Biophysical studies with AICD-47 reveal unique binding behavior characteristic of an unfolded domain

APP intracellular C-terminal domain (AICD-47), generated upon γ-secretase cleavage of amyloid precursor's protein (APP), bears the signature of a classical intrinsically unstructured domain (IUD). Comparing the recent crystal structures of AICD-47 peptides bound to its different adaptors such as protein-tyrosine-binding domain-2 (PTB2) of Fe65 and Src homology 2 (SH2) domain of growth factor receptor binding protein 2 (Grb2), the "conformational switching" of AICD-47 becomes evident. In order to understand different binding processes undertaken by this flexible molecule, upon recognizing different interfaces resulting in different 3D conformations, spectroscopic and calorimetric studies have been done. CD spectroscopy has revealed an overall random coil like structure in different pHs while TFE (2'-2'-2'-trifluoro ethanol) and HFIP (Hexa fluoro isopropanol) induced α-helicity to a certain extent. Binding of Tyr phosphorylated AICD-47 ((P)AICD-47) to Grb2-SH2 domain was carried out by a favorable enthalpic change (ΔH=-197.5±6.2kcalmole(-1) at 25°C) and an unfavorable entropic contribution (ΔS=-631calmol(-1)deg(-1) at 25°C). Alternative conformation of AICD-47 in different biological contexts is another remarkable feature of IUDs which presumably has definitive roles in regulating Alzheimer's disease phenotype.     

Characterization of cell membrane parameters of clinical isolates of Staphylococcus aureus with varied susceptibility to alpha-melanocyte stimulating hormone

Staphylococcus aureus (S. aureus), a major human pathogen of hospital and community acquired infections, is becoming resistant to almost all commercially available antibiotics. This has prompted development of antimicrobial peptides as therapeutic options. Alpha melanocyte stimulating hormone (α-MSH) is one such peptide known to possess antimicrobial properties. In the present study, we analyzed the antimicrobial activity of α-MSH against 75 clinical strains of S. aureus including both methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) strains. Results of our previous study showed that membrane damage is the major mechanism of staphylocidal activity of α-MSH. In this context, we compared the various bacterial membrane parameters, viz., membrane fluidity, lipid composition, and surface charge of a few selected MSSA and MRSA strains that showed variable susceptibility to the melanocortin peptide. Our results showed that α-MSH killed both type of strains efficiently (≥70% killing in 84% clinical strains after exposure with 6μM of α-MSH for 1h). It was observed that compared to the α-MSH-susceptible strains, the α-MSH-non-susceptible strains had a different membrane order and phospholipid pattern. There was no consistent pattern of cell surface charge to distinguish α-MSH-susceptible strain from a non-susceptible strain. In conclusion, α-MSH possessed potential staphylocidal activity for both against MSSA and MRSA strains. S. aureus strains not susceptible to the peptide exhibited a rigid membrane and a higher amount of the cationic phospholipid as compared to the α-MSH-susceptible strains.     

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens

Aims: To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. Methods and Results: LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross-examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID(50) ml(-1) , whereas the detection limit of the PCR was 1000 TCID(50) ml(-1) . Conclusions: The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. Significance and Impact of the Study: The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost-effective field-based method for direct detection of CPV from the suspected faecal samples of dogs.     

Reduction of Photoautotrophic Productivity in the Cyanobacterium Synechocystis sp. Strain PCC 6803 by Phycobilisome Antenna Truncation

Truncation of the algal light-harvesting antenna is expected to enhance photosynthetic productivity. The wild type and three mutant strains of Synechocystis sp. strain 6803 with a progressively smaller phycobilisome antenna were examined under different light and CO(2) conditions. Surprisingly, such antenna truncation resulted in decreased whole-culture productivity for this cyanobacterium.     

Exploring the world of phospholipids and their interactions with proteins: the work of William Dowhan

