Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Ciliary Neuronotrophic Factor Stimulates Choline Acetyltransferase Activity in Cultured Chicken Retina Neurons

It has been demonstrated that cultured cholinergic retinal neurons from 8-day-old chicken embryos respond to a polypeptide factor present in retinal cell-conditioned medium (RCM) and in retinal extracts. Compared with control cultures, the activity of acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; ChAT) is enhanced more than twofold in neuronal retinal cultures grown for 7 days in the presence of RCM. The present study demonstrates that both ciliary neuronotrophic factor (CNTF), which is characterized by its trophic activity on parasympathetic ciliary neurons, and RCM exhibit identical stimulatory effects on ChAT activity in retinal monolayer cultures. Similarly, RCM supports the in vitro survival of ciliary neurons to the same extent as CNTF. The active species in RCM has a molecular weight (20,900 +/- 1,000) identical to that of CNTF, as determined by preparative sodium dodecyl sulfate gel electrophoresis. The results indicate that cholinergic retinal neurons represent a central neuronal target for CNTF or a closely related protein.     

Lithium Stimulation of Membrane-Bound Phospholipase C from PC 12 Cells Exposed to Nerve Growth Factor

LiCl stimulated the formation of inositol monophosphate in PC12 cells that had been exposed to nerve growth factor (NGF) for 4-5 days. Half-maximal accumulation was observed at approximately 8 mM LiCl. Stimulation of formation of inositol bisphosphate plus inositol trisphosphate was half-maximal at approximately 1 mM LiCl. With membranes isolated from PC12 cells differentiated with NGF, the hydrolysis of added phosphatidylinositol 4,5-bisphosphate (PIP2) was stimulated by LiCl in a biphasic manner, with the first stimulation half-maximal at approximately 0.7 mM and the second half-maximal at approximately 15 mM LiCl. The apparent Km for PIP2 was lowered in the presence of 1.1 mM LiCl from approximately 200 to approximately 70 microM. Membranes from cells grown in the absence of NGF did not respond to LiCl. Although observations with intact cells are difficult to interpret without ambiguity, the results obtained with isolated membranes support our interpretation of the stimulatory action of lithium in the intact PC12 cells.     

Stimulation of Inositol Incorporation into Lipids of PC 12 Cells by Nerve Growth Factor and Bradykinin

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.     

Platelet-Activating Factor Antagonist BN52021 Decreases Accumulation of Free Polyunsaturated Fatty Acid in Mouse Brain During Ischemia and Electroconvulsive Shock

We have investigated the effects of the specific platelet-activating factor (PAF; 1-alkyl-2-acetyl-glycerophosphocholine) antagonist BN52021 on free fatty acid (FFA) and diacylglycerol (DG) accumulation and on the loss of fatty acids from phosphatidylinositol-4,5-bisphosphate (PIP2) in mouse brain. Mice were pretreated with BN52021 (10 mg/kg, i.p.) 30 min before electroconvulsive shock (ECS) or postdecapitation ischemia. These procedures cause rapid breakdown of PIP2 and accumulation of FFA and DG. Lipid extracts were prepared from microwave-fixed cerebrum and fractionated by TLC, and the fatty acid methyl esters were prepared by methanolysis and quantified by capillary GLC. In saline or vehicle (dimethyl sulfoxide)-treated mice, ECS caused marked accumulation of FFA and DG and loss of mainly stearic (18:0) and arachidonic (20:4) acids from PIP2. BN52021 pretreatment of ECS-treated mice decreased the accumulation of free palmitic (16:0), 18:0, 20:4, and docosahexaenoic (22:6) acids with no effect on the fatty acids in DG or the loss of PIP2. BN52021 had no effect on basal levels of FFA, DG, or PIP2. One minute of postdecapitation ischemia induced PIP2 loss and accumulation of FFA and DG. BN52021 attenuated the accumulation of free 20:4 and 22:6 acids, decreased the content of oleic (18:1), 20:4, and 22:6 acids in DG, but had no effect on PIP2 loss. These data indicate that BN52021 reduces the injury-induced activation of phospholipase A2 and lysophospholipase, which mediate the accumulation of FFA in brain, while having a negligible effect on phospholipase C-mediated degradation of PIP2.     

Replication of ColE2 and ColE3 plasmids: Two ColE2 incompatibility functions

Summary We have identified and localized two incompatibility determinants (IncA and IncB) within a 1.3 kb segment of ColE2 sufficient for autonomous replication. The IncA determinant is localized in a region shorter than 250 bp and expresses incompatibility against both ColE2 and ColE3. The region which determines sensitivity to the IncA determinant seems to overlap with the region specifying the IncA determinant. The expression of the trans-acting factor(s) specifically required for replication of ColE2 interferes with expression of the IncA determinant against ColE2 but not against ColE3. The IncA determinant might be at least partly responsible for the copy number control of the plasmid. The IncB determinant is localized in a 50 bp region (origin) which is sufficient for initiation of replication in the presence of the trans-acting factor(s). The IncB determinant is specific for ColE2 and seems to be due to titration of the trans-acting essential replication factor(s) by binding.     

Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1

Summary The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1. It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression. This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein. These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein. The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

The BULT Melanoma: A Spontaneous Transplantable Tumor in Mice

The BULT melanoma originated at Brown University as a spontaneous, small black nodule on the tail of an adult female mouse of the LT/Ch strain. Histological examination of a portion of the tumor indicated that it was intradermal and consisted predominantly of heavily melanized, ovoid to fusiform cells with melanin-laden macrophages scattered among them. The BULT melanoma has been maintained in LT/Ch mice for approximately 5 years by periodic transplantation, at first subcutaneously on the flanks and, more recently, intramuscularly in the hind legs. The shift in transplantation site was made following a marked decline in the growth of subcutaneous grafts. The transplants have retained the uniform deep-black melanization and general histology of the primary melanoma. Numerous melanosomes at all stages of development are found within the melanoma cells. DOPA-positive cytoplasmic vesicles are abundant. Occasional autophagic vacuoles containing clusters of melanosomes are also present. A few metastases from the transplanted melanoma have been observed in lymph nodes and on one occasion in the lungs. When grown in vitro, BULT melanoma cells do not require special growth promoting agents (e.g., TPA; cAMP) in order to proliferate. The BULT melanoma differs in one or more respects from each of the other three transplantable spontaneous mouse melanomas widely used in cancer research. In addition, it arose in a strain of mice characterized by the spontaneous death of melanocytes while the latter are engaged in synthesizing eumelanin within hair follicles. Karyotypic analysis of cultured cells showed a modal chromosome number of 68 with a range of 58–72 chromosomes.     

Purification and characterization of lymphocytic clonal growth factor (LCGF) derived from human-human hybridoma SH-76 cells

Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma.