The limited proteolysis of tumor necrosis factor-α

The limited proteolysis of human recombinant TNF- by trypsin yields two stable products resulting from cleavage after Arg6 and Arg44. In solution these two products remain associated together in a trimer with a Stokes' radius slightly greater than the radius of intact TNF- and, therefore, could not be separated from each other under nondenaturing conditions. This limited digest retains at least 20% of the activity of the original TNF- sample, and has a tertiary structure that is similar to that of the native protein by circular dichroism. On the other hand, incorrectly folded, inactive TNF- undergoes extensive digestion following similar treatment with trypsin. These results indicate that the active form of TNF- has a tight core structure which is maintained afterN-terminal cleavage and removal.     

Agarose gel electrophoresis in isozyme separation and visualization

Isozyme analysis of rodent-human somatic cell hybrids has been used frequently to detect specific human chromosomes. The majority of these isozyme systems employs starch gels, the use of which can be laborious when screening large numbers of cell lines. We describe the development of two procedures to detect the long arms of human chromosomes 1 and 2 in Chinese hamster-human cell hybrids by a rapid and reproducible method using 1-mm-thick agarose gels. Detection of human chromosome 1q was accomplished by screening for human fumarate hydratase activity, whose gene has been mapped to 1q42.1. Detection of chromosome 2q was performed by screening for the isozyme isocitrate dehydrogenase 1, which has been localized to 2q32-qter. These systems provide a basis for the further development of procedures for detecting chromosome-specific isozyme markers in agarose gels.     

Mosquito oostatic factor: a novel decapeptide modulating trypsin-like enzyme biosynthesis in the midgut

A peptide that inhibits egg development in mosquitoes (oostatic factor) has been purified from the ovaries of female Aedes aegypti. The factor is a decapeptide with a molecular mass of 1047.6. The primary sequence has been determined as NH2-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-COOH from mass spectra recorded on a quadrupole Fourier transform instrument. The amino acid sequence exhibits sequence correlation to mammalian, plant, and several viral proteins. Injection of synthetic analogs into mosquitoes, biting midges, flies, and fleas inhibited proteolytic enzyme biosynthesis in the midgut. Binding studies with [3H]oostatic factor indicated that the midgut epithelial cells have a factor-specific receptor.     

Long-term effects of lactational zinc deficiency on bone mineral composition in rats fed a commercially modified Luecke diet

The purpose of this study was threefold: 1. to determine the long-term effects of interactions between lactational zinc deficiency and gender on bone mineral composition in repleted rat offspring, 2. to determine the nutritional efficacy of the second of two commercially designed, modified Luecke diets (ML2) during the gestational and lactational stress, and 3. determine the ultratrace element contents of Ralston Rodent Laboratory Chow #5001. The ML2 basal diet, based on dextrose, sprayed egg white, and corn oil contained 0.420 μg Zn/g, was supplemented with Zn (as zinc acetate) at 0 (diet 0ML2) or 30 (diet 30ML2) μg/g, and was mixed and pelleted commercially. all rat dams were fed the 30ML2 diet ad libitum during gestation. Beginning at parturition, the dams were fed either the 1. 0ML2, 2. 30ML2 (food restricted), or 3. 30ML2 (ad libitum) diets. All pups were fed the 30ML2 diet ad libitum from 23 to 40 d of age. From d 40 to 150, all pups were fed Ralston Rodent Laboratory Chow. The 30ML2 diet was found to be nutritionally efficacious; litter size and pup growth were normal and pup mortality was only 1.2%. Pups (ZD) with access to the 0ML2 diet until 23 d of age and nursed by dams fed the 0ML2 diet, when compared to pups (PF) fed restricted amounts of the 30ML2 diet, exhibited increased mortality and decreased concentrations of tibial zinc but no change in growth. Inadequate zinc nutriture during infancy, despite postlactational zinc repletion, induced imbalances in adult bone mineral metabolism. Thus, at 150 d of age, the ZD pups exhibited increased levels of bone P and Mg and decreased concentrations of K as compared to the PF pups.     

