor results.

Development of a neuropsychological assessment in smoking. / Desarrollo de una batería de evaluación neuropsicológica en fumadores.

INTRODUCTION: Smoking is one of the causes leading to the development of disease and mortality worldwide. One of the focuses of interest in this area is the impact of smoking on neuropsychological health. However, few studies provide instruments to assess executive functioning in smokers. The purpose of this study was to examine the viability of the internal structure of a neuropsychological battery for the assessment of executive function in smokers. SUBJECTS AND METHODS: A total of 171 smokers (Mage= 47.44, SDage= 8.48) were assessed. Executive functions were assessed at baseline with measures of inhibition (go/no go task and five digit test), updating (visual search and attention test and letter-number sequencing) and shifting (delay discounting task and Iowa gambling task). RESULTS: The exploratory factor analysis obtained a three-component solution of 59.6%. Establishing a first factor composed of visual search and attention test and letter-number sequencing, a second factor composed of delay discounting task and go/no go task and a third factor with Iowa gambling task and five digit test. CONCLUSIONS: The analysis of the internal structure reflected three factors which are consistent with the structure proposed by Miyake (2000).

TITLE: Desarrollo de una batería de evaluación neuropsicológica en fumadores.Introducción. El tabaquismo es una de las causas que conducen al desarrollo de enfermedades y a la mortalidad en todo el mundo. Uno de los focos de interés en esta área es el impacto del tabaquismo en la salud neuropsicológica. Sin embargo, son pocos los estudios que proporcionan instrumentos para evaluar el funcionamiento ejecutivo en los fumadores. El propósito de este estudio fue examinar la viabilidad de la estructura interna de una batería neuropsicológica para la evaluación de la función ejecutiva en fumadores. Sujetos y métodos. Se evaluó a un total de 171 fumadores (mediaedad = 47,44; desviación estándaredad = 8,48). Las funciones ejecutivas se evaluaron en la línea de base con medidas de inhibición (tarea go/no go y prueba de los cinco dígitos), actualización (prueba de búsqueda y atención visual, y escala de inteligencia de Wechsler para adultos) y cambio (tarea de descuento por demora y tarea de juego de Iowa). Resultados. El análisis factorial exploratorio obtuvo una solución de tres componentes del 59,6%, y se estableció un primer factor compuesto por la prueba de búsqueda y atención visual y la escala de inteligencia de Wechsler para adultos; un segundo factor, por la tarea de descuento por demora y la tarea go/no go; y un tercer factor, por la tarea de juego de Iowa y la prueba de los cinco dígitos. Conclusiones. El análisis de la estructura interna reflejó tres factores que son consistentes con la estructura propuesta por Miyake (2000).

Recurrent varicose veins of the lower limbs after surgery. Role of surgical technique (stripping vs. CHIVA) and surgeon's experience.

BACKGROUND: Surgical treatment of varicose veins of the lower limbs resolves symptoms and improves quality of life. However, the high recurrence (20-80%) is a costly and complex issue. PATIENTS AND METHODS: This is a retrospective review of 1489 patients with varicose vein of the lower limbs seen at our hospital between January 1980 and December 2005. The aim is to evaluate the effect of surgical technique (stripping vs. CHIVA) and surgeon's experience in reducing recurrences. RESULTS: With experienced surgeons, CHIVA appears to be more effective than stripping in reducing the recurrence rate (p <0.05). However, when performed by an inexperienced surgeon the results are far worse than those achieved with stripping. CONCLUSION: There was a clear reduction in recurrences at 5-10 years with CHIVA than with conventional stripping. However, if performed incorrectly, results are far worse with CHIVA. In fact, good results are far more difficult to achieve with CHIVA than with stripping, which is repeatable and easy to perform.

Human adjuvant disease secondary to foreign substance injections as a cause of secondary lower extremity lymphedema.

Non-FDA approved foreign substances injected in areas such as the hips and buttocks for aesthetic purposes have resulted in significant complications including secondary lymphedema. We sought to demonstrate lymphoscintigraphic abnormalities in a group of patients with lower extremity edema following infiltration of foreign substances in but-tocks and hips to confirm secondary lymphedema. This retrospective and observational study examined 10 lower extremities for lymphoscintigraphic abnormalities from patients with history of infiltration of foreign substances and subsequent complaints about lower extremity edema. Clinical evaluation, lymphedema index, lymphoscintigraphy, and Transport Index (TI) were evaluated. The average lymphedema index documented in each limb was 236.45 categorizing most of our patients in a lower limb lymphedema stage I. The average TI was 15.7 points (8.6 - 22.8 points) demonstrating that all patients show abnormal lymphoscintigraphy (LSG) patterns. LSG findings confirm the diagnosis of lower extremity lymphedema secondary to injection of foreign substances in the buttocks and hips in the group of patients studied.

