Differential effects of amylin on memory processing using peripheral and central routes of administration.

Amylin is a peptide hormone secreted from the beta cells of the pancreatic islets. Amylin was administered peripherally or centrally following weak or strong training on footshock avoidance conditioning in a T-maze. Under conditions of weak training, amylin improved memory retention in a dose-dependent manner. Under conditions of strong training, it impaired retention over the same dose range. Central administration of amylin in mice given strong training impaired retention but had no effect on the retention of mice given weak training. These findings suggest that the mechanisms of action by which amylin altered memory processing are different for peripheral and central administration. Peripherally secreted amylin may play a role in the amnesia seen in diabetes and the memory enhancement following glucose administration.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Leptin and neuropeptide Y (NPY) modulate nitric oxide synthase: further evidence for a role of nitric oxide in feeding.

Recent studies have suggested a role for nitric oxide in the regulation of food intake. Neuropeptide Y (NPY) is one of the most potent orexigenic agents. Chronic administration of leptin decreases food intake. This study examined the effects of NPY and leptin on nitric oxide synthase (NOS) in the hypothalamus. Previously it has been demonstrated that obese (ob/ob) mice have elevated NOS levels in the hypothalamus. In this study we demonstrated that the administration of leptin (6 microg/day) subcutaneously (SC) for 3 days decreased body weight (P < 0.001) and food intake P < 0.001) in obese (ob/ob) mice as expected. In addition, leptin decreased NOS in the hypothalamus nu 37% (P < 0.01) and in brown adipose tissue by 69% (P < 0.01) but not in white adipose tissue. NPY was administered intracerebroventricularly to CD-1 mice at doses of 0.25 and 0.50 microg. Mice were sacrificed 15 min after injection and NOS was measured in their hypothalami. NPY at the lower dose increased NOS in the hypothalamus by 147%. These results, taken together, with previously published studies support the concept that nitric oxide may play a role as a mediator of the effects of NPY and leptin on food intake. The alterations of NOS in brown adipose tissue following leptin administration could result in changes in blood flow or metabolism in the brown fat.     

The species–area relationship in centipedes (Myriapoda: Chilopoda): a comparison between Mediterranean island groups

The present study article examines the shapes of centipede species–area relationships (SARs) in the Mediterranean islands, compares the results of the linear form of the power model between archipelagos, discusses biological significance of the power model parameters with other taxa on the Aegean archipelago, and tests for a significant small‐island effect (SIE). We used 11 models to test the SARs and we compared the quality‐of‐fit of all candidate models. The power function ranked first and Z‐values was in the range 0.106–0.334. We assessed the presence of SIEs by fitting both a continuous and discontinuous breakpoint regression model. The continuous breakpoint regression functions never performed much better than the closest discontinuous model as a predictor of centipede species richness. We suggest that the relatively low Z‐values in our data partly reflect better dispersal abilities in centipedes than in other soil invertebrate taxa. Longer periods of isolation and more recent island formation may explain the somewhat lower constant c in the western Mediterranean islands compared to the Aegean islands. Higher breakpoint values in the western Mediterranean may also be a result of larger distance to the mainland and longer separation times. Despite the differences in the geological history and the idiosyncratic features of the main island groups considered, the overall results are quite similar and this could be assigned to the ability of centipedes to disperse across isolation barriers. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 146–159.     

Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10² CFU/ml in pure cultures and ≥10³ CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Effects of systemic pancreastatin on memory retention

Pancreastatin, a peptide isolated from the pancreas, was shown to enhance memory retention after peripheral administration in mice when administration following T-maze footshock avoidance training. The effect of pancreastatin on memory retention, one week after training, was time dependent showing enhancement of retention when pancreastatin was administered 0 and 30 min but not 60 min after training. Pancreastatin reversed the amnesia produced by scopolamine. The pancreastatin fragment (33-49) also enhanced memory. Pancreastatin did not increase glucose in vivo. We conclude that peripherally administered pancreastatin modulates memory processing.     