Transforming growth factor-beta is a potent immunosuppressive agent that inhibits IL-1-dependent lymphocyte proliferation

Transforming growth factor-beta (TGF-beta), a product of neoplastic and hemopoietic cells, is a bifunctional regulator of the immune response. At femtomolar concentrations, TGF-beta stimulates monocyte migration, and picomolar quantities induce synthesis of monocyte growth factors, including IL-1, that may promote tissue repair by regulating fibrosis and angiogenesis. Paradoxically, TGF-beta at picomolar concentrations also blocks the ability of IL-1 to stimulate lymphocyte proliferation. At 0.01 to 1.0 ng/ml, TGF-beta 1 and its homologue, TGF-beta 2, suppress the IL-1-dependent murine thymocyte proliferation assay. TGF-beta also inhibits human peripheral blood T lymphocyte mitogenesis. Inhibition of cell division appears to occur after activation of the lymphocytes inasmuch as neither gene expression nor translation of IL-2R is suppressed. Furthermore, TGF-beta does not block synthesis of IL-2. Therefore, TGF-beta 1 and TGF-beta 2 likely act at a site distal to IL-1 to block lymphocyte DNA synthesis. These findings suggest that TGF-beta secreted in an inflammatory site may be beneficial in diminishing lymphocyte function while promoting fibrosis and tissue repair. However, TGF-beta generated by neoplastic tissues may provide a mechanism for unrestricted tumor cell growth through its selective immunosuppressive effects.     

Failure of non-selective inhibition of arachidonic acid metabolism to ameliorate hyperoxic lung injury

We have previously reported that bronchoalveolar lavage fluid cyclo-oxygenase products of arachidonic acid (AA) metabolism increase prior to the development of significant hyperoxic lung injury. To further assess the role of AA metabolites in the development of hyperoxic lung injury, we have utilized this same model of hyperoxic lung injury and administered either indomethacin (an inhibitor of the cyclo-oxygenase pathway of AA metabolism) or dexamethasone (inhibitor of AA release). A total of 46 adult rabbits were exposed to greater than 95% oxygen for 65 hours. Fourteen animals were given either 2 or 3 mg/kg/day indomethacin, 7 served as controls: 18 animals were given either 0.5 or 1.0 mg/kg/day of dexamethasone, 7 served as controls. The surviving animals were sacrificed after 65 hours of hyperoxia and bronchoalveolar lavage of the left lung was done; the right lung was examined by light microscopy. Treatment with indomethacin or dexamethasone failed to ameliorate the hyperoxic lung injury process. However, in both the indomethacin and dexamethasone treatment groups, significant suppression of 6-keto-PGF1 alpha, a PGI2 metabolite, was observed. Some suppression of TXB2 production was observed, but there was no evidence of any decrease in leukotriene production. We postulate that failure to ameliorate hyperoxic lung injury with either indomethacin or dexamethasone therapy was related to significant suppression of PGI2, a potentially protective AA metabolite, and/or to failure to significantly decrease production of potential pathogenic participants, such as TXA2 or LTB4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of upper gastrointestinal prostaglandin synthesis by dietary fatty acids

The effects of dietary iols on gastric, duodenal mucosa and liver were investigated ina rat model. Unsaturated fatty acid profles and in vitro prostaglandin (PG) synthesis (PGE₂, PGF_2α, 6-oxo-PGF_1α and thromboxane B₂). were measured after 14 days of dietary oil supplements.There were no significant differences in prostanoid synthesis between rats fed coconut oil (high saturated fat content) and standard diet. After fish oil supplement, tissue eicosapentaenoic acid and docosahexaenoic acid levels were higher, arachidonic acid levels were lower, and prostanoid synthesis was reduced in both stomach and duodenum. After corn oil and evening primrose oil, linoleic acid levels were variaby increased, bt there were no significant differences in arachidonic acid or prostanoid synthesis. Dihomogamma-linolenic acid levels were slightly increased after evening primrose oil.Dietary incorporation of fatty acids into gastroduodenal tissue is not uniform. When incorporated, fatty acids can modify prostaglandin synthesis.     

Several distinct types of sequence elements are required for efficient mRNA 3'' end formation in a pea rbcS gene.

We have conducted an extensive linker substitution analysis of the polyadenylation signal from a pea rbcS gene. From these studies, we can identify at least two, and perhaps three, distinct classes of cis element involved in mRNA 3' end formation in this gene. One of these, termed the far-upstream element, is located between 60 and 120 nt upstream from its associated polyadenylation sites and appears to be largely composed of a series of UG motifs. A second, termed the near-upstream element, is more proximate to poly(A) sites and may be functionally analogous to the mammalian polyadenylation signal AAUAAA, even though the actual sequences involved may not be AAUAAA. The third possible class is the putative cleavage and polyadenylation site itself. We find that the rbcS-E9 far-upstream element can replace the analogous element in another plant polyadenylation signal, that from cauliflower mosaic virus, and that one near-upstream element can function with either of two poly(A) sites. Thus, these different cis elements are largely interchangeable. Our studies indicate that a cellular plant gene possesses upstream elements distinct from AAUAAA that are involved in mRNA 3' end formation and that plant genes probably have modular, multicomponent polyadenylation signals.