Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates

The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.

Here, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.

We have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.

We further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (γH2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.

Enzyme induction in Neurospora crassa

I. Studies on Nicotinamide Adenine Dinucleotide Glycohydrase (NADase)

NADase, like tyrosinase and L-amino acid oxidase, is not present in two day old cultures of wild type Neurospora, but it is coinduced with those two enzymes during starvation in phosphate buffer. The induction of NADase, like tyrosinase, is inhibited by puromycin. The induction of all three enzymes is inhibited by actinomycin D. These results suggest that NADase is synthesized de novo during induction as has been shown directly for tyrosinase. NADase induction differs in being inhibited by certain amino acids.

The tyrosinaseless mutant ty-1 contains a non-dialyzable, heat labile inhibitor of NADase. A new mutant, P110A, synthesizes NADase and L-amino acid oxidase while growing. A second strain, pe, fl;cot, makes NADase while growing. Both strains can be induced to make the other enzymes. These two strains prove that the control of these three enzymes is divisible. The strain P110A makes NADase even when grown in the presence of Tween 80. The synthesis of both NADase and L-amino acid oxidase by P110A is suppressed by complete medium. The theory of control of the synthesis of the enzymes is discussed.

II. Studies with EDTA

Neurospora tyrosinase contains copper but, unlike other phenol oxidases, this copper has never been removed reversibly. It was thought that the apo-enzyme might be made in vivo in the absence of copper. Therefore cultures were treated with EDTA to remove copper before the enzyme was induced. Although no apo-tyrosinase was detected, new information on the induction process was obtained.

A treatment of Neurospora with 0.5% EDTA pH 7, inhibits the subsequent induction during starvation in phosphate buffer of tyrosinase, L-amino acid oxidase and NADase. The inhibition of tyrosinase and L-amino acid oxidase induction is completely reversed by adding 5 x 10^-5M CaCl₂, 5 x 10^-4M CuSO₄, and a mixture of L-amino acids (2 x 10^-3M each) to the buffer. Tyrosinase induction is also fully restored by 5 x 10^-4M CaCl₂ and amino acids. As yet NADase has been only partially restored.

The copper probably acts by sequestering EDTA left in the mycelium and may be replaced by nickel. The EDTA apparently removes some calcium from the mycelium, which the added calcium replaces. Magnesium cannot replace calcium. The amino acids probably replace endogenous amino acids lost to the buffer after the EDTA treatment.

The EDTA treatment also increases permeability, thereby increasing the sensitivity of induction to inhibition by actinomycin D and allowing cell contents to be lost to the induction buffer. EDTA treatment also inhibits the uptake of exogenous amino acids and their incorporation into proteins.

The lag period that precedes the first appearance of tyrosinase is demonstrated to be a separate dynamic phase of induction. It requires oxygen. It is inhibited by EDTA, but can be completed after EDTA treatment in the presence of 5 x 10^-5M CaCl₂ alone, although no tyrosinase is synthesized under these conditions.

The time course of induction has an early exponential phase suggesting an autocatalytic mechanism of induction.

The mode of action of EDTA, the process of induction and the kinetics of induction are discussed.

Induction of mitotic and meiotic gynogenesis and production of genetic clones in rohu, Labeo rohita Ham.

Studies were undertaken to produce genetic clones derived from all homozygous mitotic gynogenetic individuals in rohu, Labeo rohita Ham. ln view of this, attempts were made to interfere with the normal functioning of the spindle apparatus during the first mitotic cell division of developing eggs using heat shocks, there by leading to the induction of mitotic gynogenetic diploids in the F1 generation. Afterwards, viable mitotic gynogenetic alevins were reared and a selected mature female fish was used to obtain ovulated eggs which were fertilized later with UV-irradiated milt. Milt was diluted with Cortland’s solution and the sperm concentration was maintained at 10⁸/ml. The UV-irradiation was carried out for 2 minutes at the intensity of 200 to 250 µW/cm² at 28± 1°C. The optimal heat shock of 40°C for 2 minutes applied at 25 to 30 minutes a.f. was used to induce mitotic gynogenesis in first (F1) generation and at 3 to 5 minutes a.f. to induce meiotic gynogenesis in the second (F2) generation. The results obtained are presented and the light they shed on the timing of the mitotic and meiotic cell division in this species is discussed.

