Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities

A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.     

Trolox C, a lipid-soluble membrane protective agent, attenuates myocardial injury from ischemia and reperfusion

The lipophilic antioxidant Trolox C, a vitamin E analog, was administered to isolated, buffer-perfused rabbit hearts subjected to 25 min of global stop-flow ischemia and 30 min of reperfusion. In six hearts, Trolox C (200 microM) was infused for 15 min immediately prior to ischemia and for the first 15 min of reperfusion. Six control hearts received only vehicle. Gas chromatography analysis confirmed that effective myocardial levels of Trolox were attained. At 30 min reperfusion, the recovery of left ventricular developed pressure was 56 +/- 3% of baseline in control hearts versus 70 +/- 4% in Trolox-treated hearts (p < .01). There was also significant improvement in recovery of Trolox-treated hearts in diastolic pressure and both maximum and minimum values of the first derivative of left ventricular pressure (dP/dt). Creatine phosphokinase release into the coronary effluent at 30 min of reperfusion was 16.5 +/- 8.4 IU/min in untreated and 6.3 +/- 1.0 IU/min (p < .05) in Trolox-treated hearts. Thus Trolox C, a lipophilic antioxidant, attenuated myocardial injury during stop-flow ischemia and reperfusion.     

The theory of the frequency response of ellipsoidal biological cells in rotating electrical fields

In this paper we have presented in as compact a form as possible the theoretical formalism that is needed to predict the frequency response of a biological cell of arbitrary ellipsoidal shape to a frequency dependant rotating external field. The formalism is much more complicated than that for a spherical or cylindrical cell where the radial vector is always parallel to the surface normal at each point of the surface. In addition to providing the theory we have demonstrated that the spin rate and its frequency dependance is very intimately related to the electrical properties of the cell interior and to that of the suspending fluid. It is possible to probe these properties of the cell and its environment by utilizing this technique. This aspect has been demonstrated by examining rotational changes as a function of the conductivity of both the cell interior and its suspending liquid. We also have shown, by considering a very simple model for the cell and the two dielectric constants, that the frequency spectrum is shape dependant. All our calculations have been carried out for "lossy" systems with frictional dissipation where energy minimization methods are no longer applicable. The invariant form of the Poynting vector forms the basis of the method.     

Pathogenicity of Pasteurella multocida A:3 in Flemish giant and New Zealand white rabbits.

Pasteurella multocida A:3 was isolated during an outbreak of pasteurellosis in Flemish Giant (FG) rabbits. Since New Zealand White (NZW) rabbits housed in the same room were not as severely affected as FG rabbits, experimental inoculation was undertaken to determine if FG rabbits were more susceptible than NZW rabbits to pasteurellosis induced by this isolate. Rabbits of each breed were inoculated with P. multocida A:3 and observed for 3 weeks. Four of 5 FG rabbits developed severe clinical disease on days 6, 9, 12 and 14 after inoculation; whereas, the one affected NZW rabbit became ill 14 days after inoculation. All rabbits with clinical disease developed fibrinosuppurative pleuritis, pyothorax and pneumonia which was more severe in FG than NZW rabbits. At necropsy, P. multocida A:3 was isolated from multiple sites of the diseased rabbits. No significant difference (P = 0.099) in the prevalence of lesions between the two breeds was found; however, the score of pneumonia and pleuritis was 3 times greater in FG rabbits than NZW rabbits.     

Genetic assay of misincorporation

A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13-bearing lacI gene can be detected by plaque color. Mutants in the 5'-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13-lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i- (blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3'-terminus of the primer. Nearly all of the mutants induced by '-A' conditions were T----C base substitutions, and those induced by '-G' conditions were C----T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i- phenotype and unobserved by others) were isolated by the procedures described in this paper.     

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

Summary The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a -Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho ⁰ strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.     

Endoparasitic flies,pollen-collection by bumblebees and a potential host-parasite conflict

Summary Conopid flies (Conopidae, Diptera) are common larval parasites of bumblebees. The larva develops inside the abdomen of workers, queens and males. Development is completed within 10–12 days after oviposition when the host is killed and the parasite pupates in situ. Development results in parasitised bees becoming unable to carry large loads of nectar, as the conopid larvae reside where the honey crop is normally located. Furthermore, an addition to the bee's unloaden body mass is likely (average larval weight reached at pupation by the common parasite species Sicus ferrugineus: ±SD 36.3±12.3 mg, n=59; by Physocephala rufipes: 55.8±16.9 mg, n=108). We here asked whether the propensity of workers of the bumblebee Bombus pascuorum to collect nectar rather than pollen is related to the presence of conopid larvae. For samples of bees (n=2254 workers) collected over 3 years of field studies in northwestern Switzerland, there was no difference in the frequency of bees caught as pollen collectors among parasitised (38.1% of cases, n=210) as compared to non-parastised bees (43.9%, n=360) ( ²=1.83, n.s.). However, compared to the non-parasitised bees (n=360), those hosts containing a third (last) instar larva (n=9) were less likely to collect pollen than expected by chance ²=6.91, P=0.003. Similarly, hosts with short survival time between capture and being killed by the developing larva (which hence must have harboured a late instar parasite at time of capture) were less likely to collect pollen (8%, n=25) than those found not parasitised (37.6%, n=891 ²=9.16, P<0.001). Late instar larvae grow so big that they fill the entire abdomen. Although there was also a tendency for presumably older bees to collect less pollen, this is unlikely to explain the observations. We also discuss whether these changes in foraging behaviour of bumblebees may reflect a host-parasite conflict over the type of resource to be collected.