Cytotoxic and topographical properties of 6-arylidene-2-dimethylaminomethylcyclohexanone hydrochlorides and related compounds.

A number of 2-arylidenecyclohexanones (1a-h) were converted into the corresponding Mannich bases (2a-h) and (3a,f). Evaluation against murine L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes revealed the marked cytotoxicity of the Mannich bases and also the fact that almost invariably these compounds were more potent than the precursor enones (1a-h). Further evaluation of most of the Mannich bases towards a panel of nearly 60 human tumour cell lines confirmed their utility as potent cytotoxins. In this assay, the compounds showed growth-inhibiting properties greater than the anticancer alkylator melphalan. QSAR studies revealed that in some cell lines compounds possessing small electron-attracting aryl substituents showed the greatest potencies. Molecular modeling and X-ray crystallography demonstrated that various interatomic distances and torsion angles correlated with cytotoxicity. A representative compound (2a) demonstrated weak inhibiting properties towards human N-myristoyltransferase and stimulated a tyrosine protein kinase. A single dose of 100 mg/kg of most of the compounds did not prove to be lethal in mice.

Mamestra configurata serpin-1 homologues: cloning, localization and developmental regulation.

A screen of a Mamestra configurata (bertha armyworm) midgut cDNA library identified three types of cDNA clones that resemble the Manduca sexta serpin-1 gene family. Two serpins, 1b and 1c, possess a common conserved serpin amino terminal scaffold domain but bear no similarity to any members of the M. sexta gene family within the reactive centre loop. These serpins differ from one another by only two amino acids in the reactive centre loop (S(363)-->P) and serpin signature (M(369)-->T) regions. The other member, denoted serpin-1a, is closely related to the M. sexta serpin-1Z. M. configurata serpins as a group were expressed in all insect developmental stages including eggs, larvae and adult moths. Within larvae, serpin gene expression was restricted to the early to middle instar developmental phase and mainly in the fat body and hemocytes. Stress imposed by starvation strongly induced expression in fat body and to a lesser degree in alimentary organs, nervous system and Malphigian tubules. Conversely, starvation decreased expression in hemocytes. Wounding or inoculation with bacteria did not induce serpin gene transcription but did lead to the formation of higher and lower molecular weight forms, presumably serpin-protease complexes and resultant truncated serpin, respectively. Two dimensional PAGE and western blotting analysis revealed at least 12 distinct serpins consisting primarily of neutral, but also highly acidic and basic isoforms, as well as additional high and low molecular weight immuno-reactive species. Serpins-1b/1c are the more prominent serpin isoforms and are expressed predominantly in the fat body and subsequently exported to the hemolymph as revealed by western blotting and immunolocalization. The serpin-1b/1c isoform was found only as the fully glycosylated species within the hemolymph. Hemolymph protease activity was comprised mostly of serine proteases whose overall activity increased dramatically at the onset of the molt concomitant with a sharp decline in serpin gene expression.

Changes in cysteine protease activity and localization during midgut metamorphosis in the crucifer root maggot (Delia radicum).

We show that differential localization and/or activation of two cysteine protease activities occur at the onset of dipteran midgut metamorphosis. A 26 kDa cysteine protease activity was associated specifically with midgut tissues of late third instar larvae. Starvation of mid third instar larvae simulated the onset of prepupation and resulted in loss of the 26 kDa protease activity. A cDNA clone encoding a cysteine protease, termed DrCP1, was isolated and shown to be highly similar to those from Sarcophaga peregrina and Drosophila melanogaster (DmCP1). DrCP1 mRNA was present in all developmental stages including eggs, larvae, pupae and adults, but was highly induced at the onset of the larval-pupal transition and thereafter. The DrCP1 protein is localized to the exterior of the midgut tissues during the onset of the prepupal transition period, possibly in response to ecdysone. Analysis of transcription factor binding sites associated with the DmCP1 promoter indicated that elements exist that allow for both ecdysone-mediated as well as tissue-specific regulation. Based upon these and other studies we propose: (1) that the expression, activity and localization of the DrCP1-like cysteine proteases are highly regulated throughout development; and, (2) that cysteine protease activities are involved in aspects of tissue reconstruction at the onset of and during metamorphosis.