[Scientific Presentations at the meetings of the Spanish Pediatric Nephrology Association (AENP), 1988-2007]. / Comunicaciones científicas en los congresos de la Asociación Española de Nefrología Pediátrica (AENP), 1988-2007.

UNLABELLED: OBJECTIVES AND STUDY: To find out that characteristics of the scientific presentations given at the AENP's meetings in the past 20 years. METHODS: We reviewed in the scientific programs of the AENP's meetings of the past 20 years: number of presentations, number of participating institutions, institutions that provided the majority of the presentations, presentation format, number of studies involving experimental nephrology, topics most commonly presented. RESULTS: There have been 1,119 presentations in the past 20 years, 45/year between 88-92 and 67/year between 03-07. Ninety-one institutions participated in the meetings, 17/year between 88-92 and 34/year between 03-07. Pediatric Nephrology unit from the H. La Paz (Madrid) contributed the most presentations. Poster presentations were accepted at the ANEP meetings after 1995. Since then, 369 of the 815 presentations followed this format. Between 88-07 only 16 presentations dealt with experimental nephrology. The most common topics of presentation were glomerular disease (203) and urinary tract infection/VUR (132). Fifty-one presentations dealt with dialysis (almost 2/3 peritoneal). Transplantation was the topic of 123 presentations. Of the 21 presentations on molecular genetics only one happened before 1998. CONCLUSIONS: The poster is a useful alternative in scientific presentations which has allowed an increase in presentations, authors and institutions participating in the ANEP meetings. The main topic of presentation was glomerular disease. The frequency of presentations dealing with transplantation has increased in the last years. The past decade has seen more presentations on molecular genetics, but presentations dealing with experimental nephrology are still infrequent.

Recommendations for the reopening and activity resumption of the neurogastroenterology units in the face of the COVID-19 pandemic. Position of the Sociedad Latinoamericana de Neurogastroenterología. / Recomendaciones para la reapertura y reinicio de actividades de las Unidades de Neurogastroenterología ante la pandemia por COVID-19. Posicionamiento de la Sociedad Latinoamericana de Neurogastroenterología.

The COVID-19 pandemic has forced the establishment of preventive measures against contagion during the performance of diagnostic and therapeutic tests in gastroenterology. Digestive tract motility tests involve an intermediate and elevated risk for the transmission of COVID-19 infection. Given their elective or non-urgent indication in the majority of cases, we recommend postponing those tests until significant control of the infection rate in each Latin American country has been achieved during the pandemic. When the health authorities allow the return to normality, and in the absence of an effective treatment for or preventive vaccine against COVID-19 infection, we recommend a strict protocol for classifying patients according to their infectious-contagious status through the appropriate use of tests for the detection of the virus and the immune response to it, and the following of protective measures by the healthcare personnel to prevent contagion during the performance of a gastrointestinal motility test.

[Mature cystic teratoma of the testis (dermoid cyst). Case report and literature review]. / Teratoma quístico maduro testicular (quiste dermoide). Aportación de un caso y revisión de la literatura.

SUMMARY OBJECTIVES: Case report of a mature cystic teratoma (dermoid cyst), pseudopilomatrixoma like variant. METHOD: 53-year old patient with a left testicular mass, diagnosed as mature cystic teratoma of the testis(dermoid cyst), the pilomatrixoma-like variant, after radical orchiectomy. Tumoral markers were negative and no extension was observed. The patient is disease-free after 6 months follow-up. RESULTS: Testicular teratoma is a tumour composed of cells derived from the three embryonic layers (ectoderm, endoderm and mesoderm). They can appear at any age, but they are much more prevalent in childhood. They appear at any age, but are much more common in childhood, where they comprise up to 30% of all tumours. They are much less prevalent in adults,representing only 7% of all testicular germ cell tumours CONCLUSIONS: Prepubertal cases are invariably benign in nature, regardless of their histology. However, cases which appear after puberty are potentially malignant, even if histologically pure. Mature cystic teratoma (dermoid cyst) is the only exception to this rule, as no malignant degeneration of these types of tumours has been published.

[Secondary bilateral testicular plasmacytoma. Case report and review of the literature]. / Plasmocitoma testicular bilateral secundario. Aportación de un caso y revisión de la literatura.

