Effect of base sequence on the DNA cross-linking properties of pyrrolobenzodiazepine (PBD) dimers

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA minor-groove cross-linking agents that possess two electrophilic imine moieties (or their equivalent) capable of forming covalent aminal linkages with guanine C2-NH₂ functionalities. The PBD dimer SJG-136, which has a C8–O–(CH₂)₃–O–C8′′ central linker joining the two PBD moieties, is currently undergoing phase II clinical trials and current research is focused on developing analogues of SJG-136 with different linker lengths and substitution patterns. Using a reversed-phase ion pair HPLC/MS method to evaluate interaction with oligonucleotides of varying length and sequence, we recently reported (JACS, 2009, 131, 13 756) that SJG-136 can form three different types of adducts: inter- and intrastrand cross-linked adducts, and mono-alkylated adducts. These studies have now been extended to include PBD dimers with a longer central linker (C8–O–(CH₂)₅–O–C8′), demonstrating that the type and distribution of adducts appear to depend on (i) the length of the C8/C8′-linker connecting the two PBD units, (ii) the positioning of the two reactive guanine bases on the same or opposite strands, and (iii) their separation (i.e. the number of base pairs, usually ATs, between them). Based on these data, a set of rules are emerging that can be used to predict the DNA–interaction behaviour of a PBD dimer of particular C8–C8′ linker length towards a given DNA sequence. These observations suggest that it may be possible to design PBD dimers to target specific DNA sequences.     

Lead Dysregulates Serine/Threonine Protein Phosphatases in Human Neurons

It is well established that lead (Pb) exposure in humans leads to learning and memory impairment. However, the biological and molecular mechanisms are still not clearly understood. When over activated, serine/threonine protein phosphatases are known to function as a constraint on learning and memory. Activation of these phosphatases can also result in cytoskeletal changes that will adversely affect learning and memory. We investigated the effects of Pb exposure on these phosphatases in primary cultures of human neurons. Neurons were exposed to physiologically relevant concentrations of Pb (5, 10, 20 and 40 μg/dL) and total phosphatase and PP2A activities were determined in neuronal lysate using para-nitrophenyl phosphate (pNPP), and a PP2A-specific phosphopeptide as substrates. Expression of various serine/threonine phosphatases, tau and its phosphorylation state were determined by Western blot (WB) and immunocytochemistry (ICC). We found that the total phosphatase activity in the neuronal lysate was increased by 30–50% by all the concentrations of Pb tested. PP2A activity was increased by 5 μg/dL Pb only. PP1 expression was increased (ranging from 25–50%) by 10, 20 and 40 μg/dL of Pb. PP2B expression was increased substantially (up to 2.5-fold) by 10 μg/dL Pb, whereas, higher concentrations did not show any effect. On the other hand, Pb (at all concentrations used) decreased expression of PP2A and PP5. Pb exposure induced substantial hyperphosphorylation of tau at serine 199/202 by 5 and 10 μg/dL Pb, and Threonine 231 at higher doses. Expression of total tau was mostly unaffected by lead. Immunocytochemistry data confirmed the WB results of expression of PP1, PP2A, tau protein and the phosphorylation of tau. These results support our hypothesis that Pb exposure up regulates some of the serine/threonine phosphatases (PP1 and PP2B) that are known to impair memory formation, and suggest a novel mechanism of Pb neurotoxicity.     

Chromium and Manganese Levels in Biological Samples of Pakistani Myocardial Infarction Patients at Different Stages as Related to Controls

It has been speculated that trace elements may play a role in the pathogenesis of heart diseases In the present study, we aimed to access the levels of chromium (Cr) and manganese (Mn) in biological samples (whole blood, urine, and scalp hair) of myocardial infarction (MI) patients of both gender age ranged (45?C60?years) at first, second, and third heart attack (n?=?130), hospitalized in cardiac ward of National Hospital of Hyderabad city (Pakistan). For comparison, healthy age-matched referent subjects (n?=?61), of both gender were also selected. The Cr and Mn in biological samples were measured by electrothermal atomic absorption spectrometry, prior to microwave-assisted acid digestion. The validity of the methodology was checked by the biological certified reference materials. During this study, 78% of 32 registered patients of third MI attack (aged >50?years) were died. In these subjects the concentration of Cr and Mn were decreased by 24.7% and 19.8% in scalp hair, while in blood samples 17.9% and 12.4%, respectively, as compared to those who tolerated third MI attack (p?=?0.063). Although these data do not prove a causal relationship, these results are consistent with the hypothesis that heart disease may cause deficiencies of certain essential trace elements. The excretion levels of Cr and Mn in urine samples of first MCI were higher than controls at p values (0.029 and 0.011), respectively, whereas the excretion rates of both elements were further enhance after second myocardial infarction attack. The Cr and Mn concentration was inversely associated with the risk of myocardial infarction attacks in both genders. These results add to an increasing body of evidence that, Cr and Mn are importance for cardiovascular health.     

