A role for clathrin in reassembly of the Golgi apparatus

The Golgi apparatus is a highly dynamic organelle whose organization is maintained by a proteinaceous matrix, cytoskeletal components, and inositol phospholipids. In mammalian cells, disassembly of the organelle occurs reversibly at the onset of mitosis and irreversibly during apoptosis. Several pharmacological agents including nocodazole, brefeldin A (BFA), and primary alcohols (1-butanol) induce reversible fragmentation of the Golgi apparatus. To dissect the mechanism of Golgi reassembly, rat NRK and GH3 cells were treated with 1-butanol, BFA, or nocodazole. During washout of 1-butanol, clathrin, a ubiquitous coat protein implicated in vesicle traffic at the trans-Golgi network and plasma membrane, and abundant clathrin coated vesicles were recruited to the region of nascent Golgi cisternae. Knockdown of endogenous clathrin heavy chain showed that the Golgi apparatus failed to reform efficiently after BFA or 1-butanol removal. Instead, upon 1-butanol washout, it maintained a compact, tight morphology. Our results suggest that clathrin is required to reassemble fragmented Golgi elements. In addition, we show that after butanol treatment the Golgi apparatus reforms via an initial compact intermediate structure that is subsequently remodeled into the characteristic interphase lace-like morphology and that reassembly requires clathrin.     

Superior quality agar from <Emphasis Type="Italic">Gracilaria</Emphasis> species (Gracilariales,Rhodophyta) collected from the Gulf of Mannar,India

Gracilaria edulis, G. crassa, G. foliifera, and G. corticata are naturally occurring agarophytes of Indian waters. These agarophytes were evaluated for their agar contents using an improved process recently reported by us (US Patent 2005/0267296A1). The effect of different concentrations of NaOH in the alkali treatment was studied for optimizing the extraction conditions. These Gracilaria species of Indian waters produced agars, both native and alkali treated, with different properties confirming the heterogeneity of the agar polymers in this genera, as one would expect. Among these, G. edulis and G. crassa produced agar polymers having high gel strengths of 490 ± 8.16 and 800 ± 15.4 g cm⁻², respectively, with 8% NaOH treatment as opposed the low gel strength agars that have been reported in the literature to date.     

Distribution and Determinants of Cytomegalovirus Induced End Organ Disease/s among People Living with HIV/AIDS in a Poor Resource Setting: Observation from India

BackgroundIn India, despite well-established anti-retroviral treatment programs, Cytomegalovirus (CMV) infection-related end-organ diseases (EODs) still remain a major concern resulting in exacerbation of morbidity and mortality among HIV/AIDS patients. A prospective study was designed to understand the distribution and prognosis of CMV associated EODs and to determine a standardized cut-off value for serum CMV viral load associated with the development of EODs amongst HIV/AIDS subjects.MethodsIn a cohort of 400 late-diagnosed HAART naïve HIV/AIDS subjects attending anti-retroviral centers of Kolkata during 2008–2014, the median duration of follow-up was 560 days, and at least 3 visits subsequent to the baseline were mandatory for eligibility. HIV-1 and CMV viral load were estimated by performing Real-Time Polymerase Chain Reactions (PCR).ResultsAmong subjects, 40.5% (162/400) had CMV EODs which were more common at lower CD4 counts. Poor prognosis and higher death rate were associated with a low CD4 count and increased HIV-1 and CMV viral loads. Subjects having higher CD4 count responded better to therapy [for CD4 = 60–100: Risk Ratio:RR = 1.48 (95% Confidence Interval: 95%CI = 1.18–1.82) and for CD4 = 30–59: RR = 1.64 (95%CI = 1.18–2.27)]. The cut off value of the serum CMV viral load (expressed as log10DNA/ml serum) associated with the development of EODs and disseminated CMV EODs was determined as 5.4 (p<0.0001) and 6.4 (p<0.0001) respectively. These cut offs were found to have satisfactorily high sensitivity, specificity, positive and negative predictive values.ConclusionPrognosis of CMV EOD was poor as indicated by higher death rates among subjects with lower CD4 count, and specific cut-off values were found to have useful potential for identification and treatment of CMV infected HIV/AIDS patients in due time to avoid CMV EODs among HIV/AIDS subjects. Targeted intervention programs seemed to be required urgently to make these cut-offs operational in order to minimize the burden of CMV EOD in this vulnerable population.     

