Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: II. characterization of cells cultured ex vivo and their contribution to the in vivo immunomodulation

Summary Burkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines (LCL). We studied three groups of BL lines for their capacity to interact with allogeneic lymphocytes. The results showed that as long as the lines have the group I phenotype, they do not stimulate allogeneic T lymphocytes irrespective whether they carry the EBV genome. The group II and III cells are stimulatory. Generally there was no correlation between sensitivity to lymphocyte-mediated lysis and the phenotype of the lines. In one set of lines, the group I cells had higher sensitivity to both natural killer and lymphokine-activated killer effectors compared to the group II or III lines. However, such correlation could not be seen with the other two sets of lines. Among the phenotypic features investigated, expression of the adhesion molecules LFA-1 and LFA-3 correlated with the tendency for cell aggregation.     

Familial porphyria cutanea tarda: hybridization analysis of the uroporphyrinogen decarboxylase locus

Familial porphyria cutanea tarda (PCT) results from a deficiency of uroporphyrinogen decarboxylase (URO-D) activity. Hybridization analysis of genomic DNA from unrelated normal individuals and PCT pedigree members failed to detect any major deletions, rearrangements or restriction fragment length polymorphisms at the URO-D locus.     

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.     

Observations on egg production rates and seasonal changes in the internal morphology of Mediterranean populations of Acartia clausi and Centropages typicus

Egg production rates in wild populations of Acartia clausi and Centropages typicus, sampled biweekly in the Gulf of Naples from October 1985 to July 1987, showed marked seasonal fluctuations with maximum values in early spring that proceeded the annual maxima for adult female densities in summer. A positive correlation between chlorophyll a concentrations and egg production was evident only during the early spring phytoplankton bloom. A strong diminution in egg deposition occurred later in spring and continued throughout the summer notwithstanding high chlorophyll concentrations. In winter, when population abundances for adult females were lowest, egg production rates were always higher than in summer. Differences in egg production rates coincided with pronounced morphological changes between summer and winter populations of both species. The most striking of these changes consisted, in winter, in the presence of a dark brown fluid-like mass of granular material that seemed to freely bathe the gonads. The presence of this substance only during periods of elevated egg production suggests that it may enhance egg production rates when the adult population reaches minimum annual levels. Such a mechanism of self-regulation may operate to dampen the effects of environmental variability thereby contributing to maintain a conservative structure in coastal copepod communities.     

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.     

Carbonyl 13C NMR spectrum of basic pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding

The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering.     

Characterization of a high molecular weight glycoprotein secreted by the peri-implantation bovine conceptus

Cow conceptuses were flushed from uteri on Day 17 of pregnancy and cultured with [3H]glucosamine and [14C]leucine. A high molecular weight glycoprotein (HMWG) having an Mr = 765,000 was isolated by a combination of anion-exchange and gel-filtration chromatography. Selective chemical and enzymatic degradations were performed. The HMWG was resistant to Pronase and peptide: N-glycanase F. Only endo-beta-galactosidase and harsh alkaline reducing conditions were successful in dissociating carbohydrate from the protein core, suggesting that carbohydrate chains are N-linked to Asn and contain beta-galactosidic linkages. The intact molecule could bind to an affinity column of Datura stramoniom lectin, suggesting the presence of beta(1-4)-linked oligomers of N-acetylglucosamine. The susceptibility of HMWG to endo-beta-galactosidase suggests that at least some of these oligomers are substituted with galactose to form N-acetyllactosamine. Binding of HMWG to lectin could be inhibited partially with N-acetyllactosamine or completely with a mixture of N, N'-diacetylchitobiose and N, N', N"-triacetylchitotriose. In summary, properties of the HMWG suggest it contains lactosaminoglycan components and is almost identical to an HMWG secreted by the Day 16 ovine conceptus. Thus, embryos of these two ruminant species secrete similar molecules during early pregnancy.     

Secretory proteins of the bovine conceptus alter endometrial prostaglandin and protein secretion in vitro

The conceptus is believed to produce factors that regulate endometrial function and prevent luteolysis during early pregnancy. Endometrial tissues were collected from cyclic (n = 8) and pregnant (n = 2) cows at Day 17 post-estrus and cultured for 24 and 48 h with bovine conceptus secretory proteins (bCSP) (0%, 10%, 100%), where the amount of protein produced by a bovine conceptus during 24 h of culture is 100%. Incorporation of [3H]leucine into secreted proteins was determined and examined qualitatively by trichloroacetic acid precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Levels of an intracellular endometrial inhibitor of prostaglandin synthesis were determined with a cotyledonary microsomal test system. Treatment with 10% and 100% bCSP reduced incorporation of [3H]leucine into secreted proteins. However, bCSP selectively induced two secreted proteins (13 and 10 kDa) from endometrium of cyclic cows. Prostaglandin F (PGF) secretion was decreased by bCSP treatment while prostaglandin E2 secretion was unaltered. An intracellular endometrial inhibitor of prostaglandin synthesis was induced by bCSP; synthesis of PGF by the cotyledonary prostaglandin-generating system was decreased when incubated with cytosol of endometrium treated with bCSP, but unaltered by cytosol from control tissues. In conclusion, products produced by the bovine conceptus are capable of regulating endometrial protein and prostaglandin biosynthesis in a fashion that could act to prevent luteolysis in vivo and provide endometrial secretory products for embryonic development.     

Characterization and immunohistochemical localization of rat salivary cobalamin-binding protein and comparison with human salivary haptocorrin

Rat saliva contains a cobalamin-binding protein that binds cobalamin as well as cobinamide. The protein binds cobalamin with an affinity constant of 8 X 10(10) l X mol-1, and it binds cobalamin over a more narrow pH range (pH 7.5-10) than does human haptocorrin. It has a Stokes radius of 2.45 nm as compared to the Stokes radius of 4.50 nm for human haptocorrin. Upon isoelectricfocusing it dissociates into four strong bands with pI between 7 and 8, while human haptocorrin dissociates into acid isoproteins. Since human haptocorrin binds to concanavalin A while rat haptocorrin does not, we suggest that rat haptocorrin lacks carbohydrate. The substance concentration of rat saliva haptocorrin is 0.04-12.9 nmol X l-1 (median 7.5 nmol X l-1, n = 9) for control animals. After stimulation with isoproterenol, a beta-adrenergic agent, the substance concentration is 46.4-96.6 nmol X l-1 (median 69.7 nmol X l-1, n = 8). Immunohistochemical studies show haptocorrin in the secretory acini of the submandibular and parotid glands of the rat. In the human submandibular gland, the protein is detected both in the mucous secretory acini and in the intercalated ducts.