The stress experience dependent long-term depression disassociated with stress effect on spatial memory task

Behavioral stress can either block or facilitate memory and affect the induction of long-term potentiation (LTP) and long-term depression (LTD). However, the relevance of the stress experience-dependent long-term depression (SLTD) to spatial memory task is unknown. Here we have investigated the effects of acute and sub-acute elevated platform (EP) and foot shock (FS) stress on LTD induction in CA1 region of the hippocampus of anesthetized rats and spatial memory in Morris water maze. We found that LTD was facilitated by acute EP stress, but not by sub-acute EP stress that may be due to the fast adaptation of the animals to this naturalistic mild stress. However, FS stress, an inadaptable strong stress, facilitated LTD induction both in acute and sub-acute treatment. In addition, with the same stress protocols, acute EP stress impaired spatial memory but the sub-acute EP stressed animals performed the spatial memory task as well as the controls, may due to the same reason of adaptation. However, acute FS stress slightly impaired learning but sub-acute FS even enhanced memory retrieval. Our results showed that SLTD was disassociated with the effect of stress on memory task but might be related to stress experience-dependent form of aberrant memory. (C) 2003 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved.

Stress-facilitated LTD induces output plasticity through a synchronized-spikes and spontaneous unitary discharges the CA1 region of the hippocampus

Long-term potentiation (LTP) and long-term depression (LTD) of the excitatory synaptic inputs plasticity in the hippocampus is believed to underlie certain types of learning and memory. Especially, stressful experiences, well known to produce long-lasting strong memories of the event themselves, enable LTD by low frequency stimulation (LFS, 3 Hz) but block LTP induction by high frequency stimulation (HFS, 200 Hz). However, it is unknown whether stress-affected synaptic plasticity has an impact on the output plasticity. Thus, we have simultaneously studied the effects of stress on synaptic plasticity and neuronal output in the hippocampal CA1 region of anesthetized Wistar rats. Our results revealed that stress increased basal power spectrum of the evoked synchronized-spikes and enabled LTD induction by LFS. The induction of stress-facilitated LTD but not LFS induced persistent decreases of the power spectrum of the synchronized-spikes and the frequency of the spontaneous unitary discharges; However, HFS induced UP in non-stressed animals and increased the power spectrum of the synchronized-spikes, without affecting the frequency of the spontaneous unitary discharges, but HFS failed to induce UP in stressed animals without affecting the power spectrum of the synchronized-spikes and the frequency of the spontaneous unitary discharges. These observations that stress-facilitated LTD induces the output plasticity through the synchronized-spikes and spontaneous unitary discharges suggest that these types of stress-related plasticity may play significant roles in distribution, amplification and integration of encoded information to other brain structures under stressful conditions. (C) 2004 Elsevier Ireland Ltd and The Japan Neuroscience Society. All rights reserved.

Glucocorticoid receptor and protein/RNA synthesis-dependent mechanisms underlie the control of synaptic plasticity by stress

Learning and memory are exquisitely sensitive to behavioral stress, but the underlying mechanisms are still poorly understood. Because activity-dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as the hippocampus we have examined the means by which stress affects synaptic plasticity in the CA1 region of the hippocampus of anesthetized rats, Inescapable behavioral stress (placement on an elevated platform for 30 min) switched the direction of plasticity, favoring low frequency stimulation-induced decreases in synaptic transmission (long-term depression, LTD), and opposing the induction of long-term potentiation by high frequency stimulation, We have discovered that glucocorticoid receptor activation mediates these effects of stress on LTD and longterm potentiation in a protein synthesis-dependent manner because they were prevented by the glucocorticoid receptor antagonist RU 38486 and the protein synthesis inhibitor emetine. Consistent with this, the ability of exogenously applied corticosterone in non-stressed rats to mimic the effects of stress on synaptic plasticity was also blocked by these agents, The enablement of low frequency stimulation-induced LTD by both stress and exogenous corticosterone was also blocked by the transcription inhibitor actinomycin D, Thus, naturally occurring synaptic plasticity is liable to be reversed in stressful situations via glucocorticoid receptor activation and mechanisms dependent on the synthesis of new protein and RNA, This indicates that the modulation of hippocampus-mediated learning by acute inescapable stress requires glucocorticoid receptor-dependent initiation of transcription and translation.

