Efficient and chemoselective N-acylation of 10-amino-7-ethyl camptothecin with poly(ethylene glycol)

A new poly(ethylene glycol) (PEG) conjugate of 10-amino-7-ethyl camptothecin, a potent antitumor analogue of camptothecin, has been synthesized and preliminary in vivo tests have been performed. Successful chemoselective N-acylation of 10-amino-7-ethyl camptothecin was accomplished using phenyl dichlorophosphate, a coupling reagent used in esterification of alcohols, while other coupling methods failed, due to the low nucleophilicity of the amino group in position 10. The conjugate was tested against P388 murine leukemia cell lines and resulted equipotent to CPT-11, a camptothecin analogue already in clinical use.     

Anchimeric assistance effect on regioselective hydrolysis of branched PEGs: a mechanistic investigation

Branched poly(ethylene glycols) (PEG2) are nowadays widely used for protein and peptides bioconjugation, for their favourable properties (such as the ability to protect the protein surface in an 'umbrella like' fashion). The discovery that mPEG(2)-LysMetbeta AlaOEt lost one mPEG chain during standard base-catalysed ester hydrolysis conditions prompted us to investigate the hydrolytic stability of such systems and the mechanism involved in the PEG chain loss. A series of branched PEGs, substituted with different aminoacids and dipeptides, have been prepared to test the influence of steric hindrance, chain lengths, ramification and Lys-AA amide substitution on hydrolysis. Unexpected results reveal an anchimeric assistance of the Lys-AaA amide proton to the hydrolysis of the carbamoyl moiety joining mPEG to the alpha-amino group of lysine through the formation of an hydantoin system.     

Ester synthesis from trimethylammonium alcohols in dry organic media catalyzed by immobilized Candida antarctica lipase B

Twenty-one different organic solvents were assayed as possible reaction media for the synthesis of butyryl esters from trimethylammonium alcohols in dry conditions catalyzed by immobilized Candida antarctica lipase B. The reactions were carried out following a transesterification kinetic approach, using choline and L-carnitine as primary and secondary trimethylammonium alcohols, respectively, and vinyl butyrate as acyl donor. The synthetic activity of the enzyme was strictly dependent on the water content, the position of the hydroxyl group in the trimethylammonium molecule, and the Log P parameter of the assayed solvent. Anhydrous conditions and a high excess of vinyl butyrate over L-carnitine were necessary to synthesize butyryl-L-carnitine. The synthetic reaction rates of butyryl choline were practically 100-fold those of butyryl-L-carnitine with all the assayed solvents. In both cases, the synthetic activity of the enzyme was dependent on the hydrophobicity of the solvent, with the optimal reaction media showing a Log P parameter of between -0.5 and 0.5. In all cases, 2-methyl-2-propanol and 2-methyl-2-butanol were shown to be the best solvents for both their high synthetic activity and negligible loss of enzyme activity after 6 days.     

The snake venom metalloproteases berythractivase and jararhagin activate endothelial cells

PIII snake venom metalloproteases (SVMPs) are metalloproteases structurally related to ADAMs (a disintegrin and metalloprotease human family of proteins). Berythractivase and jararhagin are PIII SVMPs with 69% homology that have different hemostatic properties. In order to clarify these differences and further characterize the biological effects of these proteins, we have analyzed the effect of both proteases on human umbilical-vein endothelial cell functions. We found that both proteins enhanced nitric oxide generation, prostacyclin production and interleukin-8 release. Berythractivase but not jararhagin increased the expression of decay accelerating factor. Jararhagin decreased cell viability in a concentration-dependent manner and induced cellular apoptosis, while berythractivase did not modulate cell survival. Our results show for the first time that, besides the known anti-aggregating or procoagulant effects of PIII SVMPs, these proteins trigger endothelial cell effector responses. Although structurally related, berythractivase and jararhagin induce a dissimilar generation and release of endothelial molecules that may account for their different hemorrhagic activity.     

Echocardiographic [correction of Ehocardiographic] evaluation of regional left atrial function after rapid atrial pacing

Rapid regular atrial pacing (RAP) produces changes in atrial function similar to those caused by atrial fibrillation in animal models. Left atrial appendage (LAA) function represents regional atrial function. The aim of our study was to investigate the influence of RAP on left atrial regional function and to evaluate the reversibility of changes after termination of pacing in a canine model. Eight dogs were subjected to RAP (400 bpm) for 16 days. Transesophageal echocardiography was performed at baseline, immediately after RAP and 4 weeks after the termination of RAP. The LAA peak late emptying velocity (LAA-E) and filling wave (LAA-f) were measured. LAA-E velocities were significantly reduced and filling wave velocities (LAA-f) were significantly less negative after RAP compared with the baseline values. Four weeks after termination of pacing, the LAA-E and LAA-f velocities were normal. RAP results in impaired regional atrial systolic and diastolic function. The changes were completely reversible 4 weeks after termination of pacing. These results suggest that the LAA is mechanically stunned after RAP.     

