Mutation of Threonine 34 in Mouse Podoplanin-Fc Reduces CLEC-2 Binding and Toxicity in Vivo While Retaining Anti-lymphangiogenic Activity

The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn) is a small, transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells (LEC). During mouse development, binding of Pdpn to the C-type lectin-like receptor 2 (CLEC-2) on platelets is critical for the separation of the lymphatic and blood vascular systems. Competitive inhibition of Pdpn functions with a soluble form of the protein, Pdpn-Fc, leads to reduced lymphangiogenesis in vitro and in vivo. However, the transgenic overexpression of human Pdpn-Fc in mouse skin causes disseminated intravascular coagulation due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonine 34, which is considered essential for CLEC-2 binding, was mutated to alanine (PdpnT34A-Fc). Indeed, PdpnT34A-Fc displayed a 30-fold reduced binding affinity for CLEC-2 compared with Pdpn-Fc. This also translated into fewer side effects due to platelet activation in vivo. Mice showed less prolonged bleeding time and fewer embolized vessels in the liver, when PdpnT34A-Fc was injected intravenously. However, PdpnT34A-Fc was still as active as wild-type Pdpn-Fc in inhibiting lymphangiogenesis in vitro and also inhibited lymphangiogenesis in vivo. These data suggest that the function of Pdpn in lymphangiogenesis does not depend on threonine 34 in the CLEC-2 binding domain and that PdpnT34A-Fc might be an improved inhibitor of lymphangiogenesis with fewer toxic side effects.     

Selective enzymatic hydrolysis of aromatic diesters using esterase from Brevibacterium imperiale B222

Summary A practical procedure has been developed for the chemoselective microbial hydrolysis of aromatic dicarboxylic esters to give the corresponding monoesters, using cellular lysate and whole cell of Brevibacterium imperiale B222. The produced monoesters can be transformed into hydroxyacids, useful intermediates in the synthesis of polyesters.     

From individual behaviour to population pattern: weather-dependent foraging and breeding performance in black kites

The links between weather and animal behaviour in population processes have received relatively little attention. I studied the effect of weather conditions on foraging and breeding performance of a medium-sized raptor, the black kite, Milvus migrans. The frequency of prey capture attempts and their likelihood of success increased with temperature and declined with rainfall. Kites used flight styles involving a higher energy expenditure in less favourable weather. Nestling provisioning rates declined during rain spells. More kites hunted during periods of favourable weather and after periods with a high frequency of successful prey capture attempts by conspecifics; this result suggested that individuals may fine-tune their foraging effort to the expected foraging reward. Such compensatory behavioural adjustments may increase species resilience to climate change. The behaviourally mediated effects of weather on prey availability translated into population effects. Yearly weather conditions during the last stage of the prelaying period affected population-level productivity, probably through an effect on female body condition mediated by male provisioning capability and hunting yield. The predicted effects of climate change on kites may already be occurring, with progressively earlier laying and northward range expansion. These results confirm the need to pay greater attention to behaviourally mediated effects of climate on populations, particularly when individuals make compensatory adjustments that may enhance resilience to climate change. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.      

Unstable chromosome sites and evolutionary rearrangements in akodont rodents (Cricetidae)

Evolutionary rearrangements producing changes in chromosome 1 of Akodon molinae were traced by comparing the G banding patterns of the karyotypes from six species of akodont rodents. It was possible to subdivide chromosome 1 of A. molinae into unstable and stable regions. Most of the spontaneous rearrangements of chromosome 1 appearing in passages 116-128 of a continuous line of A. molinae cells (AKm line) occurred in the unstable regions which comprise repetitive DNA sequences favouring the setting up of heteroduplexes leading to rearrangements. When AKm cells were irradiated with UV light it was observed that unstable regions of chromosome 1 showed higher rates of unscheduled DNA synthesis (UD) than stable areas. A differential degree of condensation making certain regions of the chromatin fibril more accessible to repair enzymes or a better target for damage, is probably the cause of the variable response to UV light, and perhaps to most clastogenic agents (including those responsible for spontaneous rearrangements). Thus, the distribution of repetitive DNA sequences, the structure of the chromatin fibril and the efficiency of the DNA repair machinery may be important factors in the origin of spontaneous chromosomal rearrangements.     

Hsp40 is involved in cilia regeneration in sea urchin embryos.

In a previous paper we demonstrated that, in Paracentrotus lividus embryos, deciliation represents a specific kind of stress that induces an increase in the levels of an acidic protein of about 40 kD (p40). Here we report that deciliation also induces an increase in Hsp40 chaperone levels and enhancement of its ectodermal localization. We suggest that Hsp40 might play a chaperoning role in cilia regeneration.     

