Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.     

Adenylate cyclase activity during growth and encystment of Acanthamoeba castellanii

The adenylate cyclase activity of Acanthamoeba castellanii (Neff) was studied in extracts prepared after breaking cells in the Potter-Elvehjem homogenizer. The adenylate cyclase activity of cells is low during the exponential growth phase, but then rises 2–4-fold during the stationary phase to a peak, roughly at the time that cyst forms are detectable in the culture. A 2–4-fold activity rise to a peak also occurs 4–8 h after late log cells are transferred to a non-nutrient encystment medium, a time which is shortly before numbers of cyst forms can be detected in the culture. The pattern of activity observed when stationary phase cells are transferred to encystment medium is complex and depends in part on whether the cultures have exhibited the peak of cyclase activity and have begun to initiate cyst formation prior to the transfer. Within the usual time frame after transfer to encystment medium, early logarithmic phase cells do not exhibit a 2–4-fold rise of cyclase activity and they do not encyst. The results suggest a relationship between encystment and the pattern of rise and fall in cyclase specific activity. Fractionation of the homogenate of trophozoites indicated that adenylate cyclase activity was associated with the particulate microsomal fraction.     

Fungal foliar diseases in Withania somnifera and its effect on secondary metabolites

Withania somnifera is a promising revitalizing medicinal herb. The plant is affected by foliar diseases in Lakkavalli forest region of Bhadra Wildlife Sanctuary. The symptomatology of foliar fungal disease incidence, severity and distribution in the study area was examined during 2006–2009. The seedborne nature and transmission of the causal pathogen and its management with seed dressing fungicides were studied. The results of the study indicated that Alternaria alternata caused severe leaf spot disease, while Myrothecium roridum and Fusarium oxysporum caused minor diseases. Based on the internal transcribed spacer (ITS1 and ITS2) regions of rDNA, the major pathogen was identified as A. alternata. The disease is homogeneously distributed in Lakkavalli forest region and high severity is recorded during November. Alternaria alternata and Fusarium oxysporum were the dominant seedborne pathogens that are transmitted to seedlings. Among the seed dressing fungicides used, Hyzeb was the most effective, followed by Captra, Antracol and Bavistin, in reducing the incidence of A. alternata and other seedborne fungi. The infected W. somnifera foliages had decreased steroids and alkaloids and increased phenolics and flavonoids. Analysis of alkaloids in diseased foliages by high performance thin layer chromatography indicated the occurrence of transformed compounds at R_f = 0.1, 0.77 (254 nm) and 0.2 (366 nm).     

Sonication as a tool for the study of adenylyl cyclase activity.

The technique of sonication was applied in studying adenylyl cyclase activity of cultured fibroblasts. Exposure of BHK 21 c/13 to brief periods of low power sonication gives cell preparations with greater basal, fluoride and hormone sensitive adenylyl cyclase activites than those of broken cell preparations of homogenized cells. The sonicated cells provide a convenient method to study adenylyl cyclase since they are added directly to the adenylyl cyclase reaction vessels without further processing. Maximal epinephrine stimulated activity in sonicated cells is nearly equivalent to that activated by sodium fluoride, but the apparent affinity of the enzyme system is similar to that of broken cell preparations. Furthermore, broken cell preparations of sonicated cells possess greater adenylyl cyclase activity than broken cell preparations of unsonicated cells. This procedure may provide a useful tool for the analysis of the hormonal regulation of adenylyl cyclase activity of isolated cells.     