Testicular plasmacytoma is a very unfrequent plasma cells neoplasm. It is usually an incidental autopsy finding devoid of clinical expression, but it presents sometimes as part of a multiple myeloma and is exceptionally its only location. Bilateral involvement is extremely unfrequent. We report the case of a 58-years-old man diagnosed with multiple myeloma in complete remission who complains about progressive enlargement of both testicles. After ultrasonography and PAAF, bilateral testicular plasmacytoma is diagnosed. Treatment includes surgery (orchiectomy), radiotherapy and chemotherapy. Prognostic is worse when multiple myeloma is associated.

Desarrollo de una batería de evaluación neuropsicológica en fumadores / Development of a neuropsychological assessment in smoking

Introducción: El tabaquismo es una de las causas que conducen al desarrollo de enfermedades y a la mortalidad en todo el mundo. Uno de los focos de interés en esta área es el impacto del tabaquismo en la salud neuropsicológica. Sin embargo, son pocos los estudios que proporcionan instrumentos para evaluar el funcionamiento ejecutivo en los fumadores. El propósito de este estudio fue examinar la viabilidad de la estructura interna de una batería neuropsicológica para la evaluación de la función ejecutiva en fumadores. Sujetos y métodos: Se evaluó a un total de 171 fumadores (mediaedad = 47,44; desviación estándaredad = 8,48). Las funciones ejecutivas se evaluaron en la línea de base con medidas de inhibición (tarea go/no go y prueba de los cinco dígitos), actualización (prueba de búsqueda y atención visual, y escala de inteligencia de Wechsler para adultos) y cambio (tarea de descuento por demora y tarea de juego de Iowa). Resultados: El análisis factorial exploratorio obtuvo una solución de tres componentes del 59,6%, y se estableció un primer factor compuesto por la prueba de búsqueda y atención visual y la escala de inteligencia de Wechsler para adultos; un segundo factor, por la tarea de descuento por demora y la tarea go/no go; y un tercer factor, por la tarea de juego de Iowa y la prueba de los cinco dígitos. Conclusiones: El análisis de la estructura interna reflejó tres factores que son consistentes con la estructura propuesta por Miyake (2000).(AU)

Introduction: Smoking is one of the causes leading to the development of disease and mortality worldwide. One of the focuses of interest in this area is the impact of smoking on neuropsychological health. However, few studies provide instruments to assess executive functioning in smokers. The purpose of this study was to examine the viability of the internal structure of a neuropsychological battery for the assessment of executive function in smokers. Subjects and methods: A total of 171 smokers (Mage= 47.44, SDage= 8.48) were assessed. Executive functions were assessed at baseline with measures of inhibition (go/no go task and five digit test), updating (visual search and attention test and letter-number sequencing) and shifting (delay discounting task and Iowa gambling task). Results: The exploratory factor analysis obtained a three-component solution of 59.6%. Establishing a first factor composed of visual search and attention test and letter-number sequencing, a second factor composed of delay discounting task and go/no go task and a third factor with Iowa gambling task and five digit test. Conclusions: The analysis of the internal structure reflected three factors which are consistent with the structure proposed by Miyake (2000).(AU)

Identification of a point mutation in the thyrotropin receptor of the hyt/hyt hypothyroid mouse.

The hyt/hyt hypothyroid mouse has an autosomal recessive, fetal-onset, severe hypothyroidism related to TSH hyporesponsiveness and associated with elevated TSH. Our previous work has suggested that the hypothyroidism and TSH hyporesponsiveness may result from a mutation in the hyt/hyt TSH receptor (TSHr) of the thyroid gland. Based on DNA sequencing of the entire coding region of the TSHr gene from the wild-type BALB/cBY +/+ mouse, the +/+ TSHr is 92% and 94% identical at the nucleotide and amino acid residue levels, respectively, compared to the rat TSHr gene. The coding region of the hyt/hyt TSHr, compared to that of the +/+ TSHr, has a single base change, CCG to CTG, at nucleotide position 1666, which leads to the replacement of a highly conserved proline at amino acid position 556 with a leucine in transmembrane domain IV. This mutation was introduced by site-directed mutagenesis into the wild-type human TSHr and transiently expressed in COS-7 cells. Although the size and abundance of the mutant TSHr mRNA suggested that there was no effect on the nature of the mRNA, TSH binding and the response to TSH in transfected cells were abolished. Further studies are necessary to clarify how the Pro to Leu replacement interferes with receptor expression on the cell surface or influences TSH binding. These functional consequences of the mutation appear to account for the observed TSH hyporesponsiveness and hypothyroidism in the hyt/hyt mouse.

Some aspects of iron metabolism during erythropoietic depression in the mouse.

