Elevated rate of DNA synthesis and its correlation to cAMP-phosphodiesterase activity during induction of polydactyly in mouse embryos heterozygous for Hemimelia-extra toe (Hmx)

The induction of polydactyly in mouse embryos heterozygous for Hemimelia-extra toe (Hmx) is associated with aberrant outgrowth of the developing autopod on day 12 of gestation. We have quantitated the rate of DNA synthesis and the activity of cAMP-phosphodiesterase (PDE) that is characteristic of the prospective polydactylous region. Mid-stage 18 hind-limb buds were labeled with [3H]dThd either in situ using whole embryo culture, or as isolated preaxial autopod fragments cultured on a membrane substratum. The mean specific activities of incorporation were compared for normal (+/+) and mutant (Hmx/+) genotypes. A significant (P less than or equal to 0.01) 19% increase, peculiar to the prospective polydactylous region, was measured after 4 hours in embryo culture. The same increment was detected after 4 hours in organ culture, but was amplified linearly to 55% when incubation was extended to 20 hours. During this period, continuous exposure to 1.0 mM IBMX (3-isobutyl-1-methyl xanthine), an inhibitor of cAMP-PDE activity, "slowed down" the rate of DNA synthesis to untreated +/+ proportions. When cAMP-PDE activity was assayed in uncultured autopods, a significant (P less than or equal to 0.01) 18% increase was detected within the prospective polydactylous region specifically on stage 18 of gestation. This is the developmental phase during which polydactylous outgrowth is induced in situ. Thus, uncontrolled cAMP-PDE activity may, in part, provoke the enhanced rate of cell proliferation.     

Molecular link between membrane cholesterol and Na+/H+ exchange within human platelets.

Incubation of human platelets with cholesterol-poor, cholesterol-normal and cholesterol-rich liposomes revealed that: (i) acquisition or depletion of platelet membrane cholesterol was highly selective; (ii) variation in membrane cholesterol was highly selective. Variation in membrane cholesterol content (cholesterol-to-phospholipid molar ratio from 0.15-1.2) with respect to values found in unmodified normal platelets, was paralleled by the observed changes in amiloride-sensitive cytoplasmic pH, as well as phospholipase A2 activity. However, a decrease in cytoplasmic pH was accompanied by an increase in phospholipase A2 activity; (iii) membrane cholesterol-modulated changes in intra-platelet pH, as well as phospholipase A2 activity, was completely inhibited when platelets were pretreated with quinacrine (a specific phospholipase A2 inhibitor) before exposure to various types of liposomes. Although exposure of platelets (pretreated with amiloride) with various types of liposomes resulted in the inhibition of Na+/H+ exchange it had no noticeable effect upon the observed phospholipase A2 activity. Based upon these results we suggest that membrane cholesterol-modulated phospholipase A2 activity may be the basic mechanism responsible for the nature of Na+/H+ exchanger activity observed in cholesterol-enriched platelets, leading these platelets to a hypersensitized state.     

Active site studies on Bacillus amyloliquefaciens α-amylase (I)

Summary Modification of liquefying -amylase by diethylpyrocarbonate or its photo-oxidation in the presence of rose bengal caused rapid loss of enzyme activity. The photo-oxidation followed pseudo-first-order kinetics giving maximal value at pH 8.0. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm which was directly proportional to the extent of inactivation. Diethylpyrocarbonate at low concentration at pH 6.0 and 30 ° C completely inactivated a-amylase. Inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by diethylpyrocarbonate was one, thus indicating modification of a single histidine per mole of the enzyme. Diethylpyrocarbonate-modified enzyme showed increased absorbance at 240 nm which was reversed completely upon treatment with NH₂OH at 30 °C for 16 hr. Calculating the histidine residues being modified from the increase in absorbance at 240 nm showed that three residues were ethoxyformylated on treatment with diethylpyrocarbonate, of which only one was found at the active site. Substrate and competitive inhibitor protects the enzyme against both, photo-oxidation, and modification by diethylpyrocarbonate, confirming that histidine plays an essential role at the -amylase active site.     

The use of in vitro procedures in teratology.

The capabilities of investigators in the fields of teratology and toxicology are greatly enhanced by the use of tissue culture procedures in unraveling the mechanisms of drug action. Techniques currently available for the culture of postimplantation mammalian embryos permit evaluation of their metabolic responses to potential teratogens even when the length of time embryos survive and develop in culture is too short to allow a conventional teratologic survey of malformations. A simple procedure for culturing mouse embryos during early organogenetic stages is described in this report that will be of value to teratologists; it avoids the requirements of special glassware and equipment by using ordinary capped test tubes which are rotated tomaintain and efficient nutritional and gaseous evnironment. Some studies concucted with this procedure to monitor the metabolism of embryo during the first 24 h of culture are summarized. Another aspect of tissue culture, organ culture, provides further manipulative capability by which embryonic organs can be maintained for long periods of time during which they develop and differentiate to an extent that their morphological and biochemical responses to a teratogen can usually be made. Comparative effects of several teratogenic agents and the relative concentration of each that produces a similar degree of response are summarized. It is concluded that organs are more sensitive to teratogens in culture than they are in vivo, and that different teratogens possess enough specificity to isolate their simple growth-retarding effect from the role they play in distrubing other specific developmental events.     