Naphthoquinone-mediated Inhibition of Lysine Acetyltransferase KAT3B/p300, Basis for Non-toxic Inhibitor Synthesis

Hydroxynaphthoquinone-based inhibitors of the lysine acetyltransferase KAT3B (p300), such as plumbagin, are relatively toxic. Here, we report that free thiol reactivity and redox cycling properties greatly contribute to the toxicity of plumbagin. A reactive 3rd position in the naphthoquinone derivatives is essential for thiol reactivity and enhances redox cycling. Using this clue, we synthesized PTK1, harboring a methyl substitution at the 3rd position of plumbagin. This molecule loses its thiol reactivity completely and its redox cycling ability to a lesser extent. Mechanistically, non-competitive, reversible binding of the inhibitor to the lysine acetyltransferase (KAT) domain of p300 is largely responsible for the acetyltransferase inhibition. Remarkably, the modified inhibitor PTK1 was a nearly non-toxic inhibitor of p300. The present report elucidates the mechanism of acetyltransferase activity inhibition by 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor.     

Nuclear pool of phosphatidylinositol 4 phosphate 5 kinase 1α is modified by polySUMO-2 during apoptosis

Phosphatidylinositol 4 phosphate 5 kinase 1α (PIP5K) is mainly localized in the cytosol and plasma membrane. Studies have also indicated its prominent association with nuclear speckles. The exact nature of this nuclear pool of PIP5K is not clear. Using biochemical and microscopic techniques, we have demonstrated that the nuclear pool of PIP5K is modified by SUMO-1 in HEK-293 cells stably expressing PIP5K. Moreover, this SUMOylated pool of PIP5K increased during apoptosis. PolySUMO-2 chain conjugated PIP5K was detected by pull-down experiment using affinity-tagged RNF4, a polySUMO-2 binding protein, during late apoptosis.     

On the properties of agar gel containing ionic and non-ionic surfactants

Rheological and thermal properties of agar sol and gel in presence of various cationic, anionic and non-ionic surfactants are reported. The agar used was from the red seaweed Gelidiella acerosa. The gel strength, viscosity, rigidity (G'), gelling temperature and melting temperature were observed to decrease in presence of non-ionic surfactants whereas these were enhanced in presence of ionic surfactants. TGA studies showed that 1.5% agar gels containing non-ionic surfactants lose water at a lower temperature than the control agar gel whereas gels containing ionic surfactants hold on to water more tenaciously. DSC studies, on the other hand, show that the gel to sol transition occurs at lower temperatures in presence of non-ionic surfactants and at higher temperature in presence of ionic surfactants when compared with the control gel. The non-ionic surfactants, Triton X-100 and Brij 35, enabled relatively concentrated agar extractive to be filtered readily, as a result of which water usage in the process could be reduced by 50%. The surfactant was subsequently removed through freeze-thaw operations to restore the gelling capacity of the agar. The finding that 0.3-0.4% (w/v) sodium lauryl sulfate (SLS) lowers the sol-gel transition temperature from 41 to 36 degrees C without adversely affecting gel strength is another useful outcome of the study that may enable better formulations of bacteriological agar to be prepared.     