Spleen and bone marrow patterns of response differ in mice subjected to erythropoietic depressors. Radioiron injected a few hours after a high dose of cyclophosphamide or x-irradiation is retained in the bone marrow. The magnitude of medullary retention is closely related to the number of cells able to synthesize hemoglobin at the moment of iron administration, and to the rate of cell death provoked by the cytotoxic agent. Depressors such as Actinomycin and transfusion, that block the differentiation of stem cells while allowing normal maturation of the erythroid cohort, do not induce marrow entrapment of iron. By contrast, retention is never observed in the spleen, where the 59-Fe turnover is not influenced by the mechanism and magnitude of aplasia. A functional lack of homogeneity of splenic and bone marrow erythropoiesis, hemoglobin metabolism and/or handling of iron stores is proposed. These results would be in agreement with other data from the literature reporting physiological differences among the various sectors of the reticuloendothelial system.

[Treatment of obesity in a hospital endocrinology clinic. Influence of parental body mass index]. / Tratamiento de la obesidad en la consulta de endocrinología de un hospital. Influencia del índice de masa corporal de los padres.

INTRODUCTION: Parental obesity is a risk factor for childhood obesity. The aim of this study was to determine if parental obesity influences the adherence and success of obesity treatment in a hospital paediatric endocrinology clinic. MATERIAL AND METHODS: An analytical, prospective, longitudinal study was conducted on obese children aged 4-14. An initial body mass index (BMI), and again at 6 months after receiving health, hygiene and dietary recommendations. Success was considered as a decrease of 0.5 in the BMI Z-score, and adherence to attending the 6-month review. Parental BMI was determined to identify overweight. The χ(2) test was used for qualitative variables and the T-Student test for quantitative (significance, p<<.05). RESULTS: The study included 100 children (52 male), 9.9±2.7 years old, BMI 28.1± 4.5kg/m(2) and BMI Z-Score 3.11±0.98. (85% had a BMI Z-score>3). More than half (59%) of the children had one or both parents obese (41 fathers and 37 mothers were obese). Treatment was not adhered to by 25 children. Adherence was worse if both parents were obese OR 3.65 (1.3 to 10.5) (P<=.01) and adherence was better if the mother was not obese, although the father was (P=.01). The treatment had significant success in 40 patients. If the mother was the only obese one in the family, the possibility of treatment failure was greater OR 5.6 (1.4 to 22.4) (P<.01) CONCLUSIONS: A high percentage of children with severe obesity have obese parents. The mother has an important influence on adherence and response to treatment for the severely obese child.

[Evaluation of isometric force in lower limbs and body composition in preterm infants]. / Valoración de fuerza isométrica en extremidades inferiores y composición corporal en prematuros.

INTRODUCTION: Strength is a physical quality with a clear influence on quality of life. It is determined by the structure of the musculoskeletal system, and depends on the muscular structure. It has been described that prematurity conditions both qualities. The aims of this study are to determine whether prematurity is associated with strength or body composition and evaluate the relationship between prematurity, strength and muscle mass. MATERIAL AND METHODS: Case-control study. Participants were premature 7-to-11 year-old children and full-term birth controls. Strength was measured by a strength gauge and body composition from DEXA (duel-energy X-ray absorptiometry) scans. A total of 89 subjects were included and divided into three groups: 30 prematures with birth-weight ≤ 1500g, 29 prematures with birth-weight 1500-2000g, and 30 controls. RESULTS: Weight and BMI z-score was lower in the premature group. No differences were found in muscular mass or strength between groups. A ratio was established between strength and weight or muscular mass. It was observed that it was possible for them to move four times their weight, without finding any differences between groups or a relationship with birth-weight. CONCLUSIONS: Between 7 and 11 years of age, children who were premature have lower weight and BMI than the rest of the children. However, there were no differences in body composition or strength between preterm children and controls.

[Amount of sleep and changes in its patterns in children less than two years old]. / Cuantificación del sueño y presencia de alteraciones en la duración del sueño en niños menores de 2 años.