Two-drug combinations of memory enhancers: effect of dose ratio upon potency and therapeutic window, in mice

Two-drug combinations have been reported to enhance retention more effectively than when either drug was administered alone at the same dose. Some combinations of cholinergic drugs enhance retention even though the total drug dosage is reduced by as much as 97% compared to the dose needed to improve retention when the same drugs are administered singly. The choice of dose ratio is usually arbitrary or based on empirical results. The present study systematically varied the ratio of two drugs in a combination and at the same time varied the dosage of each drug. The drug combinations were administered to mice immediately after training on T-maze footshock avoidance task. Retention was tested one week later. Three two-drug combinations were selected for presentation because they differed considerably as to (a) the lowest effective total dose that improved memory-retention, (b) the optimal ratio that improved retention and (c) the width of the therapeutic window. The effect of a drug combination on retention was found to be dependent on the particular drugs in the combination, the ratio and the dose administered.     

Immunoregulatory role of Ig isotypes. II. Activation of cells that block induction of contact sensitivity responses by antibodies of IgG2a and IgG2b isotypes

The influence that the isotype of Ag-specific antibody has on the induction of contact hypersensitivity (CS) has been investigated. Injection (i.v.) of mice with haptenated peritoneal exudate cells (PEC) pretreated with anti-hapten mAb of the IgG2a and IgG2b isotypes results in the activation of Ag-specific afferent acting Ts cells (Ts-aff). These suppressor cells are not generated when animals are injected with anti-hapten antibodies of other isotypes. The Ts-aff cells function to inhibit the generation of CS responses when injected into naive animals. Suppression is due to the induction of both Lyt-1+,2- I-J+ and Lyt-1-,2+ I-J+ T cells, both of which adhere to the lectin Vicia villosa. Attachment of both TNP and 4-ethoxymethylene-2-phenyloxazolone haptens to the same PEC, followed by treatment with an IgG2a anti-TNP antibody, generates Ts-aff cells specific for both 4-ethoxymethylene-2-phenyloxazolone and TNP. The MHC haplotype of the PEC is irrelevant, as allogeneic PEC will also induce Ts-aff cells when injected by using an identical protocol. Ts-aff cells cannot be generated in B cell-depleted mice, nor does the Ts-aff cells generated in normal mice suppress CS responses in B cell-depleted mice. These results show that Ag-antibody complexes bound on the surface of a PEC can induce potent afferent suppression in vivo. A possible general role for antibody isotypes in directing regulatory activities is discussed.     

Growth and pelagic larval duration of presettlement and newly settled neon damselfish, Pomacentrus coelestis, at multiple spatial scales

Variation in planktonic larval duration (PLD) and growth of Pomacentrus coelestis was investigated on the southern Great Barrier Reef at multiple spatial scales. A tetracycline experiment, using presettlement fish, demonstrated that increments were formed daily. Variation in PLD was low between reef clusters (0%), reefs within clusters (0.4–8.5%) and sites within reefs (13.1%), but high among individuals within sites (86.5–91.5%); PLD ranged from 15 to 27 days. It was predicted that PLD would vary at greater spatial scales, but differences were low and a review of all studies on P. coelestis in tropical waters had a similar range of PLDs to our study. Contrary to a hypothesis that fish with slower growth would have longer PLDs, there was no significant relationship between mean presettlement increment width of otoliths and PLD for fish from the two reef clusters examined. There were, however, differences in presettlement growth rates between reef clusters (over 100 km apart) over the last 5–6 days of planktonic life. Warmer waters at the Swain Reefs (0.1–1°C) may have contributed to these differences in growth. Stochastic transport of larvae, habitat choice by presettlement fish in a reef mosaic, and variable conditions in the plankton may contribute to variation in PLD, presettlement growth and size-at-settlement in P. coelestis. We propose that prolonged periods of settlement choice may obscure simple relationships between PLD and size-at-settlement.