In vitro and in silico studies on membrane interactions of diverse Capsicum annuum flower γ-thionin peptides

Thionins are small, cysteine-rich peptides that play an important role in plant defense, primarily through their interactions with membranes. Eight novel γ-thionin peptides (CanThio1-8) were isolated from the flower of Capsicum annuum. Sequence analysis revealed that the peptides cluster into three groups. A representative peptide from each group (CanThio1, 2, and 3) was used for experimental characterization. Interestingly, peptides were found to possess some cytotoxic activity against normal human embryonic kidney cell line but higher cytotoxicity against cancer cell line MCF-7. CanThio3 peptide was chosen as a representative peptide to study the molecular mechanism of action on membranes. Microsecond timescale atomistic simulations of CanThio3 were performed in the presence of a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer. Simulations revealed that CanThio3 interacts with the bilayer and causes lipid thinning in the vicinity. Nonpolar amino acids specific to the α-core region of CanThio3 along with nonpolar residues in the γ-core region are seen to interact with the lipid tails. The differences in the amino acid sequence of CanThio peptides in these regions explain the variability in cytotoxic activities. In summary, our results demonstrate the membrane-mediated activity of a novel series of γ-thionin peptides from C. annuum.     

Preparation of superior quality products from two Indian agarophytes

The effect of alkali pre-treatments on the properties of agar obtained from Gelidiella acerosa and Gelidium pusillum growing along the West and South coast of India was studied in order to evaluate its potential for industrial use. Agar samples were extracted by hot water extraction and termed native agar, and alkali pre-treatment was carried out with various NaOH concentrations e.g. 4%, 6%, 8%, 10% and 15%, to optimise the alkali concentration for preparation of alkali-treated agar. Gelidiella acerosa and Gelidium pusillum produced superior quality agar polymers having greater gel strengths 2,000 ± 50 and 1,400 ± 50 g cm⁻² and lower sulphate contents ≤0.30% and ≤0.50%, respectively, as opposed the low gel strength and high sulphate content agars reported in the literature. Furthermore, the physicochemical properties of agars were identical with the commercially available agars. The results of this study revealed that Indian agarophytes may be used for production of superior quality agar and agaroses, which may be used for food and biological applications.     

Galactans from Gracilaria millardetii and G textorii (Gracilariales,Rhodophyta) of Indian waters

Galactans containing methylated galactan moieties were extracted from Indian agarophytes, namely, Gracilaria millardetii and Gracilaria textorii growing naturally along the west coast of India. The galactans were treated with α‐amylase to remove floridean starch. These were characterized by Fourier Transform‐Infrared (FT IR), ¹³C‐Nuclear Magnetic Resonance (C NMR), Gas Chromatography‐Mass Spectrometry (GC MS), Inductively Coupled Plasma spectroscopy (ICP) and Gel Permeation Chromatography (GPC) and were found to be composed of d ‐galactose, 6‐O‐methyl‐D‐galactose and 3,6‐anhydro‐L‐galactose. Methylation analyses revealed that both the polysaccharides consisted of 3‐, 2,3‐, 4,6‐linked galactose as well as four‐linked 3,6‐anhydro galactose residues. Both the Gracilaria species produced low gelling (<100 g cm^?2) and highly sulfated (2.1% to 4.8%) galactans containing very low heavy metal contents (ICP). These galactans may be of potential utility in food and biological applications.     

Profiling of cellulose content in Indian seaweed species

Cellulose contents were estimated in 12 seaweed samples belonging to different families e.g. red, brown and green, growing in Indian waters. Each cellulose sample was fractionated to yield alpha (α) and beta (β) celluloses. Characterization was done using various analytical tools and results were validated by comparison with those of the cellulose obtained from Whatman filter paper No. 4. The greatest yields of cellulose (crude), α- and β-cellulose were obtained from Gelidiella acerosa (13.65%), Chamaedoris auriculata (9.0%) and G. acerosa (3.10%). G. acerosa was also found to contain relatively high amount of α-cellulose (8.19%). The lowest cellulose contents were recorded from Kappaphycus alvarezii (2.00%) and Sarconema scinaioides (2.1%), while the latter contained the lowest α-, and β-celluloses (1.0% and 0.30%, respectively). It appears that agarophytic and alginophytic algae contain high cellulose and α-cellulose contents, while the carrageenophyte contains low cellulose. The brown algae, in general contain high cellulose as well as α- and β-celluloses.