Species specificity of iron delivery in hybridomas

Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that HxH hybridomas do not respond to bovine transferrin a⁺ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.     

The biomechanical effects of deep tissue support as related to brow and facelift procedures

The adverse effects of increased tension across a healing wound are well known. However, the effect of closing a wound in layers in order to decrease tension on the epidermis has been a source of controversy. It is hypothesized that deep tissue support decreases skin tension upon wound closure. In order to clarify this issue, a two-part study was designed to address the immediate effects of deep tissue support in vitro using fresh-frozen cadavers and in vivo on patients undergoing scheduled surgery. Closing skin tension was measured at standard reference points in coronal brow lift and rhytidectomy procedures performed with and without galeal closure and superficial musculoaponeurotic system (SMAS) procedures, respectively. Deep tissue support was found to significantly (p less than 0.05) decrease skin tension at the time of skin closure at standard reference points in coronal brow lift and rhytidectomy procedures performed on fresh-frozen cadavers. Similar significant (p less than 0.05) decreases in closing skin tension also were found in vivo in patients undergoing similar surgical procedures. Stress relaxation was not found to play a significant role in contributing to this immediate decrease in closing skin tension. It would appear, therefore, that deep tissue support, in the form of galeal closure and an SMAS procedure in coronal brow lift and rhytidectomy procedures, respectively, provides increased viscoelastic support, producing immediate significant decreases in closing skin tension in these procedures. The beneficial effects on wound healing, scar formation, tension-related trophic skin changes, and possible improved long-term results are discussed.     

Co-immunolocalization of topoisomerase II and chloroplast DNA in developing,dividing and mature wheat chloroplasts

This paper describes the first localization of immunofluorescence of topoisomerase II in developing chloroplasts. In order to investigate the relationship between topoisomerase II and chloroplast DNA (ctDNA) replication during chloroplast development the 7-day-old wheat leaf was used. Topoisomerase II was immunolabelled and fluorescein tagged and the ctDNA simultaneously stained with 4,6-diamidino-2-phenylindole (DAPI) in the same sections. Topoisomerase II was detected at every stage of chloroplast development and maximal levels of topoisomerase II were found in chloroplasts at the time of ctDNA replication. Topoisomerase II was localized around the plastid periphery, exactly mirroring the position of the ctDNA. After chloroplast division both topoisomerase II and ctDNA are seen to be restricted to small discrete areas within the plastid, but at different sites. These findings strongly suggest a role for topoisomerase II in ctDNA decatenation prior to chloroplast division.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Effect of melphalan in vitro on induction of murine suppressor T cells by ConA

Summary The effect of treatment with melphalan in vitro on the activity of spleen cells from BALB/c mice was investigated. Incubation of spleen cells with 1.5–5 g melphalan/1×10⁷ inhibited subsequent mitogenic stimulation by ConA or PHA and the allogeneic response of BALB/c spleen cells against C57B1 target spleen cells. Incubation of spleen cells with ConA led to induction of suppressor T cells which when added to fresh cultures inhibited the allogeneic response. Preincubation of spleen cells with melphalan even at low concentrations (0.15–0.5 g 1×10⁷ cells) which do not directly affect mitogenic stimulation or allogeneic response partially inhibited the generation of suppressor T cells by ConA. Treatment with melphalan had no effect on already induced suppressor T cells as shown by incubation of spleen cells with melphalan (0.15–5 g/1×10⁷ cells) after incubation with ConA. Addition of cells treated with melphalan alone (without ConA) to fresh cultures led to an increase in the allogeneic response.     

Hemodynamics and vascular sensitivity to circulating norepinephrine in normal skin and delayed and acute random skin flaps in the pig

Cutaneous circulation in 4 X 10 cm skin samples and delayed and acute random skin flaps constructed on the flanks of castrated Yorkshire pigs (13.3 +/- 0.7 kg; n = 12) were studied during intravenous infusion (0.5 ml per minute) of 5% dextrose solution (vehicle) and 5% dextrose containing norepinephrine (1 microgram/kg per minute). Total and capillary blood flow and A-V shunt flow were measured by the radioactive microsphere technique 6 hours after the raising of 4 X 10 cm single-pedicle acute and delayed random skin flaps using the technique and calculations published previously. Fluorescein dye test was also performed to assess vascular perfusion. It was observed that the capillary blood flow in the single-pedicle delayed skin flaps was similar to that in the normal skin, and the maintenance of this normal skin blood flow was not due to the closing of A-V shunt flow in the delayed skin flaps. Similarly, the significant (p less than 0.01) decrease in capillary blood flow and distal perfusion in the acute skin flaps compared with the delayed skin flaps was not due to the opening of A-V shunts in the acute skin flaps. There was no evidence to indicate that A-V shunt flow per se was the primary factor for the regulation of capillary blood flow in the acute and delayed skin flaps in the pig. Our data seemed to indicate that tissue ischemia in the distal portion of acute skin flaps was likely the result of vasoconstriction of the small random arteries which supplied blood to arterioles and A-V shunts, and locally released neurohumoral substances may play an important role in the pathogenesis of vascular resistance and ischemia in the acute skin flaps.     

