Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

Impedance of goat lens as a function of frequency at DC voltages 0, 50, 100, 200 mV

Impedance characteristics of lens tissue has been studied using the AC impedance system (EG&G PARC, model 318) at different low voltage excitations using a Cole-Cole Plot. The extracellular resistance (Re), intracellular resistance (Ri), depressed angle (theta), total impedance (/Z/), and phase angle (phi) of the tissue were measured. The impedance locus between the real part (Z') and imaginary part (Z') of the complex impedance of lens was examined at discrete frequencies ranging from 10 mHz to 10 Hz. A decrease in extra-cellular resistance (Re) and increase in distribution of the factor (alpha) of 56.8 KOmega, 48.1 KOmega, 32.8 KOmega, 13.4 KOmega, and 0.40, 0.43, 0.46, 0.53 were found at 0 mV, 50 mV, 100 mV, and 200 mV, respectively. The total impedance (/Z/) and phase angle (phi) were also evaluated and the observed frequency dependent for the frequency range was tested as a function of excitation voltage. An attempt has been made to explain the effect of voltage stress on lens impedance.     

Hypoxia induced non-apoptotic cellular changes during aerenchyma formation in rice (Oryza sativa L.) roots

The stress of low oxygen concentrations in a waterlogged environment is minimized in some plants that produce aerenchyma, a tissue characterized by prominent intercellular spaces. It is produced by the predictable collapse of root cortex cells, indicating a programmed cell death (PCD) and facilitates gas diffusion between root and the aerial environment. The objective of this study was to characterize the cellular changes take place during aerenchyma formation in root of rice that accompany PCD. Scanning electron microscopy and transmission electron microscopy were used for cellular analysis of roots. Aerenchyma development was observed in both aerobic and flooded conditions. Structural changes in membranes and organelles were examined during development of root cortex cells to compare with previous examples of PCD. There was an initial collapse which started at a specific position in the mid cortex, indicating loss of turgor, and the cytoplasm became more electron dense. These cells were distinct in shape from those located towards the periphery. Mitochondria and endoplasmic reticulum appeared normal at this early stage though the tonoplast lost its integrity. Subsequently it underwent further degeneration while the plasmalemma retracted from the cell wall followed by death of neighboring cells followed a radial path. However, pycnosis of the nucleus, blebbing of plasma membrane and production of apoptotic bodies were not found which in turn indicated nonapoptotic PCD during aerenchyma formation in rice.     

Effect of 2-benzoxazolinone (BOA) on seedling growth and associated biochemical changes in mung bean (Phaseolus aureus)

BOA (2-benzoxazolinone) is a potent phytotoxin present in several graminaceous crops such as rye, maize and wheat. Due to its wide range of phytotoxicity, it is considered as a potential pesticide. A study was conducted to explore the impact of BOA on the radicle and plumule elongation of mung bean (Phaseolus aureus) and associated changes in the macromolecular content - proteins and carbohydrates - and activities of enzymes like amylases, proteases, polyphenol oxidases and peroxidases. BOA significantly reduced the radicle and plumule length of P. aureus, and the contents of proteins and carbohydrates in both root and leaf tissue. On the other hand, activities of hydrolytic enzymes - proteases, amylases, polyphenol oxidases and peroxidases - increased substantially in both root and leaf tissue of P. aureus upon BOA exposure. This indicated that BOA treatment induced stress in P. aureus and enhanced enzyme activities to counter the induced stress and continue the growth. In other words, BOA-induced stress altered the plant biochemical status and related enzyme activities resulting in increased metabolism that serves to provide protection against cellular injury. Such studies providing information about the biomolecular content and enzymatic activities in response to natural products serve as clues for furtherance of knowledge about the modes of action of natural compounds of commercial interest.     

Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-mediated pathway

Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, to regress 7,12-dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway.     

