Immunoregulation in cancer-bearing hosts. Down-regulation of gene expression and cytotoxic function in CD8+ T cells.

The causes of the decreased immune responsiveness in tumor-bearing hosts are incompletely understood. The impact of a decreased immune response in cancer patients on the clinical response in immunotherapy trials has not been evaluated. The present report demonstrates a marked decrease in the therapeutic efficacy of adoptively transferred T lymphocytes obtained from murine hosts bearing tumor for greater than 30 days [late tumor-bearing mice (TBM)] as compared with normal mice and mice bearing tumor for less than 21 days (early TBM). In vitro analysis of the functions of the T lymphocytes from late TBM showed an apparently normal proliferative response to anti-CD3 and IL-2 with adequate lymphokine production from CD4+ cells, but a significant decrease in the cytotoxic function of CD8+ cells. The decreased cytotoxicity was not because of cell-mediated suppression. The expression of granzyme B mRNA was significantly delayed and decreased in magnitude in CD8+ cells from late TBM. Culture supernatants from two unrelated tumor cell lines were able to inhibit the cytotoxic activity of normal CD8+ cells in vitro. The tumor-derived suppressive factor is not transforming growth factor-beta (TGF-beta), but it has not been further characterized. The data suggest that one potential mechanism responsible for immunologic defects in patients with large tumor burdens is a tumor-induced defect that compromises the function of CD8+ effector T cells.     

Dissimilar phloem loading in leaves with symplasmic or apoplasmic minor-vein configurations

Plant species were selected on the basis of abundant or no symplasmic continuity between sieveelement-companion-cell (SE-CC) complexes and adjacent cells in the minor veins. Symplasmic continuity and discontinuity are denoted, respectively, as symplasmic and apoplasmic minor-vein configurations. Discs of predarkened leaves from which the lower epidermis had been removed, were exposed to ¹⁴CO₂. After 2 h of subsequent incubation, phloem loading in control discs and discs treated with p-chloromercuribenzenesulfonic acid (PCMBS) was recorded by autoradiography. Phloem loading was strongly suppressed by PCMBS in minor veins with symplasmically isolated SE-CC complexes (Centaurea, Impatiens, Ligularia, Pelargonium, Pisum, Symphytum). No significant inhibition of phloem loading by PCMBS was observed in minor veins containing sieve elements with abundant symplasmic connections (Epilobium, Fuchsia, Hydrangea, Oenothera, Origanum, Stachys). Phloem loading in minor veins with both types of SE-CC complex (Acanthus) had apoplasmic features. The results provide strong evidence for coincidence between the mode of phloem loading and the minor-vein configuration. The widespread occurrence of a symplasmic mode of phloem loading is postulated.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE-CC complex sieve-element-companion-cell complex     

Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.     

Isolation of bovine Y-derived sequence: potential use in embryo sexing.

To obtain bovine Y-derived probes, we have constructed a bovine plasmid library enriched for Y-specific DNA sequences by the deletion enrichment method. The resulting clones were analyzed by hybridization to Southern blots of male and female genomic DNA. From 200 clones tested, two (BC1.2 and BC1.34) were entirely male specific, six gave a male-female differential hybridization pattern, and the remaining reacted similarly with male and female DNA. Interspecies somatic cell hybrid studies and chromosomal in situ hybridization confirmed that the BC1.2 sequence was derived from the Y chromosome. This 54-bp fragment is present at about 2000-2500 copies in the bovine male genome. No polymorphism was revealed with any of the restriction enzymes used, suggesting enzyme site conservation within blocks of repeats. Evolutionary study has shown that the BC1.2 sequence is conserved within Bos and Bison genera and remains male specific. The male specificity and repeated nature of the BC1.2 sequence have enabled us to use it as a molecular probe for sex determination on small numbers of cells by in situ hybridization.     

Roberts' — SC phocomelia syndrome with cytogenetic findings

Summary This paper reports the physical and cytogenetic findings in an eight-year-old severely mentally retarded female child with the following features: tetraphocomelia; weight, lenght, and head circumference below the third percentile; microcephaly with prominent frontal bones; hypertelorism; shallow orbits; prominent eyes; bilateral corneal opacities; micrognathia; hypoplastic alae nasi; small, low set ears; short neck; sparse silvery blond hair; severe flexion deformities of both knees and wrist joints; a cardiac murmur. Cytogenetic studies revealed premature centromere separation.     

Rotational dynamics of monoclonal anti-dansyl immunoglobulins

The rotational motions of monoclonal mouse anti-dansyl immunoglobulins were studied by nanosecond fluorescence emission anisotropic spectroscopy using a mode-locked argon-ion laser as the pulsed excitation source. Three homogeneous antibodies of the immunoglobulin Gl (IgGl) subclass containing different V regions were prepared. The fluorescence emission maxima of these antibodies (designated as DNS1, DNS2 and DNS3) are at 515, 480 and 500 nm, respectively. Their mean rotational correlation times, 〈φ〉, are 84, 109 and 96 ns, respectively. The binding of protein A or a monoclonal anti-allotype antibody to the F_c unit of DNS1 increased 〈φ〉 to 142 and 150 ns, respectively, whereas reduction of the disulfide bond between the heavy chains decreased 〈φ〉 to 48 ns. These nanosecond measurements show that the rotational motion of the F_ab arms in mouse IgGl is restricted.     

