Pyrrolizidine alkaloids crosslink DNA with actin

Pyrrolizidine alkaloids (PAs) are toxic constituents of hundreds of plant species, some of which people are exposed to in herbal products and traditional remedies. The bioactivity of PAs are related, at least in part, to their ability to form DNA-protein complexes (DPC). Previous studies from our laboratory indicated a possible role for actin in PA-induced DPCs. Nuclei prepared from Madin-Darby bovine kidney (MDBK) and human breast carcinoma (MCF-7) cells were treated with the pyrrolic PAs dehydrosenecionine (DHSN) and dehydromonocrotaline (DHMO). DPCs were purified and then analyzed by Western immunoblotting. Actin was found in DPCs induced by both DHSN and DHMO, but not in those from control nuclei. Actin was also present in DPCs induced by cisplatinum and mitomycin C, two bifunctional cross-linkers. In separate experiments, DHSN and DHMO were crosslinked to a mixture of HindIII digested lambda phage with varying amounts of glutathione (GSH), cysteine, or methionine to identify the stoichiometry of competition between DNA and alternate nucleophiles for crosslink formation with pyrroles. GSH and cysteine, but not methionine, competed with lambda phage for DNA crosslinking, indicating that reduced thiols may have a role in nucleophilic reactions with pyrroles in the cell. While actin involvement in cisplatinum-induced DPCs is documented, the discovery of actin crosslinking in PA or mitomycin C-treated cells or nuclei is, to our knowledge, novel. Pyrrole-induced DPC formation with actin, a protein with structural and/or regulatory importance proteins, may be a significant mechanism for PA toxicity and bioactivity.     

DARPP-32: regulator of the efficacy of dopaminergic neurotransmission

Dopaminergic neurons exert a major modulatory effect on the forebrain. Dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein (32 kilodaltons) (DARPP-32), which is enriched in all neurons that receive a dopaminergic input, is converted in response to dopamine into a potent protein phosphatase inhibitor. Mice generated to contain a targeted disruption of the DARPP-32 gene showed profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication. The results show that DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission.     

Costimulation pathways in host immune responses to allogeneic hepatocytes

BACKGROUND: This study investigated the role of the CD28/B7 (blocked by CTLA4Ig) and CD40/CD40L (blocked by MR1) costimulation pathways on in vivo host T-cell- mediated immune responses to allogeneic hepatocytes. METHODS: Survival of allogeneic hepatocytes (H-2q) in C57BL/6 (H-2b) mice untreated or treated with MR1, CTLA4Ig, L6 (control fusion protein), or a combination of MR1 and CTLA4Ig fusion protein was determined. RESULTS: Median survival time for hepatocellular allografts was 10, 84, 10, 10, and 84 days in untreated (n= 10), MR1-treated (n=7) (P<.0001), CTLA4Ig-treated (n=7) (P=0.02), L6-treated (n=3) (P, not significant), and the combination of MR1- and CTLA4Ig-treated (n=6) (P=0.0003) groups, respectively. CONCLUSIONS: Host treatment with MR1, but not CTLA4Ig, prolonged hepatocellular allograft survival. These data suggest that CD28/B7 interactions appear relatively unimportant, whereas CD40/CD40L interactions provide critical costimulator signals for T-cell-dependent immune responses to allogeneic hepatocytes.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.     

Synthesis and metabotropic glutamate receptor activity of a 2-aminobicyclo[3.2.0]heptane-2,5-dicarboxylic acid, a molecule possessing an extended glutamate conformation

A photochemical approach to the synthesis of the aminobicycloheptane 6a is reported. This compound assumes an extended glutamate conformation, and for this reason was created to further probe the structural features relevant to achieving selectivity for the subtypes of the metabotropic glutamate family of receptors.     

Value of breast imaging in women with painful breasts: observational follow up study

OBJECTIVES: To determine the value of breast imaging in patients with localised or diffuse pain in the breast in whom physical examination shows no abnormalities. DESIGN: Observational follow up study. SETTING: Radiology department of a teaching hospital in the Netherlands. SUBJECTS: Altogether 987 women referred for radiological breast imaging because of pain alone and a control group of 987 asymptomatic women referred for a screening mammogram. MAIN OUTCOME MEASURES: Correlation of the radiological findings with clinical and pathological findings over two years of follow up. RESULTS: Radiological examination of the painful breast(s) showed the following: normal findings in 854 (86.5%) women, benign abnormalities in 85 (8.6%; mainly small cysts or mastopathy), abnormalities that were probably benign in 36 (3.6%), suspicious findings in 8 (0.8%), and malignancy in 4 (0.4%). Biopsy of the painful area was performed in 10 of the 939 women with normal findings or benign abnormalities, in two of 36 women with radiological abnormalities that were probably benign, and in all women with suspicious or malignant findings. Only the four lesions that had been classified radiologically as malignant were found to be malignant at surgery. The prevalence of breast cancer was similar in symptomatic and control women. CONCLUSIONS: Breast imaging in women who present with pain alone is of value only in providing reassurance--no abnormalities are usually found in the painful area, radiological abnormalities classified as benign do not generally have any clinical consequences, and the prevalence of cancer is low in these women. Biopsy of the painful area should be performed only where radiological findings are suspicious.     

Endothelial-dependent dynamic and antithrombotic properties of porcine aortic and pulmonary valves

BACKGROUND: In the present study, the endothelium-dependent antithrombotic and dynamic properties of porcine aortic (AoV) and pulmonary valves (PuV) were investigated. METHODS: Fifteen fresh AoV and 15 fresh PuV were obtained from 25 9-month-old swines. The valves were examined for endothelial function by pharmacologic evaluation (with and without endothelium) of both the endothelial-releasing capacity of prostacyclin and the endothelial-dependent dynamic response to relaxing (acetylcholine from 10[-10] mol/L to 10[-4] mol/L in AoV and PuV segments precontracted with norepinephrine [3 x 10(-6) mol/L]) and contracting (endothelin-1, from 10[-11] mol/L to 10[-5] mol/L; and NG-monomethyl-L-arginine, 10[-4] mol/L) drugs. The ultrastructural integrity of the endothelial valve layer was also examined with transmission electron microscopy. RESULTS: Acetylcholine caused potent relaxation in both AoV and PuV specimens with, but not in those without, endothelium. Endothelin-1 produced a concentration-dependent tension increase in AoV and PuV with and without endothelium. However, the intrinsic activity of the peptide significantly increased in tissues without endothelium. NG-monomethyl-L-arginine evoked a progressive increase in resting tension of the preparations, but the AoV and PuV without endothelium were less sensitive to the inhibition of the nitric oxide generation. Aortic and pulmonary valves with an intact endothelium showed a spontaneous ability to release prostacyclin. The basal release of this lipidic autacoid significantly decreased in cardiac valves without endothelium. This phenomenon was observed in both basal conditions, and under stimulation with the aforementioned drugs. Transmission electron microscopy showed the perfect preservation of endothelial cells in all the preparations examined. CONCLUSIONS: Valvular endothelium of AoV and PuV seems to have similar antithrombotic and dynamic functions of vascular endothelium, actively participating in valvular homeostasis.