Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.     

Soil invertebrate and plant responses to mowing and carbofuran application in a North American tallgrass prairie

Relationships between soil invertebrate populations and primary production of a tallgrass prairie were investigated using the insecticide-nematicide carbofuran and a range of mowing intensities to manipulate invertebrate densities and resource quantity and quality. The trophic composition of nematode populations was monitored through each of two growing seasons. Earthworm and macroarthropod densities and primary production were assessed at the end of the second season. Invertebrate densities were generally reduced in carbofuran-treated plots, although individual weights of surviving macroarth-ropod herbivores increased significantly (p<0.05). Carbofuran failed to affect estimates of above- or belowground plant biomass after two years of treatment. Changes in resource quantity and quality resulted in rapid responses by dominant invertebrate consumer populations. A 28% reduction in live root mass and a 24% increase in root detritus following two years of mowing was associated with a 54% decrease in herbivorous nematode densities, a 47% increase in microbivorous nematode densities, and a 41% increase in native earthworm biomass.     

Response of macroinvertebrates to blooms of iron-depositing bacteria

In field measurements and laboratory experiments we assessed the influence of high levels of iron, manganese, and concurrent blooms of iron-depositing bacteria, Leptothrix ochracea, on macroinvertebrates. Macroinvertebrate communities in five of six streams were depauerate inside blooms. Reasons for the decreased abundance vary among taxa, with our experiments demonstrating the importance, for one or more species, of (1) direct toxic effects, and,/or smothering, (2) behavioral avoidance of bacterial-coated substrates, and (3) an inability to use bacteria as food. Three mayfly species showed increased mortality when caged inside the blooms, but five trichopterans and one plecopteran did not. Five invertebrates avoided Leptothrix-coated substrate in choice trials, while three did not. Stenonema fuscum could not ingest Leptothrix, and Neophylax nacatus had reduced growth feeding on it, but Heptagenia umbratica grew equally well on diets of Leptothrix or diatoms. This study demonstrates the important role epilithic organisms play in modifying substrates, and how these changes may act to influence benthic abundance and distribution in streams.     

Eimeria and sarcocystis in raccoons in Illinois

Eimeria nuttalli oocysts were found in 58% (21/36) and E. procyonis oocysts in 25% (9/36) of raccoons Procyon lotor in Illinois, and sporocysts of Sarcocystis sp. in 17% (2/12) of other raccoons in Illinois. The oocysts of E. nuttalli were ellipsoidal to ovoid, 15-21 X 12-17 micrometer, with a one-layered, smooth, colorless wall. The oocysts of E. procyonis were 22-28 X 18-22 micrometer, with a rough, striated, brownish, two-layered wall. The sporulated sporocysts of Sarcocystis sp. were 11-13 X 8-10 micrometer. Attempts to infect baby pigs by feeding them sporocysts of Sarcocystis sp. from the reaction failed.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Distribution of beta-glucanases within the genus Bacillus.

Representative strains (368) from 36 species in the genus Bacillus were screened for the secretion of beta-glucanases. (1 leads to 6)-beta-glucanases active on pustulan were produced by a minority of the organisms studied (4%), but (1 leads to 3)-beta-glucanases which hydrolyzed laminarin and pachyman were more widespread and were secreted by 56 and 44% of the strains, respectively.     

Effects of six fasciolicides against Fascioloides magna In white-tailed deer

Thirty-three white-tailed deer (Odocoileus virginianus) of various ages, both sexes, and in good physical condition were captured for anthelmintic evaluation of six compounds against the large American liver fluke, Fascioloides magna. Based on fluke mortality, hexachlorophene administered at the rate of 12 to 26 mg/kg of body weight was lethal to 5 of 10 mature flukes in seven deer. Nitroxynil at 11 to 24 mg/kg inhibited egg production, but did not kill mature flukes in eight deer. Rafoxanide at 12 to 25 mg/kg killed 6 of 8 (75%) immature flukes in eight deer, but was not effective against 17 mature flukes. Clioxanide at 16 to 38 mg/kg, diamphenethide at 255 to 280 mg/kg, and hexachloroethane at 463 to 629 mg/kg were not effective against F. magna in four, two and four deer, respectively. There was no indication that treatment with fasciolicides at the higher dose rates was more efficacious than at the lower dose rates. Detection of fluke eggs in the feces was a reliable method for diagnosing the presence of mature F. magna in deer prior to treatment, but was not reliable for measuring fasciolicidal activity of all compounds tested.     

Haemolysis complicating viral hepatitis in patients with glucose-6-phosphate dehydrogenase deficiency.

Out of 20 patients with viral hepatitis whose glucose-6-phosphate dehydrogenase (G-6-PD) levels were normal, 14 had clinical evidence of a mild to moderate degree of haemolysis but in all the patients studied the half life of chromium-51-labelled red cells was shortened. Out of 18 viral hepatitis patients deficient in G-6-PD 17 had clinical evidence of haemolysis, and in eight this was more severe than in the group with normal G-6-PD values. Massive intravascular haemolysis occurred in four, three of whom died. The massive haemolysis was attributed to the presence of additional drug-induced oxidative stress to the G-6-PD-deficient red cells.     

HIV-protease inhibitors induce expression of suppressor of cytokine signaling-1 in insulin-sensitive tissues and promote insulin resistance and type 2 diabetes mellitus

Insulin resistance, hyperglycemia, and type 2 diabetes are among the sequelae of metabolic syndromes that occur in 60-80% of human immunodeficiency virus (HIV)-positive patients treated with HIV-protease inhibitors (PIs). Studies to elucidate the molecular mechanism(s) contributing to these changes, however, have mainly focused on acute, in vitro actions of PIs. Here, we examined the chronic (7 wk) in vivo effects of the PI indinavir (IDV) in male Zucker diabetic fatty (fa/fa) (ZDF) rats. IDV exposure accelerated the diabetic state and dramatically exacerbated hyperglycemia and oral glucose intolerance in the ZDF rats, compared with vehicle-treated ZDF rats. Oligonucleotide gene array analyses revealed upregulation of suppressor of cytokine signaling-1 (SOCS-1) expression in insulin-sensitive tissues of IDV rats. SOCS-1 is a known inducer of insulin resistance and diabetes, and immunoblotting analyses revealed increases in SOCS-1 protein expression in adipose, skeletal muscle, and liver tissues of IDV-administered ZDF rats. This was associated with increases in the upstream regulator TNF-alpha and downstream effector sterol regulatory element-binding protein-1 and a decrease in IRS-2. IDV and other PIs currently in clinical use induced the SOCS-1 signaling cascade also in L6 myotubes and 3T3-L1 adipocytes exposed acutely to PIs under normal culturing conditions and in tissues from Zucker wild-type lean control rats administered PIs for 3 wk, suggesting an effect of these drugs even in the absence of background hyperglycemia/hyperlipidemia. Our findings therefore indicate that induction of the SOCS-1 signaling cascade by PIs could be an important contributing factor in the development of metabolic dysregulation associated with long-term exposures to HIV-PIs.