The role of polycyclic aromatic hydrocarbon-DNA adducts in inducing mutations in mouse skin

Polycyclic aromatic hydrocarbons (PAH) form stable and depurinating DNA adducts in mouse skin to induce preneoplastic mutations. Some mutations transform cells, which then clonally expand to establish tumors. Strong clues about the mutagenic mechanism can be obtained if the PAH-DNA adducts can be correlated with both preneoplastic and tumor mutations. To this end, we studied mutagenesis in PAH-treated early preneoplastic skin (1 day after exposure) and in the induced papillomas in SENCAR mice. Papillomas were studied by PCR amplification of the H-ras gene and sequencing. For benzo[a]pyrene (BP), BP-7,8-dihydrodiol (BPDHD), 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DB[a,l]P), the codon 13 (GGC to GTC) and codon 61 (CAA to CTA) mutations in papillomas corresponded to the relative levels of Gua and Ade-depurinating adducts, despite BP and BPDHD forming significant amounts of stable DNA adducts. Such a relationship was expected for DMBA and DB[a,l]P, as they formed primarily depurinating adducts. These results suggest that depurinating adducts play a major role in forming the tumorigenic mutations. To validate this correlation, preneoplastic skin mutations were studied by cloning H-ras PCR products and sequencing individual clones. DMBA- and DB[a,l]P-treated skin showed primarily A.T to G.C mutations, which correlated with the high ratio of the Ade/Gua-depurinating adducts. Incubation of skin DNA with T.G-DNA glycosylase eliminated most of these A.T to G.C mutations, indicating that they existed as G.T heteroduplexes, as would be expected if they were formed by errors in the repair of abasic sites generated by the depurinating adducts. BP and its metabolites induced mainly G.C to T.A mutations in preneoplastic skin. However, PCR over unrepaired anti-BPDE-N(2)dG adducts can generate similar mutations as artifacts of the study protocol, making it difficult to establish an adduct-mutation correlation for determining which BP-DNA adducts induce the early preneoplastic mutations. In conclusion, this study suggests that depurinating adducts play a major role in PAH mutagenesis.     

Diterpenoids from Jatropha multifida

Chemical investigation on the stems of Jatropha multifida yielded two diterpenoids, multifolone and (4E)-jatrogrossidentadione acetate along with five known diterpenoids, a flavone and a coumarino-lignan. The structures of the compounds were settled by detailed analysis of their 1D and 2D NMR spectra. The X-ray crystallographic analysis of (4E)-jatrogrossidentadione acetate was also accomplished.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Local circuit input to the medullary reticular formation from the rostral nucleus of the solitary tract

The intermediate reticular formation (IRt) subjacent to the rostral (gustatory) nucleus of the solitary tract (rNST) receives projections from the rNST and appears essential to the expression of taste-elicited ingestion and rejection responses. We used whole cell patch-clamp recording and calcium imaging to characterize responses from an identified population of prehypoglossal neurons in the IRt to electrical stimulation of the rNST in a neonatal rat pup slice preparation. The calcium imaging studies indicated that IRt neurons could be activated by rNST stimulation and that many neurons were under tonic inhibition. Whole cell patch-clamp recording revealed mono- and polysynaptic projections from the rNST to identified prehypoglossal neurons. The projection was primarily excitatory and glutamatergic; however, there were some inhibitory GABAergic projections, and many neurons received excitatory and inhibitory inputs. There was also evidence of disinhibition. Overall, bath application of GABA(A) antagonists increased the amplitude of excitatory currents, and, in several neurons, stimulation of the rNST systematically decreased inhibitory currents. We have hypothesized that the transition from licks to gapes by natural stimuli, such as quinine monohydrochloride, could occur via such disinhibition. We present an updated dynamic model that summarizes the complex synaptic interface between the rNST and the IRt and demonstrates how inhibition could contribute to the transition from ingestion to rejection.     