Effect of Auxins and Light on Rooting Stem Cuttings of Populus nigra Salix tetrasperma, Ipomea fistulosa and Hibiscus notodus in Relation to Polarity

The apical and basal ends of stem cuttings of Populus nigra, Salix tetrasperma, Ipomoea fistulosa and Hibiscus notodus were treated with 10 mg/l solutions of IAA and IBA for 24 hours and were planted either erect or inverted both in light and dark. Observations for the number of cuttings that rooted and the roots produced on them were recorded at weekly intervals. In Salix, Ipomoea and Hibiscus rooting was more on cuttings planted erect, while in populus it did not differ much with the manner of planting. The reduced rooting in inverted cuttings may be ascribed to the low level of endogenous auxin at the apex due to polar transport. An exogenous application of auxins enhanced rooting on inverted cuttings. In dark, roots on Populus and Salix cuttings were produced both above and within the rooting medium. The weak polarity of these two plants may be due to the potential root primordia reported in their stem. The formation of callus occurred on the top of Populus cuttings whether planted erect or inverted but it differentiated into branches on erect cuttings only. In those planted in an inverted position the callus failed to differentiate in spite of the application of kinetin, auxins, TIBA, coumarin and sucrose, and dried ultimately.     

An NMR- and MS-based metabonomic investigation of saliva metabolic changes in feline odontoclastic resorptive lesions (FORL)-diseased cats

The feline odontoclastic resorptive lesion (FORL) is a common oral problem in cats. The disease has increased steadily since the domestication of cats and etiology of this disease has not been fully determined although several theories have been proposed. Feeding practices, vaccination, and neutering programs have all been suspected to be associated with FORL. The aim of the current study is to assess the feasibility of metabonomics to detect at an early stage the onset of the disease. The diagnostic biomarkers could then be used as “efficacy markers” for nutritional intervention in preventing and/or slowing the progression of FORL. ¹H-NMR- and LC/MS-based metabonomic analysis of saliva samples obtained from a group of 21 cats (11 healthy and 10 FORL diseased) showed clear differences in the metabolic composition of saliva from healthy and FORL-diseased cats. To identify biomarkers, the spectroscopic data was processed using partial least-squares discriminant analysis (PLS-DA) and validated by leave-one-subject-out cross validation. The PLS-DA model predicted FORL- diseased cats with over 60% accuracy. The maximum value of Q² of the random permutation sets was less than 0.3. The diseased cats showed increased levels of many organic and amino acids, such as acetate, lactate, propionate, isovalerate, tryptamine, and phenylalanine suggesting changes in oral microflora in the disease situation. This study is preliminary and a larger study with more samples to further validate the biomarker profile predictive of an early FORL pathophysiological status is in progress.     

A serpin from the gut bacterium Bifidobacterium longum inhibits eukaryotic elastase-like serine proteases

Serpins form a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Recently identified prokaryotic members of this protein family may provide a key to the evolutionary origins of the unique serpin fold and the associated inhibitory mechanism. We performed a biochemical characterization of a serpin from Bifidobacterium longum, an anaerobic Gram-positive bacterium that naturally colonizes human gastrointestinal tract. The B. longum serpin was shown to efficiently inhibit eukaryotic elastase-like proteases with a stoichiometry of inhibition close to 1. Porcine pancreatic elastase and human neutrophil elastase were inhibited with the second order association constants of 4.7 x 10(4) m(-1) s(-1) and 2.1 x 10(4) m(-1) s(-1), respectively. The B. longum serpin is expected to be active in the gastrointestinal tract, because incubation of the purified recombinant serpin with mouse feces produces a stable covalent serpin-protease adduct readily detectable by SDS-PAGE. Bifidobacteria may encounter both pancreatic elastase and neutrophil elastase in their natural habitat and protection against exogenous proteolysis may play an important role in the interaction between these commensal bacteria and their host.     

The approximate entropy of the electromyographic signals of tremor correlates with the osmotic fragility of human erythrocytes

Background  

The main problem of tremor is the damage caused to the quality of the life of patients, especially those at more advanced ages. There is not a consensus yet about the origins of this disorder, but it can be examined in the correlations between the biological signs of aging and the tremor characteristics.     

In vitro production of cattle-water buffalo (Bos taurus--Bubalus bubalis) hybrid embryos

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using inter species gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.