Continued withdrawal from the cell cycle and regulation of cellular genes in mouse erythroleukemia cells blocked in differentiation by the c-myc oncogene.

Constitutive expression of the c-myc oncogene blocks dimethyl sulfoxide (DMSO)-induced differentiation of mouse erythroleukemia (MEL) cells. During the first 12 h of treatment with DMSO, MEL cells undergo a temporary decrease in the level of c-myc mRNA, followed by a temporary withdrawal from the cell cycle. We found the same shutoff of DNA synthesis during the first 12 to 30 h after DMSO induction in normal MEL cells (which differentiate) and in c-myc-transfected MEL cells (which do not differentiate). We also examined whether deregulated c-myc expression grossly interfered with the regulation of gene expression during MEL cell differentiation. We used run-on transcription assays to monitor the rate of transcription of four oncogenes (c-myc, c-myb, c-fos, and c-K-ras); all except c-K-ras showed a rapid but temporary decrease in transcription after induction in both c-myc-transfected and control cells. Finally, we found the same regulation of cytoplasmic mRNA expression in both types of cells for four oncogenes and three housekeeping genes associated with growth. We conclude that in the MEL cell system, the effects of deregulated c-myc expression do not occur through a disruption of cell cycle control early in induction, nor do they occur through gross deregulation of gene expression.

Calcium phosphate supplementation increases faecal Lactobacillus spp. in a randomised trial of young adults.

The aim of the studies was to determine the effects of calcium carbonate and calcium phosphate supplementation on faecal Lactobacillus spp., with and without a probiotic supplement, in healthy adults. Study 1 comprised of a randomised, double-blind, crossover design; participants (n=15) received 2 capsules/d of 250 mg elemental calcium as calcium carbonate (Ca1) and calcium phosphate (Ca2) each for 2-week periods, with 2-week baseline and washout periods. Study 2 was a randomised, double-blind, crossover design; participants (n=17) received 2 capsules/d of Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011 (probiotic) alone, the probiotic with 2 capsules/d of Ca1, and probiotic with 2 capsules/d of Ca2 each for 2-week periods with 2-week baseline and washout periods. In both studies, stools were collected during the baseline, intervention and washout periods for Lactobacillus spp. quantification and qPCR analyses. Participants completed daily questionnaires of stool frequency and compliance. In Study 1, neither calcium supplement influenced viable counts of resident Lactobacillus spp., genome equivalents of lactic acid bacteria or stool frequency. In Study 2, faecal Lactobacillus spp. counts were significantly enhanced from baseline when the probiotic was administered with Ca2 (4.83±0.30, 5.79±0.31) (P=0.02), but not with Ca1 (4.98±0.31) or with the probiotic alone (5.36±0.31, 5.55±0.29) (not significant). Detection of L. helveticus R0052 and L. rhamnosus R0011 was significantly increased with all treatments, but did not differ among treatments. There were no changes in weekly stool frequency. Calcium phosphate co-administration may increase gastrointestinal survival of orally-administered Lactobacillus spp.

Increased retinoblastoma gene expression is associated with late stages of differentiation in many different cell types.

The retinoblastoma (Rb) gene is a recessive oncogene or tumor-suppressor gene whose inactivation leads to the development of tumors. Recently, evidence pointing to a role for the Rb gene in the differentiation of certain human cell types has been presented. We have studied three mouse cell lineages to determine whether there is a correlation between Rb gene expression and differentiation. We find that induction of mouse erythroleukemia cell differentiation with either dimethylsulfoxide (DMSO) or hexamethylene bisacetamide (HMBA) leads to increased expression of Rb mRNA. Increased expression of Rb mRNA was also found in S2 myoblasts induced by mitogen depletion to become differentiated myotubes. In the B-cell lineage, Rb expression is low in pre-B and B cell lines but high in plasmacytomas, which represent late stages of B cell differentiation. Thus, in all three lineages (erythroid, muscle, and B-cell) late stages of differentiation are associated with increased amounts of Rb mRNA.

Purification and characterization of ternary complexes containing accurately initiated RNA polymerase II and less than 20 nucleotides of RNA.

We have previously demonstrated that transcription of the adenovirus type 2 (Ad2) late promoter in vitro under UTP-limiting conditions results in pauses by the elongating RNA polymerase II between positions +6 and +17. We report here the purification of complexes between the paused RNA polymerase and a 260 base-pair Ad2 promoter-bearing DNA fragment. The procedure involves sedimentation through sucrose gradients, electrophoresis in agarose gels, and electroelution from the gels; the final complex pool is completely active in chain elongation. We observe a sharp discontinuity in complex stability during purification as a function of the number of bases added to the growing chains: complexes in which the polymerase has added more than ten bases are stable and are active in chain elongation even after the electroelution step, whereas complexes containing seven or fewer bases dissociate very easily. When purified complexes are extensively digested with proteinase K their electrophoretic mobility is increased considerably, yet they remain fully active in chain elongation. If purified complexes are digested with DNase I their electrophoretic mobility does not change. When the nuclease-treated complexes are allowed to continue chain elongation, they are able to add approximately 20 more bases to the nascent chains.

