Treating tumor-bearing mice with vitamin D3 diminishes tumor-induced myelopoiesis and associated immunosuppression,and reduces tumor metastasis and recurrence

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colonystimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1,25-dihydroxyvitamin D₃ reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D₃ reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D₃ treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D₃ after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D₃ treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.This study was supported in part by grants from the Medical Research Service of the Department of Veterans Affairs and by grants CA-45080 and CA-48080 from the National Institutes of Health     

Construction and characterization of an absolute deletion mutant of Escherichia coli ribonuclease II

Abstract The degradation of mRNA plays a central role in the control of protein synthesis. In Escherichia coli , the rnb gene encodes ribonuclease II (RNase II), one of the two main exonucleases involved in mRNA decay. We have constructed strain CMA201, in which the rnb promoter region and the gene were deleted from the chromosome and replaced by a tet^tr cassette. This is the first rnb absolute deletion mutant that shows the complete absence of rnb -specific mRNA. This strain has growth characteristics similar to the wild-type, even though it has no RNase II activity, and it should be useful in studies of mRNA metabolism.     

Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE

Homopolymeric α-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.     

Localization of corticotropin-releasing factor-containing neurons in the brain of the domestic fowl

Summary The corticotropin-releasing factor (CRF)-containing neurons were investigated in the brain of the domestic fowl by means of the peroxidase-antiperoxidase technique at the light-microscopic level. The detection of CRF-immunoreactivity was facilitated by silver intensification. CRF-containing perikarya were found in the paraventricular, preoptic and mammillary nuclei of the hypothalamus and in some extrahypothalamic areas (nuclei dorsomedialis and dorsolateralis thalami, nucleus accumbens septi, lobus parolfactorius, periaqueductal gray of the mesencephalon, nucleus oculomotorius ventralis). Immunoreactive nerve fibers and terminals were demonstrated in the external zone of the median eminence and the organum vasculosum of the lamina terminalis. These results indicate that an immunologically demonstrable CRF-neurosecretory system also exists in the avian central nervous system.     

Discriminative stimulus,antagonist, and rate-decreasing effects of cyclorphan: Multiple modes of action

The discriminative effects of cyclorphan were studied in pigeons trained to discriminate 0.32 mg/kg ethylketazocine, 1.8 mg/kg cyclazocine, or 32 mg/kg naltrexone from saline. A fourth group of pigeons was administered 100 mg/kg/day morphine and trained to discriminate 0.1 mg/kg naltrexone from saline. Cyclorphan produced dose-related ethylketazocine-appropriate responding that reached a maximum of 83% of the total session responses at 0.3 mg/kg. Higher cyclorphan doses produced less ethylketazocine-appropriate responding. In pigeons trained to discriminate cyclazocine from saline, maximum drug-appropriate responding of greater than 90% occured at 5.6–10.0 mg/kg cyclorphan. In narcotic-naive pigeons trained to discriminate 32 mg/kg naltrexone from saline, cyclorphan produced a maximum of less than 50% drug-appropriate responding. In contrast, in pigeons chronically administered morphine and trained to discriminate 0.1 mg/kg naltrexone from saline, 1.0 mg/kg cyclorphan resulted in 100% drug-appropriate responding. In pigeons responding under a multiple fixed-interval, fixed-ratio schedule of food delivery, cyclorphan produced a complete dose-related reversal of the rate-decreasing effects of 10 mg/kg morphine, the maximally effective antagonist doses being 1.0–3.2 mg/kg. Higher cyclorphan doses (10 mg/kg) resulted in response rate decreases that were not reversed by naloxone (1 mg/kg). Thus, cyclorphan has discriminative effects that are similar to those of both ethylketazocine and, at 20-fold higher doses, cyclazocine. In addition, in morphine-treated pigeons, cyclorphan, across the same range of doses that produce ethylketazocine-appropriate responding, has discriminative effects that are similar to those of naltrexone, an effect that is probably related to the antagonist action of the drug.     

