Perioperative surgical outcome of conventional and robot-assisted total laparoscopic hysterectomy.

BACKGROUND: To evaluate surgical outcome in a consecutive series of patients with conventional and robot assisted total laparoscopic hysterectomy. METHODS: A retrospective cohort study was performed among patients with benign and malignant indications for a laparoscopic hysterectomy. Main surgical outcomes were operation room time and skin to skin operating time, complications, conversions, rehospitalisation and reoperation, estimated blood loss and length of hospital stay. RESULTS: A total of 294 patients were evaluated: 123 in the conventional total laparoscopic hysterectomy (TLH) group and 171 in the robot TLH group. After correction for differences in basic demographics with a multivariate linear regression analysis, the skin to skin operating time was a significant 18 minutes shorter in robot assisted TLH compared to conventional TLH (robot assisted TLH 92m, conventional TLH 110m, p0.001). The presence or absence of previous abdominal surgery had a significant influence on the skin to skin operating time as did the body mass index and the weight of the uterus. Complications were not significantly different. The robot TLH group had significantly less blood loss and lower rehospitalisation and reoperation rates. CONCLUSIONS: This study compares conventional TLH with robot assisted TLH and shows shorter operating times, less blood loss and lower rehospitalisation and reoperation rates in the robot TLH group.

[Haemoglobinopathy in the 21st century: incidence, diagnosis and heel prick screening]. / Hemoglobinopathie in de 21e eeuw: incidentie, diagnose en hielprikscreening.

OBJECTIVE: To determine the incidence of severe haemoglobinopathy, to evaluate the effect of heel prick screening, and to identify those children who do not benefit from this early diagnosis. DESIGN: Prospective descriptive study. METHOD: Registration of all symptomatic and asymptomatic children who between 2003-2009 were newly diagnosed with the a severe form of a hereditary disorder concerning the formation of the alpha haemoglobin chain (HbH disease), or the beta haemoglobin chain (sickle cell disease or beta thalassaemia major) in the Netherlands. Registration was done by collecting anonymised reports from the Dutch Paediatric Surveillance Unit and TNO, and by additional questionnaires. RESULTS: During the study period, 48 children (range: 36-76) per year were diagnosed with severe haemoglobinopathy. The overall incidence was 2.5 per 10,000 live births. The incidence of sickle cell disease diagnosed by heel prick screening was 2.1 per 10,000 live births and of thalassaemia major 0.6 per 10,000 live births. In 7% of the children with sickle cell disease who were diagnosed without any form of screening, the diagnosis was made on (a life threatening) infection. Twenty-two percent of the children with a severe form of haemoglobinopathy were not born in the Netherlands. The parents of almost half of the children with sickle cell disease originally came from West- or Central Africa. The parents of children with thalassaemia major were mainly from Morocco or various Asiatic countries. CONCLUSION: The number of children with severe haemoglobinopathy in the Netherlands has trebled since 1992. In order for all children to benefit from early diagnosis and preventive treatment, it is advisable that children who originate from risk areas should be tested for haemoglobinopathy when they first arrive in the Netherlands.

Screening for chronic hepatitis B and C in migrants from Afghanistan, Iran, Iraq, the former Soviet Republics, and Vietnam in the Arnhem region, The Netherlands.

Migrants born in hepatitis B virus (HBV) and hepatitis C virus (HCV) endemic countries are at increased risk of being infected with these viruses. The first symptoms may arise when liver damage has already occurred. The challenge is to identify these infections early, since effective treatment has become available. In 2011 we conducted a screening project in first-generation migrants (FGMs) born in Afghanistan, Iran, Iraq, the former Soviet Republics, and Vietnam and living in Arnhem and Rheden. All participants were offered free blood screening for HBV and HCV. In total 959 participants were tested, with the country of origin known for 927, equating to 28·7% of all registered FGMs from the chosen countries. Nineteen percent (n = 176) had serological signs of past or chronic HBV infection and 2·2% (n = 21) had chronic HBV infection. The highest prevalence of chronic HBV infection was found in the Vietnamese population (9·5%, n = 12). Chronic HCV was found in two persons from the former Soviet Republics and one from Vietnam. Twenty-four percent (n = 5) of the newly identified patients with chronic HBV and one of the three patients with chronic HCV received treatment. Three of the patients, two with HCV and one with HBV, already had liver cirrhosis. The highest (9·5%) HBV prevalence was found in FGMs from Vietnam, indicating a high need for focusing on that particular immigrant population in order to identify more people with silent HBV infection. The fact that three patients already had liver cirrhosis underlines the necessity of early identification of HBV and HCV infection in risk groups.