Alkaloid studies. 8. Isolation and characterization of alkaloids of Tabernaemontana heyneana Wall and antifertility properties of coronaridine.

PIP: In this study, the roots of Tabernaemontana heyneana Wall were examined and the isolation and identification of additional indole alkaloids and some pharmacological properties of coronaridine are described. Extraction of the roots yielded the alkaloids coronaridine, voacangine, ibogamine, 19-oxocoronaridine, and the pseudoindoxyl of voacangine. An acqueous ethanolic extract of the roots was found to prevent fertilization of adult female rats when administered orally. After the residue of this extract was treated chromatographic fractionation on silica gel yielded the alkaloid coronaridine. When administered to adult female rats, orally, coronaridine hydrochloride at levels of 5 mg/kg/day or above prevented pregnancy. Voacangine, assayed by the same procedure, did not prevent pregnancies. Data indicated that coronaridine was weakly estrogenic.^ieng

A new orally active male antifertility agent.

PIP: CL 88,236 (1-1-amino-3-chloro-2-propanol HC1) was tested orally for antifertility in male rats, mice and hamsters. It was given orally in propylene glycol either daily for 14 days with mating on Days 7-14, (or for 7 days with mating on Days 2, 4, and 6, or for 7 days every other week for 3 weeks in withdrawal tests. In male rats, 5 mg per kg for 14 days reduced number of pregnant females 50%, and at 10-80 mg per kg males were completely sterile. Libido, coitus, and ejaculation were normal, but fragmented sperm, spermatogenic arrest, and granuloma-like epididymal lesions appeared at 40 and 80 mg per kg. Serial matings showed that sterility developed within 6 days and lasted for 1 week after withdrawal. Sterility was maintained by treating rats with CL 88,236 on alternating weeks. Male mice and hamsters were sterilized by 300 mg per kg per day for 14 days, without toxicity. Sterility was apparently mediated by affecting epididymal and vas sperm stores.^ieng

Constitutive c-myc oncogene expression blocks mouse erythroleukaemia cell differentiation but not commitment.

Mouse erythroleukaemia cells (also called Friend cells) can be isolated from the spleen of certain strains of mice that have been infected with the Friend virus complex. The cells resemble proerythroblasts and, when exposed to dimethyl sulphoxide (DMSO) or a variety of other chemicals, can be induced to undergo a programme of differentiation which closely resembles the final stages of normal erythropoiesis. This includes the cessation of proliferation and large increases in the production of messenger RNA for both alpha- and beta-globin. In addition, DMSO induces a rapid (less than 2 h) decrease in c-myc mRNA levels. The c-myc oncogene is expressed in the majority of proliferating normal cells and altered expression of the gene has been implicated in the genesis of a wide variety of tumours. To study the influence of oncogene activation on differentiation, we have transfected viral-promoter-driven c-myc genes into mouse erythroleukaemia cells. Constitutive c-myc expression was found to block DMSO-induced differentiation.

Promoter-proximal pausing by RNA polymerase II in vitro: transcripts shorter than 20 nucleotides are not capped.

We have synthesized RNA from cloned adenovirus 2 late promoter DNA in an in vitro transcription extract under UTP-limiting conditions. Under these circumstances, almost all of the alpha-amanitin-sensitive RNA produced is shorter than 20 nucleotides; most of these short transcripts are present in four species, 6, 7, 13, and 17 nucleotides. These short RNAs are initiated at the adenovirus 2 promoter, as judged by partial sequence analysis and by the abolition of their synthesis upon cleavage of the template DNA at sites which also abolish the production of full-length transcripts. All of the short transcripts can be chased, with excess UTP, into 197-base run-off transcripts; thus, these RNAs are precursors of full-length transcripts and not synthetic "dead ends." Significantly, none of these short RNAs is capped or 2'-O-methylated. However, 79-base run-off transcripts synthesized from this promoter with nonlimiting NTP levels are fully capped.

Transcription initiation by RNA polymerase II in vitro. At least two nucleotides must be added to form a stable ternary complex.

We have prepared RNA polymerase II preinitiation complexes by incubating templates containing the adenovirus 2 major late promoter with HeLa cell nuclear extracts in the absence of nucleoside triphosphates. These preinitiation complexes are partially purified by gel filtration and are then provided with the appropriate substrates to allow either one or two phosphodiester bonds to be formed. When substrates that allow only one bond to form are used, no stable ternary complex is obtained and no RNA is made that can be incorporated into longer RNA chains. A stable complex is obtained, however, if the RNA polymerase can make two bonds. The production of a stable ternary complex requires ATP or dATP and is inhibited by alpha-amanitin. In the course of exploring the energy requirement for initiation we found that dATP may be incorporated, in the absence of ATP, as the initial base of the RNA. However, deoxyribonucleotides are not appreciably incorporated into the body of the transcript after the first two bases have been added to the growing chain.