Induction of cardiac cytochrome p450 in cocaine-treated mice

Cytochrome P450 (P450) is a ubiquitous family of enzymes responsible for the metabolism of a wide variety of drugs and their metabolites, including cocaine. To investigate the effects of cocaine on myocardial injuries and cardiac P450 expression, BALB/c mice were injected daily intraperitoneally with cocaine (30 mg/kg) or cocaine plus pretreatment of P450 inhibitors for 14 days. Tumor necrosis factor-alpha (TNF-alpha) content and creatine phosphokinase (CPK) activity in mice hearts and serums were significantly increased after long-term treatment with cocaine. Pretreatment with the P450 inhibitor, cimetidine (Cime, 50 mg/kg) or metyrapone (Mety, 40 mg/kg) abolished or significantly attenuated the effects of cocaine on TNF-alpha and CPK activity. Western blot analysis shows that mouse cardiac tissues express the P450 isoforms CYP1A1, CYP1A2, and CYP2J2. The protein levels normalized with cyclophilin A were 1.20 plus minus 0.07, 0.67 plus minus 0.03, and 1.48 plus minus 0.01 for CYP1A1, CYP1A2, and CYP 2J2, respectively. After cocaine administration, CYP2J2 increased by 43.6% and CYP1A1 increased by 108.5%, but CYP1A2 was not significantly altered. However, the cytochrome P450 inhibitors Cime and Mety suppressed the cocaine-induced increase in CYP1A1 and CYP2J2 expression. Moreover, application of Cime or Mety alone did not alter the level of cardiac TNF-alpha or the expression of P450. Our results demonstrate that long-term exposure to cocaine causes an increase in cardiac CYP1A1 and CYP2J2 concentration. We speculate that induction of P450 isoforms may cause cardiac injury due to cocaine metabolites locally catalyzed by P450 or the increase in P450 expression itself.     

The novel Streptomyces olivaceoviridis ABC transporter Ngc mediates uptake of N-acetylglucosamine and N,N'-diacetylchitobiose

During cultivation in the presence of N-acetylglucosamine or chitin, Streptomyces olivaceoviridis mycelium efficiently takes up [(14)C]-labelled N-acetylglucosamine. Uptake of the labelled compound can be completely inhibited by unlabelled N-acetylglucosamine and partially by chitobiose. After extraction of the membrane with Triton X-100, two forms of a protein that binds to N-acetylglucosamine and N, N'-diacetylchitobiose (chitobiose) were purified to homogeneity by two consecutive rounds of anionic exchange chromatography. The protein was named NgcE. Using surface plasmon resonance, its binding parameters were determined. It showed highest affinity for N-acetylglucosamine (K(D)=8.28 x 10(-9) M) and for chitobiose (K(D)=2.87 x 10(-8) M). Varying equilibrium dissociation constants in the micromolecular range were ascertained for chitotetraose (K(D)=4.5 x 10(-6) M), chitopentaose (K(D)=1.03 x 10(-6) M) and chitohexaose (K(D)=3.02 x 10(-6) M); the lowest value was measured for chitotriose (K(D)=19.4 x 10(-6) M). After having determined the sequences of several internal peptides from the binding protein by Edman degradation, the corresponding ngcE gene, which encodes a predicted lipid-anchored protein, was identified by reverse genetics. Using a genomic phage library of S. olivaceoviridis genes encoding two other membrane proteins (named NgcF and NgcG) were identified adjacent to ngcE. Each of these is predicted to have six membrane-spanning helices and a consensus motif for integral membrane proteins characteristic of ABC transporters. In addition, the gene for a predicted regulator protein (NgcR) was detected. The ngcEFG operon lacks a gene for an ATP-hydrolysing protein. NgcE is a new member of the CUT-1 family of ABC transporters for carbohydrates. Comparative studies of the wild-type and a mutant strain carrying an insertion within the ngc operon clearly demonstrate that the Ngc system mediates the uptake of N-acetylglucosamine and chitobiose in vivo.     

