A novel multistep method for generating precise unidirectional deletions using BspMI, a class-IIS restriction enzyme

A novel approach is described that permits the introduction of unidirectional deletions into a cloned DNA fragment, in a precisely controlled manner. The method is based on the use of a special vector and a class-IIS restriction endonuclease, BspMI, which produces staggered cuts 4 and 8 nucleotides (nt) to the 3' from its recognition site 5'-ACCTGC-3'. The DNA fragment is inserted into the pUC19-based plasmid, which contains a unique BspMI recognition site, and the appropriate number of cleavage-and-deletion cycles is performed, each cycle removing 4 bp. Since the recognition site is not affected by the BspMI cleavage, no recloning of the DNA fragment is necessary. Each cycle consists of BspMI cleavage, removal of the 4-nt single-stranded cohesive ends with mung bean nuclease (MB), and blunt-end ligation to recircularize the plasmid. The shortened plasmid is reintroduced into the host, after one or after several such 4-bp deletion cycles. When DNA is inserted into the multiple cloning site in the lacZ alpha gene, the progress of 4-bp removal can be followed by determining the Lac phenotype, since removal of multiples of 3 bp retains the reading frame while other kinds of deletions distort (or restore) the reading frame. Loss of pre-existing restriction sites or creation of new ones also permits monitoring the progress of the deletion process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of epidermal metabolism and DNA-binding of benzo[a]pyrene by ellagic acid

Ellagic acid, a common plant phenol, was shown to be a potent inhibitor of epidermal microsomal aryl hydrocarbon hydroxylase (AHH) activity in vitro, and of benzo[a]pyrene (BP)-binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. The in vitro addition of ellagic acid (0.25-2.0 microM) resulted in a dose-dependent inhibition of AHH activity in epidermal microsomes prepared from control or carcinogen-treated animals. The I50 of ellagic acid for epidermal AHH was 1.0 microM making it the most potent inhibitor of epidermal AHH yet identified. In vitro addition of ellagic acid to microsomal suspensions prepared from control or coal tar-treated animals resulted in 90% inhibition of BP-binding to calf thymus DNA. Application of ellagic acid to the skin (0.5-10.0 mumol/10 gm body wt) caused a dose-dependent inhibition of BP-binding to epidermal DNA. Our results suggest that phenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer from environmental chemicals.     

9-Hydroxydodecanoic acid,an acid from Blepharis sindica seed oil

A hitherto unknown hydroxy acid has been isolated from Blepharis sindica seed oil has been characterized as 9-hydroxydodecanoic acid by IR, NMR and mass spectral studies. The structure of this acid was further supported by its chemical transformations.     

Cutaneous metabolism of benzo[a]pyrene: comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague--Dawley rats

The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.     

Growth ofDiacrisia obliqua [Lep.: Arctiidae] with low doses ofBacillus thuringiensis var.Kurstaki

Early 3rd instarDiacrisia obliqua Walk. larvae were treated with concentrations ofBacillus thuringiensis var.kurstaki (Dipel^®) and the growth of treated larvae was assessed. All the doses reduced significantly the weight and survival of the insects (p<0.001).     

The effects of brain-stem section on the breathing and behavioural response to morphine in the fetal sheep

In the unanesthetized fetal sheep the administration of morphine causes initial apnoea followed by hyperpnoea. We thought that a section of the brain at midcollicular level might separate these two effects. Therefore we sectioned the brain stem of five fetuses at 132 +/- 1 (SEM) days of gestation and compared their responses to morphine (17 experiments) with that observed in seven intact fetuses at similar gestational ages (15 experiments). Brain stem sections were confirmed morphologically and histologically. Morphine, 1 mg/kg was injected in the fetal jugular vein during low-voltage electrocortical activity (ECoG). We measured ECoG, eye movements, diaphragmatic activity, blood pressure and amniotic pressure. Sectioned fetuses before the administration of morphine had a complete dissociation between ECoG and breathing activity. With the administration of morphine we found: (i) the length of the apnoea was 139.8 +/- 15.5 min in sectioned fetuses and 17.0 +/- 5.8 min in intact fetuses (P less than 0.01); and (ii) there was no hyperpneic response in the sectioned fetus whereas the length of hyperpnoea in the intact group was 99.1 +/- 11.8 min (P less than 0.001). The results support the idea of two central distinct areas of action of morphine in the fetal brain. The absence of hyperpnoea in the sectioned fetuses suggests that neurons inhibiting the 'respiratory neurons' are located rostrally to the mid-collicular line.     

In vitro flowering of Fortunella hindsii (Champ.)

Branch internodes of mature plants and stem internodes of seedlings of Fortunella hindsii flowered in vitro on half-strength MT (Murashige and Tucker 1969) basal medium supplemented with benzyladenine, adenine, 6---dimethylallylaminopurine and kinetin. The highest percentage of flowering was achieved with explants originating from branch internodes of flowering plants close to the apex on half-strength MT basal medium containing 5% sucrose and 0.01 mg 1^–1 BA in light. Exposure to darkness for more than 3 weeks followed by re-exposure to light reduced flowering. Flowering required a 4-day exposure to BA, but shoot formation could be initiated even without exposure to BA. First branch internode segments on MT basal medium containing 5% sucrose were prolific in flower (85%) production. The sucrose treatment affected the flower bud size distribution. There were about 13 flower buds per culture in the largest size category (>5 mm).     

Sterol and ochratoxin biosynthesis and miconazole action onAspergillus ochraceus activity

Miconazole (MC), an effective drug used in medicine as a topical broad-spectrum antifungal agents for treating tinea, aspergilloses and candidoses, was used for studying its effect on sterols, ochratoxin A and B biosynthesis byAspergillus ochraceus in vitro. Sterol biosynthesis and production of ochratoxin A and B were inhibited by MC when added into the culture medium together with the inoculum. MC, addition 2 d after culture inoculation, inhibited sterol biosynthesis after 5, 9 and 14 d, ochratoxin A production after 5 and 9 d and ochratoxin B production after 9 d of incubation. However, at 10, 50 and 100 ppm concentrations it induced a significant accumulation of both toxins after 14 d of incubation.