The Saccharomyces cerevisiae mre11(ts) allele confers a separation of DNA repair and telomere maintenance functions.

The yeast Mre11 protein participates in important cellular functions such as DNA repair and telomere maintenance. Analysis of structure-function relationships of Mre11 has led to identification of several separation-of-function mutations as well as N- and C-terminal domains essential for Mre11 meiotic and mitotic activities. Previous studies have established that there is a strong correlation between Mre11 DNA repair and telomere maintenance functions and that Mre11-Rad50-Xrs2 complex formation appears to be essential for both of these activities. Here we report that the mre11(ts) allele, previously shown to cause temperature-dependent defects in DNA repair and meiosis, confers a temperature-independent telomere shortening, indicating that mre11(ts) is a separation-of-function mutation with respect to DNA repair and telomere maintenance. In a yeast two-hybrid system, Mre11(ts) fails to form a homodimer or interact with Rad50 and Xrs2 irrespective of experimental temperatures. These observations collectively suggest that the Pro(162)Ser substitution in Mre11(ts) confers a novel separation of Mre11 mitotic functions. Moreover, we observed that while overexpression of the 5'-3' exonuclease gene EXO1 partially complements the MMS sensitivity of mre11, rad50, and xrs2 null mutants, it has no effect on telomere shortening in these strains. This result provides additional evidence on possible involvement of distinctive mechanisms in DNA repair and telomere maintenance by the Mre11-Rad50-Xrs2 complex.

Formation of the yeast Mre11-Rad50-Xrs2 complex is correlated with DNA repair and telomere maintenance.

The yeast Mre11 is a multi-functional protein and is known to form a protein complex with Rad50 and Xrs2. In order to elucidate the relationship between Mre11 complex formation and its mitotic functions, and to determine domain(s) required for Mre11 protein interactions, we performed yeast two-hybrid and functional analyses with respect to Mre11 DNA repair and telomere maintenance. Evidence presented in this study indicates that the N-terminal region of Mre11 constitutes the core homo-dimerization and hetero-dimerization domain and is sufficient for Mre11 DNA repair and maintaining the wild-type telomere length. In contrast, a stretch of 134 amino acids from the extreme C-terminus, although essential for achieving a full level of self-association, is not required for the aforementioned Mre11 mitotic functions. Interestingly, deletion of these same 134 amino acids enhanced the interaction of Mre11 with Rad50 and Xrs2, which is consistent with the notion that this region is specific for meiotic functions. While Mre11 self-association alone is insufficient to provide the above mitotic activities, our results are consistent with a strong correlation between Mre11-Rad50-Xrs2 complex formation, mitotic DNA repair and telomere maintenance. This correlation was further strengthened by analyzing two mre11 phosphoesterase motif mutants ( mre11-2 and rad58S ), which are defective in DNA repair, telomere maintenance and protein interactions, and a rad50S mutant, which is normal in both complex formation and mitotic functions. Together, these results support and extend a current model regarding Mre11 structure and functions in mitosis and meiosis.

Molecular cloning and genetic characterization of the Saccharomyces cerevisiae NGS1/MRE11 gene.

The Saccharomyces cerevisiae ngs1-1 mutant was previously identified by its enhanced sensitivity to simple DNA-alkylating agents such as methyl methanesulfonate but not to UV. Molecular cloning and sequencing of NGS1 as a putative DNA-alkylation repair gene revealed that it isidentical to MRE11, a gene that is involved in DNA recombinational repair. In order to investigate functional domains of the Mre11 protein, nucleotide-sequence alterations of a number of mre11 mutant alleles, including ngs1-1, mre11-1 (ts), mre11-2, mre11-3 and mre11-58, were determined. Most of these mutations map to the N-terminus ofMre11, emphasizing the importance of this highly conserved domain. The ngs1-1 and mre11-3 mutants carry nonsense mutations resulting in truncated proteins. Missense mutations were found in mre11-1 (ts), mre11-2 and mre11-58, of which mre11-2 and mre11-58 mapped to the conserved phosphoesterase domains, indicating the involvement of these motifs in the formation and/or processing of DNA double-strand breaks. Finally, mitotic-recombination assays show that the mre11 delta mutation enhances inter-chromosomal recombination but decreases the intra-chromosomal deletion frequency. In addition, MRE11 appears to play different roles during spontaneous and alkylation-induced homologous mitotic recombination.

Cytotoxic activities of Mannich bases of chalcones and related compounds.

Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity toward murine P388 and L1210 leukemia cells as well as a number of human tumor cell lines. The most promising lead molecule was 21 that had the highest activity toward L1210 and human tumor cells. In addition, 21 exerted preferential toxicity to human tumor lines compared to transformed human T-lymphocytes. Other compounds of interest were 38, with a huge differential in cytotoxicity between P388 and L1210 cells, and 42, with a high therapeutic index when cytotoxicity to P388 cells and Molt 4/C8 T-lymphocytes were compared. In general, the Mannich bases were more cytotoxic than the corresponding chalcones toward L1210 but not P388 cells. A ClusCor analysis of the data obtained from the in vitro human tumor screen revealed that the mode of action of certain groups of compounds was similar. For some groups of compounds, cytotoxicity was correlated with the sigma, pi, or molar refractivity constants in the aryl ring attached to the olefinic group. In addition, the IC50 values in all three screens correlated with the redox potentials of a number of Mannich bases. X-ray crystallography and molecular modeling of representative compounds revealed various structural features which were considered to contribute to cytotoxicity. While a representative compound 15 was stable and unreactive toward glutathione (GSH) in buffer, the Mannich bases 15, 18, and 21 reacted with GSH in the presence of the pi isozyme of glutathione S-transferase, suggesting that thiol alkylation may be one mechanism by which cytotoxicity was exerted in vitro. Representative compounds were shown to be nonmutagenic in an intrachromosomal recombination assay in yeast, devoid of antimicrobial properties and possessing anticonvulsant and neurotoxic properties. Thus Mannich bases of chalcones represent a new group of cytotoxic agents of which 21 in particular serves as an useful prototypic molecule.

Isolation of hMRE11B: failure to complement yeast mre11 defects due to species-specific protein interactions.

The Saccharomyces cerevisiae MRE11 gene plays an important role in meiotic recombination, mitotic DNA repair and telomere maintenance. We present the isolation of hMRE11B cDNA from a human HeLa cell cDNA library as an MRE11 homolog. Compared to the previously identified hMRE11, hMRE11B contains an additional 84bp sequence that results in a 28 amino-acid insertion close to the C-terminus. The expression pattern of hMRE11B in different tissues shows the presence of two mRNA species of approx. 2.6 and 7.5kb. Overexpression of hMRE11B does not complement the alkylation sensitivity of the mre11 null and temperature-sensitive mutant strains. In this study, we examine factors that may explain this lack of complementation. First, both Northern and Western analyses rule out the lack of hMRE11B transcription and/or translation in yeast. Second, we demonstrate that hMre11B, like the yeast Mre11 protein, dimerizes in vivo in a yeast two-hybrid system. This dimerization requires the C-terminal one-third of hMre11B protein, which includes the 28 amino acids absent in hMre11. However, hMre11B does not interact with Mre11, Rad50 and Xrs2. Hence, the lack of protein-protein interaction between hMre11B and the yeast Mre11, Rad50, and Xrs2 may explain the inability of hMRE11B to complement the yeast mre11 mutants. We rule out the hypothesis that the lack of interaction and, in turn of complementation, is due to the absence of sequence homology at the C-terminal domain of hMre11B compared to the yeast Mre11. Instead, we propose that the C-terminus of hMre11B participates in protein-protein interaction and functions in a species-specific manner.

Synthesis and cytotoxic evaluation of some Mannich bases of alicyclic ketones.

A number of Mannich bases of alicyclic ketones containing one and two basic centres were prepared in order to evaluate the theory of sequential cytotoxicity and develop structure-activity relationships in these series of compounds. The compounds were evaluated in vitro against murine P388 D1 lymphocytic leukemia cells. The data generated supported the theory of sequential cytotoxicity and in general, compounds containing alicyclic rings of five and six carbon atoms possessed greater activity than the corresponding dodecyl analogues. Those Mannich bases containing dialkylamino groups were associated with greater cytotoxicity than related compounds possessing a basic heterocycle. Calculations of the atomic charges of the enone groups from selected compounds afforded some rationalization for the cytotoxic screening results.