Non-specific depolymerization of chitosan by pronase and characterization of the resultant products.

Pronase (type XXV serine protease from Streptomyces griseus) efficiently depolymerizes chitosan, a linear beta-->1,4-linked polysaccharide of 2-amino-deoxyglucose and 2-amino-2-N-acetylamino-D-glucose, to low-molecular weight chitosans (LMWC), chito-oligomers (degree of polymerization, 2-6) and monomer. The maximum depolymerization occurred at pH 3.5 and 37 degrees C, and the reaction obeyed Michaelis-Menten kinetics with a Km of 5.21 mg.mL(-1) and Vmax of 138.55 nmoles.min(-1).mg(-1). The molecular mass of the major product, LMWC, varied between 9.0 +/- 0.5 kDa depending on the reaction time. Scanning electron microscopy of LMWC showed an approximately eightfold decrease in particle size and characterization by infrared spectroscopy, circular dichroism, X-ray diffractometry and 13C-NMR revealed them to possess a lower degree of acetylation, hydration and crystallinity compared to chitosan. Chitosanolysis by pronase is an alternative and inexpensive method to produce a variety of chitosan degradation products that have wide and varied biofunctionalities.     

Uptake of 45Ca by mitochondria of Trigonella foenum-graecum as influenced by selenium and mimosine—Detailed kinetic analyses

Mitochondria from Trigonella foenum-graecum seedlings grown independently in the presence of either selenium (0.75 ppm) or mimosine (0.1 mM) exhibited respiration-stimulated energy-dependent uptake of Ca²⁺. Uptake studies were carried out independently at a series of Ca²⁺ concentrations at two different levels: (1) 1–20 μM and (2) 25–1500 μM. Levels of uptake were 50–100% higher in the mitochondria of seedlings of both the Se and mimosine groups. Detailed kinetic analyses revealed negative cooperative effects operative during uptake of Ca²⁺ at 25–1500 μM given in the medium. Hill coefficients for Ca²⁺ uptake by the mitochondria of different groups remained unchanged (nH, 0.75). Biphasic Scatchard plots were concave upward, suggestive of two classes of binding sites. High-affinity binding sites were estimated to be 16 nmol/mg protein with dissociation constant (K _Ca) of 2.5×10⁹ L/mol. In contrast, graphical analyses of the uptake of Ca²⁺ in the range 1–20 μM in the medium revealed cooperative effects of positive nature. The present study demonstrates mixed cooperative effects during Ca²⁺ uptake by mitochondria from seedlings of T. foenum-graecum     

Activity of the major staphylococcal autolysin Atl

The major autolysin of Staphylococcus aureus (AtlA) and of Staphylococcus epidermidis (AtlE) are well-studied enzymes. Here we created an atlA deletion mutant in S. aureus that formed large cell clusters and was biofilm-negative. In electron micrographs, the mutant cells were distinguished by rough outer cell surface. The mutant could be complemented using the atlE gene from S. epidermidis. To study the role of the repetitive sequences of atlE, we expressed in Escherichia coli the amidase domain encoded by the gene, carrying no repeat regions (amiE) or two repeat regions (amiE-R1,2), or the three repeat regions alone (R1,2,3) as N-terminal His-tag fusion proteins. Only slight differences in the cell wall lytic activity between AmiE and AmiE-R1,2 were observed. The repetitive sequences exhibit a good binding affinity to isolated peptidoglycan and might contribute to the targeting of the amidase to the substrate. AmiE and AmiE-R1,2 have a broad substrate specificity as shown by similar activities with peptidoglycan lacking wall teichoic acid, O-acetylation, or both. As the amidase activity of AtlA and AtlE has not been proved biochemically, we used purified AmiE-R1,2 to determine the exact peptidoglycan cleavage site. We provide the first evidence that the amidase indeed cleaves the amide bond between N-acetyl muramic acid and L-alanine.