A new regioselective method of macrobicyclic Schiff bases synthesis

A new regioselective method of di-gem-thio-substituted PNP-crown derivatives synthesis is presented. The geminalmercaptoethanolanetricyclophosphaza-PNP-lariat ether structure has been determined by X-ray crystallography and characterised by ab initio calculations. The 16-membered PNP-crown ether ring exists in unique conformation: ac⁻ap ac⁻sc⁺sc⁺ap sc⁺ap ap sc⁻ap sc⁻sc⁻ac⁺ap ac⁺. All the ether oxygen atoms are directed into the interior of the ring. All endocyclic P-N bond lengths are equal within experimental error with the mean value 1.578(2) Å. The P-S bond properties have been characterised in terms of natural bond orbital (NBO) analysis, and its interactions with other NBO have been described. The spirocyclisation mechanism at cyclophosphazene phosphorus atom has been proposed.     

Radiation therapy with curative intention in men with de novo metastatic prostate carcinoma: shoot‘em all!

BackgroundAbout 5% of prostate cancer cases are metastatic at diagnoses. Radiotherapy of both primary tumor and secondary lesions can be, in addition to systemic treatments, a radical alternative for selected patients.Materials and methodsPatients with de novo prostate carcinoma with bone or lymph node metastases were retrospectively reviewed. All patients received moderate hypofractionated IMRT/VMAT up to 63 Gy in 21 daily fractions of 3 Gy to prostate and metastases with neoadjuvant and concurrent androgen deprivation therapy (ADT). According to known advances some patients also received abiraterone, enzalutamide, or docetaxel.ResultsBetween 2015–2020, we attended 26 prostate cancer patients (median age 69.5 years, range 52–84) with simultaneous oligometastases [mean 2.1 metastases, median 1.5 metastases (range 1–6)]. Eighteen patients (69%) presented lymph node metastases, 4 (15.5%) bone metastases and 4 (15.5%) both lymph node and bone metastases. With a median follow-up of 15.5 months (range 3–65 months), 16 patients (62%) are alive and tumor free while 10 (38%) are alive with tumor. Four patients (17%) developed tumor progression, out of irradiated area in all cases, with a median time to progression of 43.5 months (range 27–56 months). Actuarial progression-free survival (PFS) rates at 12 and 24 months were 94.1% and 84.7%, respectively. No grade > 2 acute or late complications were recorded.ConclusionsSimultaneous directed radical hypofractionated radiation therapy for prostate and metastases is feasible, well tolerated and achieves an acceptable PFS rate. However, further studies with longer follow-up are necessary to definitively address these observations.     

Cellular stress induced by resazurin leads to autophagy and cell death via production of reactive oxygen species and mitochondrial impairment

Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin‐induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL‐60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL‐60 cells which were also more sensitive and contained autophagic vesicles. Studies in Rho⁰ cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti‐proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy, and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance. J. Cell. Biochem. 111: 574–584, 2010. © 2010 Wiley‐Liss, Inc.     

Early-onset renal cell carcinoma as a novel extraparaganglial component of SDHB-associated heritable paraganglioma

Hereditary paraganglioma syndrome has recently been shown to be caused by germline heterozygous mutations in three (SDHB, SDHC, and SDHD) of the four genes that encode mitochondrial succinate dehydrogenase. Extraparaganglial component neoplasias have never been previously documented. In a population-based registry of symptomatic presentations of phaeochromocytoma/paraganglioma comprising 352 registrants, among whom 16 unrelated registrants were SDHB mutation positive, one family with germline SDHB mutation c.847-50delTCTC had two members with renal cell carcinoma (RCC), of solid histology, at ages 24 and 26 years. Both also had paraganglioma. A registry of early-onset RCCs revealed a family comprising a son with clear-cell RCC and his mother with a cardiac tumor, both with the germline SDHB R27X mutation. The cardiac tumor proved to be a paraganglioma. All RCCs showed loss of the remaining wild-type allele. Our observations suggest that germline SDHB mutations can predispose to early-onset kidney cancers in addition to paragangliomas and carry implications for medical surveillance.     

A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides

The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the trans-Golgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8-10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins.     