Hepatitis B prevalence in the Turkish population of Arnhem: implications for national screening policy?

Despite the increased prevalence of hepatitis B and C in most migrant groups in The Netherlands, a national screening policy for these infections is not available. In order to estimate the prevalence of hepatitis B and C in the largest group of first-generation migrants (FGM) in The Netherlands, we conducted a screening project in the Turkish community of Arnhem. In a separate project we identified patients from the target population with chronic hepatitis B and C from hospital records (1990-2008). Educational meetings concerning hepatitis were organized, with all participants being offered a blood screening test. Participants were tested for hepatitis B surface antigen (HBsAg), antibodies to hepatitis B core antigen (anti-HBc) and antibodies to hepatitis C virus (anti-HCV). In total 709 persons were tested, a complete dataset was available for 647 patients. We found that 3·0% and 0·4% of Turkish FGM aged >24 years in Arnhem had active hepatitis B, defined as HBsAg positive, and tested positive for anti-HCV, respectively. The hospital records revealed another 32 patients, 28 with hepatitis B and four with hepatitis C representing 0·7% for hepatitis B and 0·1% for hepatitis C in relation to the total number of Turkish FGM in Arnhem. We believe that active hepatitis screening of FGM from Turkey should be part of the national health policy as it will benefit the individual and public health.

Distribution of extended-spectrum beta-lactamase genes using a commercial DNA micro-array system.

Extended-spectrum beta-lactamase (ESBL) genes are distributed worldwide and their epidemiology is complex. Using the Check-ESBL assay, the distribution of class A ESBL genes in clinical isolates of aerobic Gram-negative bacilli from three laboratories in the East of The Netherlands was determined. Four patient categories were distinguished: (i) patients admitted to an intensive care unit (ICU); (ii) non-ICU inpatients; (iii) outpatients admitted less than a year before collection of the isolate, (<1); (iv) outpatients admitted more than one-year prior to isolate collection or who had never been hospitalized (>1). From February 2009 until March 2010, out of 491 putative ESBL-positive isolates detected by the Vitek2 or Phoenix automated sensitivity testing systems, ESBL genes were detected in 247 (50.3%) by the Check-ESBL assay. Of these, 116 were from hospitalized patients (35 ICU, 81 non-ICU) and 131 were from outpatients (43 <1, 88 >1). In all, 274 ESBL genes were identified in these 247 isolates: 153 CTX-M-1 group (predominantly in E. coli and K. pneumoniae, 70.4% and 51.6% respectively), 67 CTX-M-9 group (predominantly in E. cloacae, 57.9%), 32 SHV, 14 TEM and 8 CTX-M-2 group. ESBL-producing E. cloacae were significantly more common in hospitalized patients than in outpatients, 20.7% and 3.8% respectively (P=0.001). CTX-M-9 group ESBLs were significantly more prevalent in ICU patients (P=0.003), whereas SHV ESBLs were more common in hospitalized patients than in outpatients (P<0.001). There was no significant difference in distribution of ESBL genes between the two outpatient groups.

Conventional surgery and endovenous laser ablation of recurrent varicose veins of the small saphenous vein: a retrospective clinical comparison and assessment of patient satisfaction.

OBJECTIVES: Recurrences of varicosities of the small saphenous vein (SSV) are common. Surgical reintervention is associated with increased complication rates. The aim of the study was to assess the feasibility of endovenous laser ablation (EVLA) in recurrent varicose veins of the SSV and to compare this technique with surgical reintervention. METHODS: All case files of patients treated for SSV varicosities between May 2006 and October 2008 were evaluated and recurrences were selected. Demographics, duplex findings, clinical, aetiological, anatomical and pathological classification, perioperative and follow-up data were all registered. Additionally, a questionnaire focusing on patient satisfaction was taken. RESULTS: Two hundred and eighty-one patients were treated for varicosities of the SSV, of which 42 were for recurrences. Twenty-six of these were treated with EVLA, all under local anaesthesia, and 16 were surgically treated. Most surgically treated patients were treated under regional anaesthesia (88%). Technical success was achieved in 94% of surgically treated patients and in all EVLA-treated patients. Complications in both groups were mostly minor and self-limiting. Sural nerve neuralgia appeared to be more frequent in the surgically treated group (20% versus 9%). After correction for length of follow-up, the incidence of rerecurrences was not statistically significant between groups. CONCLUSION: EVLA is feasible in patients with recurrent varicose veins of the SSV with possibly a lower incidence of sural nerve injury. Patient satisfaction is high for both treatment modalities. Studies with larger samples are indicated to confirm these observations.

