Repetitive sequences of the sea urchin genome: Distribution of members of specific repetitive families

Three repetitive sequence families from the sea urchin genome were studied, each defined by homology with a specific cloned probe one to a few hundred nucleotides long. Recombinant λ-sea urchin DNA libraries were screened with these probes, and individual recombinants were selected that include genomic members of these families. Restriction mapping, gel blot, and kinetic analyses were carried out to determine the organization of each repeat family. Sequence elements belonging to the first of the three repeat families were found to be embedded in longer repeat sequences. These repeat sequences frequently occur in small clusters. Members of the second repeat family are also found in a long repetitive sequence environment, but these repeats usually occur singly in any given region of the DNA. The sequences of the third repeat are only 200 to 300 nucleotides long, and are generally terminated by single copy DNA, though a few examples were found associated with other repeats. These three repeat sequence families constitute sets of homologous sequence elements that relate distant regions of the DNA.     

The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca²⁺ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.     

Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.     

Thermoregulation during prolonged exercise in heat: alterations with beta-adrenergic blockade

Thermoregulation and cardiovascular drift were studied under conditions of prolonged exercise in a warm environment (dry bulb temperature 31.7 +/- 0.3 degrees C, rh 44.7 +/- 4.7%) during beta-adrenergic blockade. Fourteen subjects performed 90-min rides on a cycle ergometer at a work rate equivalent to 40% of their control maximal O2 uptake under each of three treatments provided in a randomized double-blind manner: atenolol (100 mg/day), propranolol (160 mg/day), and a placebo. Exercise during the propranolol trial resulted in significantly higher forearm vascular resistance values and significantly lower forearm blood flows (FBF) compared with the placebo trial. However, the significantly lower FBF during propranolol did not significantly alter the rectal temperature (Tre) response to prolonged exercise. In addition, both beta-blockers produced lower FBF for any given Tre, suggesting that beta-adrenergic blockade affects FBF through nonthermal factors. The slight differences in Tre, despite the large differences in FBF between the various treatments, are apparently the result of an enhanced sweat loss and a lower mean skin temperature during exercise with beta-blockade. The uncoupling of FBF and sweat loss provides evidence of independent regulation. The reduction in FBF at any given Tre was concomitant to lower blood pressure values during beta-blockade and suggests that baroreflexes provide significant input to the control of skin blood flow when both pressure and temperature maintenance are simultaneously challenged.     

Clinical importance of enteric communication with abdominal abscesses.

The dynamics of leucocytes in abdominal abscesses were studied using indium-111 autologous leucocyte scanning in 30 patients. Thirteen patients showing enteric drainage of leucocytes on delayed scans were characterised by a lack of abdominal localising signs and a low detection rate by ultrasound (25%). By contrast, 16 of 17 patients without enteric drainage had abdominal signs, and in these patients ultrasound was associated with a higher detection rate (58%). Despite the presence of an enteric route of drainage for the abscess 10 of the 13 patients needed surgical intervention. These results help explain the wide variation in clinical presentation of abdominal abscesses; suggest that 111In leucocyte scanning should be the initial investigation in those patients without focal signs; and show that formal surgical drainage is needed in patients recognised as having enteric communication with abscesses.