Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3'' processing signal AAUAAA.

Cleavage and polyadenylation factor (CPF) is required for the cleavage as well as for the subsequent polyadenylation reaction during 3' processing of messenger RNA precursors. Here, we have investigated the interaction of CPF and poly(A) polymerase with short RNA substrates. CPF activates poly(A) polymerase to elongate RNA primers carrying the canonical hexamer recognition signal AAUAAA. CPF specifically binds to such RNA as shown by gel mobility shift assays and competition experiments. Upon binding of CPF, two polypeptides of 35 kDa and 160 kDa can be covalently crosslinked to the RNA by irradiation with UV light. These polypeptides may correspond to the smallest and the largest subunit contained in purified CPF fractions. In addition, chemical modification-exclusion experiments demonstrate that CPF interacts directly with the AAUAAA recognition signal in the RNA. The entire hexamer signal is involved in binding of CPF since modification of any of its bases interferes with complex formation.     

Thymidine photoproducts of non-cyclobutane type as a crucial event in the conformation of UV-irradiated DNA

The UV-light induced conformational effects in deoxyoligonucleotides and polynucleotides have been analyzed by CD measurements and isolation of the photoproducts. The results demonstrate that the essential photoproduct formed on irradiation of thymidylyl-thymidine at 254 nm is of non-cyclobutane type and may be correlated to the primary photoproduct formed in DNAs at low doses Formation of thymine dimers of cyclobutane-type structure appears to be a secondary product generated by treatment with formic acid.     

An evaluation of analgesia associated with the immobility response in laboratory rabbits

The immobility response (IR) was studied in rabbits to evaluate its analgesic properties and reliability as a method of restraint. The participation of the endogenous opioid system in IR was studied indirectly by evaluating the effects of the narcotic antagonist naloxone on this phenomenon. Twenty-four adult New Zealand White rabbits were subjected to six noxious stimuli while restrained by IR and while restrained under control conditions. Testing on each animal was repeated under both conditions following the administration of naloxone. The noxious stimuli consisted of three levels of electric shock (10 volts, 30 volts, and 50 volts) applied to the shaved forearm, and mechanical pressure applied to the pinna, front toe, and hind toe. Withdrawal and changes in blood pressure, heart rate, and respiration were used as indicators of pain perception. Distress associated with noxious electrical and pressure stimulation was significantly reduced by IR, which suggested that the phenomenon does have a significant analgesic component. However, the rabbits showed wide variability in their susceptibility to IR induction, and even animals which did not withdraw in response to noxious stimulation under IR sometimes exhibited physiological changes suggestive of distress. Therefore, IR should not be considered as a reliable or humane alternative to analgesic/anesthetic drugs for laboratory rabbits. Naloxone had little effect on IR or IR-associated analgesia.     

Contrasting responses of oligochaete communities to the abatement of eutrophication in Lake Geneva

Tubificid and lumbriculid worms were used to monitor, at depths of 150 m, the recovery of Lake Geneva (Switzerland) from eutrophication. As predicted from the decrease of phosphorus concentrations, relative abundance of oligotrophic species was higher from 1988 to 1993 than in 1983, i.e. before the abatement of eutrophication. However, this trend towards oligotrophication can be reversed, as indicated by a decrease of oligotrophic species, recorded in 1993. But this change corresponded to the effects of an increase of water temperature on the abundance of the mesotrophic species Potamothrix vejdovskyi rather than to a deterioration of the profundal. In addition to this short-term setback, oligochaete communities located at a depth of 150 m responded more slowly and less clearly to the decrease of phosphorus concentrations than those located at a depth of 40 m. However, the zoobenthos indicated more clearly the recovery of Lake Geneva than the phytoplankton.     

Molecular binding scaffolds increase local substrate concentration enhancing the enzymatic hydrolysis of VX nerve agent

Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in K_M and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased K_M by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.     

Evaluation of Repellency Effect of Two Natural Aroma Mosquito Repellent Compounds,Citronella and Citronellal*

Repellent efficacies of two natural aroma compounds, citronella and citronellal, against mosquitoes, Culex pipiens pallens, were evaluated both in field and in vitro. In vitro, the experiment was conducted with three controlled bands impregnated with 30% citronella extract, 15% citronella extract and 30% citronellal extract, and with bands impregnated 30% citronella in field. Data was obtained by the means of counting numbers bitten by mosquitoes per unit time, namely human bait method. Percentage repellencies of above three controlled bands were calculated at 86%, 73%, and 78%, respectively in vitro, and 80% in field, showing high repellent effectiveness against mosquitoes. This estimation was also confirmed by t‐test compared between control group and each experimental group.     

Immunological identity of a 60 kd oncofetal protein induced in rats by chemical carcinogens and released by transformed cells

A 60,000 dalton (60 kd) oncofetal protein was previously shown to be produced by tumors in tumor-bearing rats and by target tissues within 3 weeks of carcinogen treatment. The factor is released to and accumulates in the blood in vivo and in the conditioned medium of cultured transformed cells in vitro. A polyclonal antibody produced against the 60 kd factor purified from the plasma of a rat carrying the N-2-fluorenylphthalamic acid-induced transplantable Hepatoma 7777, was tested against the 60 kd factor from various sources. Based on the results of immunoprecipitation of biochemical activity associated with the 60 kd factor, it was determined that these anti-60 kd antibodies cross-reacted with the factor released by a dimethylbenzanthracene-induced rat mammary carcinoma, with the factor in rat tumor cytosol and with rat spontaneous lymphoma cells, but not with a 60 kd factor isolated from pooled cancer patient plasma. Furthermore, these antibodies cross-reacted with the 60 kd factor induced within 21 days of treatment of the rats with a range of carcinogens from 8 chemical structural groups. The anti-60 kd factor antibodies did not cross-react with a 35 kd factor having similar biochemical activity found in normal adult cells.     

Flufenamic acid, mefenamic acid and niflumic acid inhibit single nonselective cation channels in the rat exocrine pancreas.

The non-steroidal anti-inflammatory drugs, flufenamic acid, mefenamic acid and niflumic acid, block Ca2(+)-activated non-selective cation channels in inside-out patches from the basolateral membrane of rat exocrine pancreatic cells. Half-maximal inhibition was about 10 microM for flufenamic acid and mefenamic acid, whereas niflumic acid was less potent (IC50 about 50 microM). Indomethacin, aspirin, diltiazem and ibuprofen (100 microM) had not effect. It is concluded that the inhibitory effect of flufenamate, mefenamate and niflumate is dependent on the specific structure, consisting of two phenyl rings linked by an amino bridge.