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.     

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.     

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.     

Chromosomal radiosensitivity of pig leucocytes in relation to sampling time

Pig blood cultures were used to analyse the sensitivity to X-rays (measured as frequency of induced dicentrics) of lymphocytes sampled at variable times. By using the BrdU-Giemsa method it was possible to identify the lymphocytes that were performing their first division at early (less than 30% of cells in second division), intermediate (30–50% of cells in second or subsequent divisions) and late stages (more than 50% of cells in second or subsequent divisions). No difference was found in the radiosensitivity of these 3 varieties of lymphocyte. It was also observed that: (a) the combination of radiation followed by BrdU treatment did not increase the clastogenic action of X-rays, (b) X-rays in the dose used in our cultures did not increase the frequency of SCEs, and (c) minor changes in culture conditions probably influence the basal frequency of SCEs.     

DNA conformation in N,N-dimethyl formamide-H2O solutions

Optical density, viscosity, and light scattering measurements for calf thymus DNA in water–N,N dimethyl formamide (DMF) solutions are presented. DMF content varied between 0 and 60% (v/v) and DNA molecular weight varied between 15 × 10⁶ and 0.5 × 10⁶. Complementary measurements of the solubility of adenine, thymine, guanine, and cytosine in H₂O–DMF mixtures are presented. The denaturation temperature of DNA, manifested by about a 35% increase of optical density, is gradually depressed by increasing DMF content. However, a significant increase of OD occurs even before (and even after) the denaturation point, when DMF content is increased isothermally. The intrinsic viscosity also exhibits a large decrease when DMF content is increased both before and after the denaturation point. Light scattering data for high-molecular-weight DNA in the predenaturation range indicate a decrease of the mean-square radius and a constant molecular weight on increasing DMF content. The results, interpreted in terms of the wormlike chain of Kratky and Porod, indicate a large decrease of the persistence length of DNA. For low-molecular-weight DNA, radius and molecular weight increase with DMF content, indicating intermolecular aggregation. The formation of compact structures of native DNA is discussed in terms of an increased solubility of uncharged bases, and a decreased solubility of phosphate and deoxyribose groups, when a less polar environment is provided by the addition of DMF.