Molecular basis and species specificity of high affinity binding of vasoactive intestinal polypeptide by the rat secretin receptor

The affinity and specificity of the binding interaction between ligands and their receptors are key for appropriate hormonal regulation of target tissues. However, it is now apparent that vasoactive intestinal polypeptide (VIP) binds to the rat secretin receptor with similar affinity to that for its natural ligand, secretin (Holtmann et al., 1995). In this report, we establish that this is not a characteristic of the human secretin receptor, and use rat-human secretin receptor chimeras, site mutants and truncated receptor constructs to establish the molecular basis for this unusual binding interaction. Of note, isolated N-terminal domains of the rat secretin and the VIP receptors are capable of high affinity binding of VIP. In the recently recognized secretin family of receptors, this domain has six conserved cysteine residues and disulfide bonds that are likely important to achieve the complex conformation critical for this binding. A single acidic residue (Asp98) present in the rat secretin receptor appears to be critical, because a site-mutant changing this to the polar, but uncharged residue present in that position in the human receptor (Asn) eliminates the high affinity binding of VIP. Of interest, a previously identified critical basic residue in VIP (Lys15) provides a candidate for charge-pairing with this residue, potentially aligning the peptide ligand in a nonproductive orientation within this receptor.     

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1)

BACKGROUND: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop. RESULTS: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme. CONCLUSIONS: We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.     

A questionnaire study among 171 medical candidates enrolled in a PhD program. Three elements in the PhD education: supervision, research courses and international relations

One hundred and seventy-one medical doctors (median age 34 years) registered as Ph.D.-students at the Medical Faculty, University of Aarhus, were given a questionnaire concerning the Ph.D-program (91% reply rate). The Ph.D.-students had typically graduated four years before enrollment and had gained basic clinical experience. Eighty-four percent had been involved in research projects prior to their formal research education. In general, the Ph.D.-students found the supervision offered by senior researchers adequate, although, more Ph.D.-students in clinical than in preclinical departments would have liked their main supervisor to be more enthusiastic and have more specific expertise. By tradition, the Medical Faculty in Aarhus offers a broad introductory course on research methodology, this was appreciated by the Ph.D.-students. However, they found that too much time was allocated for this purpose. The Ministry of Education recommends that Ph.D.-students gain experience from international collaboration, preferably from a stay abroad. However, only 24% of Ph.D.-students had stayed at an international collaborating institution. Although the overall evaluation of the medical Ph.d.-program was positive, the Ph.D.-students pointed out weaknesses and conflicts requiring adjustment.     

A novel k-space trajectory measurement technique

OBJECTIVE: To report our experience with erosion of permanent suture or mesh material after abdominal sacrocolpopexy. METHODS: A retrospective chart review was performed to identify patients who underwent sacrocolpopexy by the same surgeon over 8 years. Demographic data, operative notes, hospital records, and office charts were reviewed after sacrocolpopexy. Patients with erosion of either suture or mesh were treated initially with conservative therapy followed by surgical intervention as required. RESULTS: Fifty-seven patients underwent sacrocolpopexy using synthetic mesh during the study period. The mean (range) postoperative follow-up was 19.9 (1.3-50) months. Seven patients (12%) had erosions after abdominal sacrocolpopexy with two suture erosions and five mesh erosions. Patients with suture erosion were asymptomatic compared with patients with mesh erosion, who presented with vaginal bleeding or discharge. The mean (+/-standard deviation) time to erosion was 14.0+/-7.7 (range 4-24) months. Both patients with suture erosion were treated conservatively with estrogen cream. All five patients with mesh erosion required transvaginal removal of the mesh. CONCLUSION: Mesh erosion can follow abdominal sacrocolpopexy over a long time, and usually presents as vaginal bleeding or discharge. Although patients with suture erosion can be managed successfully with conservative treatment, patients with mesh erosion require surgical intervention. Transvaginal removal of the mesh with vaginal advancement appears to be an effective treatment in patients failing conservative management.     

The Drug Evaluation Classification Program: using ocular and other signs to detect drug intoxication

BACKGROUND: A systematic approach to determining drug intoxication has been developed for use by police officers. By considering specific physiological signs, trained officers can detect the effects of seven major drug types. METHODS: Officers follow a 12-step testing sequence and evaluate signs such as pupil sizes and responses, eye movements, heart rate, body temperature, mental timing, and balance. A matrix is then used to compare that subject's signs to those that would be produced by the seven types of drugs. If a pattern match is found, the officer concludes that the subject is under the influence of a drug and specifies the drug type. RESULTS: Several field and laboratory validation studies have been conducted using these procedures. In general, officers were 70% to 90% accurate in determining intoxication status and drug classification, but poly-drug use and drug rebound effects can sometimes cause problems in interpretation. CONCLUSION: Ocular and other physiological signs can be used to detect drug intoxication and classify the type of drug taken. Knowledge of the procedures used in the Drug Recognition Program can enable optometrists to serve as consultants to the police and as expert witnesses in cases involving the use of ocular signs that indicate illicit drug use.     

Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor

We used KO mice lacking either TNF receptor 1 (TNFR-1) or receptor 2 (TNFR-2) to determine whether signaling at the start of liver regeneration after partial hepatectomy (PH) involves only one or both TNF receptors and to analyze in more detail the abnormalities caused by lack of TNFR-1 receptor, which is required for the initiation of liver regeneration. Lack of TNFR-2 had little effect on NF-kappaB and STAT3 binding, and no effect in interleukin-6 production after PH, but caused a delay in AP-1 and C/EBP binding and in the expression of c-jun and c-myc messenger RNA (mRNA). In contrast to mice lacking TNFR-1, which had deficient hepatocyte DNA synthesis and massive lipid accumulation in hepatocytes, TNFR-2 KO mice had normal liver structure and similar levels of hepatocyte DNA replication as those of wild type mice. We conclude that TNFR-1, but not TNFR-2, is necessary for liver regeneration, and that NF-kappaB and STAT3 binding are activated by signals transduced by TNFR-1. Inhibition of AP-1 and C/EBP binding and in the expression of c-jun and c-myc mRNA in the first 4 hours after PH, as well as the apparent lack of Fos in AP-1 complexes, had no effect on the timing or extent of DNA replication.     

Investigation of the presynaptic effects of quinine and quinidine on the release and uptake of monoamines in rat brain tissue

Quinine and quinidine are reported to potentiate the behavioural effects of serotonergic agents and monoamine uptake inhibitors. We have therefore investigated the presynaptic actions of quinine and quinidine on monoamine uptake and release in rat brain tissue in vitro. Quinidine evoked the release of [3H]5-HT, [3H]noradrenaline and [3H]dopamine from pre-loaded rat brain slices in a concentration dependent manner with EC50 values of 175, 486 and 150 microM, respectively. Quinine induced [3H]monoamine release with similar potencies. Both quinine and quinidine also inhibited the active uptake of [3H]5-HT, [3H]noradrenaline and [3H]dopamine into rat brain synaptosomes with IC50 values in the range 0.13-12.4 microM. The potency of each drug to inhibit [3H]5-HT uptake was significantly higher than that for [3H]noradrenaline or [3H]dopamine. The relative potency of quinidine compared to quinine was more marked in the case of [3H]5-HT (58-fold) than for [3H]noradrenaline (3-fold) or [3H]dopamine (4-fold). The inhibition of [3H]5-HT uptake by quinine and quinidine was competitive in nature and corresponded with the potencies of these drugs to inhibit [3H]paroxetine binding. No correlation was observed between the potencies of quinine and quinidine to induce the release of [3H]monoamines and to inhibit their uptake, suggesting that these effects are mediated by two distinct mechanisms. We conclude that the presynaptic actions of quinine and quinidine on monoamine uptake and release may be implicated in their potentiation of the effects of serotonergic agents and uptake blockers.     

Determination of the in situ forces and force distribution within the human anterior cruciate ligament

The in situ forces and their distribution within the human anterior cruciate ligament (ACL) can clarify this ligament's role in the knee and help to resolve controversies regarding surgical treatment of ACL deficiency. We used a universal force-moment sensor (UFS) to determine the magnitude, direction, and point of application of the in situ forces in the ACL in intact human cadaveric knees. Unlike previous studies, this approach does not require surgical intervention, the attachment of mechanical devices to or near the ACL, or a priori assumptions about the direction of in situ force. Anterior tibial loads were applied to intact knees, which were limited to 1 degree of freedom at 30 degrees flexion. The in situ forces developed in the ACL were lower than the applied force for loads under 80 N, but larger for applied loads of more than 80 N. The direction of the force vector corresponded to that of the anteromedial (AM) portion of the ACL insertion on the tibial plateau. The point of force application was located in the posterior section of the anteromedial portion of the tibial insertion site. The anterior and posterior aspects of the anteromedial portion of the ACL supported 25% and 70% of the in situ force, respectively, with the remainder carried by the posterolateral portion. We believe that the data obtained with this new UFS methodology improves our understanding of the role of the ACL in knee function, and that this methodology can be easily extended to study the function of other ligaments.     

Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of Candida spp. (50 C. albicans, 50 C. glabrata, 10 C. kefyr, 20 C. krusei, 10 C. lusitaniae, 20 C. parapsilosis, and 20 C. tropicalis isolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of C. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.