Chronobiologically interpreted ambulatory blood pressure monitoring: past,present, and future

Research at the Halberg Chronobiology Center focused to a large extent on the monitoring of blood pressure (BP) and heart rate (HR). Self-measurements and later ambulatory BP monitoring yielded new knowledge of interest to basic science and clinical practice. After a brief review of BP measurement, we outline developments in methods of data analysis that paralleled technological advances in the measurement of BP. We review work done in cooperation with colleagues worldwide to illustrate how a chronobiological approach led to the mapping of spontaneous circadian and other rhythms for the derivation of refined reference values and to the assessment of response rhythms underlying chronotherapy. BIOCOS members work in different fields, spanning from cardiology and nutrition to obesity, diabetes, exercise physiology and rehabilitation, but all strive for “pre-habilitation”. The early recognition of increased risk can prompt the timely institution of prophylactic intervention. As technology continues to improve, studies on groups are complemented by longitudinal self-surveillance for health maintenance. Longitudinal records serve for the investigation of environmental influences on human physiology, the topic of chronomics. As current advances in technology and wireless communication will likely impact the future of healthcare, chronobiological methods and concepts should be an integral part of this seachange.     

Diversity of Phytases in the Rumen

Examples of a new class of phytase related to protein tyrosine phosphatases (PTP) were recently isolated from several anaerobic bacteria from the rumen of cattle. In this study, the diversity of PTP-like phytase gene sequences in the rumen was surveyed by using the polymerase chain reaction (PCR). Two sets of degenerate primers were used to amplify sequences from rumen fluid total community DNA and genomic DNA from nine bacterial isolates. Four novel PTP-like phytase sequences were retrieved from rumen fluid, whereas all nine of the anaerobic bacterial isolates investigated in this work contained PTP-like phytase sequences. One isolate, Selenomonas lacticifex, contained two distinct PTP-like phytase sequences, suggesting that multiple phytate hydrolyzing enzymes are present in this bacterium. The degenerate primer and PCR conditions described here, as well as novel sequences obtained in this study, will provide a valuable resource for future studies on this new class of phytase. The observed diversity of microbial phytases in the rumen may account for the ability of ruminants to derive a significant proportion of their phosphorus requirements from phytate.     

Glomerular morphometry in biopsy evaluation of minimal change disease, membranous glomerulonephritis, thin basement membrane disease and Alport's syndrome

OBJECTIVE: To assess the role of glomerular morphometry in biopsy evaluation in renal disorders in addition to conventional diagnostic procedures. STUDY DESIGN: The study includes 10 cases each of minimal change disease (MCD), idiopathic membranous glomerulonephritis (idiopathic MGN), thin basement membrane disease (TBMD) and Alport's syndrome. Renal biopsies for normal study were obtained from age- and sex-matched autopsy cases without any renal disorder, confirmed histologically and ultrastructurally. Glomerular morphometry was performed by semiautomatic procedure using Quantimet-600 image analysis system (Leica, Cambridge, United Kingdom). RESULTS: Morphometric findings revealed significant increase in glomerular "diameter and area" and "tuft diameter and area" in patients of idiopathic MGN, but no significant difference was found in patients of MCD, TBMD and Alport's syndrome. Evaluation of glomerular volume fractions revealed a decrease in capillary space volume fraction and an increase in "membranes and mesangial matrix" volume fraction in patients with idiopathic MGN. Significant decrease in capillary space volume fraction was also observed in patients of MCD. Patients with Alport's syndrome showed variable changes. CONCLUSION: Glomerular morphometry could be considered as an adjunct to the diagnostic armamentarium of light microscopy, immunofluorescence and electron microscopy because it provides deep insight into quantitative parameters.     

Small and lethal: searching for new antibacterial compounds with novel modes of action.

The discovery of drugs used to combat infectious diseases is in the process of constant change to address the ever-worsening problem of antibiotic resistance in pathogens and a lack of recent success in discovering new antibacterial drugs. In the past 2 decades, research in both academia and industry has made use of molecular biology, genetics, and comparative genomics, which has led to the development of key technologies for the discovery of novel antibacterial agents. Genome-scale efforts have led to the identification of numerous molecular targets. Chemical diversity from synthetic combinatorial libraries and natural products is being used to screen for new molecules. A wide variety of approaches are being used in the search for novel antibiotics, and these can be categorized as being either biochemically focused or cell based. The over-riding goal of all methods in use today is to discover new chemical matter with novel mechanisms of action against drug-resistant pathogens.     

4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization

The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM < c < mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9.     