Effects of unconditioned and conditioned aversive stimuli in an intense fear conditioning paradigm on synaptic plasticity in the hippocampal CA1 area in vivo

Repeated vivid recalls or flashbacks of traumatic memories and memory deficits are the cardinal features of post-traumatic stress disorder (PTSD). The underlying mechanisms are not fully understood yet. Here, we examined the effects of very strong fear conditioning (20 pairings of a light with a 1.5-mA, 0.5-s foot shock) and subsequent reexposure to the conditioning context (chamber A), a similar context (chamber B), and/or to the fear conditioned stimulus (CS) (a light) on synaptic plasticity in the hippocampal CA1 area in anesthetized Sprague-Dawley rats. The conditioning procedure resulted in very strong conditioned fear, as reflected by high levels of persistent freezing, to both the contexts and to the CS, 24 h after fear conditioning. The induction of long-term potentiation ON was blocked immediately after fear conditioning. It was still markedly impaired 24 h after fear conditioning; reexposure to the conditioning chamber A (CA) or to a similar chamber 13 (CB) did not affect the impairment. However, presentation of the CS in the CA exacerbated the impairment of LTP, whereas the CS presentation in a CB ameliorated the impairment so that LTP induction did not differ from that of control groups. The induction of long-term depression (LTD) was facilitated immediately, but not 24 h, after fear conditioning. Only reexposure to the CS in the CA, but not reexposure to either chamber A or B alone, or the CS in chamber B, 24 h after conditioning, reinstated the facilitation of LTD induction. These data demonstrate that unconditioned and conditioned aversive stimuli in an intense fear conditioning paradigm can have profound effects on hippocampal synaptic plasticity, which may aid to understand the mechanisms underlying impairments of hippocampus-dependent memory by stress or in PTSD. (c) 2005 Wiley-Liss, Inc.

Amplitude/frequency of spontaneous mEPSC correlates to the degree of long-term depression in the CA1 region of the hippocampal slice

Prior synaptic or cellular activity influences degree or threshold for subsequent induction of synaptic plasticity, a process known as metaplasticity. Thus, the continual synaptic activity, spontaneous miniature excitatory synaptic current (mEPSC) may correlate to the induction of long-teen depression (LTD). Here, we recorded whole-cell EPSC and mEPSC alternately in the Schaffer-CA1 synapses in brain slice of young rats, and found that this recording configuration affected neither EPSC nor mEPSC. Low frequency stimulation (LFS) induced variable magnitudes of LTD. Remarkably, larger magnitudes of LTD were significantly correlated to smaller amplitude/lower frequency of the basal mEPSC. Furthermore, under the conditions reduced amplitude/frequency of the basal mEPSC by exposure to behavioral stress immediately before slice preparation or low concentration of calcium in bath solution, the magnitudes of LTD were still inversely correlated to mEPSC amplitude/frequency. These new findings suggest that spontaneous mEPSC may reflect functional and/or structural aspects of the synapses, the synaptic history ongoing metaplasticity. (C) 2005 Elsevier B.V. All rights reserved.

Ovulation in the tree shrew (Tupaia belangeri) induced by gonadotrophins

The induction of ovulation by exogenous gonadotrophins is an important approach for recovering oocytes used for studies on the reproductive biology of some mammals. In the present study, pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadot

Apoptosis induction on human hepatoma cells Hep G2 of decabrominated diphenyl ether (PBDE-209)

Polybrominated diphenyl ethers (PBDEs) are an important class of halogenated organic brominated flame retardants. Because of their presence in abiotic and biotic environments widely and their structural similarity to polychlorinated biphenyls (PCBs), concern has been raised on their possible adverse health effects to humans. This study was designed to determine the anti-proliferative, apoptotic properties of decabrominated diphenyl ether (PBDE-209), using a human hepatoma Hep G2 line as a model system. Hep G2 cells were cultured in the presence of PBDE-209 at various concentrations (1.0-100.0 mu mol/L) for 72 h and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that PBDE-209 inhibited the cells viability in time and concentration-dependent characteristics at concentrations (10.0-100.0 mu mol/L). We found that anti-proliferative effect of PBDE-209 was associated with apoptosis on Hep G2 cells by determinations of morphological changes, cell cycle and apoptosis. Mechanism study showed that PBDE-209 could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. The mechanism for its hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation. In addition, activity of lactate dehydrogenase (LDH) release increased when the cells incubated with PBDE-209 at various concentrations and times. These results suggested that PBDE-209 had the toxicity activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Linear energy transfer dependence of the effects of carbon ion beams on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta

Purpose: To determine the effects of carbon ion beams with five different linear energy transfer (LET) values on adventitious shoots from in vitro leaf explants of Saintpaulia ionahta Mauve cultivar with regard to tissue increase, shoots differentiation and morphology changes in the shoots. Materials and methods: In vitro leaf explant samples were irradiated with carbon ion beams with LET values in the range of 31 similar to 151 keV/mu m or 8 MeV of X-rays (LET 0.2 keV/mu m) at different doses. Fresh weight increase, surviving fraction and percentage of the explants with regenerated malformed shoots in all the irradiated leaf explants were statistically analysed. Results: The fresh weight increase (FWI) and surviving fraction (SF) decreased dramatically with increasing LET at the same doses. In addition, malformed shoots, including curliness, carnification, nicks and chlorophyll deficiency, occurred in both carbon ion beam and X-ray irradiations. The induction frequency with the former, however, was far more than that with the X-rays. Conclusions: This work demonstrated the LET dependence of the relative biological effectiveness (RBE) of tissue culture of Saintpaulia ionahta according to 50% FWI and 50% SF. After irradiating leaf explants with 5 Gy of a 221 MeV carbon ion beam having a LET value of 96 keV/mu m throughout the sample, a chlorophyll-deficient (CD) mutant, which could transmit the character of chlorophyll deficiency to its progeny through three continuous tissue culture cycles, and plantlets with other malformations were obtained.

Survival and initial chromatid breakage in normal and tumour cells exposed in vitro to gamma rays and carbon ions at the HIRFL

Human hepatoma and normal liver cells were irradiated with C-12(6+) ion beams (linear energy transfer (LET) = 96 keV mu m(-1)) and gamma-rays at the Heavy Ion Research Facility in Lanzhou (HIRFL). The numbers and types of chromatid breaks were detected using the premature chromosome condensation technique. Irradiation with C-12(6+) ions produced a majority of isochromatid break types, while chromatid breaks were dominant for irradiation with gamma-rays. Experimental results showed that the initial level of chromatid breaks is clearly related to the absorbed dose from C-12(6+), ions and gamma-rays. The (12)C(6+)ions are relatively more effective at inducing initial chromatid breaks when compared with the gamma-rays. A relative biological effectiveness (RBE) of about 2.5 resulted for the induction of initial chromatid breaks by C-12(6+) ions relative to gamma-rays in both cell lines.

Copolymerization of carbon dioxide and cyclohexene oxide catalyzed by aluminum porphyrin-quaternary ammonium salt in the presence of bulky lewis acid

Chloro( 5,10,15,20-tetraphenyl-porphyrinato)-aluminum/tetraethylammonium bromide ( Et4NBr) in combination with bulky Lewis acid was used for the copolymerization of CO2 and cyclohexene oxide ( CHO). Bulky Lewis acid having substituents at the ortho positions of the phenolate ligands, like methylaluminum bis(2,6-di-tert-butyl-4-methylphenolate), significantly shortened the induction period and raised the catalytic activity, the corresponding turnover frequency reached 44.9 h(-1) in 9 h, which was 23.8% higher than that from ( TPP)AlCl/Et4NBr binary catalyst. The resulting polycarbonate has carbonate linkage over 93% with number average molecular weight of ( 4.5-6.5) x 10(3) and polydispersity index below 1.10.

The first synthesis of a water-soluble alpha-cyclodextrin/C-60 supramolecular complex using anionic C-60 as a building block

A novel supramolecular inclusion complex of alpha-CD/C-60 was synthesized using anionic C-60. The reaction progress was monitored in situ by visible and near-IR spectroscopy. The obtained complex was characterized by UV-vis, C-13 NMR, MALDI-TOF, and cyclic voltammetry. The induction and dispersion forces are considered to be the major driving forces for the formation of a resulting alpha-CD/C-60(.-) inclusion complex.

Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.