The Gene Encoding the Phosphatidylinositol Transfer Protein Is Essential for Cell Growth (Aitken, J. F., van Heusden, G. P., Temkin, M., and Dowhan, W. (1990) J. Biol. Chem. 265, 4711–4717)A Phospholipid Acts as a Chaperone in Assembly of a Membrane Transport Protein (Bogdanov, M., Sun, J., Kaback, H. R., and Dowhan, W. (1996) J. Biol. Chem. 271, 11615–11618)William Dowhan''s curiosity about the connections between phospholipids and proteins associated with them goes back as far as his days as a graduate student with Esmond Snell at the University of California, Berkeley. In these two JBC Classics, his group''s ability to manipulate biochemical and molecular genetics tools to answer fundamental questions about lipid biology shines through. “William Dowhan and his research group have made many contributions to the biochemistry of phospholipid metabolism and membrane biogenesis,” says Robert Simoni at Stanford University.Open in a separate windowBill Dowhan (right) is shown here with the late Chris Raetz (left), who was a longtime collaborator and friend, and his former postdoctoral advisor, the late Gene Kennedy, on the occasion of Kennedy''s 90th birthday in 2009 (photo courtesy of William Dowhan).The first paper, published in 1990, documented the importance of phosphatidylinositol/phosphatidylcholine transfer proteins in vivo. Dowhan''s group, which has been based at the University of Texas Medical School since 1972, used a combination of biochemistry and genetics to clone the protein''s gene. Dowhan had first heard of phospholipid transfer proteins in 1969, when he began his postdoctoral training with Eugene (Gene) Kennedy at Harvard Medical School. At his very first Kennedy lab meeting, the discussion centered around a publication that had just come out (1). The paper described “one of the first observations of proteins in the soluble phase that transferred lipids between bilayers,” recalls Dowhan. “No one could figure out what these proteins really did in vivo, but they knew the proteins had this function” of transferring lipids between membranes.As he moved through his career, Dowhan focused on cloning and characterizing genes and purifying enzymes responsible for phospholipid metabolism in Escherichia coli. Then came a sabbatical in 1983 with Gottfried (Jeff) Schatz at the Biozentrum of the University of Basel in Switzerland, that expanded Dowhan''s research directions into yeast genetics. He says the opportunity to work with Schatz “got me into the possibility of looking for this phosphatidylinositol/phosphatidylcholine transfer protein (PI-TP) in yeast, which I probably would have never done if I hadn''t taken this sabbatical.”Fresh from his sabbatical, Dowhan started tracking down the protein and its gene in vivo. “I submitted a grant at that time with some preliminary data that we had begun to purify to homogeneity the PI-TP from yeast, which had never been done before. Fortunately, we got the grant,” he says.The Dowhan group managed to purify PI-TP from yeast. “The most important part was using basic biochemistry and understanding how to purify proteins before the advent of genetically tagging proteins for affinity chromatography,” explains Dowhan.For the next step in the process of finding the gene for the protein, Dowhan and colleagues had to apply reverse genetics because the yeast genome was not available in the late 1980s. They sequenced the amino terminus of the protein, made the corresponding oligonucleotide probes, tested yeast cDNA libraries with those probes, and pulled out the gene. “We still didn''t know the role PI-TP played in cell function. But now we had the sequence of the gene and the knock-out mutant was not viable,” notes Dowhan. “So we published” the findings.At the same time, Vytas Bankaitis, now at the University of North Carolina, had been working on cloning the SEC14 gene in yeast, which is necessary for vesicular transport. “It turns out we had missed the DNA sequence,” Dowhan says. From Bankaitis'' work, it was obvious that “PI-TP was the product of the SEC14 gene. It all came together in a joint report in Nature. Now we had a function associated with the SEC14 gene, which we didn''t have before,” Dowhan explains (2). “We had a phenotype of a mutant lacking this phospholipid transfer protein, which then stopped vesicular transport.”This initial link between phospholipid metabolism and vesicular transport opened up the field to characterization of the Sec14 protein superfamily in a broad range of biological systems. These proteins contain lipid-binding domains, which sense membrane lipid composition and integrate lipid metabolism and lipid-mediated signaling with an array of cellular processes.The second JBC Classic focused on a different feature of phospholipids: their role in protein folding. Dowhan was fascinated by membrane proteins ever since he was a graduate student and had gone to the Kennedy laboratory as a postdoctoral fellow, intending to purify the membrane component expressed by the lac operon for lactose transport in E. coli. He was unsuccessful because, at that time, the necessary detergents were not available. Once the lactose permease was purified (3), Dowhan noticed in the literature that other researchers mentioned that when the protein was reconstituted in liposomes missing phosphatidylethanolamine, the protein was defective in energy-dependent uphill transport. Dowhan recalls that he wondered, “Was that an artifact of the liposome system or was that also true in vivo?”To get to the bottom of this observation, Dowhan''s group used E. coli to generate null mutants of what were considered to be absolutely essential genes for phospholipid synthesis and cell viability. They created a null mutant of the pssA gene, which encodes the committed step to the synthesis of the major phospholipid, phosphatidylethanolamine. By establishing conditions in which bacterial cells lacking phosphatidylethanolamine remained viable, the investigators were able to identify and characterize different cell phenotypes caused by the missing phospholipid both in vivo and in vitro. In collaboration with Ronald Kaback at UCLA, Dowhan''s group showed that phosphatidylethanolamine was essential for the proper folding of an epitope of lactose permease that was also necessary to support the energy-dependent uphill transport of lactose. “Studies by others have since shown a similar chaperone role for lipids in other bacteria, plants and mammalian cells,” notes Simoni.To obtain their data, the investigators developed a new technique, the Eastern-Western blot. In this method, membrane proteins were delipidated and partially denatured by SDS. The proteins underwent gel electrophoresis and then were transferred to a solid support by Western blotting. A series of individual lipids were then laid over the proteins at a 90° angle so that the investigators could see, after incubating with conformation-specific antibodies, which lipids helped which membrane proteins regain proper conformation.This technique was used to establish that phosphatidylethanolamine was necessary in a late step of folding of lactose permease, but was not necessary to maintain the final folded state. This observation suggested that lipids act as molecular chaperones in helping protein maturation. “This paper set the stage for understanding how lipids affect the topological organization of wild-type proteins in the membrane,” notes Dowhan.Dowhan and his collaborator Mikhail Bogdanov have continued using bacterial mutants in phospholipid metabolism to systematically manipulate the native membrane lipid compositions during the cell cycle. They have analyzed the transmembrane domain orientation of membrane proteins to establish the molecular basis for lipid-dependent organization of lactose permease and other secondary transporters (4).Dowhan says his work has two take-home messages. One is that “Lipids aren''t just glorified biological detergents,” he says. “They have specific roles” in the cell. The other message is in the power of numbers. Dowhan says the more techniques applied to solve a biological mystery, the more likely the mystery will be successfully solved.     

Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defence against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen‐activated protein kinase, LmjMPK2. Leishmania parasites coexpressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo‐osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr‐197 and this phosphorylation requires LmjMPK2 activity. Lys‐42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild‐type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. Leishmania mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild‐type cells. This is the first report where a parasite aquaglyceroporin activity is post‐translationally modulated by a mitogen‐activated protein kinase.     

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line

Background

The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.

Methodology

Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.

Principal Findings

The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.

Conclusions

We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.