Discovery and development of a synthetic peptide derived from lactoferrin for clinical use

There is an urgent need to develop new antimicrobial drugs especially for combating the rise of infections caused by multi-resistant pathogens such as MRSA and VRSA. The problem of antibiotic resistant micro-organisms is expected to increase disproportionally and controlling of infections is becoming difficult because of the rapid spread of those micro-organisms. Primary therapy with classical antibiotics is becoming more ineffective. Combinational therapy of antibiotics with antimicrobial peptides (AMP's) has been suggested as an alternative approach to improve treatment outcome. Their unique mechanism of action and safety profile makes AMP's appealing candidates for simultaneous or sequential use in different cases of infections. In this review, for antimicrobial treatment the application of synthetic antimicrobial peptide hLF(1-11), derived from the first 11 amino acids of human lactoferrin is evaluated in both pre-clinical and clinical settings. Present information indicates that this derivate from lactoferrin is well tolerated in pre-clinical tests and clinical trials and thus hLF(1-11) is an interesting candidate for further exploration in various clinical indications of obscure infections, including meningitis. Another approach of using AMP's is their use in prevention of infections e.g. as coating for dental or bone implants or in biosensing applications or useful as infection specific radiopharmaceutical.     

Spectral modulation attenuates molecular, endocrine, and neurobehavioral disruption induced by nocturnal light exposure

The human eye serves distinctly dual roles in image forming (IF) and non-image-forming (NIF) responses when exposed to light. Whereas IF responses mediate vision, the NIF responses affect various molecular, neuroendocrine, and neurobehavioral variables. NIF responses can have acute and circadian phase-shifting effects on physiological variables. Both the acute and phase-shifting effects induced by photic stimuli demonstrate short-wavelength sensitivity peaking ≈450-480 nm. In the current study, we examined the molecular, neuroendocrine, and neurobehavioral effects of completely filtering (0% transmission) all short wavelengths <480 nm and all short wavelengths <460 nm or partially filtering (~30% transmission) <480 nm from polychromatic white light exposure between 2000 and 0800 in healthy individuals. Filtering short wavelengths <480 nm prevented nocturnal light-induced suppression of melatonin secretion, increased cortisol secretion, and disrupted peripheral clock gene expression. Furthermore, subjective alertness, mood, and errors on an objective vigilance task were significantly less impaired at 0800 by filtering wavelengths <480 nm compared with unfiltered nocturnal light exposure. These changes were not associated with significantly increased sleepiness or fatigue compared with unfiltered light exposure. The changes in molecular, endocrine, and neurobehavioral processes were not significantly improved by completely filtering <460 nm or partially filtering <480 nm compared with unfiltered nocturnal light exposure. Repeated light-dark cycle alterations as in rotating nightshifts can disrupt circadian rhythms and induce health disorders. The current data suggest that spectral modulation may provide an effective method of regulating the effects of light on physiological processes.     

The CW domain, a new histone recognition module in chromatin proteins

Post-translational modifications of the N-terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin-related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me-marked genes, allowing for ASHH2-dependent H3K36 tri-methylation, which contributes to sustained expression of tissue-specific and developmentally regulated genes. This suggests that ASHH2 is a combined 'reader' and 'writer' of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states.     

Distribution of naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone in chitin-induced and uninduced Nepenthes khasiana: molecular events in prey capture

Prey capture and digestion in Nepenthes spp. through their leaf-evolved biological traps involve a sequence of exciting events. Sugar-rich nectar, aroma chemicals, narcotic alkaloid secretions, slippery wax crystals, and other biochemicals take part in attracting, capturing, and digesting preys in Nepenthes pitchers. Here we report the distribution of three potent naphthoquinones in Nepenthes khasiana and their roles in prey capture. Plumbagin was first detected in N. khasiana, and its content (root: 1.33 ± 0.02%, dry wt.) was the highest found in any natural source. Chitin induction enhanced plumbagin levels in N. khasiana (root: 2.17 ± 0.02%, dry wt.). Potted N. khasiana plants with limited growth of roots and aerial parts, showed higher levels of plumbagin accumulation (root: 1.92 ± 0.02%; root, chitin induction: 3.30 ± 0.21%, dry wt.) compared with field plants. Plumbagin, a known toxin, insect ecdysis inhibitor, and antimicrobial, was also found embedded in the waxy layers at the top prey capture region of N. khasiana pitchers. Chitin induction, mimicking prey capture, produced droserone and 5-O-methyl droserone in N. khasiana pitcher fluid. Both these naphthoquinone derivatives provide antimicrobial protection to the pitcher fluid from visiting preys. A two-way barrier was found between plumbagin and its two derivatives. Plumbagin was never detected in the pitcher fluid whereas both its derivatives were only found in the pitcher fluid on chitin induction or prey capture. The three naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone, act as molecular triggers in prey capture and digestion in the carnivorous plant, N. khasiana.