Impaired sinus node function and global atrial conduction time after high rate atrial pacing in dogs

In animal and human studies, it has been shown that atrial fibrillation shortens the atrial refractory period and impairs its rate adaptation. The objective of this study was to evaluate the effects of high-rate pacing on sinus node function and intra-atrial conduction. Eight dogs were subjected to rapid atrial pacing (AP) at 400 bpm for 16 days. Sinus node recovery time (SNRT) and P-wave duration were measured at baseline, immediately after AP and four weeks after the termination of AP. SNRT immediately after AP was significantly prolonged at all pacing rates compared to the baseline values. P-wave duration was significantly longer after AP relative to the baseline values. All the variables were completely reversible four weeks after the termination of pacing. Rapid AP induces sinus node dysfunction and prolongs the intra-atrial conduction time. It is possible that the electrical remodelling extends to the sinus node as well.     

Nackt (nkt), a new hair loss mutation of the mouse with associated CD4 deficiency

 A spontaneous recessive mutation named nackt (symbol: nkt) affecting hair growth and T-cell development was discovered in a moderately inbred stock of mice. Skin lesions were characterized by sparse rough coat, bare patches around the eyes and neck, and a scratching behavior throughout life. Fluorescence-activated cell sorter analysis indicated a deficiency in the CD4⁺ 8^– T-cell subset in the thymus and a marked decrease in CD4⁺ T cells in peripheral lymphoid organs. Linkage analysis using a set of molecular markers and an F₂ intersubspecific cross indicated that the mutation maps to the central region of mouse chromosome 13, in a region homologous to human chromosome 5q22-q35. Received: 15 July 1998 / Revised: 27 October 1998     

Molecular approaches of a mitochondrial mutation inCandida utilis

Analysis of the cytochrome spectra of a mitochondrial mutant ofCandida utilis showed complete absence of apocytochromeb; this suggests a certain degree of damage, probably a small deletion in themit genes of mitochondrial DNA. Oxygen uptake measurements with and without cyanide of the respiratory-competentCandida utilis parent strain and its derivative mitochondrial mutant P_1,2 indicated the absence of the cyanide-sensitive or normal respiratory chain and a lowered rate of cyanide-insensitive or alternate respiration. Mitochondrial profiles and distribution of parental and mutant cells account for an altered mitochondrial DNA which affects mitochondria in the latter cell shape and function. The mutant cells ofCandida utilis were considered asmit ^– mutants from the observations reported here.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration and cryopreservation of <Emphasis Type="Italic">Arachis glabrata</Emphasis> (Fabaceae) using leaflet explants

Arachis glabrata Benth (perennial peanut) is a rhizomatous legume with high forage value and great potential for soil conservation as well as it displays valuable plant genetic resources for the cultivated edible peanut improvement. In this study, we developed for the first time successful protocols for micropropagation and cryopreservation of A. glabrata. First fully expanded leaflets from greenhouse-growing plants were efficiently established in vitro (93%) and displayed high frequency of bud induction (58%) on MS medium with 6 mg L^?1 1-fenil-3-(1,2,3-tiadiazol-5-il)urea [TDZ]. Whole plant regeneration was achieved via direct organogenesis by transferring the induced buds to MS media. Immature unexpanded leaves from micropropagated plants were effectively cryopreserved by using the droplet-vitrification technique. Maximum survival (~ 70%) and further regeneration (60–67%) were obtained by preconditioning immature leaves on semisolid MS with 0.3 M sucrose (1 d), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (30 min) followed by glycerol-sucrose plant vitrification solution PVS3 (150 min in ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on cryoplates. Tissues were rewarmed by plunging the aluminum foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and plant regeneration were efficiently achieved via shoot organogenesis, and somatic embryogenesis by culturing cryostored explants on MS added with 6 mg L^?1 TDZ. Genetic stability of plants derived from cryopreserved leaves was confirmed by random amplified polymorphic DNA markers. The protocols established in this study have great potential for rapid multiplication and conservation of selected A. glabrata genotypes.     

Palynofacies associated to hyperpycnite deposits of the Miocene,Cabo Viamonte Beds,Austral Basin,Argentina

The Miocene deposits in the Punta Basílica locality, southernmost Argentina, are included within the Cabo Viamonte Beds, Cabo Domingo Group, in the Austral foreland basin of Tierra del Fuego province. The prograding clinoform systems were accumulated during a weak compressional tectonic regime that allowed the development of a narrow shelf. Paleoenvironmental reconstructions suggest that these clinoforms comprise two dominant architectural elements, channel-levee and lobe complexes, formed mainly by density hyperpycnal currents in outer shelf to depositional slope environments. The transitional and recurrent (vertical and lateral) alternation between sedimentary structures without rheologic boundaries associated with the co-occurrence of plant remains (Nothofagus) are diagnostic criteria for the recognition of hyperpycnites. This type of density flow typically transports large volumes of sediment and organic matter from proximal to deep-marine settings. Four palynofacies types were recognized in a cluster analysis. In general, the palynofacies show predominance of spores and pollen grains, tissues, cuticles, and spongy to fibrous amorphous organic matter (plant and/or freshwater to brackish algae derived), which reflect different positions within the depositional system (e.g., levee-channel and lobe deposits). The co-occurrence of inshore (Batiacasphaera spp., Lingulodinium sp.) with relatively more oceanic (Operculodinium centrocarpum, Spiniferites spp.) dinoflagellates is a strong indication that shallow-water assemblages have been displaced into deep-water settings. Due to the presence of Lingulodinium hemicystum (first appearance data: 23.0 Ma.) and Pentadinium laticinctum (last appearance data: 11.6 Ma.) an age not older than Miocene and not younger than the Serravallian/Tortorian boundary for the Punta Basílica section is proposed.