OBJECTIVES: The aim is to study the sleep duration and the 24-hour sleep distribution pattern in children under 2 years-old from two different areas of Spain, as well as to determine the prevalence of abnormal sleep duration and the influence of age on this prevalence, and compare them with the already published data. MATERIAL AND METHODS: An observational, descriptive and cross-sectional study on children from two health centers: one in Castilla y León and the other in Asturias. Their parents completed the Brief Infant Sleep Questionnaire (BISQ). Daytime and nighttime sleep duration was assessed, with percentiles being used to evaluate the results. Two groups were created: one according to the age (children under or above 6 months), and the other according to the area. RESULTS: A total of 125 children were studied (73 males). The nighttime sleep duration increased from 8.28±2.06 hours to 10.43±1.21 hours (P=.0001) and the daytime sleep decreased from 4.61±2.66 to 1.96±0.79 hours (P=.0001) for children under 6 months and above 6 months, respectively. The children from Castilla y León slept more during daytime than those from Asturias (3.91±2.67 vs 3.00±2.09 hours, P=.041). Nineteen children had a total sleep duration below the 2nd percentile. A sleep duration below normal percentiles was observed in children under 6 months (14/72 vs 5/53, P=.006). CONCLUSIONS: As children grow older, they substitute daytime sleep for nighttime sleep. Approximately 15% of the children under 2 years-old may have changes in sleep duration, and is more common in children under 6 months.

Evidence for an important functional role of intracellular loop II of the lutropin receptor.

The lutropin receptor (LHR) is a G protein-coupled receptor in which high affinity ligand binding occurs to the relatively large extracellular N-terminal domain. Various portions of the receptor have been mapped for their relative importance in localization and in hormone-mediated signaling. There is, however, a paucity of information available on the intracellular loops (ICL), where, along with the C-terminal cytoplasmic tail, G protein coupling is expected to occur. Site-directed mutagenesis was used to investigate the role of several conserved ionizable groups and one tyrosyl residue in ICLs I-III of the rat LHR. The pSVL expression vector, containing the LHR cDNA (wild-type and mutants), was transiently transfected into COS-7 cells, and human choriogonadotropin (hCG) binding and hCG-mediated cAMP production were determined. Several point mutants of amino acid residues in ICL II were prepared and characterized with the following results: replacements of Lys-455 and of His-460 with Glu gave mutant LHRs that failed to localize or fold properly at the cell surface as evidenced by the lack of significant binding to intact cells, although hCG binding could be detected in broken cell preparations, and a neighboring Arg-459 --> Glu replacement had no apparent effect on receptor trafficking, hCG binding or hCG-mediated cAMP-production. A reversal mutant in ICL II in which Glu-441, at the boundary of transmembrane helix III and ICL II, and His-460, at the interface between ICL II and transmembrane helix IV, were interchanged, exhibited hCG binding to intact cells, but the maximal cAMP level at high concentrations of ligand was less than that obtained with COS-7 cells transfected with wild-type LHR. The total number of cell surface receptors determined with the reversal mutant was less than that found with wild-type LHR. This difference, however, is not believed to be responsible for the reduced signaling, since maximal cAMP responses to hCG were obtained with comparable receptor densities of wild-type and various mutant LHRs. Other single replacements in ICL I, Lys-368 --> Glu and to Gln, and in ICL III, Arg-526 --> Glu and Tyr-528 --> Ser, resulted in mutant LHRs with characteristics of wild-type LHR in trafficking, hCG binding and hCG-mediated cAMP production. These findings suggest an important functional role of several amino acid residues in ICL II of LHR.

Replacement of the invariant tyrosine in the CAGY region of the human chorionic gonadotropin beta subunit.

The mammalian glycoprotein hormone beta subunits contain a highly conserved amino acid sequence, Cys-Ala-Gly-Tyr-Cys (residues 34-38 of human chorionic gonadotropin beta), that is denoted as the 'CAGY region'. Using site-directed mutagenesis we have replaced Tyr-37 in hCG beta, i.e., the invariant Tyr in all known mammalian CG, LH, FSH, and TSH beta subunits, with two hydrophobic amino acid residues, Phe and Leu. The resultant mutant forms were characterized for alpha subunit binding and the resulting heterodimers were analyzed for biological activity using two in vitro assays with transformed murine Leydig cells (MA-10). Chinese hamster ovary cells containing a stably integrated gene for bovine alpha were transiently transfected with a eukaryotic expression vector containing a Rous sarcoma viral promoter and the wild-type and mutant cDNAs. The hCG beta(Phe-37) mutant bound to alpha essentially to the same extent as hCG beta wild-type, while the hCG beta(Leu-37) mutant formed somewhat less heterodimer. The heterologous heterodimeric mutant and wild-type gonadotropins were equipotent in a competitive binding assay with [125I]hCG. In a steroidogenic assay, the mutant hormones were active, but they appeared slightly less potent than the wild-type form. Thus, this invariant Tyr can be replaced with another aromatic amino acid residue or with a hydrophobic, but not aromatic amino acid residue in hCG beta without any dramatic effect on function. These results indicate that Tyr-37 in hCG beta, while not obligatory, may participate, either directly or indirectly, in subunit assembly and that the hydroxyl group may function in a modulatory role in signaling.