A novel sphingophosphonolipid head group 1-hydroxy-2-aminoethyl phosphonate in Bdellovibrio stolpii.

Members of the bacterial genus Bdellovibrio include strains that are free-living, whereas others are known to invade and parasitize larger Gram-negative bacteria. The bacterium can synthesize several sphingophospholipid compounds including those with phosphoryl bonds as well as phosphonyl bonds. In the present study, the dominant sphingophosphonolipid component was isolated by column chromatography, and the long-chain bases, fatty acids, and polar head groups were identified by thin-layer and gas-liquid chromatographic procedures. The definitive structural identity of the sphingolipid was established by nuclear magnetic resonance and mass spectrometry of hydrolysis products and the intact compound. The compound was identified as N-2'-hydroxypentadecanoyl-2-amino-3,4-dihydroxyheptadecan-1-phosphono-(1-hydroxy-2-aminoethane).

Determination of temporal expression patterns for multiple genes in the rat carotid artery injury model.

Vascular injury induces extensive alteration to the extracellular matrix (ECM). These changes contribute to lesion formation and promote cell migration and proliferation. To elucidate ECM response to arterial injury, we used real-time polymerase chain reaction monitoring to quantitate the expression levels of 81 genes involved in the synthesis and breakdown of ECM as well as receptors and signaling proteins that communicate and respond to ECM molecules. The temporal regulation of gene expression in the carotid was measured at 1, 3, 5, 7, 9, 14, and 28 days postinjury. Among the 68 genes that showed detectable expression by our method, 47 (69%) were significantly induced or repressed over time, confirming the extensive ECM gene response in this model. More ECM-related genes (31) were regulated at day 1 than at any other time point, and the number of regulated genes decreased over time. However, 14 of the genes were still induced or repressed at day 28, indicating that return to preinjury expression patterns did not occur and no new steady state was achieved over 28 days. In spite of the large number of changes in gene expression, only a small number of expression patterns was observed, suggesting that ECM-related genes could potentially be coregulated.

Pneumocysterol [(24Z)-ethylidenelanost-8-en-3beta-ol], a rare sterol detected in the opportunistic pathogen Pneumocystis carinii hominis: structural identity and chemical synthesis.

Pneumocystis carinii pneumonia (PcP) remains among the most prevalent opportunistic infections among AIDS patients. Currently, drugs used clinically for deep mycosis act by binding ergosterol or disrupting its biosynthesis. Although classified as a fungus, P. carinii lacks ergosterol. Instead, the pathogen synthesizes a number of distinct Delta7, 24-alkylsterols, despite the abundance of cholesterol, which it can scavenge from the lung alveolus. Thus, the pathogen-specific sterols appear vital for organism survival and proliferation. In the present study, high concentrations of a C32 sterol were found in human-derived P. carinii hominis. The definitive structural identities of two C-24 alkylated lanosterol compounds, previously not reported for rat-derived P. carinii carinii, were determined by using GLC, MS, and NMR spectroscopy together with the chemical syntheses of authentic standards. The C31 and C32 sterols were identified as euphorbol (24-methylenelanost-8-en-3beta-ol) and pneumocysterol [(24Z)-ethylidenelanost-8-en-3beta-ol], respectively. The identification of these and other 24-alkylsterols in P. carinii hominis suggests that (i) sterol C-24 methyltransferase activities are extraordinarily high in this organism, (ii) 24-alkylsterols are important components of the pathogen's membranes, because the addition of these side groups onto the sterol side chain requires substantial ATP equivalents, and (iii) the inefficacy of azole drugs against P. carinii can be explained by the ability of this organism to form 24-alkysterols before demethylation of the lanosterol nucleus. Because mammals cannot form 24-alkylsterols, their biosyntheses in P. carinii are attractive targets for the development of chemotherapeutic strategies against this opportunistic infection.

CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation.

The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC50 of 0.5 microM in vitro. Inhibition was competitive with respect to ATP (Ki = 95 nM), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8.5- and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G1/S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC50 for growth arrest ranging from 1.25 to 20 microM. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation.

Characterization of the human cyclin-dependent kinase 2 gene. Promoter analysis and gene structure.

Cyclin-dependent kinase 2 is a serine/threonine protein kinase essential for progression of the mammalian cell cycle from G1 to S phase. CDK2 mRNA has been shown to be induced by serum in several cultured cell types. Therefore, we set out to identify elements that regulate the transcription of the human CDK2 gene and to characterize its structure. This paper describes the cloning of approximately 2.4-kilobase pair genomic DNA fragment from the upstream region of the human CDK2 gene. This fragment contains five transcription initiation sites within a 72-nucleotide stretch. A 200-base pair sub-fragment that confers 70% of maximal basal promoter activity was shown to contain two synergistically acting Sp1 sites. However, a much larger DNA fragment containing approximately 1.7 kilobase pairs of upstream sequence is required for induction of promoter activity following serum stimulation. The intron exon boundaries of seven exons in this gene were also identified, and this information will be useful for analyzing genomic abnormalities associated with CDK2.

Functional analysis of the human cyclin D2 and cyclin D3 promoters.

