Effect of vanadate of PHA-induced proliferation of human lymphocytes from young and old subjects

The effect of sodium orthovanadate on mitogen-induced proliferation of lymphocytes from young and old human subjects is reported. We found that vanadate is not mitogenic per se; it has an inhibitory effect during the first 3 days of culture, when both differentiation and proliferation take place; it enhances DNA synthesis, acting as a co-mitogen, in the following days of culture, when proliferation prevails. In spite of the fact that lymphocytes from the two groups differ in their responsiveness to PHA and in the activity of (Na+,K+)ATPase, no difference was found as for the effects of vanadate.     

Diverse effects of three furocoumarins on human lymphocyte proliferation

The biological effects of three furocoumarins on the proliferation of human normal peripheral blood lymphocytes have been investigated. Mitogen-stimulated human lymphocytes were assayed "in vitro" by measuring 3H-thymidine (3H-TdR) incorporation in the presence and in the absence of 15-30 microM 3-carbethoxypsoralen (3-CPs), trimethylangelicin (TMA) and psoralen (PSR) with and without UV-A irradiation (365 nm). The three furocoumarins differ in their ability to form mono- and bi-functional adducts with DNA pyrimidine bases and in producing reactive species of oxygen. At low furocoumarin doses and short times of UV-A irradiation (15-30 sec) used in the present study, 3-CPs did not affect 3H-TdR incorporation in PHA-stimulated human lymphocytes, TMA strongly inhibited 3H-TdR incorporation, while, unexpectedly, PSR increased 3H-TdR incorporation in the absence of irradiation, likely acting, under these experimental conditions, as a co-mitogen.     

DNA repair after gamma irradiation in lymphocytes exposed to low-frequency pulsed electromagnetic fields

The effect of exposure to extremely low-frequency pulsed electromagnetic fields (EMFs) on DNA repair capability and on cell survival in human lymphocytes damaged in vitro with gamma rays was studied by two different micromethods. In the first assay, which measures DNA repair synthesis (unscheduled DNA synthesis, UDS), lymphocyte cultures were stimulated with phytohemagglutinin (PHA) for 66 h and then treated with hydroxyurea (which blocks DNA replication), irradiated with 100 Gy of 60Co, pulsed with [3H]thymidine ([3H]TdR), and then exposed to pulsed EMFs for 6 h (the period in which cells repaired DNA damage). In the second assay, which measures cell survival after radiation or chemical damage, lymphocytes were first irradiated with graded doses of gamma rays or treated with diverse antiproliferative agents, and then stimulated with PHA, cultured for 72 h, and pulsed with [3H]TdR for the last 6 h of culture. In this case, immediately after the damage induced by either the radiation or chemicals, cultures were exposed to pulsed EMFs for 72 h, during which cell proliferation took place. Exposure to pulsed EMFs did not affect either UDS or cell survival, suggesting that this type of nonionizing radiation--to which humans may be exposed in the environment, and which is used for both diagnostic and therapeutic purposes--does not affect DNA repair mechanisms.     

Formation of wheat protein bodies: Involvement of the Golgi apparatus in gliadin transport

Developing wheat (Triticum aestivum L.) endosperm was examined using ultrathin sections prepared from tissues harvested at 5, 9, 16 and 25 d after flowering. Protein bodies were evident by 9 d and displayed a variety of membranous structures and inclusions. The Golgi apparatus was a prominent organelle at all stages, and by 9 d was associated with small electron-dense inclusions. By immunocytochemical techniques, gliadin (wheat prolamine) was localized within these vesicles and in homogeneous regions of protein bodies, but not in the lumen of the rough endoplasmic reticulum. The protein bodies appear to enlarge by fusion of smaller protein bodies resulting in larger, irregular-shaped organelles. The affinity of the Golgi-derived vesicles for gliadin-specific probes during the period of maximal storage-protein synthesis and deposition indicates that this organelle includes the bulk, if not all, of the gliadin produced. The involvement of the Golgi apparatus in the packaging of gliadins into protein bodies indicates a pathway which differs from the mode of prolamine deposition in other cereals such as maize, rice and sorghum, and resembles the mechanism employed for the storage of rice glutelin and legume globulins.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - DAF days after flowering     

Purification and partial characterization of serum monocytotropic factor, a platelet-derived cyclooxygenase-inducing polypeptide

It has previously been shown that a heat- and acid-stable component of human and animal sera was capable of stimulating prostanoid biosynthesis in human blood monocytes, very probably by a mechanism involving cyclooxygenase induction. Many physico-chemical characteristics of this factor are similar to those of identified platelet factors. Here we show that human platelets are a rich source of this factor (serum monocytotropic factor) and that results from experiments using arachidonic acid or thrombin as releasers are consistent with its presence in platelet membranes. Serum monocytotropic factor has been purified 1500-fold by three chromatographic steps. Purification was more difficult when starting from platelet releasates or lysates. The purified serum monocytotropic factor had an apparent molecular mass of 70,000 as judged by Sephadex G-75 chromatography and by polyacrylamide gel electrophoresis; however, when subjected to HPLC on a gel permeation column in the presence of 6 M urea, one major peak corresponding to a relative molecular mass (Mr) of 30,000-35,000 was observed, which suggests a homodimeric structure. It is therefore very likely that human platelets store, in addition to the two well-identified polypeptide growth factors, platelet-derived growth factor and transforming growth factor-beta, a third polypeptide capable of regulating prostanoid production in monocytes.     