2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2

Protein kinase CK2 is a highly pleiotropic enzyme whose high constitutive activity is suspected to be instrumental to the enhancement of the tumour phenotype and to the propagation of infectious diseases. Here we describe a novel compound, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), which is superior to the commonly used specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in several respects. DMAT displays the lowest K(i) value ever reported for a CK2 inhibitor (40 nM); it is cell permeable and its efficacy on cultured cells, both in terms of endogenous CK2 inhibition and induction of apoptosis, is several fold higher than that of TBB. The selectivity of DMAT assayed on a panel of >30 protein kinases is comparable to that of TBB, with the additional advantage of being ineffective on protein kinase CK1 up to 200 microM. These properties make DMAT the first choice CK2 inhibitor for in vivo studies available to date.     

Experimental infections and colonization of porcine skin samples with B. anthracis

In this study there was demonstrated the process of appearance and intensity of colonisation of the swine's skin samples by exposing them on agar plate cultures or immersed in liquid cultures of B. anthracis. The results of research were documented with photos of coloured histological preparations. As a result of the research there was demonstrated the possibility of infection and colonisation of skin consignment by B. anthracis from accidental located skin of died or killed animals because of anthrax. This colonisation may occur in the period 3 to 24 hours in optimal conditions. Anthrax bacteria are starting to penetrate and colonize the skin samples during 3 hours with exposing them from the hypodermic side; whereas penetration and colonisation from epidermis side appears after 24 hours. The results demonstrated that in natural conditions for optimal cultivation of B. anthracis there can be a possibility of infection and colonization of health parts of the skin's in storage skin for the longer period of time or longer transport.     

Caspase-mediated cleavage converts the tumor necrosis factor (TNF) receptor-associated factor (TRAF)-1 from a selective modulator of TNF receptor signaling to a general inhibitor of NF-kappaB activation

The role of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-1 in NF-kappaB activation by various members of the TNF receptor family is not well understood, and conflicting data have been published. Here, we show that TRAF1 differentially affects TRAF2 recruitment and activation of NF-kappaB by members of the TNF receptor family. Interestingly, a naturally occurring caspase-derived cleavage product of TRAF1 solely comprising its TRAF domain (TRAF1-(164-416)) acted as a general inhibitor of NF-kappaB activation. In contrast, a corresponding fragment generated by cleavage of TRAF3 showed no effect in this regard. In accordance with these functional data, TRAF1, but not TRAF3, interacted with the IKK complex via its N-TRAF domain. Endogenous TRAF1 and the overexpressed TRAF domain of TRAF1 were found to be constitutively associated with the IKK complex, whereas endogenous receptor interacting protein was only transiently associated with the IKK complex upon TNF stimulation. Importantly, the caspase-generated TRAF1-fragment, but not TRAF1 itself inhibited IKK activation. Our results suggest that TRAF1 and TRAF1-(164-416) exert their regulatory effects on receptor-induced NF-kappaB activation not only by modulation of TRAF2 receptor interaction but especially TRAF1-(164-416) also by directly targeting the IKK complex.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Summary Neb-TMOF, the trypsin modulating oostatic factor of gray fleshflyNeobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 byd-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X=NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) andd-Asn (XII). The influence of these peptides on trypsin biosynthesis inN. bullata was determinedin vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [d-His)⁶]- and [d-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performedin vitro on heart ofTenebrio molitor were found that-TMOF and [Phe(p-NH₂)⁶]-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

New Proctolin Analogues: Synthesis and Biological Investigation in Insects

We have extended our work on structure/activity relationship studies of the neuropeptiden proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) by evaluating the effects of the following proctolin analogues: H-X¹-Tyr-Leu-Pro-Thr-OH, where X¹ = D-Arg (I), N-Me-Arg (II), Can (III), Orn(di-Me) (IV), Orn(iPr) (V), Lys(N, N-di-Me) (VI), Lys(iPr) (VII), Lys(Nic) (VIII) and D-Lys(Nic) (IX). In analogues I–IX, the N-terminal Arg residue was replaced by basic amino acid derivatives with peptides containing amino acid residues with an isosteric system on the back side chain relative to Arg (compounds III, V and VI) or homo-Arg (compound VII). Analogues I–IX were evaluated for myotropic activity on the in vitro heart preparation of Tenebrio molitor, whereas peptides II, V, and VII–IX were tested for contractile activity on the isolated foregut of locust Schistocerca gregaria. Peptide II and III showed full cardiotropic activity in T. molitor while peptides V and VII showed 40% and 15%, respectively, locust-gut contracting activity of proctolin.