The D-type cyclins promote progression through the G1 phase of the cell cycle and may provide a link between growth factors and the cell cycle machinery. We determined the nucleotide sequence of the 5'-flanking region of the human cyclin D2 and cyclin D3 genes and identified the transcription start sites. Analysis of the upstream sequences required for transcription of the cyclin D2 and cyclin D3 genes in continuously dividing cells revealed marked differences in their regulatory elements. In the cyclin D2 gene positive elements were localized between positions -306 and -114 relative to the ATG codon at +1. Additional positive elements were localized between -444 and -345, whereas sequences that reduced transcription were identified between nucleotides -1624 and -892. In the cyclin D3 gene all of the positive elements required for maximal transcription were localized between nucleotides -366 and -167, and no negative elements were found. The activities of a reporter gene linked to the upstream regulatory sequences of the cyclin D2 gene but not the cyclin D3 gene were induced when starved cells were serum stimulated. This suggests that although the abundance of both the cyclin D2 and cyclin D3 mRNAs is increased by serum stimulation, only the cyclin D2 gene is up-regulated at the transcriptional level. Sequences between nucleotides -306 and -1624 of the cyclin D2 gene were necessary for serum inducibility.

Kinetics of the creatine kinase reaction in neonatal rabbit heart: an empirical analysis of the rate equation.

Here we define the kinetics of the creatine kinase (CK) reaction in an intact mammalian heart containing the full range of CK isoenzymes. Previously derived kinetic constants [Schimerlik, M. I., & Cleland, W. W. (1973) J. Biol. Chem. 248, 8418-8423] were refit for the reaction occurring at 37 degrees C. Steady-state metabolite concentrations from 31P NMR and standard biochemical techniques were determined. 31P magnetization transfer data were obtained to determine unidirectional creatine kinase fluxes in hearts with differing total creatine contents and differing mitochondrial CK activities during KCl arrest and isovolumic work for both the forward reaction (MgATP synthesis) and reverse reaction (phosphocreatine synthesis). The NMR kinetic data and substrate concentration data were used in conjunction with a kinetic model based on MM-CK in solution to determine the applicability of the solution-based kinetic models to the CK kinetics of the intact heart. Our results indicated that no single set of rate equation constants could describe both the KCl-arrested and working hearts. We used our experimental data to constrain the solution-derived kinetic model and derived a second set of rate equation constants, which describe the isovolumic work state. Analysis of our results indicates that the CK reaction is rate limited in the direction of ATP synthesis, the size of the guanidino substrate pool drives the measured CK flux in the intact heart, and during isovolumic work the CK reaction operates under saturating conditions; that is, the substrate concentrations are at least 2-fold greater than the Km or Kim for each substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

An in vivo model for the study of Bordetella avium adherence to tracheal mucosa in turkeys.

An in vivo model was developed for studies characterizing the adherence of Bordetella avium to the tracheal mucosa of turkeys. Three-week-old turkeys were anesthetized, and the cervical part of the trachea was isolated after tracheostomy was done. A hemostat was applied craniad to the tracheostomy site to occlude the tracheal lumen. Isolated tracheal segments were filled with an aqueous bacterial inoculum for 1 minute, and then excess inoculum and the hemostat were removed. After 1 hour, a 1-cm section was excised from each tracheal segment, and adherent viable bacteria were quantified. Modifications of the procedure were evaluated to produce a model that was technically simple to do, economical, and reproducible. To examine the validity of the model, adherence of B avium was compared with that of Escherichia coli and Staphylococcus aureus. Adherence of B avium to tracheal mucosa was 17 times greater than that with E coli and 1,550 times greater than that with S aureus. Colonization of the tracheal mucosa by B avium was demonstrated in tracheal sections obtained 6 hours after filling with bacterial inoculum. Because the ciliary clearance mechanism of the tracheal segments remained functional, washing of the tracheal lumen had no effect on numbers of associated bacteria. An important advantage of this model over in vitro models is the excellent preservation of the tracheal mucosal surface.

Detection of a bulk water structure related conformational change in horse heart cytochrome c in Cl- -H2O solutions utilizing spectrofluoroelectrochemical techniques.

Utilizing the ratio of the fluorescence intensities of the reduced and oxidized forms of horse heart cytochrome c (cyt c), it is possible to monitor conformational changes of the protein upon reduction. The temperature dependence from 25 to 50 degrees C of the ratio is sigmoidal in nature, indicative of a conformational transition with the midpoint being 43 degrees C in 0.10 M NaCl, 0.10 M PO4 buffer, pH 7.0, solution. This transition is consistent with the previously postulated biphasic model used to explain the nonlinearity in E0' vs T in Cl- -H2O solutions [C.W. Anderson, H.B. Halsall , W.R. Heineman , and G.P. Kreishman (1977) Biochem. Biophys. Res. Commun. 76, 339-344]. In addition, the chemical shift of the bulk water proton in tetramethylammonium chloride solution shows a nonlinearity at 42 degrees C and it is postulated that the conformational changes of cyt c are the result of the behavior of the bulk water structure.

Synergistic, additive, and antagonistic mutagenic responses to binary mixtures of benzo(a)pyrene and benzo(e)pyrene as detected by strains TA98 and TA100 in the Salmonella/microsome assay.

Binary mixtures of benzo(a)pyrene (B(a)P) and benzo(e)pyrene (B(e)P) produce synergistic mutagenic (comutagenic) responses in Salmonella typhimurium strain TA98 (a frameshift detector). The optimum enhancement (25 X) was found at B(a)P concentration of 0.3 microgram/plate and B(e)P concentration of 1.5 microgram/plate. The response of strain TA100 (mostly a base-substitution detector) is opposite that of TA98, showing antagonism and additivity in similar concentration ranges.