Molecular fate of thidiazuron and its effects on auxin transport in hypocotyls tissues of Pelargonium × hortorum Bailey

Thidiazuron, a synthetic phenylurea-type cytokinin, has previously beenfound to induce somatic embryogenesis and organogenesis in a wide range ofplantspecies and to modulate the metabolism of endogenous auxins and cytokinins. Inspite of these findings, the precise mode of action of TDZ remainsundetermined.The current studies were undertaken to determine the fate of the TDZ moleculeand the effects of TDZ exposure on auxin transport in plants. The fate of tworadiolabelled versions of thidiazuron, [¹⁴C-5-thidiazol]-TDZ and[¹⁴C-U-phenyl]-TDZ, was investigated in sterile hypocotyl culturesofgeranium (Pelargonium×hortorumBailey). Radiolabelled TDZ was recovered from the tissue explants inethanol-insoluble, ethanol-soluble and chloroform fractions as well as inacidic, basic and neutral eluants from Dowex resins. Hypocotyl sections thathadbeen exposed to TDZ were found to accumulate more ¹⁴C-IAA from theculture medium and to translocate the auxin over a greater distance within thetissues. These data provide the first evidence that the TDZ molecule remainsintact in both a free and conjugated form within the plant tissues and providesome indication that TDZ-exposure enhances the accumulation and translocationofauxin within the tissues.     

Impact of Dissolved Oxygen during UV-Irradiation on the Chemical Composition and Function of CHO Cell Culture Media

Ultraviolet (UV) irradiation is advantageous as a sterilization technique in the biopharmaceutical industry since it is capable of targeting non-enveloped viruses that are typically challenging to destroy, as well as smaller viruses that can be difficult to remove via conventional separation techniques. In this work, we investigated the influence of oxygen in the media during UV irradiation and characterized the effect on chemical composition using NMR and LC-MS, as well as the ability of the irradiated media to support cell culture. Chemically defined Chinese hamster ovary cell growth media was irradiated at high fluences in a continuous-flow UV reactor. UV-irradiation caused the depletion of pyridoxamine, pyridoxine, pyruvate, riboflavin, tryptophan, and tyrosine; and accumulation of acetate, formate, kynurenine, lumichrome, and sarcosine. Pyridoxamine was the only compound to undergo complete degradation within the fluences considered; complete depletion of pyridoxamine was observed at 200 mJ/cm². Although in both oxygen- and nitrogen-saturated media, the cell culture performance was affected at fluences above 200 mJ/cm², there was less of an impact on cell culture performance in the nitrogen-saturated media. Based on these results, minimization of oxygen in cell culture media prior to UV treatment is recommended to minimize the negative impact on sensitive media.     

Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Light plays an important role in almost all plant developmental processes and provides the fundamental building blocks for growth, development, primary and secondary metabolism. The effects of light on growth rate and caffeic acid derivative (CADs) biosynthesis in hairy root cultures of Echinacea purpurea (Moench) were assessed. Light-grown hairy roots accumulated increased levels of anthocyanins, which became visible in outer cell layer of the cortex as a ring of purple color. The light-grown root cultures also had radially thickened morphology compared with the dark-grown controls. The growth rate and cell viability of the hairy root cultures in light did not show obvious difference in comparison with those in dark. However, biosynthesis of CADs including cichoric acid, caftaric acid, chlorogenic acid and caffeic acid was significantly increased in hairy root cultures grown in the light. The enhanced accumulation of CADs and anthocyanins in E. purpurea hairy root cultures was correlated to an observed light-stimulated activity of phenylalanine ammonium lyase (PAL).     

Thidiazuron-induced regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Micropropagation in solid and liquid culture systems

The goals of this study were to investigate thidiazuron (TDZ)-induced morphogenesis of Echinacea purpurea L. and to assess the possibility of developing a liquid-based protocol for rapid micropropagation. Callus development and root organogenesis were observed on leaf explants cultured on media containing 2,4-dicholorophenoxyacetic acid or dicamba, but no plantlets were regenerated. Addition of TDZ to the culture medium as the sole growth regulator resulted in the production of regenerable callus cultures. The highest rate of regeneration was observed for explants cultured on medium with TDZ at 2.5 μM or higher. Tissue derived from 1.0 μM TDZ treatments was used to initiate liquid cultures. All liquid treatments produced a similar number of regenerants but significantly more healthy plants were obtained from cultures grown in the presence of 0.1 and 1.0 μM TDZ. This TDZ-based micropropagation system is the first liquid, large-scale propagation protocol developed for the mass production of E. purpurea plants.     

Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.     

Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates

Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ～100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignment of human osteopontin

Osteopontin (OPN) is a 33.7 kDa intrinsically disordered protein and a member of the SIBLING family of proteins. OPN is bearing a signal peptide for secretion into the extracellular space, where it exerts its main physiological function, the control of calcium biomineralization. It is often involved in tumorigenic processes influencing proliferation, migration and survival, as well as the adhesive properties of cancer cells via CD44 and integrin signaling pathways. Here we report the nearly complete NMR chemical shift assignment of recombinant human osteopontin.     

Paramagnetic behaviour of the differentiating reproductive apex of wheat

Summary A study of the physiological significance of EPR signal was undertaken in the developing apical organs as well as in the top most leaf of a determinate type of plant,Triticum aestivum Cv. S 227 at various stages of vegetative and reproductive differentiation. Pour types of signals are reported: (a) a weak asymmetrical signal having 700 Gauss of width and g=2, the origin of which, is not clear; (b) a broad six peak signal also having g=2 which, as available evidence suggests, may be due to Mn⁺⁺; (c) in association with Mn⁺⁺ signal there are also other signals caused either by paramagnetic metals or by organic free radicals (FR); (d) at the centre of Mn⁺⁺ signal there appears a free radical signal with g=2.0023. It is observed that the amplitude of Mn⁺⁺ and free radical signal increases sharply in the shoot apex during its transformation from vegetative to reproductive state. The leaf also consistently records higher Mn⁺⁺ and FR contents at all stages of reproductive differentiation. Synchronously with the above mentioned enhanced paramagnetic behaviour of the apex and of the leaf there is an upsurge in metabolic activity of the plant. The possible role of free radicals and Mn⁺⁺ in energy transfer is discussed in relation to ascorbic acid turnover.     

Chitinase production by Streptomyces viridificans: its potential in fungal cell wall lysis

R. GUPTA, R.K. SAXENA, P. CHATURVEDI AND J.S. VIRDI. 1995. Streptomyces viridificans was found to be a good chitinase producer among nine species of Streptomyces screened. Minimum levels of constitutive enzyme were observed with both simple and complex carbon substrate. Arabinose doubled the enzyme production amongst the various pentoses and hexoses used with chitin. However, with glucose end-product inhibition and catabolite repression were observed. The enzyme tolerated a wide range of temperature (30–55°C) and pH (3–7˙5). Among various divalent cations Mn²⁺ and Hg²⁺ completely inhibited the purified enzyme while β-mercaptoethanol stimulated its activity. Crude and purified enzyme had potential for cell wall lysis of many fungal pathogens tested.