Influence of osmolarity and pH increase to achieve a reduction of monoclonal antibodies aggregates in a production process

Anti PSA monoclonal antibodies for diagnostic use were produced in an in vitro system. After purification using Protein G affinity chromatography a percentage of about 10% of antibody aggregates remained. The use of monoclonal antibodies containing aggregates as a capture antibody in a diagnostic kit reduces the performance of the test making it often unacceptable. The aggregates could be eliminated using gel filtration chromatography but, in that way, the final recovery of the whole production process was only about 50%. Aggregation is favoured when the working pH is near to the isoelectric point of the antibody. We varied the culture medium composition, modifying pH and osmolarity. We tested different values of pH and osmolarity: 7.1, 7.5, 8.0, 8.5 for pH, and 300, 340, 367, 395 mOsm/kg H2O for osmolarity. By modification of the cell culture medium we obtained a significant decrease of monoclonal antibody aggregates in the production cycle. In this way we achieved higher recovery rate and could avoid gel filtration polishing step. The experiments were performed in two stages: first in culture flasks changing one parameter in each experiment, and then in spinner bottle using the best conditions obtained in the first stage. During scale up we used the modifications achieved from the experiment showed in this paper in our production by hollow fibre bioreactor with positive results.     

Gastroscopic evaluation of anti-inflammatory agents

Gastroscopy was performed in 164 patients with rheumatoid arthritis (RA) and 85 with osteoarthritis (OA) to assess the effects of anti-inflammatory agents on the stomach. The main criterion for entry into the trial was the absence of active gastric lesions on pretreatment endoscopy. The patients were divided into groups to receive one of 12 anti-inflammatory drugs or combinations of these. Gastroscopy repeated at three to six and at 12 months disclosed gastric lesions in 78 cases (31%), patients in both disease categories being similarly affected. Lesions occurred in 41 of the 177 patients (23%) receiving a single drug and in 37 of the 72 (51%) receiving combined treatment. All the anti-inflammatory drugs caused gastric damage, the greatest offender being aspirin (13 out of 26 patients) and the least sulindac and diflunisal (two out of 19 (11%) and two out of 20 (10%) patients respectively). Corticosteroids caused gastric damage in only three out of 21 patients (14%), a lower incidence than expected.The indiscriminate prescribing of anti-inflammatory drugs to patients with OA is to be deplored. A lack of correlation between the patients'' subjective complaints of gastric discomfort and the gastroscopic findings emphasises the unreliability of patients'' complaints and the importance of gastroscopy in assessing gastric tolerance. It was not possible to assess minimal prescribing doses or minimum periods of treatment below which gastric damage may be guaranteed not to occur.     

Relationship between laying dates of black kites Milvus migrans and spring temperatures in Italy: rapid response to climate change?

Many Holarctic bird species have been shown to be laying progressively earlier in response to global warming. Most studies have been conducted on small-bodied species at northern latitudes. In Italy, black kite Milvus migrans populations are concentrated near large pre-Alpine lakes, whose water temperatures have been increasing steadily in recent decades. Annual black kite laying dates have become 10–11 days earlier during the last nine years. This shift was related to temporal variations in spring air temperatures, with earlier laying in warmer springs. The progressive advancement in laying dates was not associated with an increase in population level productivity, despite the fact that productivity declined with laying date at the individual territory level within years. Avian response to climate change may be occurring more rapidly than previously thought, even in relatively long-lived species, and not only at the most northern latitudes or highest elevations. Because this change has occurred so rapidly, and because laying dates match annual variations in spring temperatures, the response must be facultatively driven by prevailing conditions, rather than genetic in response to natural selection.     

Optimization of 2-piperidin-4-yl-acetamides as melanin-concentrating hormone receptor 1 (MCH-R1) antagonists: Designing out hERG inhibition

Herein, we disclose the discovery and optimization of 2-piperidin-4-yl-acetamide derivatives as MCH-R1 antagonists. Structural investigation of piperidin-4-yl-amide and piperidin-4-yl-ureas identified 2-piperidin-4-yl-acetamide-based MCH-R1 antagonists with outstanding in vivo efficacy but flawed with high affinity towards the hERG potassium channel. While existing hERG SAR information was employed to discover highly potent MCH-R1 antagonists with minimized hERG inhibition, additional hurdles prevented their subsequent clinical exploration.