An active precursor in assembly of yeast nuclear ribonuclease P

The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand. Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P. Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage. The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized. The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.     

间种蔬菜对甘蔗地生态环境和甘蔗生长的影响

甘蔗 (ＳａｃｃｈａｒｕｍｏｆｆｉｃｉｎａｒｕｍＬ .)是广西重要的经济作物 ,种植面积达 4 5 33× 10 6ｈａ[1] ,甘蔗的前期生长比较缓慢 ,行间有比较大的可利用空间 ,到6月下旬～ 7月上旬才进入迅速生长期并逐渐封行。有研究表明 ,在甘蔗园间种玉米对甘蔗的生长发育和产量有一定的影响[5] ,而合理间种豆科作物 (黄豆和花生 )不仅不影响甘蔗的正常生长发育 ,还可以增加甘蔗的产量[4 ,6] 。但间种作物对甘蔗园生态环境的影响未见报道 ,我们于 1997- 1999年在桂西北开展了甘蔗园间种经济效益比较高的蔬菜作物的试验 ,并对间种甘蔗园生态环…     

Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block

The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a denned cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.     

Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth

We examined whether lung growth after pneumonectomy (PNX) invokes normal signaling pathways of postnatal development. We qualitatively and quantitatively assessed the immunoexpression of epidermal growth factor (EGF), its receptor (EGFR), surfactant proteins (SP) [SP-A and -D and surfactant proproteins (proSP)-B and -C] and proliferating cell nuclear antigen (PCNA) in immature and mature dog lung. We also assayed these proteins in lungs of immature dogs 3 wk or 10 mo after they underwent right PNX compared with simultaneous matched sham controls. During maturation, alveolar cell proliferation is regionally regulated in parallel with EGF and EGFR levels and inversely correlated with SP-A and proSP-C levels. In contrast, post-PNX lung growth is not associated with EGF or EGFR upregulation but with markedly increased SP-A level and moderately increased SP-D level; proSP-B and proSP-C levels did not change. We conclude that 1) signaling of EGF axis and differential regulation of SPs persist during postnatal lung development, 2) post-PNX lung growth is not a simple recapitulation of maturational responses, and 3) SP-A and SP-D may modulate post-PNX lung growth.     

Utility of thiol-cross-linked fluorescent dye labeled terminators for DNA sequencing

Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by directly coupling 5-carboxyfluorescein NHS ester with 18-ddUTP 9. These terminators (10, 11, and 13) in combination with the Thermo Sequenase II DNA polymerase, in thermal cycle sequencing experiments, revealed that the thioether cross-conjugated terminator 10 and amide cross-conjugated terminator 13 served as good terminating substrates, generating satisfactory single-color gel images and electropherograms, while the other thioether cross-conjugated and maleimide derived one 11 underwent unexpected pH and temperature induced decomposition without showing fluorescent signatures for incorporation.     

Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens

The structure and infectivity of the oocysts of a new species of Cryptosporidium from the feces of humans are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts. and measure 4.4-5.4 microm (mean = 4.86) x 4.4-5.9 microm (mean = 5.2 microm) with a length to width ratio 1.0-1.09 (mean 1.07) (n = 100). Oocysts were not infectious for ARC Swiss mice, nude mice. Wistar rat pups, puppies, kittens or calves, but were infectious to neonatal gnotobiotic pigs. Pathogenicity studies in the gnotobiotic pig model revealed significant differences in parasite-associated lesion distribution (P = 0.005 to P = 0.02) and intensity of infection (P = 0.04) between C. parvum and this newly described species from humans. In vitro cultivation studies have also revealed growth differences between the two species. Multi-locus analysis of numerous unlinked loci, including a preliminary sequence scan of the entire genome demonstrated this species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further support for its species status. Based on biological and molecular data, this Cryptosporidium infecting the intestine of humans is proposed to be a new species Cryptosporidium hominis n. sp.