Regulation of thromboxane A2 biosynthesis in platelet-free human monocytes and the possible role of polypeptide growth factor(s) in the induction of cyclooxygenase system

It has previously been shown that platelet-free human monocytes, when properly incubated in the presence of animal and human sera, became capable of producing large amounts of thromboxane A2 and prostaglandin E2. The characteristics of these processes are reported here. Prostaglandin biosynthesis was time and cell concentration dependent; 24 h of incubation at 37 degrees C and 0.5 X 10(6) cells per ml medium were found to give the most reproducible results. Human monocytes produced thromboxane A2 and prostaglandin E2 in a typical ratio which ranged from 2.0 to 5.0 (28 experiments). Animal and human sera were similarly effective, while serum obtained from platelet-free blood was much less active. The activity of all sera tested was stable to heating (100 degrees C for 2-10 min) and extreme pH values (pH 2 and 11). It was unstable when the serum was heated at pH 11 and after 2-mercaptoethanol treatment. These observations prompted us to check the effect of polypeptide growth factors having properties similar to those reported above, such as platelet-derived growth factor, fibroblast growth factor, epidermal growth factor as well as insulin and transferrin. None of these, alone or in various combinations, was capable of eliciting a stimulation comparable with that of serum. Stimulation due to sera was, as expected, dose dependently inhibited by acetylsalicylic acid and more efficiently by indomethacin; unexpectedly it was also inhibited by protein synthesis inhibitors such as actinomycin D and cycloheximide in conditions under which no toxic effect of the drugs was evident. On the basis of these results we conclude that: (a) polypeptide growth factor(s) with a molecular weight at least 30 000 (as judged by Amicon ultrafiltration) is involved in the regulation of prostaglandin biosynthesis); (b) such a factor(s) acts by inducing rather than by activating the cyclooxygenase system.     

Microheterogeneity of the mammalian high mobility group (HMG) proteins 1 and 2 investigated by reverse-phase high performance liquid chromatography

Microheterogeneity within the high mobility group (HMG)-1 and HMG-2 groups of nonhistone chromatin proteins has been investigated using reverse-phase high-performance liquid chromatography (RP-HPLC) under conditions (acetonitrile elution with 0.1% trifluoroacetic acid (TFA) as the counter ion) which separate proteins primarily on the basis of differences in their overall hydrophobicity. RP-HPLC proved to be a fast and efficient means for separating multiple subspecies of both the HMG-1 and HMG-2 proteins from both crude nuclear extracts and from ion-exchange column "purified" protein samples obtained from different types of mammalian cell nuclei. In crude nuclear extracts at least eight different HMG-2 protein species (two major and six minor), but only one major HMG-1 species, could be resolved by RP-HPLC. Three of the minor HMG-2 protein species could be isolated in "pure" form from crude extracts in one RP-HPLC step whereas under the same conditions the two major HMG-2 peaks (as well as the other minor species) were contaminated with either HMG-1 or HMG-3 (a degradation product of HMG-1). In crude extracts the major HMG-1 fraction always seems to be contaminated with one of the HMG-2 subfractions. RP-HPLC analysis of apparently "pure" protein preparations isolated by ion-exchange chromatography techniques revealed that "pure" HMG-1 can be resolved into at least three different protein species and "pure" HMG-2 into at least four different species. Amino acid analyses of different resolvable forms of the HMG proteins were not inconsistent with the suggestion that at least some of these may be primary sequence variants of the individual proteins, but other possibilities also exist.     

Inhibition of cell proliferation by D-ribose and deoxy-D-ribose

D-Ribose and deoxy-D-ribose inhibited DNA, RNA, and protein synthesis in a wide variety of cells (dividing and nondividing, normal and neoplastic, freely floating and substrate adhering, human and murine) at concentrations at which other monosaccharides have little or no effect. Inhibition was irreversible and proportional to the sugar concentration and time of contact. However, the first effects were seen only after 24 hr of incubation and progressed slowly to cell death. Whether the two sugars share the same mechanisms of action is not known. In any case, they deeply disturb metabolic processes in both dividing and nondividing cells.