Life difficulties,coping, and the use of medical services

A retrospective study of 124 healthy females was designed to explore the components of high use of medical services. Analysis of data from medical records and responses to survey and in-depth interview questions showed that poor, less educated, separated or divorced women were likely to feel that their social resources were only nominally effective in mediating between them and the pressures of life difficulties. It was found that such women used health services for vague, unorganized symptoms more frequently than women whose situations were less burdened.Michigan State University     

The fine structure of distal receptors on the labium of the aphid,Brevicoryne brassicae L. (Homoptera)

Summary Short peg receptors located at the distal tip of the aphid labium have the structure of mechanoreceptors. Each peg is innervated by a single sensory nerve which is anchored eccentrically to a basal cuticular tube and terminates in electron-dense material in the base of the peg. The arrangement and eccentric insertion of the eight pegs in the labial wall on one side of the stylet groove, with the eccentric insertions of their innervating neurones, provide a mirror image of the receptors on the opposite side. On the basis of a comparison of the structure of these receptors with that of tactile receptors for which electrophysiological data on sensitivity are available, it is possible to predict that the receptors detect both surface contact (pressure) and surface profile; and that the bilateral symmetry in the receptor arrangement facilitates the detection of vein contours which are preferred settling sites on the leaf. The structure of the dendritic terminal and its insertion is that of a well reinforced cytoskeleton designed to transmit tension to the cell membrane, in agreement with the concept that transduction is a membrane related phenomenon. The distal microtubules, fifty per-cent of which originate as well as terminate in the tubular body, are packed in electron-dense material which binds to the cell membrane. The membrane in turn is attached to cuticular components of the receptor. Abrupt changes in dimension of the dendritic outer segment may be designed to modulate the conduction of a membrane potential. On the other hand, lack of continuity in the microtubules makes these organelles poor candidates for the transduction of excitation from a distal site of stimulation to a proximal region.Supported by operating grants Nos. A 6063 and A 9856 from NRCC     

Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors

Using artificial electron donors and acceptors, it is shown here that the major HCO₃^? effect in the Hill reaction is after the “primary” electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acceptor Q^? to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q^?, show no significant bicarbonate stimulation of electron flow. However, a 6–7-fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.     

An early enlargement of the putrescine pool is required for growth in L1210 mouse leukemia cells under hypoosmotic stress

Hypoosmotic stress is a potent inducer of ornithine decarboxylase (ODC) activity in a variety of mammalian cells, but the physiological relevance of this response has not been determined. To test whether an increased putrescine content confers a growth advantage at lower osmolarities, we compared the ability of L1210 mouse leukemia cells and of ODC-overproducing variants obtained from this cell line (D-R cells) to proliferate after a hypotonic shock (325----130 mosmol/kg). The growth rate of D-R cells at 130 mosmol/kg was greater than or equal to 5-fold higher than in L1210 cells; and unlike the ODC-overproducing strain, L1210 cells underwent up to a 90% loss of viability over time as seen after restoration of normosmotic growth conditions and by trypan blue exclusion tests. The addition of putrescine or L-ornithine stimulated the proliferation of both cell sublines up to 5-fold in a concentration-dependent manner, with a maximal effect observed at about 10 and 100 microM, respectively. Putrescine restored virtually normal growth rates in both sublines at osmolarities as low as 190 mosmol/kg. No other alpha,omega-diamine was active in that respect whereas spermidine was markedly inhibitory. Furthermore, D-R cells incubated at 130 mosmol/kg showed a marked growth inhibition by 1-aminooxy-3-aminopropane (potent ODC inhibitor to which they are resistant in isotonic media) as a result of putrescine but not spermidine depletion. Whereas ODC was strongly and rapidly induced by hypotonic shock there was a precipitous decline in S-adenosylmethionine decarboxylase activity. Putrescine synthesis and accumulation were nevertheless reduced in D-R cells incubated at 130 mosmol/kg because of a decreased availability of L-ornithine. When either putrescine or L-ornithine was added to hypotonic media, D-R cells accumulated putrescine massively for extended periods together with a reduction in spermidine and spermine contents. Putrescine transport patterns were altered by hypotonic shock, net excretion of the diamine being reduced by about 80%, with a concurrent enlargement of the intracellular pool. Finally, parental L1210 cells incubated with an irreversible inhibitor of S-adenosylmethionine decarboxylase for 24 h until hypotonic shock and supplemented with putrescine in the presence of the drug thereafter exhibited a greatly exaggerated growth stimulation by the diamine. These results demonstrate an essential role for an early increase in putrescine content in the growth adaptation of a mammalian cell line to a lower osmolarity.     

Phylogenetic depth ofThermotoga maritima inferred from analysis of thefus gene: Amino acid sequence of elongation factor G and organization of theThermotoga str operon

Summary The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacteriumThermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). TherpsG, fus, andtuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in thestr operon ofEscherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrogram, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies betweenThermotoga and the other bacterial lineages.