microscope. For the electron microscopy analysis, animals were fixed with glutaraldehyde and osmium tetraoxide (34). Five or six L4 or young adult animals were aligned within a small agar block, embedded, and sectioned together. Sections were poststained with uranyl acetate and lead citrate. 4. R. Durbin, thesis. University of Cambridge, Cambridge, England (1987). 5. C. Garriga, C. Desai, H. R. Horvitz. Development 117, 1071 (1993. 6. S. Kim and W. G. Wadsworth, data not shown. 7. The nid-7(RNAi) animals were generated as described previously [A. Fire et al., Nature 391. 806 (1998)l by using a 1-kb sequence from exon 8, which was cloned into pBluescript (Stratagene) as template for RNA synthesis. RNA was produced by both T3 and T7 RNA polymerase, and the reactions were pooled before being injected into the intestines of edls2O(unc-779::CFP) transgenic animals. Phenotypes of the nervous system were observed under epifluorescence microscopy. 8. Seven PCR fragments, including the whole coding sequence and intron region, were amplified from the genomic DNA of nid-l(ur47) animals. PCR fragments were cloned into a pBluescript vector and subsequently sequenced by automatic sequencer. The mutation was confirmed by sequencing two independent PCR fragments. . 16. lmmunostaining was performed by using freeze-fracture and methanol-acetone fixation as previously described (35). Polyclonal antibodies raised against mouse nidogen and a mouse monoclonal antibody against myosin heavy chain B (UNC-54) were used. For costaining, anti-rabbit fluorescein-conjugated and anti-mouse rhodamine-conjugated secondary antibodies were used. . 24. Transgenic strains were generated by standard methods (36). plM#194, an expression construct for nid-7, was constructed by cloning the 2.5-kb 5' flanking region of nid-7 into pPD 95.77 vector (from A. Fire). This CFP construct was coinjected at 10 pg/ml with pRF4 at 100 pg/ml. To establish a stable line, lM329 urls757 [plM#194, pRF4], transgenes were integrated by y-ray irradiation. For the ectopic expression construct of nid-7, constructs plM#195, plM#196, and plM#197, were made by using the 7-kb genomic nid-7 coding region, which was amplified by high-fidelity PCR, ligated to Nhe I-Bgl Il-digested vectors, pPD96.41, pPD49.83, and pPD96.52 (from A. Fire). These vectors contained 5' flanking regulatory sequences of mec-7, hsp76-47, and myo-3, respectively (36). The unc-779 regulatory sequence was amplified by using plM175 as template (23), and cloned into the pPD49.26 vector (from A. Fire) to construct plM#198. These constructs were injected at 10 pg/ml, with pRF4 into nid-l(ur47); kyls723 (zc27::CFP) animals. The resulting strains are IM330 urEx752 [plM#195]; nid-7 (ur47); kys123(zcZI::GFP); IM331 urEx753 [plM#196]; nid-l(ur47); kyls723 (zc27::tFP). lM332 urEx754 [plM#197]; nid-l(ur47); kyls723(zc27::CFP), 11' 1333 urEx755 [plM#198]; nidl(ur47); kyIs723(zc27::CFP). Ectopic expression of nid-7 was checked by in situ hybridization. IM331 emblyos collected 1 to 6 hours after being laid were heat-shock...

The ability to optimize behavioral performance when confronted with continuously evolving environmental demands is a key element of human cognition. The dorsal anterior cingulate cortex (dACC), which lies on the medial surface of the frontal lobes, plays an important role in regulating cognitive control. Hypotheses regarding its function include guiding reward-based decision making1, monitoring for conflict between competing responses2, and predicting task difficulty3. Precise mechanisms of dACC function remain unknown, however, due to the limited number of human neurophysiological studies. Here we demonstrate with functional imaging and human single-neuron recordings that the firing of individual dACC neurons encodes current and recent cognitive load. We show that the modulation of current dACC activity by previous activity produces a behavioral adaptation that accelerates reactions to cues of similar difficulty as previous ones, and retards reactions to cues of differing difficulty. Furthermore, this conflict adaptation, or Gratton effect2,4, is abolished after surgically targeted dACC ablation. Our results demonstrate that the dACC provides a continuously updated prediction of expected cognitive demand to optimize future behavioral responses. In situations with stable cognitive demands, this signal promotes efficiency by hastening responses, but in situations with changing demands, it engenders accuracy by delaying responses.

Niemann-Pick C disease (NP-CWe conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.

The demonstration of a defect of cholesterol esterification in a mutant strain of BALB/c mice with an attendant reduction of sphingomyelinase activity [Pentchev, P.