Role of Minor Groove Width and Hydration Pattern on Amsacrine Interaction with DNA

Amsacrine is an anilinoacridine derivative anticancer drug, used to treat a wide variety of malignancies. In cells, amsacrine poisons topoisomerase 2 by stabilizing DNA-drug-enzyme ternary complex. Presence of amsacrine increases the steady-state concentration of these ternary complexes which in turn hampers DNA replication and results in subsequent cell death. Due to reversible binding and rapid slip-out of amsacrine from DNA duplex, structural data is not available on amsacrine-DNA complexes. In the present work, we designed five oligonucleotide duplexes, differing in their minor groove widths and hydration pattern, and examined their binding with amsacrine using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Complexes of amsacrine with calf thymus DNA were also evaluated for a comparison. Our results demonstrate for the first time that amsacrine is not a simple intercalator; rather mixed type of DNA binding (intercalation and minor groove) takes place between amsacrine and DNA. Further, this binding is highly sensitive towards the geometries and hydration patterns of different minor grooves present in the DNA. This study shows that ligand binding to DNA could be very sensitive to DNA base composition and DNA groove structures. Results demonstrated here could have implication for understanding cytotoxic mechanism of aminoacridine based anticancer drugs and provide directions to modify these drugs for better efficacy and few side-effects.     

Exacerbation of Brain Pathology After Partial Restraint in Hypertensive Rats Following SiO2 Nanoparticles Exposure at High Ambient Temperature

This investigation examines the possibility that exposure to silica dust of hypertensive individuals may exacerbate brain pathology and sensory motor dysfunction at high environmental temperature. Hypertension was produced in rats (200–250 g) by two-kidney one clip (2K1C) method, and in these animals, SiO₂ nanoparticles (NPs; 50 to 60 nm) were administered at 50 mg/kg, i.p. daily for 1 week. On the 8th day, these rats were subjected to partial restraint in a Perspex box for 4 h either at room temperature (21 °C) or at 33 °C in a biological oxygen demand incubator (wind velocity, 2.6 cm/s; relative humidity, 65 to 67 %). In these animals, behavioral functions, blood–brain barrier (BBB) permeability to Evans blue albumin (EBA) and radioiodine (^[131]-Iodine), brain water content and neuronal injuries were determined. Hypertensive rats subjected to 4 h restraint at room temperature did not exhibit BBB dysfunction, brain edema, neural injury, or alterations in rotarod or inclined plane angle performances. However, when these hypertensive rats were subjected to restraint at 33 °C, breakdown of the cortical BBB (EBA, +38 %; radioiodine, +56 %), brain water (+0.88 %), neuronal damages (+18 %), and behavioral impairment were exacerbated. Interestingly, SiO₂ exposure to these rats further exacerbated BBB breakdown (EBA, 280 %; radioiodine, 350 %), brain edema (4 %), and neural injury (30 %) after identical restraint depending on the ambient temperature. SiO₂ treatment also induced brain pathology and alteration in behavioral functions in normotensive rats after restraint at high temperature. These observations clearly show that hypertension significantly enhances restraint-induced brain pathology, and behavioral anomalies particularly at high ambient temperature and SiO₂ intoxication further exacerbated these brain pathologies and cognitive dysfunctions.     

A Structural Insight into Major Groove Directed Binding of Nitrosourea Derivative Nimustine with DNA: A Spectroscopic Study

Nitrosourea therapeutics occupies a definite place in cancer therapy but its exact mechanism of action has yet to be established. Nimustine, a chloroethyl nitrosourea derivative, is used to treat various types of malignancy including gliomas. The present work focuses on the understanding of nimustine interaction with DNA to delineate its mechanism at molecular level. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) has been used to determine the binding sites of nimustine on DNA. Circular dichroism (CD) spectroscopy has been used to confirm conformational variations in DNA molecule upon nimustine-DNA interaction. Thermodynamic parameters of nimustine-DNA reaction have been calculated by isothermal titration calorimetry. Results of the present study demonstrate that nimustine is not a simple alkylating agent rather it causes major grove-directed-alkylation. Spectroscopic data suggest binding of nimustine with nitrogenous bases guanine (C6 = O6) and thymine (C4 = O4) in DNA major groove. CD spectra of nimustine-DNA complexes point toward the perturbation of native B-conformation of DNA and its partial transition into C-form. Thermodynamically, nimustine-DNA interaction is an entropy driven endothermic reaction, which suggests hydrophobic interaction of nimustine in DNA-major groove pocket. Spectral results suggest base binding and local conformational changes in DNA upon nimustine interaction. Investigation of drug-DNA interaction is an essential part of rational drug designing that also provides information about the drug’s action at molecular level. Results, demonstrated here, may contribute in the development of new nitrosourea therapeutics with better efficacy and fewer side effects.