13C NMR Analysis of some simple tetrahydroisoquinolines

¹³C NMR resonances of 15 simple tetrahydroisoquinolines have been assigned on the basis of chemical shift theory, ¹³C-¹H coupling constants     

Cyclic AMP dependent regulation of mitosis in human lymphoid cells

Intracellular levels of cyclic AMP (cAMP), cAMP-dependent phosphodiesterase activity, and adenylate cyclase activity are examined in an established line of human lymphoid cells synchronized by either excess thymidine or by colcemid treatment. cAMP levels and adenylate cyclase activities during the two G periods are high when compared with the values in M. cAMP-dependent phosphodiesterase activity, which is low during early G 2, is shown to increase during G 2 and reach a maximum activity during M. Agents such as dibutyryl cAMP, 1-methyl-3-isobutyl xanthine, noradrenaline, and isopropyl noradrenaline, which increase the levels of intracellular cAMP were examined to determine their effects on mitosis and on DNA synthesis. In thymidine-synchronized cells the onset of mitosis is prevented by increasing or maintaining high levels of cAMP during G 2. The specificity of inhibition of DNA synthesis or mitosis by dibutyryl cAMP is a function of the time, during the cell cycle, when the analogue is added. The elevation of cAMP by methyl xanthine results in a more general inhibition of nucleic acid synthesis and mitosis. Although both catecholamine hormones inhibit mitosis, isopropylnoradrenaline also inhibits DNA synthesis while noradrenaline treatment does not result in such inhibition.     

Studies on the polyphenol metabolism of tissue cultures derived from the tea plant (Camellia sinensis L.)

1. The growth characteristics on various media of solid and liquid suspension cultures derived from the stem of the tea plant are described; chlorophyll and anthocyanin synthesis occurred in the light. 2. Only the simplest catechins and leucoanthocyanins were present in callus tissue, although oligomeric and polymeric leucoanthocyanin fractions were also represented. Light caused an increase in all monomeric components analysed, but inhibited polymerization of the leucoanthocyanins. 3. The polyphenol oxidase activity of cultures was comparable with that of the apical regions of the intact plant, and was inversely correlated with growth rate. 4. Growth was stimulated by hormonal variation, and inhibited by high concentrations of sucrose and by high light-intensity; polyphenol concentrations were generally inversely correlated with growth rate. 5. From the inability of callus tissue and of cultured root apices to synthesize complex catechins, it is inferred that complex catechin formation in intact plants is associated with the process of cell vacuolation.     

Chloroplast and cytoplasmic low-molecular-weight ribonucleic acid components of the leaf of Vicia faba L

1. A method for the extraction of plant nucleic acids and their separation on methylated-serum-albumin-kieselguhr columns is described. It is demonstrated that the characteristics of the elution profiles of material from the same source are consistently reproducible. 2. Major dissimilarities were found in the elution profiles of nucleic acids from root and from leaves of Vicia faba L. These dissimilarities were confirmed by polyacrylamide-gel electrophoresis. 3. Four distinct types of low-molecular-weight RNA were demonstrated to be present in leaves, clearly distinguished by their behaviour when chromatographed on methylated-serum-albumin-kieselguhr columns. (a) Both cytoplasmic and chloroplast ribosomes contained a low-molecular-weight RNA, and these components were distinct from each other. (b) The chloroplast possessed a unique ;soluble' RNA (i.e. RNA that is not precipitated by centrifugal forces that sediment ribosomes) which was not present in the rest of the cell. (c) A soluble component, probably transfer RNA, was found in both the chloroplasts and in the cytoplasm. 4. The components distinguishable by methylated-serum-albumin-kieselguhr column chromatography could not be distinguished by sucrose-density-gradient centrifugation.