Effectiveness of ultrasound screening for developmental dysplasia of the hip.

OBJECTIVE: To determine the effectiveness of ultrasound screening for developmental dysplasia of the hip (DDH) after the neonatal period. DESIGN: Prospective cohort study. SETTING: Child health care centres. PARTICIPANTS: Infants attending the child health care centres. INTERVENTIONS: The intervention group (n = 5170) was screened by ultrasound at 1, 2, and 3 months of age. The control group (n = 2066) was screened by routine physical examination as part of the programme for child health surveillance at the child health care centres (CHC screening). For evaluation of the screening, the children in both the intervention and control group received an ultrasound examination after 6 months of age to detect any abnormality that might have been missed by the screening. RESULTS: The sensitivity of the ultrasound screening was 88.5%, and the referral rate 7.6%. As a result of the ultrasound screening, 4.6% of the children were treated. The sensitivity of the CHC screening was 76.4%, with a referral rate of 19.2%. The treatment rate was 2.7%. Of the treated children in the ultrasound screening group, 67% were referred before the age of 13 weeks, whereas in the CHC screening group only 29% were referred before this age. CONCLUSIONS: This study shows that ultrasound screening detects more children with DDH than CHC screening and that more of them are detected at an earlier age. To accomplish this, even fewer children have to be referred. However, even general ultrasound screening seems not to eradicate late cases of DDH. The higher treatment rate in the population screened by ultrasound may be a result of overtreatment.

Ultrasonographic screening for developmental dysplasia of the hip in infants. Reproducibility of assessments made by radiographers.

We studied the reproducibility of ultrasonographic screening examination of the hip when read by diagnostic radiographers. In order to determine interobserver variability, 200 ultrasonograms were classified according to Graf's method by five observers (four radiographers and one radiologist). The kappa values for interobserver variability indicated moderate agreement (kappa 0.47) for the exact Graf classification and substantial agreement (kappa 0.65) for the classification of normal (type I) versus abnormal (type IIa-IV). Agreement was significantly different for normal, immature and abnormal hips. Comparison of the findings in our interobserver study with existing information based on other examinations and treatment revealed that only a small number of infants with mildly dysplastic hips would have been typed as normal by some observers as a result of observer variability. In conclusion, the interobserver agreement on the ultrasound assessment of the hip was good enough for screening purposes. Observer variability did not result in any severe cases being missed.

HLA haplotypes and susceptibility to rheumatoid arthritis. More than class II genes.

Our aim was to examine, using microsatellite (ms) markers, the contribution of the telomeric part of the HLA region to rheumatoid arthritis (RA) predisposition in the Spanish population. We have looked at the distribution of DQB1, DRBI and five ms loci (D6S1014, D6S273, D6STNFa, MIB and C1-2-5) within the HLA region in 147 Spanish RA patients and 202 control subjects. A total of 19 conserved ms configurations were observed, twelve of them in linkage disequilibrium with particular DQB1-DRB1 haplotypes. Interestingly, haplotype c1 (DQB1*0201-DRB1*0301-D6S1014*143-D6S273*139-D6STNFa*99-MIB*350-C1-2-5*196) was significantly associated with RA predisposition. As part of this haplotype, the MIB*350 allele was found to be a risk factor independently of the RA-predisposing haplotypes. The present results along with data from others prove the existence of a second predisposing locus located inside the MHC region, and suggest that might be located within the TNFa-HLA-B region.

The telomeric part of the HLA region predisposes to rheumatoid arthritis independently of the class II loci.

We have evaluated the possible contribution of genes besides DQ and DR to the association of HLA with rheumatoid arthritis (RA). To this end, we have looked at the allele distributions of six microsatellites, D6S1014, D6S2673, TNFalpha, MIB, C1-2-5, and C1-3-2 among 132 RA patients and 254 controls. We have defined 19 microsatellite clusters corresponding to previously described ancestral haplotypes. One of them was D6S1014*143-D6S273*139-TNFalpha*99-MIB*350-C1-2-5*196-C1-3-2*354, often found associated with DQB1*0201-DRB1*0301. As part of this microsatellite cluster, the allele MIB*350 was found to be a RA-predisposing factor, independent of DRB1*0301 and RA-predisposing haplotypes DQB1*03-DRB1*04 and DQB1*0501-DRB1*01. We conclude that the telomeric part of the HLA region contains a locus conferring predisposition to RA independently of HLA class II.

