In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology.
Mushrooms are considered an important food for their traditionally famous nutritional and medicinal values, although much information about their potential at the molecular level is unfortunately unknown. Edible mushrooms include fungi that are either collected wild or cultivated. Many important species are difficult to cultivate but attempts have been made with varying degrees of success, with the results showing unsatisfactory economical cultivation methods. Recently, proteomic analysis has been developed as a powerful tool to study the protein content of fungi, particularly basidiomycetes. This mini-review article highlights the contribution of proteomics platforms to the study of edible mushrooms, focusing on the molecular mechanisms involved in developmental stages. This includes extracellular and cytoplasmic effector proteins that have potential or are involved in the synthesis of anticancer, antidiabetic, antioxidant, and antibiotic, in blood pressure control, in the supply of vitamins and minerals, and in other responses to environmental changes. The contribution of different proteomics techniques including classical and more advanced techniques is also highlighted.
A unique feature of influenza A virus (IAV) life cycle is replication of the viral genome in the host cell nucleus. The nuclear import of IAV genome is an indispensable step in establishing virus infection. IAV nucleoprotein (NP) is known to mediate the nuclear import of viral genome via its nuclear localization signals. Here, we demonstrate that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of incoming IAV viral ribonucleoproteins (vRNPs) and is important for efficient IAV replication. Hsp40 was found to interact with NP component of IAV RNPs during early stages of infection. This interaction is mediated by the J domain of Hsp40 and N-terminal region of NP. Drug or RNAi mediated inhibition of Hsp40 resulted in reduced nuclear import of IAV RNPs, diminished viral polymerase function and attenuates overall viral replication. Hsp40 was also found to be required for efficient association between NP and importin alpha, which is crucial for IAV RNP nuclear translocation. These studies demonstrate an important role for cellular chaperone Hsp40/DnaJB1 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins.
To date, the genus Mangrovimonas consists of only one species, Mangrovimonas yunxiaonensis strain LY01 that is known to have algicidal effects against harmful algal blooms (HABs) of Alexandrium tamarense. In this study, the whole genome sequence of three Mangrovimonas-like strains, TPBH4(T)(=LMG 28913(T),=JCM 30882(T)), ST2L12(T)(=LMG 28914(T),=JCM 30880(T)) and ST2L15(T)(=LMG 28915(T),=JCM 30881(T)) isolated from estuarine mangrove sediments in Perak, Malaysia were described. The sequenced genomes had a range of assembly size ranging from 3.56 Mb to 4.15 Mb which are significantly larger than that of M. yunxiaonensis LY01 (2.67 Mb). Xylan, xylose, L-arabinan and L-arabinose utilization genes were found in the genome sequences of the three Mangrovimonas-like strains described in this study. In contrast, these carbohydrate metabolism genes were not found in the genome sequence of LY01. In addition, TPBH4(T) and ST2L12(T) show capability to degrade xylan using qualitative plate assay method.
Graphene is a new carbon-based material that is of interest in separation science. Graphene has extraordinary properties including nano size, high surface area, thermal and chemical stability, and excellent adsorption affinity to pollutants. Its adsorption mechanisms are through non-covalent interactions (π-π stacking, electrostatic interactions, and H-bonding) for organic compounds and covalent interactions for metal ions. These properties have led to graphene-based material becoming a desirable adsorbent in a popular sample preparation technique known as solid phase extraction (SPE). Numerous studies have been published on graphene applications in recent years, but few review papers have focused on its applications in analytical chemistry. This article focuses on recent preconcentration of trace elements, organic compounds, and biological species using SPE-based graphene, graphene oxide, and their modified forms. Solid phase microextraction and micro SPE (µSPE) methods based on graphene are discussed.
Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL(-1) at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg(-1). The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0-8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t₁/₂) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification.
Type strain Vitellibacter vladivostokensis KMM 3516(T) (=NBRC 16718(T)) belongs to the phylum Cytophaga-Flavobacterium-Bacteroides. To date, no genomes of the Vitellibacter spp. have been reported, and their metabolic pathways are unknown. This study reports the draft genome sequence of V. vladivostokensis. Moreover, mining of genes associated with proteolytic enzymes was performed to provide insights for further enzyme characterization.
Fish meal is currently the major protein source for commercial aquaculture feed. Due to its unstable supply and increasing price, fish meal is becoming more expensive and its availability is expected to face significant challenges in the near future. Therefore, feasible alternatives to fish meal are urgently required. Microalgae have been recognized as the most promising candidates to replace fish meal because the protein composition of microalgae is similar to fish meal and the supply of microalgae-based proteins is sustainable. In this study, an indigenous microalga (Chlorella vulgaris FSP-E) with high protein content was selected, and its feasibility as an aquaculture protein source was explored. An innovative photobioreactor (PBR) utilizing cold cathode fluorescent lamps as an internal light source was designed to cultivate the FSP-E strain for protein production. This PBR could achieve a maximum biomass and protein productivity of 699 and 365 mg/L/day, respectively, under an optimum urea and iron concentration of 12.4 mM and 90 μM, respectively. In addition, amino acid analysis of the microalgal protein showed that up to 70% of the proteins in this microalgal strain consist of indispensable amino acids. Thus, C. vulgaris FSP-E appears to be a viable alternative protein source for the aquaculture industry.
