Charlene M. Schaldach

Analysis of Single Bacterial Spores by Micro-Raman Spectroscopy

The spectroscopic analysis of individual living cells in their native state provides a powerful tool for the investigation of complex biological systems. Micro-Raman spectroscopy, in which confocal microscopy is combined with Raman spectroscopy, offers a promising route to achieving this, because it provides a means to study individual cells and cellular components.1–9 Here we describe the analysis of individual bacterial endospores from four species in the genus Bacillus by micro-Raman spectroscopy. Previous Raman studies on Bacillus spores resulted in spectra with strong scattering from calcium dipicolinate (CaDPA), which is the calcium chelate of dipicolinic acid (2,6-pyridinedicarboxylic acid, DPA); however, these earlier studies were conducted on concentrated samples of spores.10–14 By using micro-Raman spectroscopy, we demonstrate the ability to obtain similar information from individual spores. The Raman spectra for most spores studied were dominated by scattering from CaDPA, although Raman scattering assignable to protein bands and to phenylalanine was also observed. Approximately 4% of the spores analyzed did not exhibit Raman intensity from CaDPA, possibly due to incomplete sporulation. The results presented indicate that micro-Raman spectroscopy is a promising technique for in-

N-Methoxyindole-3-Carbinol Is a More Efficient Inducer of Cytochrome P-450 1A1 in Cultured Cells Than Indol-3-Carbinol

The well-documented reduction of cancer risk by high dietary cruciferous vegetable intake may in part be caused by modulation of cytochrome P-450 (CYP) expression and activity by indoles. The purpose of the present experiments was to study the mechanism of CYP 1A1 induction by N-methoxyindole-3-carbinol (NI3C) in cultured cells and to compare the CYP-inducing potential of NI3C and indole- 3-carbinol (I3C) administered to rats. NI3C induced 7-ethoxyresorufin-O-deethylase (EROD) activity in Hepa-1c1c7 cells in a concentration-dependent manner with 10-fold higher efficiency than I3C. Inasmuch as NI3C induced binding of the aryl hydrocarbon receptor (AhR) to the dioxin-responsive element and induced expression of endogenous CYP 1A1 mRNA and an AhR-responsive chloramphenicol acetyl transferase construct, we conclude that NI3C can activate the AhR. Besides the induction of CYP 1A1, we observed an inhibition of EROD activity, with a concentration causing 50% inhibition of 6 μM. Oral administration of NI3C at 570 μmol/kg body wt to male Wistar rats increased the hepatic CYP 1A1 and 1A2 protein levels, as well as the EROD and 7-methoxyresorufin O-demethylase activities at 8 and 24 hours after administration, but the responses were less pronounced than after administration of I3C at 570 μmol/kg body wt. Furthermore, NI3C did not induce hepatic 7-pentoxyresorufin O-depentylase activity, as I3C did. Ascorbigen, another indolylic compound formed during degradation of glucobrassicin in the presence of ascorbic acid, was tested in the same animal model, and ascorbigen only weakly induced hepatic CYP 1A1 and 1A2, but not CYP 2B1/2. In conclusion, NI3C is a more efficient inducer of CYP 1A1 in cultured cells than I3C but is less active when administered to rodents.

Non-DNA Methods for Biological Signatures

Publisher Summary This chapter focuses on the methods that can determine chemical or structural features of biological agent particles that are signatures of particular methods of growth and post-growth processing (often referred to as “weaponization”). The detection of these signatures in a sample of a bio-weapon (BW) agent can aid the attribution by indicating: (1) the level of sophistication of the producer, (2) the access to particular types of agent weaponization information, (3) the likelihood that the material could be or has been produced at a significant scale, (4) and by providing essential sample matching data for ascertaining a putative relationship with other samples obtained in other venues. An example of the use of biologicals in forensic science is DNA, amplied by the Polymerase Chain Reaction (PCR) technique, legally admissible in courtas evidence. DNA evidence is successfully used in the court to convict or clear people of crimes because each persons DNA is unique. High-resolution techniques are being applied to investigations; such as Environmental scanning electron microscopy (ESEM) is used for taking high-resolution images under hydrated conditions; this avoids any artifacts associated with the critical point drying process that is required under normal Scanning Electron Microscopy (SEM) operations. ESEM is also equipped with Energy Dispersive X-ray (EDX) microanalysis and Backscatter capabilities. SEM is a standard “workhorse” technique for characterizing particulate samples, found in many laboratories worldwide. It provides excellent imaging of the surfaces of agent particles and other material in a sample, and is used for identifying likely agent particles for analysis by other instruments. When combined with EDX, the elemental composition of the material in the imaged region can be determined. These techniques continue to signature libraries of correlations between analyses and growth and processing conditions of growth, it will be necessary to develop an information system which combines types of data to determine unique signatures.