Using cyanide to put noble gases inside C60.

After fullerenes are heated in the presence of a noble gas or an unreactive molecule at 650 degrees C and 3000 atm pressure, a small fraction of the fullerene molecules contain the atom or molecule. The incorporation fraction is greatly enhanced by adding potassium cyanide to the reaction mixture. The details of the preparation are described here.

Recovery of potato apices after several years of storage in liquid nitrogen.

A method for the systematic cryopreservation of potato apices was developed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and the Institute for Crop and Grassland Science of the Federal Agricultural Research Centre (FAL, Braunschweig). Designed specifically for routine use in genebanks, this method uses a very simple ultra-rapid freezing approach and was applied to a wide range of varieties within the Federal Centre for Breeding Research on Cultivated Plants (BAZ, Quedlinburg) Potato Collection. After several years of storage in liquid nitrogen, shoot tips from a random sample of 51 varieties were thawed and the survival and shoot regeneration percentages compared to those measured immediately after freezing. There were no major changes in either survival or recovery of frozen apices. Data presented are not the outcome of a systematic experiment but from that accumulated during our work from 1992 to 1999.

Reversible Diels-Alder addition to fullerenes: a study of equilibria using (3)He NMR spectroscopy.

3He NMR spectroscopy has been used to study the equilibria of Diels-Alder additions of 9,10-dimethyl anthracene (DMA) to (3)He@C(60) and (3)He@C(70). Spectra of a series of equilibrium mixtures showed peaks for the isomeric adducts. One monoadduct, six bis-adducts, eleven tris-adducts, and ten tetrakis-adducts of DMA to C(60) were seen. One monoadduct and three bis-adducts of C(70) were detected. Equilibrium constants were found for these reactions and values for DeltaG, DeltaH, and DeltaS were obtained.

Lung fibroblasts undergo apoptosis following alveolarization.

In the rat lung, primary saccules are transformed into alveoli from postnatal Days 4 to 13, after which time there is a 20% reduction in the number of lung fibroblasts as the interstitial volume of the alveolar walls decreases. Our objective was to determine whether apoptosis is a factor in the observed decrease in the number of interstitial lung fibroblasts beyond Day 13. We used both histologic and flow cytometric assays to detect in lung fibroblasts the DNA fragmentation and condensation that are characteristic of apoptosis. In addition, we evaluated levels of bcl-2 and BAX messenger RNAs (mRNAs) using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Apoptotic cells were quantitated in glycol methacrylate-embedded sections of neonatal rat lungs using the terminal transferase dUTP-digoxygenin nick end-labeling (TUNEL) method. Although TUNEL-positive interstitial cells were observed in the lungs of rats ranging in age from 10 to 16 d, a dramatic increase in apoptotic cells was seen on Day 17. Although diminished in number, TUNEL-positive cells were still present on Day 28. Hoechst-stained apoptotic bodies were observed in isolated lung cells that were vimentin-positive and factor VIII-negative, which identified the apoptotic cells as fibroblasts as opposed to endothelial cells. Flow cytometric analysis of freshly isolated lung fibroblasts stained with Hoechst 33342 indicated a 24% increase in chromatin condensation in cells from 17-d versus 16-d rats. DNA fragmentation was also quantitated by flow cytometry in freshly isolated fibroblasts labeled with BODIPY-conjugated dUTP in the presence of terminal deoxynucleotidyl transferase. The percentage of lung fibroblasts containing fragmented DNA was 51.4 +/- 13.4 in 17-d, 36.9 +/- 8.6 in 18-d, and 13.8 +/- 5.4 in 19-d rat pups. Finally, evaluation by RT-PCR indicated that on postnatal Day 17, mRNA for bcl-2, which inhibits apoptosis, was decreased to 73.5 +/- 11.4% (P < 0.001) of Day 5 controls; whereas mRNA for BAX, which enhances apoptosis, was increased to 243.0 +/- 102.0% (P < 0.001) of Day 5 values. These results demonstrate that rat lung fibroblasts undergo apoptosis after the completion of alveolarization, and suggest that this decrease in fibroblast number plays an important role in the thinning and remodeling of the alveolar walls of the lung.

