Characterization of the receptor for tumor necrosis factor (TNF) and lymphotoxin (LT) on human T lymphocytes. TNF and LT differ in their receptor binding properties and the induction of MHC class I proteins on a human CD4+ T cell hybridoma

TNF-alpha and lymphotoxin (LT or TNF-beta) are structurally related cytokines that share several proinflammatory and immunomodulatory activities. The shared biologic activities of TNF and LT have been attributed to their binding to a common cell surface receptor(s). We observed that rTNF enhanced the expression of MHC class I proteins on the human T cell hybridoma, II-23.D7, however LT was largely unable to regulate MHC expression. To determine the molecular basis of this disparity between LT and TNF the receptor binding characteristics of rTNF and rLT were investigated by direct and competitive radioligand assays on the II-23.D7 T hybridoma, and for comparison, anti-CD3 activated human T lymphocytes. Specific 125I-rTNF binding to the II-23.D7 line revealed a single class of sites with a Kd = 175 pM and 3000 sites/cell; anti-CD3 activated T cells exhibited specific TNF binding with similar properties. The relationship of receptor occupancy to the induction of MHC class I Ag yielded a hyperbolic curve indicating a complex relationship between rTNF binding and biologic response. LT appeared to function like a partial agonist in that rLT was 10- to 20-fold less effective than rTNF in competitively inhibiting 125I-rTNF binding on the II-23.D7 line. Scatchard type analysis revealed a single class of low affinity binding sites for 125I-rLT. No differences in the competitive binding activity of rTNF and rLT were observed on the anti-CD3-activated T cells. Receptors for rTNF and rLT were immunoprecipitated from the II-23.D7 and activated T cells with anticytokine antibodies after cross-linking of radioiodinated rTNF or rLT to intact cells by using chemical cross-linking reagents. Analysis of the cross-linked adducts by SDS-PAGE and autoradiography indicated a major adduct of 92 kDa for rTNF and 104 kDa for rLT. Enzymatic digestion with neuraminidase or V8 protease revealed a unique structure to these adducts consistent with the cross-linking of a single chain of cytokine to a cell surface glycoprotein. rTNF inhibited the formation of the 104-kDa adduct formed with 125I-rLT on the II-23.D7 line, indicating these two cytokines bind to the same receptor of approximately 80 kDa. These results suggest that the disparate activities of LT and TNF to induce MHC class I proteins on the II-23.D7 cells are, in part, associated with a modified state of a common receptor.     

Effect of high temperature on infectivity of Toxoplasma gondii tissue cysts in pork

To study the effect of high temperature on infectivity of Toxoplasma gondii tissue cysts, pork from infected pigs was mixed with infected mouse brains and homogenized thoroughly. Twenty-gram samples of infected homogenized meat were sealed in plastic pouches, pressed to a uniform thickness of 2 mm, and subjected to water-bath temperatures of 49, 52, 55, 58, 61, 64, and 67 C for 0.01, 3, 6, 9, 12, 24, 48, and 96 min. Treated samples were digested in HCl-pepsin solution and bioassayed in mice. Toxoplasma gondii tissue cysts remained viable at 52 C for 9.5 min but not for 9.5 min at 58 C; tissue cysts were generally rendered nonviable by heating to 61 C or higher temperature for 3.6 min. Tissue cysts survived once at 64 C for 3 min. These data demonstrate that T. gondii tissue cysts are less heat resistant than encysted Trichinella spiralis larvae.     

Sequence and structure of the yeast galactose transporter.

The previously cloned GAL2 gene of the Saccharomyces cerevisiae galactose transporter has been sequenced. The nucleotide sequence predicts a protein with 574 amino acids (Mr, 63,789). Hydropathy plots suggest that there are 12 membrane-spanning segments. The galactose transporter shows both sequence and structural homology with a superfamily of sugar transporters which includes the human HepG2-erythrocyte and fetal muscle glucose transporters, the rat brain and liver glucose transporters, the Escherichia coli xylose and arabinose permeases, and the S. cerevisiae glucose, maltose, and galactose transporters. Sequence and structural motifs at the N-terminal and C-terminal regions of the proteins support the view that the genes of this superfamily arose by duplication of a common ancestral gene. In addition to the sequence homology and the presence of the 12 membrane-spanning segments, the members of the superfamily show characteristic lengths and distributions of the charged, hydrophilic connecting loops. There is indirect evidence that the transporter is an N-glycoprotein. However, its only N-glycosylation site occurs in a charged, hydrophilic segment. This could mean that this segment is part of a hydrophilic channel in the membrane. The transporter has a substrate site for the cyclic AMP-dependent protein kinase which may be a target of catabolite inactivation. The transporter lacks a strong sequence enriched for proline (P), glutamate (E), aspartate, serine (S), and threonine (T) and flanked by basic amino acids (PEST sequence) even though it has a short half-life. Mechanisms for converting the poor PEST to a possible PEST sequence are considered. Like the other members of the superfamily, the galactose transporter lacks a signal sequence.     

Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12

The nucleotide sequence of a 3,162-base-pair (bp) segment of DNA containing the FNR-regulated fumB gene, which encodes the anaerobic class I fumarase (FUMB) of Escherichia coli, was determined. The structural gene was found to comprise 1,641 bp, 547 codons (excluding the initiation and termination codons), and the gene product had a predicted Mr of 59,956. The amino acid sequence of FUMB contained the same number of residues as did that of the aerobic class I fumarase (FUMA), and there were identical amino acids at all but 56 positions (89.8% identity). There was no significant similarity between the class I fumarases and the class II enzyme (FUMC) except in one region containing the following consensus: Gly-Ser-Xxx-Ile-Met-Xxx-Xxx-Lys-Xxx-Asn. Some of the 56 amino acid substitutions must be responsible for the functional preferences of the enzymes for malate dehydration (FUMB) and fumarate hydration (FUMA). Significant similarities between the cysteine-containing sequence of the class I fumarases (FUMA and FUMB) and the mammalian aconitases were detected, and this finding further supports the view that these enzymes are all members of a family of iron-containing hydrolyases. The nucleotide sequence of a 1,142-bp distal sequence of an unidentified gene (genF) located upstream of fumB was also defined and found to encode a product that is homologous to the product of another unidentified gene (genA), located downstream of the neighboring aspartase gene (aspA).     

Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.     

Purification, characterization, and amino terminal sequence of xylose reductase from Candida shehatae.

D-Xylose is a major component of the carbohydrates derived from agricultural residues and forest products. Among more than two hundred known xylose-utilizing yeasts, only a few species are known to be able to ferment xylose anaerobically. Candida shehatae is one of such xylose-fermenting yeasts. Xylose reductase (E.C. 1.1.1.21) is a key enzyme responsible for xylose metabolism in xylose-utilizing as well as xylose-fermenting yeasts. In this paper, we report the development of a convenient and reliable procedure for the purification of xylose reductase from C. shehatae to near homogeneity. The amino acid composition and N-terminal sequence of the enzyme have also been analyzed. C. shehatae seems to contain only a single xylose reductase, but the enzyme has a dual coenzyme specificity for both NADPH and NADH. The enzyme is remarkably stable at room temperature and 4 degrees C.     

Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes.

Incubation of rabbit articular chondrocytes with interleukin-1 beta caused time-dependent expression of NO synthase, determined as nitrite, after a lag period of 6h. The synthesis of nitrite was concentration-dependent and was inhibited by cycloheximide and NG-monomethyl-L-arginine, but not by dexamethasone or hydrocortisone. The synthesis of NO in the 100,000g supernatant of activated chondrocytes was inhibited by EGTA, but not by the calmodulin inhibitors W-13 or trifluoperazine. The synthesis of NO was half-maximal at approximately 20nM free Ca2+. Endotoxin also induced the expression of this NO synthase. Thus, rabbit articular chondrocytes express a novel inducible NO synthase which is Ca(2+)-dependent, and whose induction is not prevented by glucocorticoids.     

Rapid Increase of Pneumococcal Resistance to β-Lactam and Other Antibiotics in Isolates from the Respiratory Tract (Nagasaki,Japan: 1975–1994)

The susceptibility of 101 pneumococcal isolates from the respiratory tract during 1991–1994 was examined and compared with the susceptibility of isolates over the period of 1975–1990. A rapid increase of resistance was seen not only to penicillin but also other antimicrobial agents. During 1991–1994, 38% of all the isolates were resistant to penicillin. The rates of resistance during this period were 16–23% for three newer cephalosporins, 18% for imipenem, 69% for tetracycline, 31% for erythromycin, 20% for chloramphenicol and 9% for clindamycin. The use of antibiotics within one month prior to pneumococcal isolation was correlated with penicillin resistance (P < 0.05). Serotyping of the isolates by antiserum revealed differences in predominant types between penicillin-resistant (19F, 23F, 4) and -susceptible isolates (15, 4, 11A). Our data suggests that anti-pneumococcal antibiotics should be carefully chosen on the basis of susceptibility tests.     

Genetic evidence for a complex of species within Rugopharynx australis (Mönnig, 1926) (Nematoda: Stronglyloidea) from macropodid marsupials

An electrophoretic analysis using 17 enzyme loci was carried out on specimens of the gastric nematode of macropodid marsupials, Rugopharynx australis (Mönnig, 1926), collected from Macropus eugenii (Desmarest), M. fuliginosus (Desmarest), M. giganteus Shaw, M. robustus Gould, M. rufogriseus (Desmarest), M. rufus (Desmarest), Thylogale billardierii (Desmarest) and Wallabia bicolor (Desmarest) from south-eastern Australia. The extent of fixed genetic differences between nematodes from different host species ranged from 0–53%. The two distinct morphological forms of the parasite found in M. rufogriseus differed at 50% of loci. Specimens present in M. fuliginosus and M. giganteus were indistinguishable genetically, as were nematodes from M. rufus and M. robustus. Of the two morphologically distinct congeners included in the analysis as controls, Rugopharynx epsilon (Johnston & Mawson, 1939) was genetically distinct (46–69% fixed genetic differences) from all specimens of the R. australis complex while R. rufogrisea Magzoub, 1964 was closely related to one of the two species occuring in M. rufogriseus. It was concluded that R. australis is a species complex, with a genetically distinct species present in M. eugenii, M. fuliginosus/M. giganteus, M. robustus/M. rufus, W. bicolor and T. billardierii, and two species in M. rufogriseus.