No abstract

We have developed a panel of rabbit polyclonal antipeptide antibodies against the five human somatostatin receptor subtypes (hSSTR1-5) and used them to analyze the pattern of expression of hSSTR1-5 in normal human islet cells by quantitative double-label confocal fluorescence immunocytochemistry. All five hSSTR subtypes were variably expressed in islets. The number of SSTR immunopositive cells showed a rank order of SSTR1 > SSTR5 > SSTR2 > SSTR3 > SSTR4. SSTR1 was strongly colocalized with insulin in all beta-cells. SSTR5 was also an abundant isotype, being colocalized in 87% of beta-cells. SSTR2 was found in 46% of beta-cells, whereas SSTR3 and SSTR4 were relatively poorly expressed. SSTR2 was strongly colocalized with glucagon in 89% of alpha-cells, whereas SSTR5 and SSTR1 colocalized with glucagon in 35 and 26% of alpha-cells, respectively. SSTR3 was detected in occasional alpha-cells, and SSTR4 was absent. SSTR5 was preferentially expressed in 75% of SST-positive cells and was the principal delta-cell SSTR subtype, whereas SSTR1-3 were colocalized in only a few delta-cells, and SSTR4 was absent. These studies reveal predominant expression of SSTR1, SSTR2, and SSTR5 in human islets. Beta-cells, alpha-cells, and delta-cells each express multiple SSTR isoforms, beta-cells being rich in SSTR1 and SSTR5, alpha-cells in SSTR2, and delta-cells in SSTR5. Although there is no absolute specificity of any SSTR for an islet cell type, SSTR1 is beta-cell selective, and SSTR2 is alpha-cell selective. SSTR5 is well expressed in beta-cells and delta-cells and moderately well expressed in alpha-cells, and thereby it lacks the islet cell selectivity displayed by SSTR1 and SSTR2. Subtype-selective SSTR expression in islet cells could be the basis for preferential insulin suppression by SSTR1-specific ligands and of glucagon inhibition by SSTR2-selective compounds.

Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo-and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Both homo-and heterooligomeric receptors are occupied by two ligand molecules. We find that monomeric, homooligomeric, and heterooligomeric receptor species occur in the same cell cotransfected with two SSTRs, and that oligomerization of SSTRs is regulated by ligand binding by a selective process that is restricted to some (R5) but not other (R1) SSTR subtypes. We propose that induction by ligand of different oligomeric states of SSTRs represents a unique mechanism for generating signaling specificity not only within the SSTR family but more generally in the HHR family. H eptahelical receptors (HHRs) constitute the largest single family of transmembrane signaling molecules that respond to diverse external stimuli such as hormones, neurotransmitters, chemoattractants, odorants, and photons. Although these receptors have been generally thought to function as monomeric entities, there is growing evidence that a number of HHRs assemble as functional homo-and heterooligomers (1, 2). Dimerization seems to be necessary for function of the class C subfamily of HHRs comprising the metabotropic glutamate, calcium sensing, the GABAB, and pheromone receptors that are targeted to the plasma membrane as preformed dimers which are stabilized by ligand binding (3-7). Several HHRs such as somatostatin receptors (SSTRs), dopamine receptors, gonadotrophin-releasing hormone receptor (GnRHR), luteinizing hormone͞chorionic gonadotrophin hormone receptor, and chemokine receptors, however, which belong to the rhodopsin-like class A subfamily of HHRs, assemble on the membrane as homo-and heterooligomeric species in response to agonist activation (8-15).In the case of SSTRs, we have shown by photobleaching fluorescence resonance energy transfer (pbFRET) that the human (h) type 5 receptor (hSSTR5 or R5) exists in the basal state as a monomer, and that activation by ligand induces dose-dependent oligomerization (8). When coexpressed with another SSTR (hSSTR1 or R1) or an unrelated HHR such as the dopamine 2 receptor (D 2 R), R5 also forms a heterooligomer that displays pharmacological properties distinct from those of either of the separate receptors (9). Little is known about the stoichiometry of ligand-receptor reactions or the specificity for homoand heterooligomeric interactions between two receptors that are coexpressed in the same cell. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with R5 or R1, or cotransfected with R5 and R1, we have analyzed the ...

Background:Lyme disease is a tick-borne illness caused by the spirochete Borrelia burgdorferi. Although antibiotic therapy is usually effective early in the disease, relapse may occur when administration of antibiotics is discontinued. Studies have suggested that resistance and recurrence of Lyme disease might be due to formation of different morphological forms of B. burgdorferi, namely round bodies (cysts) and biofilm-like colonies. Better understanding of the effect of antibiotics on all morphological forms of B. burgdorferi is therefore crucial to provide effective therapy for Lyme disease.Methods:Three morphological forms of B. burgdorferi (spirochetes, round bodies, and biofilm-like colonies) were generated using novel culture methods. Minimum inhibitory concentration and minimum bactericidal concentration of five antimicrobial agents (doxycycline, amoxicillin, tigecycline, metronidazole, and tinidazole) against spirochetal forms of B. burgdorferi were evaluated using the standard published microdilution technique. The susceptibility of spirochetal and round body forms to the antibiotics was then tested using fluorescent microscopy (BacLight™ viability staining) and dark field microscopy (direct cell counting), and these results were compared with the microdilution technique. Qualitative and quantitative effects of the antibiotics against biofilm-like colonies were assessed using fluorescent microscopy and dark field microscopy, respectively.Results:Doxycycline reduced spirochetal structures ∼90% but increased the number of round body forms about twofold. Amoxicillin reduced spirochetal forms by ∼85%–90% and round body forms by ∼68%, while treatment with metronidazole led to reduction of spirochetal structures by ∼90% and round body forms by ∼80%. Tigecycline and tinidazole treatment reduced both spirochetal and round body forms by ∼80%–90%. When quantitative effects on biofilm-like colonies were evaluated, the five antibiotics reduced formation of these colonies by only 30%–55%. In terms of qualitative effects, only tinidazole reduced viable organisms by ∼90%. Following treatment with the other antibiotics, viable organisms were detected in 70%–85% of the biofilm-like colonies.Conclusion:Antibiotics have varying effects on the different morphological forms of B. burgdorferi. Persistence of viable organisms in round body forms and biofilm-like colonies may explain treatment failure and persistent symptoms following antibiotic therapy of Lyme disease.