The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H(2) receptor.

In previous studies we showed that the wild-type histamine H(2) receptor stably expressed in Chinese hamster ovary cells is constitutively active. Because constitutive activity of the H(2) receptor is already found at low expression levels (300 fmol/mg protein) this receptor is a relatively unique member of the G-protein-coupled receptor (GPCR) family and a useful tool for studying GPCR activation. In this study the role of the highly conserved DRY motif in activation of the H(2) receptor was investigated. Mutation of the aspartate 115 residue in this motif resulted in H(2) receptors with high constitutive activity, increased agonist affinity, and increased signaling properties. In addition, the mutant receptors were shown to be highly structurally instable. Mutation of the arginine 116 residue in the DRY motif resulted also in a highly structurally instable receptor; expression of the receptor could only be detected after stabilization with either an agonist or inverse agonist. Moreover, the agonist affinity at the Arg-116 mutant receptors was increased, whereas the signal transduction properties of these receptors were decreased. We conclude that the Arg-116 mutant receptors can adopt an active conformation but have a decreased ability to couple to or activate the G(s)-protein. This study examines the pivotal role of the aspartate and arginine residues of the DRY motif in GPCR function. Disruption of receptor stabilizing constraints by mutation in the DRY motif leads to the formation of active GPCR conformations, but concomitantly to GPCR instability.

In relapsed patients after lymphocyte depleted bone marrow transplantation the percentage of donor T lymphocytes correlates well with the outcome of donor leukocyte infusion.

Donor leukocyte infusions (DLI) from the original marrow donor have been shown to induce remission in patients with relapse after BMT. We analyzed factors that were associated with remission. Twenty-six patients with a relapse after T cell depleted BMT received DLI. The following pre-DLI factors were analyzed: sex and age of the patients and donors, GVHD after BMT, indication for DLI, percentage of donor T lymphocytes in the patient at the time of DLI, interval between relapse and DLI, and number of T lymphocytes infused. Remission was achieved in 11 of 15 patients (73%) treated for relapsed CML and in one of 11 patients (9%) treated for relapsed AML, ALL or RAEB-t (P = .002). Two of 13 patients (15%) with < or =40% of T lymphocytes from donor origin attained remission compared with 10 of 13 patients (77%) with >40% (P = .002). Two of 13 patients (15%) with an interval of < or =18 months between BMT and first DLI entered remission compared with 10 of 13 patients (77%) with an interval of >18 months (P = .002). Multivariate analysis demonstrated that indication for DLI (CML versus AML/ALL and RAEB-t) and the percentage T lymphocytes from donor origin (< or =40 versus >40) were significantly correlated with remission (P = .03). The occurrence of GVHD post DLI was highly associated with achievement of remission (P = .0001). DLI res ults in remission in a high percentage of patients with relapsed CML after BMT. The percentage of T lymphocytes from donor origin still present in the patient at the time of DLI is highly correlated with achievement of remission.

Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes.

Analysis of minimal residual disease (MRD) can predict outcome in acute lymphoblastic leukemia (ALL). A large prospective study in childhood ALL has shown that MRD analysis using immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets can identify good and poor prognosis groups of substantial size that might profit from treatment adaptation. This MRD-based risk group assignment was based on the kinetics of tumor reduction. Consequently, the level of MRD has to be defined precisely in follow-up samples. However, current PCR methods do not allow easy and accurate quantification. We have tested 'real-time' quantitative PCR (RQ-PCR) using the TaqMan technology and compared its sensitivity with two conventional MRD-PCR methods, ie dot-blot and liquid hybridization of PCR amplified Ig/TCR gene rearrangements using clone-specific radioactive probes. In RQ-PCR the generated specific PCR product is measured at each cycle ('real-time') by cleavage of a fluorogenic intrinsic TaqMan probe. The junctional regions of rearranged Ig/TCR genes define the specificity and sensitivity of PCR-based MRD detection in ALL and are generally used to design a patient-specific probe. In the TaqMan technology we have chosen for the same approach with the design of patient-specific TaqMan probes at the position of the junctional regions. We developed primers/probe combinations for RQ-PCR analysis of a total of three IGH, two TCRD, two TCRG and three IGK gene rearrangements in four randomly chosen precursor-B-ALL. In one patient, 12 bone marrow follow-up samples were analyzed for the presence of MRD using an IGK PCR target. The sensitivity of the RQ-PCR technique appeared to be comparable to the dot-blot method, but less sensitive than liquid hybridization. Although it still is a relatively expensive method, RQ-PCR allows sensitive, reproducible and quantitative MRD detection with a high throughput of samples providing possibilities for semi-automation. We consider this novel technique as an important step forward towards routinely performed diagnostic MRD studies.

