Differential effect of growth factors on growth stimulation and phenotypic stability of glutamine-synthetase-positive and -negative hepatocytes in primary culture

In rat liver parenchyma, two subpopulations of hepatocytes can be distinguished by the absence or presence of the marker enzyme, glutamine synthetase (GS). Hepatocytes in the perivenous zone immediately adjacent to the hepatic venules in the liver acinus are positive for GS. Using autoradiography in combination with immunocytochemistry, the response of these two hepatocyte populations (GS positive and GS negative) to a variety of growth factors (defined compounds or complex stimuli) was investigated in vitro. Irrespective of the individual growth-promoting activity (which varied considerably), all stimuli led to much higher labeling indices in GS-negative cells as compared to GS-positive cells. In GS-negative cells, the strongest effect was exerted by serum obtained from partially hepatectomized rats (labeling index, 67%) and the conditioned media of JM1 and JM2 hepatoma cells (63%-82%), followed by a combination of insulin and either norepinephrine (46%) or epidermal growth factor (EGF; 42%). In contrast, serum had the weakest influence on GS-positive cells (0.3%), while the other potent stimuli enhanced the labeling index of these cells by between 6% and 15% within 48 h. The percentage of labeled nuclei was higher in mononucleated than in binucleated GS-positive hepatocytes. The time course of thymidine incorporation was also different for the two subpopulations. Under all growth-promoting conditions, the stimulation of GS-negative cells peaked between 72 and 96 h, while it increased continuously in GS-positive cells for at least 120 h, particularly in the case of serum. In proliferating cultures, both the absolute and the relative number of GS-positive hepatocytes decreased, while no such effect was found in various nonproliferating control cultures maintained at low and high cell density. Similar results were found for GS activity. In contrast, the hormonal induction of tyrosine aminotransferase (TAT) was not affected. It is suggested that these differences in the growth response of GS-positive and -negative cells contribute to the acinar gradient in hepatocyte proliferation that occurs during liver regeneration. Furthermore, the striking phenotypic instability of GS-positive cells that have undergone DNA synthesis and mitosis supports the hypothesis that cellular reprogramming depends on passage through the cell cycle.     

Mycoplasmatales virus,MV-M1: Discovery inAcholeplasma modicum and preliminary characterization

A new virus, Mycoplasmatales virus-modicum 1 (MV-M1), was recovered from spontaneous plaques in lawns ofAcholeplasma modicum. Strain “mod” produced plaques onA. modicum strains but not on strains ofAcholeplasma laidlawii. Only MV-L3 of the three knownA. laidlawii viruses (MV-L1, MV-L2, and MV-L3) produced plaques onA. modicum. The MV-M1 virus was serologically distinct from the threeA. laidlawii viruses; filterable at 0.1 μm; partially sensitive to heat and Nonidet P-40; and chloroform labile. Spherical particles ranging from 105 to 160 nm were observed in electron micrographs of negatively stained preparations.     

Fertilization of poplar clones in the nursery

Summary Results of experiments with four poplar clones and various chemical fertilizers in a nursery in southern Greece are presented. At the end of the first growth period the heights of the four clones, without fertilizers, decreased in the order of I-214>I-262>cv. campeator > black poplar 1/64 with significant differences only between black poplar 1/64 and the rest of the clones.Of the fertilizer nutrients N, P, K and Mg only N improved heights of all clones significantly and especially of the clone I-214. One hundred and 200 kg of P fertilizer per ha had minimal or negative effect on height increase of all clones.Ammonium sulfate, ammonium nitrate and potassium nitrate all at 400 kg N per ha were found equally effective in improving height growth of the clone I-214 but ammonium nitrate is the N fertilizer of choice by its higher N content and relatively lower price.Ammonium nitrate at 200 kg N per ha, in two or three equal dosages, during the first growth period, June–July, gave the maximum height increase for two consecutive years of the clone I-214. Six hundred kgs, of N per ha reduced height increase of the same clone and increased losses of N, as NO₃ ^–, in drainage water.     

The molecular structure of (10R)-17β-hydroxy-5-methylene-4,10-cyclo-19-nor-4,5-seco-17α-pregn-20-YN-1-one

The crystal and molecular structure of (10R)-17β-hydroxy-5-methylene-4,10-cyclo-19-nor-4,5-seco-17α -pregn-20-YN-1-one has been determined by X-ray analysis in order to ascertain the configuration at C(10). As observed in most other 17α-pregn-20-yn-17β-ol structures, atom 0(17B) participates as a donor in a hydrogen bond trans to the C(16)-C(17) bond, and there is a short C(21)…0 intermolecular contact.     