Functional characterization of the insulin-like growth factor I receptor on Jurkat T cells.

Insulin-like growth factor I (IGF-I) has been shown to be important in the maintenance, development, and proliferation of various types of leukocytes, particularly T cells. Radio-receptor binding assays demonstrate that Jurkat T cells bind 125I-IGF-I with an affinity of 1.77 nM (Kd) and express approximately 230 receptors/cell. Specificity studies show insulin also binds the IGF-I receptor with an affinity 20-fold lower than that of IGF-I. Interaction of IGF-I with its receptor on Jurkat T cells induces the phosphorylation of tyrosine kinase which is detectable by Western blotting. The 95,000 MW protein detected is equivalent to the molecular weight of the beta chain of the IGF-I receptor described in other types of cells. These studies characterize the binding of IGF-I to its receptor on Jurkat T cells, demonstrate that IGF-I binding induces tyrosine phosphorylation, and support the hypothesis that IGF-I is important in the induction of T cell activation.

Respiratory burst activity associated with phagocytosis of Ehrlichia risticii by mouse peritoneal macrophages.

Peritoneal macrophages from two strains of mice, BALB/c and C3H/HeN, were analysed by flow cytometry for a respiratory burst associated with the phagocytosis of Ehrlichia risticii. Resident murine peritoneal macrophages failed to respond with a respiratory burst after phagocytosing E risticii. In contrast, mice previously infected with E risticii yielded peritoneal macrophages that did generate a respiratory burst on phagocytosing ehrlichiae.

Geographical trends within a diverse spring barley collection as identified by agro/morphological and electrophoretic data.

For ex-situ germplasm conservation purposes, the concept of genetic diversity being concentrated in certain geographic regions is useful for the conscious selection of diverse forms. Numerous studies of barley and other major corp species often confirm the concentration of simply-inherited, phenotypicallyobvious markers within the Vavilovian centres of diversity/origin. However, more recent studies of electrophoretic patterns and or more complexly-inherited traits do not always confirm the same geographic patterns. Unfortunately, few studies of world germplasm collections have screened a range of agro/morphological/electrophoretic patterns using the same germplasm collection as a consistent base for evaluation purposes, making precise genetic estimates of diverse geographic areas difficult. A diverse collection of 1 118 spring-sown barley cultivars was, therefore, evaluated for both agro/morphological and biochemical genetic markers in an effort to identify appropriate criteria for the construction of a comprehensive ex-situ germplasm collection. On the basis of both agro/morphological and biochemical data, countries whose cultivated barley germplasm was identified as diverse and genetically distinct were Algeria, Afghanistan, Argentina, Ethiopia, India, Peru and Turkey. However, within broad limits, separate cluster analyses of the agro-morphological and electrophoretic patterns identified dissimilar groups of countries, which demonstrated that a collection strategy based solely on country of origin is inappropriate.

Loss of genetic diversity from heterogeneous self-pollinating genebank accessions.

Genebank seed accessions of predominantly self-pollinating species may be stored either as bulked (mixed) seed lines or as pure line cultivars. If seed lines are bulked in storage then when considered over several regeneration cycles, loss of genetic diversity within heterogeneous self pollinating genebank accessions is shown to be severe. This within-accession loss of diversity represents opportunities foregone through the random loss of individual genotypes. Amongst working collections, the utility and repeatability of genebank accessions is paramount in the justification of the germ plasm resource. Therefore, the only practical solution to the management of predominantly self-pollinating species is to preserve individual accessions as pure lines.

Stable compounds of helium and neon: he@c60 and ne@c60.

