A Simple Mathematical Model of Second-Messenger Mediated Slow Excitatory Postsynaptic Potentials

We have developed a novel and simple mathematical model of a slow excitatory postsynaptic potential (EPSP) based on an abstraction of the processes of activation, inactivation, and summation of a cAMP, protein kinase A (PKA)-dependent second-messenger cascade. The model describes the activation of receptors, G-proteins, and production of cAMP as the first stage and uses first-order, non-rate-limited kinetics. The second stage corresponds to the release of active, PKA catalytic subunit and can use first- or higher-order kinetics. The third stage represents simple phosphorylation of ion channels and is limited by the number of channels available. The decay of each stage is based on first-order, mass-action kinetics. These equations and some variations were solved numerically and values of the parameters were determined by fitting to a variety of experimental data from myenteric neurons of the guinea-pig ileum. The model produced a slow EPSP with a nonlinear stimulus-response relationship that resulted from the underlying kinetics of the signaling cascade. This system of equations is suitable for incorporation into a large-scale computer simulation, and the methodology should be generalizable to other pathways.     

Association of butyrylcholinesterase K variant with cholinesterase-positive neuritic plaques in the temporal cortex in late-onset Alzheimer's disease

In confirmed late-onset (>65 years) Alzheimer's disease, we found a greater load, both of overall neuritic plaques and of cholinesterase-positive neuritic plaques, in the temporal cortex of carriers of the butyrylcholinesterase K variant (BCHE-K) aged <80 years than of all other patients. The differences were most striking in the case of cholinesterase-positive neuritic plaques. Among BCHE-K carriers, densities of such plaques were over six times higher in patients <80 years at death than in those >80 years (P=0.01). Furthermore, in subjects <80 years, BCHE-K carriers had nearly six-fold greater densities of these plaques than non-carriers (P=0.009). We consider three potential explanations for these findings: that the K variant binds more readily to plaque constituents, that it promotes fibril formation or that it induces aberrant neurite growth.     

Correlates and implications of breast-feeding practices in Bas Zaire

A sample of 1871 women having a child under 3 years old in Bas Zaire was studied to determine the correlates of breastfeeding practices and to examine the interrelationships among breastfeeding, contraceptive practices and desire for pregnancy. The methods of analysis applied were life table analysis and its multivariate extensions. Most of the findings in this analysis are consistent with current literature on the correlates of the duration of breastfeeding. Maternal education, economic status, age, parity, urban residence, pregnancy, and sex of the index child were significantly related to the length of breastfeeding. Among non-pregnant women, current desire for pregnancy was also related to breastfeeding status when the length of time since birth of the last child was taken into consideration. Rural women were reportedly ready for another pregnancy sooner after the birth of their last child than were urban women. Breastfeeding appears to be the most important means of contraceptive protection in the study population. The effective traditional method of extended postpartum abstinence is not widely prevalent, particularly among the urban sample, and indeed seems to be on the decline. The data presented here also suggest that breastfeeding pratices are changing in this area of Africa where little economic development has occurred in the past 20 years. This research suggests that if current trends continue, fertility levels are likely to increase significantly. The findings also indicate that Bas Zairian mothers want to space their births and for this reason may be receptive to family planning programs that use appropriate strategies.     

A general synthesis of long chain ω- and (ω-1)-hydroxy fatty acids

A method for the synthesis of long chain fatty acids substituted at the ω and ω-1 positions has been developed. The key step is the isomerization of the triple bond of an alkyn-1-ol from an internal position in the chain to the free terminus with a new, convenient reagent, sodium aminopropylamide (NaAPA). Standard functional group manipulations i.e., Jones oxidation, esterification and hydroboration of the triple bond are used to prepare ω-hydroxy fatty esters. The generality of the method is illustrated with syntheses of ω-hydroxy fatty esters with 24, 26, 28 and 30 carbon chains.In the 24 carbon series, hydration of the terminal triple bond of alkynoic ester 4a followed by reduction gave the (ω-1)-hydroxy ester.     

Acceptor substrate selectivity and kinetic mechanism of Bacillus subtilis TagA

Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of Gram-positive bacteria. Because they are essential for survival or virulence in many organisms, the enzymes involved in the biosynthesis of WTAs are attractive antibiotic targets. The first committed step in the WTA biosynthetic pathway in Bacillus subtilis is catalyzed by TagA, which transfers N-acetylmannosamine (ManNAc) to the C4 hydroxyl of a membrane-anchored N-acetylglucosaminyl diphospholipid (GlcNAc-pp-undecaprenyl, lipid I) to make ManNAc-beta-(1,4)-GlcNAc-pp-undecaprenyl (lipid II). We have previously shown that TagA utilizes an alternative substrate containing a saturated C(13)H(27) lipid chain. Here we use unnatural substrates and products to establish the lipid preferences of the enzyme and to characterize the kinetic mechanism. We report that TagA is a metal ion-independent glycosyltransferase that follows a steady-state ordered Bi-Bi mechanism in which UDP-ManNAc binds first and UDP is released last. TagA shares homology with a large family of bacterial glycosyltransferases, and the work described here should facilitate structural analysis of the enzyme in complex with its substrates.     

Identification of in vivo phosphorylation sites on human deoxycytidine kinase. Role of Ser-74 in the control of enzyme activity

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity.     

cGMP-dependent protein kinase type I inhibits TAB1-p38 mitogen-activated protein kinase apoptosis signaling in cardiac myocytes

Cardiac myocyte apoptosis during ischemia and reperfusion (I/R) is tightly controlled by a complex network of stress-responsive signaling pathways. One pro-apoptotic pathway involves the interaction of the scaffold protein TAB1 with p38 mitogen-activated protein kinase (p38 MAPK) leading to the autophosphorylation and activation of p38 MAPK. Conversely, NO and its second messenger cGMP protect cardiac myocytes from apoptosis during I/R. We provide evidence that the cGMP target cGMP-dependent protein kinase type I (PKG I) interferes with TAB1-p38 MAPK signaling to protect cardiac myocytes from I/R injury. In isolated neonatal cardiac myocytes, activation of PKG I inhibited the interaction of TAB1 with p38 MAPK, p38 MAPK phosphorylation, and apoptosis induced by simulated I/R. During I/R in vivo, mice with a cardiac myocyte-restricted deletion of PKG I displayed a more pronounced interaction of TAB1 with p38 MAPK and a stronger phosphorylation of p38 MAPK in the myocardial area at risk during reperfusion and more apoptotic cardiac myocytes in the infarct border zone as compared with wild-type littermates. Notably, adenoviral expression of a constitutively active PKG I mutant truncated at the N terminus(PKGI-DeltaN1-92) did not inhibit p38 MAPK phosphorylation and apoptosis induced by simulated I/R in vitro, indicating that the N terminus of PKG I is required. As shown by co-immunoprecipitation experiments in HEK293 cells, cGMP-activated PKG I, but not constitutively active PKG I-DeltaN1-92 or PKG I mutants carrying point mutations in the N-terminal leucine-isoleucine zipper, interacted with p38 MAPK, and prevented the binding of TAB1 to p38 MAPK. Together, our data identify a novel interaction between the cGMP target PKG I and the TAB1-p38 MAPK signaling pathway that serves as a defense mechanism against myocardial I/R injury.