Comparison of energy budgets for spruce budworm Choristoneura fumiferana (Clemens) on balsam fir and white spruce

Summary A determination was made of the differences in the utilization of energy by laboratory reared larval Choristoneura fumiferana fed either balsam fir or white spruce foliage. This enabled us to quantitatively measure the quality of these foliages vis a vis the spruce budworm.The larval strategy was to feed rapidly and develop quickly. Development time was longer on spruce than on fir. Total consumption was virtually identical on both foliage types though production was ca 20% greater on a white spruce diet. Calories/gram increased with insect development and with the development of balsam fir foliage but declined over time in white spruce. Assimilation efficiencies (A/C) were 33.9% to 40.2% while gross production efficiencies (P/C) were 9.5% to 13.3% and net production efficiencies (P/A) were 26.1% to 38.8%. The spruce fed insects gave higher values than those on fir, but all values were at the low end of the range for lepidopterous defoliators (possibly due to characteristics of conifer foliage).The enhanced growth found in spruce fed larvae contrasts the relationship between balsam fir and high insect densities. This is discussed together with the influence of spruce budworm on forest composition and the possible competitive benefits of spruce budworm induced tree mortality.Parts of this paper appeared in a Master's thesis (Koller 1978) submitted to the University of Maine     

Catecholamine-Sensitive Guanylate Cyclase from Human Caudate Nucleus

Abstract: Partial purification of soluble guanylate cyclase on DEAE-Sephacel yields two separate peaks of guanylate cyclase activity. After 10-fold purification of the soluble enzyme, guanylate cyclase is markedly inhibited by micromolar concentrations of dopamine (I₅₀= 0.2 μm). Dopamine inhibition is observed whether the reaction is conducted with Mn²¹ or with Mg²⁺, under atmosphere or N₂(g), and using enzyme from either peak from the DEAESephacel column. Other catecholamines also inhibit partially purified guanylate cyclase with an order of potency at 1 μm of: dopamine =l -DOPA > norepinephrine = isoproterenol = adrenochrome > epinephrine. The structural requirements for inhibition are two free hydroxyl groups on the phenyl ring and an ethylamine side chain. Dopamine also inhibits the Triton X-100-solubilized microsomal guanylate cyclase after partial purification on DEAESephacel. Neither chlorpromazine, propranolol, nor phentolamine at 20 μm effectively block the dopamine inhibition of partially purified soluble guanylate cyclase. Micromolar concentrations of the reducing agents dithiothreitol and glutathione also inhibit partially purified guanylate cyclase, but unlike these agents, catecholamines can inhibit whether added in the reduced or the oxidized forms. Inhibition of enzyme activity by micromolar concentrations of dopamine, adrenochrome, or dithiothreitol is rapidly reversed by dilution and the dopamine inhibition is competitive with MgGTP. Inhibition does not appear to involve covalent binding or to result from the ability of catecholamines to reduce the concentrations of oxygen or free radicals in solution.     

Mortality factors of satin moth,Leucoma salicis [Lep.: Lymantriidae], in aspen forests in Maine

Natural control agents of the satin moth,Leucoma salicis (L.) were examined in 2Populus grandidentata Michaux stands. Highest mortality occurred in overwintering larvae, with the causal agents 2 fungi,Paecilomyces sp. andHirsutella gigantea Petch, a factor causing death symptomatic of a pathogen, the parasiteEupteromalis hemipterus (Walker) and death from unknown causes. Mortality fromPaecilomyces sp. andE. hemipterus was reduced where overwintering sites were covered with burlap cloth. Parasites recovered from larvae and pupae included the braconidsApanteles melanoscelus (Ratzeburg) andMeteorus versicolor (Wesmeal), the ichneumonidCoccygomimus pedalis (Cresson), the tachinidsCompsilura concinnata (Meigen),Carcelia laxifrons Villeneuve,Tachinomyia variata Curran, and the sarcophagidsSarcophaga aldrichi Parker andAgria housei Shewell. Larval and pupal predators included the carabidCalosoma frigidum Kirby, pentatomids, and birds, particularly black-billed cuckoos,Coccyzus erythrophthaimus (Wilson). Eggs were parasitized by the scelionidTelenomus prob.californicus Ashmead and the trichogrammidTrichogramma minutum (Riley). Predators of adult satin moths included the hermit thrush,Hylocichla guttata (Pallas), and pentatomid bugs.     

Carboxyfluorescein as a probe for liposome-cell interactions effect of impurities,and purification of the dye

Impurities in 5(6)-carboxyfluorescein can affect phospholipid vesicle stability and apparent rates of carboxyfluorescein transfer into cells. Thorough purification and characterization of the dye are thus important to many applications with vesicles and/or cells. The dye can be purified by adsorption chromatography on a hydrophobic gel, following treatment with activated charcoal and precipitation from ethanol-water. The 5- and 6-carboxy-isomers can be separated from each other (though for most purposes it is not necessary to do so) by synthesis, crystallization, and hydrolysis of the diacetate derivatives. Purification is monitored by thin-layer and high pressure chromatography.