Idarubicin to intensify the conditioning regimens of autologous bone marrow transplantation for patients with acute myeloid leukemia in first complete remission.

In an effort to reduce the relapse rate after unpurged autologous bone marrow transplantation (ABMT) in patients with acute myeloid leukemia (AML) in first complete remission (CR1), the standard conditioning regimens (cyclophosphamide/busulphan and cyclophosphamide/TBI) were intensified by adding idarubicin. Seventeen patients received a continuous infusion of 21 mg idarubicin/m2/day for 2 consecutive days in addition to the standard preparative regimen. Thirteen patients served as a historical control group. The 2-year disease-free survival (DFS) of 82% in the study group was significantly (P = 0.047) better compared to 46% DFS in the control group. The relapse rate (RR) was also significantly lower (7% vs 45%; P = 0.035) in the study group. The median time to reach a white cell count (WCC) of 0.5 x 10(9)/l was 20 days in the study group vs 17 days (P = NS) in the control group. The median time until recovery of the platelet counts to 20 x 10(9)/l was 152 days in the study group vs 57 days (P = NS) in the control group. The hypoplasia in the study group resulted in a trend towards a higher need for transfusions: a median number of 38 units of erythrocytes vs 23 units in the control group (P = NS) and 23 units of platelet vs 18 units in the control group (P = NS). This pilot study suggests that addition of idarubicin to the standard conditioning regimens may improve DFS and overall survival (OS) of patients with AML treated with ABMT in CR1. These results should be confirmed in a prospective randomized study.

Allogeneic bone marrow transplantation with T-cell-depleted marrow grafts for patients with poor-risk relapsed low-grade non-Hodgkin's lymphoma.

We present the clinical results of allogeneic bone marrow transplantation (BMT) with T-cell-depleted grafts from HLA-matched sibling donors in patients with poor-risk relapsed low-grade non-Hodgkin's lymphoma (NHL). Poor risk was defined as relapse within 12 months after or progression during prior treatment. The conditioning regimen consisted of cyclophosphamide and total-body irradiation with or without additional idarubicin. Donor marrow was depleted of T lymphocytes using counterflow centrifugation. Post-BMT prophylaxis of graft-versus-host disease (GvHD) consisted of cyclosporine A. 15 patients with a median age of 47 years (range 30-57) were transplanted. All patients engrafted. After a median follow-up of 36 months (range 9-78), 10 patients were alive and in complete remission (CR). Two of them had relapsed after BMT but re-entered CR following infusions of leucocytes from the original bone marrow donor. Five patients died; causes of death were cardiomyopathy (n = 1), chronic GvHD (n = 1) and infection during chronic GvHD (n = 3). We conclude that allogeneic T-cell-depleted bone marrow transplantation is an efficacious treatment for patients with poor-risk relapsed low-grade NHL. Infusions of donor leucocytes reinduced CR in the two patients with relapse after BMT.

The C terminal tail of the histamine H2 receptor contains positive and negative signals important for signal transduction and receptor down-regulation.

To examine the role of the C terminal tail in H2 receptor regulation, three cDNAs, encoding truncated histamine H2 receptor mutants (H2T295, H2T307, and H2T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H2 receptor binding of the H2T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H2T307) did not affect the binding properties of H2 antagonists and histamine or histamine-induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H2T341), which was able to form a ternary complex but was unable to fully activate the Gs protein on histamine exposure. Agonist-induced but not the cyclic AMP-dependent H2 receptor down-regulation was more profound for the H2T307 receptor, indicating that different structural elements of the H2 receptor protein are involved in the cyclic AMP-dependent and independent pathways of H2 receptor down-regulation. Taken together, in this study we identified regions in the C terminal tail of the H2 receptor that act as positive and/or negative signals in H2 receptor signaling and down-regulation.

Inverse agonism of histamine H2 antagonist accounts for upregulation of spontaneously active histamine H2 receptors.

Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time- and dose-dependently upregulated upon exposure to the H2 antagonists cimetidine and ranitidine. This effect appears to be H2 receptor-mediated as no change in receptor density was observed after H1 or H3 antagonist treatment or after incubation with the structural analogue of cimetidine, VUF 8299, which has no H2 antagonistic effects. By using transfected CHO cells expressing different densities of wild-type H2 receptors or an uncoupled H2Leu124Ala receptor, the histamine H2 receptor was found to display considerable agonist-independent H2 receptor activity. Cimetidine and ranitidine, which both induce H2 receptor upregulation, actually functioned as inverse agonists in those cell lines displaying spontaneous agonist-independent H2 receptor activity. Burimamide, on the other hand, was shown to act as a neutral antagonist and did as expected not induce H2 receptor upregulation after long-term exposure. The displayed inverse agonism of H2 antagonists appears to be a mechanistic basis for the observed H2 antagonist-induced H2 receptor upregulation in transfected CHO cells. These observations shed new light on the pharmacological classification of the H2 antagonists and may offer a plausible explanation for the observed development of tolerance after prolonged clinical use.

Two distinct pathways for histamine H2 receptor down-regulation. H2 Leu124 --> Ala receptor mutant provides evidence for a cAMP-independent action of H2 agonists.

Pretreatment of Chinese hamster ovary cells expressing the histamine H2 receptor (CHOrH2 cells) with histamine resulted in a time-dependent (t1/2 approximately 7 h) and dose-dependent (EC50=18 nM) H2 receptor down-regulation measured as [125I]iodoaminopotentidine binding (44+/-10% down-regulation). Pretreatment of CHOrH2 cells with cholera toxin or forskolin also led to H2 receptor down-regulation. Forskolin time-dependently (t1/2 approximately 7 h) and dose-dependently (EC50 = 0.3 microM) induced H2 receptor down-regulation. Both histamine and forskolin induced rapid down-regulation of H2 receptor mRNA levels, probably caused by mRNA destabilization. Recently, Moro et al. (Moro, O. Lameh, J., Hogger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276) showed that hydrophobic amino acids in a conserved G-protein-coupled receptor motif in the second intracellular loop are implicated in G-protein coupling. To uncouple the H2 receptor from the Gs-protein, we introduced the Leu124 --> Ala mutation in the second intracellular loop of the H2 receptor. The H2 Leu124 --> Ala mutant showed altered agonist-binding parameters, attenuated histamine-induced cAMP production, and was down-regulated by concentrations of histamine that did not give rise to cAMP production. Taken together, in CHOrH2 cells, H2 receptor down-regulation appears to be induced by two distinct pathways, a cAMP-dependent and cAMP-independent pathway.

Characterization of a putative molluscan insulin-related peptide receptor.

In the pond snail Lymnaea stagnalis (Ls), growth and associated processes are likely to be controlled by a family of molluscan insulin-related peptides (MIP). Here we report on the cloning of a cDNA encoding a putative receptor for these MIP. This cDNA was isolated from Ls via PCR with degenerate oligodeoxynucleotides corresponding to conserved parts of the tyrosine kinase domain of the human insulin receptor and its Drosophila homologue. Many of the typical insulin-receptor features, including a cysteine-rich domain, a single transmembrane domain and a tyrosine-kinase domain are conserved in the predicted, 1607-amino acid (aa) protein. Comparison of the aa sequence of the molluscan receptor to other insulin-receptor sequences revealed strong variations in the percentage of sequence identity for the different domains, ranging from 70% sequence identity in the tyrosine-kinase domain to virtually no sequence identity in the C-terminal sequence. Striking differences are the absence of a clear tetrabasic cleavage site, and the extremely long C-terminus of 308 aa that contains seven Tyr residues. Southern blot analyses at varying stringencies, extensive screening of cDNA- and genomic libraries, and PCR experiments indicate the presence of a single putative MIP receptor. This suggests that the four different MIP may exert their functional role in Ls by binding to the same receptor.

A G-protein beta subunit that is expressed in the central nervous system of the mollusc Lymnaea stagnalis identified through cDNA cloning.

We have cloned cDNA encoding a G-protein beta subunit from the central nervous system (CNS) of the mollusc Lymnaea stagnalis. The deduced protein is very homologous to other metazoan beta subunits. Thus, the Lymnaea CNS can be used as a model system to study beta gamma subunits in their native setting since its large neurons can be manipulated and studied relatively easily in vivo.