A Rapid Safranin-Metal Phthalocyanine Double Staining Technique for Plants

Pure metal 4.4',4',4'-tetxa-substituted, sulfo-, carboxy- and nitrophthalocyanines were synthesized. Mounted, deparaffinized and partially dehydrated sections of plant tissues were stained with 0.5% safranin in 50% alcohol for 5-10 min. Excess safranin was removed with a series of 70%, 95% and absolute alcohol washes. The sections were then stained for 2-3 min using metal 4,4',4',4'-phthalocyanine tetracarboxylic acid (MPTC, 0.5% (V/V) containing a few drops of dilute sodium hydroxide), metal 4,4',4',4'-tetra-sulfophthalocyanine (MPTS, 0.5% (V/V)) or metal tetranitrophthalocyanine (MPTN, 0.5% (V/V) in dimethyl sulfoxide). The sections were washed with 95%, then absolute alcohol; however, the metal tetranitrophthalocyanine section was washed only with absolute alcohol. Stained sections were treated briefly with xylene, then mounted on a coverslip. Bright peacock blue (MPTC and MPTS using Cu, Co or Ni), turquoise blue (MPTN using Cu or Ni) or parrot green (zinc phthalocyanine tetracarboxylic acid-ZnPTC, zinc phthalocyanine tetranitro derivative-ZnPTN) colors were obtained. Lignin-containing cells were stained red by safranin and the remaining cell structures were stained by the metal phthalocyanine complex with color brightness superior to that of fast green. Uniform staining, no color fading after a year, reliability, brief staining times, high color contrast (log ε = 4.0-4.9) and ease of use make this double staining combination ideal for routine use and photomicrography.     

Responses of the mangroves Avicennia marina and Bruguiera gymnorrhiza to oil contamination

The effects of bunker fuel oil on morphological and physiological responses of Avicennia marina and Bruguiera gymnorrhiza were investigated in glasshouse and field experiments. In the glasshouse study, 15-month-old seedlings of A. marina were subjected to oiling or debarking treatments for 6 months. Oiling or debarking of a 5 cm ring of the basal portion of the stem, alone and in combination, reduced leaf CO₂ exchange by over 50% and resulted in the production of adventitious roots immediately above the debarked and/or oiled stem 8–12 weeks after the commencement of treatments. In the field study, sediment oiling at a single dose of 5 l m⁻² of A. marina and B. gymnorrhiza trees reduced electron transport rate (ETR) through Photosystem II (PSII) and PSII quantum yield. Oiling also reduced the photochemical efficiency of PSII (F_v/i_m) in B. gymnorrhiza, but not in A. marina. After 15 weeks of oiling, adventitious roots developed at the base of the stem in A. marina, but not in B. gymnorrhiza. Naturally occurring A. marina seedlings with adventitious roots exhibited lower leaf CO₂ exchange rates, photochemical efficiency of PSII and leaf chlorophyll content than similar seedlings without these roots. These results indicate that bunker fuel oil adversely affects photosynthetic performance of A. marina and B. gymnorrhiza mangroves. A. marina responds to oiling by producing adventitious roots at the base of the stem. Adventitious root production at the base of the stem may be a useful biological indicator of oil or other toxic pollutants in A. marina.     

Micropropagation of <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis>

An efficient protocol for micropropagation of Harpagophytum procumbens DC., an endangered African medicinal plant, was developed. Maximum shoot multiplication without callus was obtained from nodal explants cultured on Murashige and Skoog (MS) basal salts plus Gamborg’s (B₅) vitamins supplemented with 0.1 mg dm⁻³ indole-3-acetic acid and 5.0 mg dm⁻³ kinetin. The shoots were subsequently subcultured every 3 weeks on the same medium. Detached axillary shoots were transferred to MS basal salts plus B₅ vitamins supplemented with various concentrations of α-naphthalene-acetic acid or indole-3-butyric acid (IBA), ranging from 0.5 to 2.5 mg dm⁻³ and 100 % rooting and optimal subsequent acclimatization was achieved on 1.0 mg dm⁻³ IBA. After 4 weeks of culture, the rooted shoots (>5 cm) were planted in pots containing peat, vermiculite and bark (2:1:1), covered with plastic domes and maintained at 25 °C for 2 weeks before being transferred to a glasshouse. Plant survival was about 40 %.     

Purification and characterization of a pineapple crown leaf thiol protease

A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico-chemical and kinetic properties. The molecular weight of the protease (22.4-22.9 kDa), Km (97 microM) and kcat (8.8 s(-1)) for its esterolytic cleavage of the synthetic protease substrate N(alpha)-CBZ-L-lysine p-nitrophenyl ester, the concentration of its thiol activator L-cysteine required for half maximal activation A0.5 (9.9 microM), optimum pH (6.5) for its proteolytic action on azocasein, T(1/2) (60 degrees C) for inactivation by heating the enzyme (35.5 microg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH-group content (0.98 mol/mol enzyme) were determined. Most of these physicochemical and kinetic properties were found to be similar to those of the already well-characterized stem bromelain (EC 3.4.22.32). Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.