Status, invasiveness and environmental threats of three tropical American invasive weeds (Parthenium hysterophorus L., Ageratum conyzoides L., Lantana camara L.) in India

Invasive weeds have threatened the integrity of ecosystems throughout the world. They affect not only the species diversity of native areas but also their biological integrity. In India, a number of invasive exotic weeds have been reported but some viz. Parthenium hysterophorus, Lantana camara and Ageratum conyzoides, especially those from tropical America are troublesome and have caused adverse ecological, economic and social impact. These weeds can be seen growing in different landscapes but are luxuriantly localized in unattended forests and cultivated areas. Parthenium hysterophorus (Asteraceae, commonly known as congress grass) is perhaps the most troublesome and noxious weed of urban and rural India. Besides rapidly colonizing areas replacing the native vegetation, it is also known to cause a number of human health problems such as skin allergy, rhinitis and irritation to eyes of the residents in the vicinity. Likewise, it causes fodder scarcity in addition to being unpalatable and toxic to livestock. Lantana camara (Verbenaceae), another serious tropical American pest, has encroached upon large areas of land, especially the forests where it has virtually replaced the forest floor vegetation and reduced tree growth. Also because of its bushy and spreading type of growth it obstructs forest operations. The third weed, Ageratum conyzoides (Asteraceae, Billy goat weed) has invaded agricultural fields. It interferes with crops and causes yield reductions of major staple crops of India. When it invades rangeland areas, it out competes native grasses causing scarcity of fodder. These weeds have similar growth strategies such as fast growth rates, short life-cycles, greater reproductive potential, high competitive abilities and allelopathy that make them successful invaders of native habitats. Mechanical, chemical, biological and cultural control tactics have failed individually, though integrated approaches combining all these approaches along with community participation and proper land management have been relatively successful. This paper presents various aspects of biology, ecology, hazards and control measures of these weeds.     

Baroreflex mechanisms regulating mean level of SNA differ from those regulating the timing and entrainment of the sympathetic discharges in rabbits

The arterial baroreflex pathway provides the fundamental basis for the short-term control of blood pressure via the rapid regulation of the mean level of sympathetic nerve activity (SNA) in response to changes in blood pressure. A central tenet in the generation and regulation of bursts of SNA is that input from the arterial baroreceptors also regulates the timing of the bursts of sympathetic activity. With the use of an implantable telemetry-based amplifier, renal SNA was recorded in intact and arterial baroreceptor-denervated (SAD) conscious rabbits. Data were collected continuously while animals were in their home cage. Mean levels of SNA were not different between SAD and baroreceptor-intact animals. Whereas SNA was unresponsive to changes in blood pressure in SAD rabbits, the timing of the bursts of SNA relative to the arterial pulse wave was maintained (time between the diastolic pressure and the next maximum SNA voltage averaged 107+/-12 ms SAD vs. 105+/-7 ms intact). Transfer function analysis between blood pressure and SNA indicates the average gain at the heart rate frequency was not altered by SAD, indicating strong coupling between the cardiac cycle and SNA bursts in SAD animals. Further experiments in anesthetized rabbits showed that this entrainment is lost immediately after performing baroreceptor denervation surgery and remained absent while the animal was under anesthesia but returned within 20 min of turning off the anesthesia. We propose that this finding indicates the regulation of the mean level of SNA requires the majority of input from baroreceptors to be functional; however, the regulation of the timing of the bursts in the conscious animal requires only minimal input, such as a sensitive trigger mechanism. This observation has important implications for understanding the origin and regulation of SNA.     

Functional impairment of CD4 T cells despite normalization of T cell number in HIV

Using an in vitro model, we demonstrate that when CD4 T cells from HIV infected subjects are enriched from total blood lymphocytes the immune response to antigen is augmented. However, augmentation of this response is confined to HIV infected subjects with relatively preserved CD4 T cell counts. Enriching for CD4 T cells had no effect on antigen responses in patients with low CD4 lymphocyte counts. These findings support the concept that CD4 T cells in late stage HIV have inherent qualitative defects.