Methionyl-transfer ribonucleic acid deficiency during G1 arrest of Saccharomyces cerevisiae.

The mesl- mutants of Saccharomyces cerevisiae cease division and accumulate in the G1 interval of the cell cycle when deprived of methionine or shifted from 23 to 36 degrees C in the presence of methionine. Synchronous cell cycle arrest results from a deficiency of charged methionyl-transfer ribonucleic acid (methionyl-tRNAMet) as shown by direct measurement of the in vivo pools of methionine, S-adenosylmethionine, and methionyl-tRNAMet. The deficiency of methionyl-tRNAMet in these cells is the consequence of a lesion in a single gene, mes1. mes1 appears to be the structural gene for the methionyl-tRNA synthetase because some revertants of this mutation exhibited a thermolabile methionyl-tRNA synthetase in vitro. A sufficient hypothesis to explain these and previous results is that the control of cell division by S. cerevisiae in response to nutrient limitation is mediated through aminoacyl-tRNA or subsequent steps in protein biosynthesis.     

Angiotensin-like activity in resistance vessels

Summary Angiotensin II (ANG II) was localized immunocytochemically in kidney and various other organs of the chinese hamster. In the kidney ANG II-like activity was found in the epitheloid cells of the juxtaglomerular apparatus as well as in the media i.e. the smooth muscle cells of arcuate and interlobular arteries and afferent arterioles. ANG II-like activity was also observed in the medial muscle cells of resistance vessels in other organs and tissues such as submandibular gland and brown adipose tissue. The site of synthesis of ANG II needs to be investigated but the data point to the possibility of an intracellular function of ANG II in smooth muscle cells of blood vessels.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Comparison of 35S and biotin as labels for in situ hybridization: use of an HPV model system.

Colorimetric in situ hybridization is a method of potential importance in diagnosis and research. The largest criticism of the method has been a perceived loss of sensitivity compared with autoradiographic techniques. Our more positive experience with automation of colorimetric in situ hybridization led us to undertake a direct comparison of the sensitivity of 35S- and biotin-labeled probes. Serial sections of formalin-fixed, paraffin-embedded cell pellets from four human cervical carcinoma cell lines with known copies of HPV (CaSki, 400-600 copies HPV 16; HeLa, 10-50 copies HPV 18; SiHa, 1-2 copies HPV 16; HTB31, no known copies HPV) were hybridized with protocols optimized for autoradiographic or colorimetric detection. Both methods gave comparable results, with differences in each technique seen at the limits of sensitivity. The 1-2 copies of HPV 16 per SiHa cell can be detected with both methods; however, grain counting is required for interpretation of the autoradiographic result. This degree of sensitivity for colorimetric in situ hybridization in formalin-fixed, paraffin-embedded material is achieved through careful optimization of probe size and labeling, adequate tissue digestion, and removal of background. Autoradiography may be preferred in situations where quantitation is required, but colorimetric detection retains the advantages of speed, potential for automation, and improved localization of signal with comparable sensitivity.     

Gelation of xanthan with trivalent metal ions

In-situ gelation of semidilute xanthan solutions with trivalent chromium, aluminum or iron ions was studied by rheology and UV-spectroscopy. Measurements of the elastic modulus of xanthan gel cylinders prepared by dialysis against the complexing ion at pH values from 2 to 6 indicate that monomeric species of the ion are ineffective, whereas dimeric or higher oligomeric species are effective in crosslinking the polysaccharide. When chromium was used as the crosslinking species, the dependence of the gelation rate on the ionic concentration followed a power law with a coefficient of 1·7. The gelation time and the gelation rate were found to extrapolate to zero at 1 m Cr for 2·5 mg/ml xanthan. The limiting concentration of xanthan needed for gelation with 5 m Cr(III) at 20°C was estimated as 0·35 mg/ml. This critical xanthan concentration is close to the overlap concentration c^* estimated from the experimentally determined intrinsic viscosity [η] using c^* = 1·4/[η]. An apparent activation energy for crosslinking of xanthan was calculated as E_a = 42 kJ/mol and E_a = 108 kJ/mol for Cr and Al ions, respectively. The fractal dimensionality of xanthan-Cr at the sol-gel transition was estimated as 1·3 applying the Chambon-Winter criterion for gelation, thus indicating that this gelation criterion is applicable also to stiff-chain polysaccharides such as xanthan.     

Antagonistic effect of cocultivation on mitomycin C-induced aberration rate in cells of a patient with Fanconi's anemia and in Chinese hamster ovary cells

Summary The rate of spontaneous chromosomal aberrations in fibroblasts of a patient with Fanconi's anemia was slightly reduced after cocultivation with Chinese hamster ovary (CHO) cells. However, after mitomycin C treatment, a significant reduction of induced chromosomal damage was found in the FA cells while a significant increase was observed in the CHO cells. This antagonistic effect could be attributed to some diffusible agent(s). The results are discussed with respect to the underlying mechanism of the disease.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Different pathways for iNOS-mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression

Co-localization of activated microglia and damaged neurones seen in brain injury suggests microglia-induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co-cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)-derived NO aloneis responsible for neuronal death from interferon gamma (IFNgamma) +lipopolysaccharide (LPS)-activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO-mediated toxicity to immature neurones, MK801 rescues 60-70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO-donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO-mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.     

Silver enhances stamen development in female white campion (Silene latifolia [Caryophyllaceae])

Sex expression in the dioecious plant white campion (Silene latifolia Poiret subsp. alba) appears to be insensitive to exogenous applications of auxins, cytokinins, gibberellic acid, and ethylene; however, silver thiosulfate (Ag(2)S(2)O(3)), an ethylene inhibitor, enhanced stamen development in female white campion. In wild-type females, stamen development is arrested before the microspore mother cells are formed. In contrast, stamens of Ag(2)S(2)O(3)-treated females completed meiosis and produced microspores. Stamen development for these females was incomplete, however, and pollen did not mature. Ag(2)S(2)O(3) stimulated stamen development to the same extent in asexual white campion mutants that retained a Y chromosome but had lost Y-linked genes needed for early stages of stamen development. Although Ag(2)S(2)O(3) can inhibit ethylene signaling, the enhancement of stamen development in female white campion cannot be explained as a loss of ethylene response because no other ethylene inhibitor tested (1-methylcyclopropene, trans-cyclooctene, aminoethoxyvinylglycine, and cobalt chloride) caused stamens to develop in female plants. In addition, application of other metal ions could not enhance stamen development. Therefore, the effect we observed on female white campion was specifically caused by silver ions but not by their action on ethylene signaling.