It is demonstrated that fullerenes, prepared via the standard method (an arc between graphite electrodes in a partial pressure of helium), on heating to high temperatures release (4)He and (3)He. The amount corresponds to one (4)He for every 880,000 fullerene molecules. The (3)He/(4)He isotopic ratio is that of tank helium rather than that of atmospheric helium. These results convincingly show that the helium is inside and that there is no exchange with the atmosphere. The amount found corresponds with a prediction from a simple model based on the expected volume of the cavity. In addition, the temperature dependence for the release of helium implies a barrier about 80 kilocalories per mole. This is much lower than the barrier expected from theory for helium passing through one of the rings in the intact structure. Amechanism involving reversibly breaking one or more bonds to temporarily open a "window" in the cage is proposed. A predicted consequence of this mechanism is the incorporation of other gases while the "window" is open. This was demonstrated through the incorporation of (3)He and neon by heating fullerene in their presence.

Immunologic disparity in the hypopituitary dwarf mouse.

The Snell-Bagg hypopituitary dwarf mouse has been shown to be deficient in growth hormone, thyroxine, and prolactin. There are reports indicating that in addition to these neuroendocrine abnormalities, development of immune competence is also severely impaired in these animals. However, other studies indicate that the immunologic potential of these mice does not differ from their heterozygous littermate controls. Our data show that dwarf mice weaned at 21 days of age and killed at that time, or 7 days later, have reduced numbers of cells in both the spleen and thymus and the mitogen responsiveness of these cells is impaired. However, if mice weaned on day 21 are analyzed at 32 days of age or the mice are weaned at day 30 and analyzed 7 days later the ability to respond to mitogenic stimulation does not differ from controls. Further experiments show that dwarf mice weaned at 30 days of age have a normal complement of V-beta TCR as evidenced by immunofluorescence analysis as well as a primary antibody response to SRBC equivalent to that observed in normal littermates. Immunofluorescence analysis of CD4 and CD8 expression on thymocytes obtained from dwarf mice shows a distinct pattern dependent on the time of weaning and time of analysis. Initial analysis of thymocytes from dwarf mice weaned and killed at 21 days of age do not differ from controls. However, cells from dwarf mice weaned on day 21 and killed on day 28 are markedly different with a loss of immature CD4+/CD8+ cells and a corresponding increase in CD4+ and CD8+ mature thymocytes. In contrast, the phenotype of thymocytes obtained from dwarf mice weaned at 30 days of age and killed on day 37 did not differ from normal littermates. Collectively these studies indicate that hypopituitary dwarf mice lag behind their heterozygous littermates with respect to development of immunocompetence but normal immune responsiveness does develop by 32 days of age when the mice are weaned on day 21.

IBPGR morphological descriptors - their relevance in determining patterns within a diverse spring barley germplasm collection.

The International Board for Plant Genetic Resources (IBPGR) promotes a minimum set of morphological characters thought satisfactory for the custodial management of crop germplasm collections. The purpose of such conserved germplasm is as a genetic resource for future plant breeding programmes. Because future plant breeding requirements are not always known, the curator's strategy in maintaining an adequate germplasm resource is to conserve as wide a range of genetic diversity as possible. How is diversity measured to ensure a wide range of conserved germplasm? The IBPGR minimum descriptors detail genetic diversity at particular points in a genome corresponding to the observed characters. The purpose of the present study was to investigate whether diversity as identified by the IBPGR minimum set of descriptors could yield satisfactory measures of diversity in a contrasting set of genomic markers. A diverse spring-sown barley collection of 1379 cultivars was evaluated for the 12 IBPGR minimum taxonomic characters. An additional nine phenotypic characters and six biochemical markers were evaluated to enable diversity comparisons. Cluster analysis of the various sets of data revealed groups of accessions for each of the three data sets. A poor level of agreement (congruence) between data sets was observed in all comparisons indicating that, for cultivated barley at least, diverse collections according to the IBPGR minimum descriptors is not necessarily related to equivalent levels of diversity in other genetic characters. Implications of the relevance of the IBPGR descriptor list and appropriate collection strategies are discussed.

A proposed revision of the IBPGR barley descriptor list.

