Ellman's reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes.

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry of chromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

Prevalence and antifungal drug resistance of dermatophytes in the clinical samples from Pakistan.

Dermatophytosis is a major health problem all over the world including Pakistan. This is the first report of detection of dermatophytes and their antifungal drug resistance in the Northern and Western parts of Pakistan. A total of 154 samples were collected from different hospitals of Khyber Pakhtunkhwa, and out of them 136 samples were found positive. Tinea corporis (35%) was the most predominant type of infection followed by Tinea capitis (22%). The fungi identified in Tinea corporis infection types were identified as Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum. The fungi identified in Tinea capitis included Trichophyton violaceum, T. mentagrophytes, Microsporum ferrugineum. The gender wise distribution showed both males (52%) and females (48%) were infected with the fungi. More cases belonged to the rural parts of the country. Age wise distribution showed that the infection was more prevalent in the children and the prevalence decreased with the increase in age. The positive samples were checked against two antifungal agents: fluconazole and nystatin. Among 136 positive samples, none of the isolates showed resistance to nystatin while 7% of the samples showed resistance to fluconazole. The resistant isolates were then identified by amplifying the 18S rRNA gene, using universal primers (ITS1, ITS4). Among the 9 resistant isolates, 5 isolates were identified as Trichophyton spp., 3 as Microsporum spp. and 1 as Epidermophyton spp.

Structure-function analysis of manganese exporter proteins across bacteria.

Manganese (Mn) is an essential trace nutrient for organisms because of its role in cofactoring enzymes and providing protection against reactive oxygen species (ROS). Many bacteria require manganese to form pathogenic or symbiotic interactions with eukaryotic host cells. However, excess manganese is toxic, requiring cells to have manganese export mechanisms. Bacteria are currently known to possess two widely distributed classes of manganese export proteins, MntP and MntE, but other types of transporters likely exist. Moreover, the structure and function of MntP is not well understood. Here, we characterized the role of three structurally related proteins known or predicted to be involved in manganese transport in bacteria from the MntP, UPF0016, and TerC families. These studies used computational analysis to analyze phylogeny and structure, physiological assays to test sensitivity to high levels of manganese and ROS, and inductively coupled plasma-mass spectrometry (ICP-MS) to measure metal levels. We found that MntP alters cellular resistance to ROS. Moreover, we used extensive computational analyses and phenotypic assays to identify amino acids required for MntP activity. These negatively charged residues likely serve to directly bind manganese and transport it from the cytoplasm through the membrane. We further characterized two other potential manganese transporters associated with a Mn-sensing riboswitch and found that the UPF0016 family of proteins has manganese export activity. We provide here the first phenotypic and biochemical evidence for the role of Alx, a member of the TerC family, in manganese homeostasis. It does not appear to export manganese, but rather it intriguingly facilitates an increase in intracellular manganese concentration. These findings expand the available knowledge about the identity and mechanisms of manganese homeostasis proteins across bacteria and show that proximity to a Mn-responsive riboswitch can be used to identify new components of the manganese homeostasis machinery.