THE RIBOSOMAL RNA OF HAMSTER-MOUSE HYBRID CELLS

The ribosomal RNA (rRNA) of a series of hamster-mouse somatic cell hybrids was studied. Mouse 28S rRNA was separated from its hamster counterpart by a two-step procedure involving sucrose gradient centrifugation of ribosomes and polyacrylamide gel electrophoresis of rRNA. Both hamster and mouse types of rRNA were synthesized in the 11 hybrids tested, including hybrids containing only about one-half the haploid number of either mouse or hamster chromosomes. It appears that, for both hamster and mouse rRNA, when the chromosomes of one species constituted the majority of the chromosomes of a hybrid, a disproportionately higher percentage of rRNA of that species was present in the hybrid. Some hybrid clones, having a majority of mouse chromosomes, had a mouse rRNA cell concentration approximately four to five times higher than the concentration expected from linear extrapolation of the value found for the mouse parental cell line.     

Morphology,morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma

Summary The host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.The authors wish to thank Christiana Ulness and Andrea Erickson for expert technical assistance and Arnold Schmidt for the operation of the scanning electron microscope. This work was supported by grants from the U.S.P.H.S.: AI 09586, AI 10743, and AI 06720     

Cellular redox activity of coenzyme Q10: effect of CoQ10 supplementation on human skeletal muscle

In this paper, we report results obtained from a continuing clinical trial on the effect of coenzyme Q 10 (CoQ 10 ) administration on human vastus lateralis (quadriceps) skeletal muscle. Muscle samples, obtained from aged individuals receiving placebo or CoQ 10 supplementation (300 mg per day for four weeks prior to hip replacement surgery) were analysed for changes in gene and protein expression and in muscle fibre type composition. Microarray analysis (Affymetrix U95A human oligonucleotide array) using a change in gene expression of 1.8-fold or greater as a cutoff point, demonstrated that a total of 115 genes were differentially expressed in six subject comparisons. In the CoQ 10 -treated subjects, 47 genes were up-regulated and 68 down-regulated in comparison with placebo-treated subjects. Restriction fragment differential display analysis showed that over 600 fragments were differentially expressed using a 2.0-fold or greater change in expression as a cutoff point. Proteome analysis revealed that, of the high abundance muscle proteins detected (2086 ±115), the expression of 174 proteins was induced by CoQ 10 while 77 proteins were repressed by CoQ 10 supplementation. Muscle fibre types were also affected by CoQ 10 treatment; CoQ 10 -treated individuals showed a lower proportion of type I (slow twitch) fibres and a higher proportion of type IIb (fast twitch) fibres, compared to age-matched placebo-treated subjects. The data suggests that CoQ 10 treatment can act to influence the fibre type composition towards the fibre type profile generally found in younger individuals. Our results led us to the conclusion that coenzyme Q 10 is a gene regulator and consequently has wide-ranging effects on over-all tissue metabolism. We develop a comprehensive hypothesis that CoQ 10 plays a major role in the determination of membrane potential of many, if not all, sub-cellular membrane systems and that H 2 O 2 arising from the activities of CoQ 10 acts as a second messenger for the modulation of gene expression and cellular metabolism.     

Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Corpus luteum susceptibility to prostaglandin F2α (PGF2α) luteolysis in hysterectomized prepuberal and mature gilts

The susceptibility of induced corpora lutea (CL) of prepuberal gilts and spontaneously formed CL of mature gilts to prostaglandin F_2α (PGF_2α) luteolysis was studied. Prepuberal gilts (120 to 130 days of age) were induced to ovulate with Pregnant Mare Serum Gonadotropin and Human Chorionic Gonadotropin (HCG). The day following HCG was designated as Day 0. Mature gilts which had displayed two or more estrous cycles of 18 to 22 days were used (onset of estrus = Day 0). Gilts were laparotomized on Day 6 to 9, their CL marked with sterile charcoal and totally hysterectomized. On Day 20, gilts were injected IM with either distilled water (DW), 2.5 mg PGF_2α or 5.0 mg PGF_2α. An additional group of prepuberal gilts was injected with 1.25 mg PGF_2α, a dose of PGF_2α equivalent, on a per kilogram body weight basis, to the 2.5 mg PGF_2α dose given to the mature gilts. The percentages of luteal regression on Day 27 to 30 for mature and prepuberal gilts given DW, 2.5 mg PGF_2α and 5.0 mg PGF_2α were 0.0 vs 4.4, 43.5 vs 96.8 and 47.7 vs 91.6, respectively; the percentage of luteal regression for the prepuberal gilts given 1.25 mg PGF_2α was 75.1. These results indicate that induced CL of the prepuberal gilt were more susceptible to PGF_2α luteolysis than spontaneously formed CL of the mature gilt and that pregnancy failure in the prepuberal gilt could be due to increased susceptibility of induced CL to the natural luteolysin.