The International Board for Plant Genetic Resources (IBPGR) promote a set of morphological characters considered to be the minimum number for the description of crop germ plasm collections. The format and style of these IBPGR minimum descriptors are typical across numerous crop and species genera. However, no research publications have reviewed the relative usefulness of individual descriptors, nor indeed if additional descriptors should be included into the "minimum descriptor list".A diverse collection of 1,379 spring sown barleys were evaluated for the 12 IBPGR "minimum" descriptors, an "additional" 9 morphological descriptors of similar type of the minimum descriptors, and a contrasting group of 3 hordein and 3 leaf esterase biochemical markers. Multivariate analyses were performed on combinations of the "minimum", "additional" and biochemical markers to reveal detailed associations amongst these various descriptors. The IBPGR minimum descriptor list for barley was shown to be inadequate in descriminating between accessions, and a revised list is proposed.

Physiology of murine B lymphocytes. II. Life-spans of mitogen and thymus-independent antigen (type 2) reactive B lymphocytes from aged mice.

We showed earlier that life spans of murine B lymphocytes could be estimated by measuring the functional reactivities of normal B cells upon transfer into x-linked immunodeficient (xid) mice, which do not respond to anti-mouse IgM (anti-mu) antibodies and thymic-independent type-2 (TI-2) antigens. Here the same approach was adopted, to evaluate the life spans of B-lymphocytes from aged mice. Spleen cells from normal young and aged mice were transferred into young or aged xid recipients and the decay kinetics were followed by measuring the proliferative response to anti-mu and PFC response to TNP-Ficoll, a prototype TI-2 antigen. The results indicated that anti-mu reactive B cells of both young and aged mice decayed with similar non-linear kinetics. About 50% of the donor cells decayed in 8-10 days, whereas, the remaining decayed at a slower rate and it appeared that the median life-expectancy of this latter population could be at least 3 weeks. Essentially, there was no apparent difference in the decay kinetics of anti-mu reactive B cells of young and aged mice. Unlike anti-mu reactive cells, TNP-Ficoll reactive B cells showed 2-3-fold enhancement in the PFC response during the first 2 weeks, and persisted at least until 5 weeks post transfer. This result indicated that TNP-Ficoll reactive B cells are long-lived. Further, it was found that the turn over rate of TNP-Ficoll reactive B cells was also very similar in young and aged mice. The environment of the aged mice also did not appear to have any effect since the survival profiles of anti-mu reactive B cells were the same in young or aged xid recipients. Altogether, these results suggest that aging does not significantly alter the life spans of mature B lymphocytes.

The effect of the endophyte (Acremonium coenophialum) and associated toxin(s) of tall fescue on serum titer response to immunization and spleen cell flow cytometry analysis and response to mitogens.

Experiments were conducted with rats and mice to evaluate the effect of the consumption of endophyte (Acremonium coenophialum) and associated toxin(s) infected tall fescue on humoral and cellular aspects of immune function. Treatment diets were: (1) rodent chow (RC) or (2) rodent chow mixed 1:1 (w/w) with endophyte infected (E+) or (3) non-infected (E-) tall fescue seed. Rats fed the E+ diet in experiment 1 (43 days) exhibited a lower (P less than 0.05) serum titer to sheep red blood cell (SRBC) immunization than those fed the E- diet (38.4 vs 131.3). The E+ rats also had lower (P less than 0.01) white cell counts than either RC or E- groups (5225 vs 8959 and 7491/mm3). Spleen cells from mice fed the E+ diet for 37 days exhibited a reduced (P less than 0.05) response to the mitogens Concanavalin A and lipopolysaccharide. Flow cytometric analysis revealed a significant (P less than 0.01) 42% increase in T suppressor cell numbers in spleens of mice fed the E+ vs RC diets.

Transplantation of pituitary grafts fail to restore immune function and to reconstitute the thymus glands of aged mice.

There is evidence to indicate that the neuroendocrine and immune systems can interact. Thus, neuroendocrine hormones can modulate a variety of immune functions and there have been attempts to manipulate the neuroendocrine system of aged animals to enhance immune function. We have previously shown that the transplantation of a syngeneic pituitary gland under the kidney capsule of young adult mice elevates serum prolactin and enhances immune responsiveness. In the present study pituitary glands were transplanted under the kidney capsule of 22-month-old mice to determine if this maneuver can enhance a number of immunologic parameters. The results demonstrate that aged animals bearing transplanted pituitary grafts for 10 days did not exhibit any enhancement in their primary antibody response to sheep red blood cells, splenic T or B-cell mitogen responsiveness or restoration of thymic architecture. When these immunologic assessments are performed on animals bearing pituitary grafts for 28 days, the IgM and IgG primary antibody responses and splenic T-cell responsiveness are enhanced but repopulation of the thymus still does not occur. Importantly, this enhancement does not restore immunocompetence to levels observed in young mice.

Potentiation of antibody responsiveness after the transplantation of a syngeneic pituitary gland.

Transplantation of a pituitary graft under the kidney capsule and the resulting elevation of serum prolactin enhances the primary humoral antibody response to sheep red blood cells. Enhancement of the response is not due to marked changes in the percentage of T-cells and their subsets, B-cells, or the number of nucleated spleen cells. Quantitation of serum prolactin levels correlates well with the proportion of enhancement as mice with two grafts and higher levels of prolactin have increased responsiveness compared to mice with one graft. Systemic administration of mouse prolactin at the time of immunization also enhances the humoral immune response; however, if prolactin treatment is delayed and given 24 h after immunization, no potentiation of the response occurs. Thus, prolactin is enhancing the immune response by affecting an early afferent event in the induction of the immune response.

Killing lung cancer cells at cell-cycle phase by a new indium-111-bleomycin complex.

The efficacy of killing small cell lung cancer (SCLC) cells at the G1, S, and G2-M phase of the cell-cycle by a new 111In-bleomycin complex (111In-BLMC) was investigated. SCLC cells (N417, H526, H209) were synchronized by double thymidine block and assessed by DNA content with flow cytometry, and the period for the maximal accumulation of cells in S, G1, or G2-M phase was determined. Cells in different cell cycle phases were exposed to 0.9% NaCl, BLM, or 111In-BLMC for 1 hour and observed for colony formation. The survival of H526 cells treated with 111In-BLMC was 71% (for enriched S phase), 46% (G1), and 31% (G2-M). For N417 cells, it was 25% (S), 20% (G1), and 8% (G2-M) for 111In-BLMC and 18% (S), 33% (G1), and 10% (G2-M) for BLM. These results indicated that SCLC cells in G2-M were most sensitive and those in S phase were least sensitive to 111In-BLMC; cells in G1 phase were the least sensitive to BLM.

Central catecholamine depletion impairs in vivo immunity but not in vitro lymphocyte activation.

We have previously shown that depletion of central nervous system (CNS) catecholamines by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into the cisterna magna of C57B1/6 mice markedly impairs the humoral immune response to sheep red blood cells. This work extends these observations by showing that 6-OHDA treatment also inhibits the humoral antibody response to the T-cell-dependent antigen trinitrophenyl-keyhole limpet hemocyanin, but does not affect the response to the T-independent antigen trinitrophenyl-lipopolysaccharide. This treatment also impairs humoral responsiveness at peripheral lymphoid sites in addition to inhibiting natural killer cell activity. However, 6-OHDA treatment in vivo does not affect in vitro mixed lymphocyte responsiveness, mitogen-induced lymphocyte activation or antigen presentation by macrophages.

Modulation of T-suppressor cell activity by central nervous system catecholamine depletion.

This study extends our previous findings, which indicate that depletion of CNS catecholamines has a marked inhibitory effect on humoral immune responsiveness. These data show that depletion of CNS catecholamines by injection of 6-hydroxydopamine (6-OHDA) into the cisterna magna in conjunction with immunization enhances the activity of a population of splenic T-suppressor cells as evidenced by the transfer of these cells to normal recipients. Increased suppressor cell activity does not result solely from 6-OHDA treatment, but rather requires concomitant immunization. Further characterization shows that these suppressor cells are not antigen specific. Hypophysectomy abrogates the effects of 6-OHDA injection suggesting that catecholamine depletion modulates immune function via the release of pituitary hormones. Thus, depletion of CNS catecholamines impairs immune responsiveness by inducing enhanced T-suppressor cell activity, providing additional evidence of the involvement of the CNS in regulation of immune responsiveness.