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.     

An atlas of human kinase regulation

The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision‐making has been limited by the small number of simultaneously monitored phospho‐regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co‐regulation along the conditions predicts kinase–complex and kinase–substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation‐based signaling and the necessary context to better understand kinase‐driven decision‐making.     

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.     

Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways

There are several pathways for the incorporation of sugars into glycosphingolipids (GSL). Sugars can be added to ceramide that contains sphinganine (dihydrosphingosine) synthesized de novo (pathway 1), to ceramide synthesized from sphingoid bases produced by hydrolysis of sphingolipids (pathway 2), and into GSL recycling from the endosomal pathway through the Golgi (pathway 3). We reported previously the surprising observation that SW13 cells, a human adrenal carcinoma cell line, synthesize most of their GSL in pathway 2. We now present data on the synthesis of GSL in four additional cell lines. Approximately 90% of sugar incorporation took place in pathway 2, and 10% or less in pathway 1, in human foreskin fibroblasts and NB41A3 neuroblastoma cells. In contrast, approximately 50-90% of sugar incorporation took place in pathway 1 in C2C12 myoblasts. The C2C12 cells divide more rapidly and synthesize 10-14 times as much GSL as the other three cell lines. In C6 glioma cells, approximately 30% of sugar incorporation occurred in pathway 1 and 60% in pathway 2. There was no relation between the utilization of pathways for GSL and sphingomyelin synthesis in foreskin fibroblasts and C2C12 cells. In both cells pathways 1 and 2 each accounted for 50% of incorporation of choline into sphingomyelin. In five of the six cell lines that we have studied, most GSL synthesis takes place in pathway 2. We suggest that when the need for synthesis is relatively low, as in slowly dividing cells, GSL are synthesized predominantly from sphingoid bases salvaged from the hydrolytic pathway. When cells are dividing more rapidly, the need for increased synthesis is met by upregulating the de novo pathway.      

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.