The ability of Leuconostoc mesenteroides DU15 to produce antifungal peptides that inhibit growth of Aspergillus niger was evaluated under optimum growth conditions of 30 °C for 48 h. The cell-free supernatant showed inhibitory activity against A. niger. Five novel peptides were isolated with the sequences GPFPL, YVPLF, LLHGVPLP, GPFPLEMTLGPT, and TVYPFPGPL as identified by de novo sequencing using PEAKS 6 software. Peptide LLHGVPLP was the only positively charged (cationic peptides) and peptide GPFPLEMTLGPT negatively charged (anionic), whereas the rest are neutral. The identified peptides had high hydrophobicity ratio and low molecular weights with amino acids sequences ranging from 5 to 12 residues. The mode of action of these peptides is observed under the scanning electron microscope and is due to cell lysis of fungi. This work reveals the potential of peptides from L. mesenteroides DU15 as natural antifungal preservatives in inhibiting the growth of A. niger that is implicated to the spoilage during storage.
Endemic/epidemic dengue viruses (DEN) that are transmitted among humans by the mosquito vectors Aedes aegypti and Aedes albopictus are hypothesized to have evolved from sylvatic DEN strains that are transmitted among nonhuman primates in West Africa and Malaysia by other Aedes mosquitoes. We tested this hypothesis with phylogenetic studies using envelope protein gene sequences of both endemic/epidemic and sylvatic strains. The basal position of sylvatic lineages of DEN-1, -2, and -4 suggested that the endemic/epidemic lineages of these three DEN serotypes evolved independently from sylvatic progenitors. Time estimates for evolution of the endemic/epidemic forms ranged from 100 to 1,500 years ago, and the evolution of endemic/epidemic forms represents relatively recent events in the history of DEN evolution. Analysis of envelope protein amino acid changes predicted to have accompanied endemic/epidemic emergence suggested a role for domain III in adaptation to new mosquito and/or human hosts.
A panel of monoclonal antibodies specific to Hong Kong Chinese nasopharyngeal carcinoma (NPC)-associated Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) variants has been generated. These monoclonal antibodies not only differentiate the Hong Kong Chinese NPC-associated LMP1 variants from the prototype B95-8 LMP1, derived from Caucasian infectious mononucleosis, but also differentiate the 2 highly homologous LMP1 deletion variants commonly found in Hong Kong primary NPC. The predominant deletion type variant, DV-Asp335, is characterized by an aspartic acid at residue 335 located in the cytoplasmic C-terminal region, whereas the other minor deletion variant, DV-Gly335, has a glycine in the same residue position. 335D is hitherto found predominantly in LMP1 of the China 1 strain in association with NPC in the Chinese populations located in southern China and Malaysia. These antibodies, which are applicable in ELISA, immunofluorescence, immunoprecipitation, immunoblotting and immunohistochemistry on paraffin sections, are the first variant-specific anti-LMP1 monoclonal antibodies produced, and will be useful in investigating the functional significance of 335D in NPC.
We recently adopted immobilized jacalin as an affinity adsorbent to purify human serum IgA for laboratory study. In the course of our investigation, we detected a serum protein that co-eluted with IgA from jacalin-agarose affinity column. It constituted in significant quantity (24.0 +/- 0.9%, n = 30) of total jacalin-bound protein (JBP) and the yield was equivalent to 0.4 +/- 0.1 mg per ml serum. The molecular mass of this protein was 55 kDa with electromobility in the alpha 2 region as demonstrated by SDS-PAGE and immunoelectrophoresis. N-terminal microsequencing of this 55 kDa protein revealed that it is human alpha 2-HS glycoprotein (alpha 2HSG). The molecular interaction of alpha 2HSG with jacalin was characterized by competitive ELISA: human serum IgA, human colostrum secretory IgA (sIgA), and monosaccharides including D-galactose and melibiose exhibited strong inhibitory effect on its binding to jacalin. Accordingly, we propose that human alpha 2HSG binds in a similar manner as that of the bovine fetuin to jacalin. In addition, alpha 2HSG displays similar binding property to jacalin from different geographic area (India and Malaysia) and from different laboratory preparations (Sigma, Pierce and 'homemade' jacalin).
Many proteins derived from the latex of Hevea brasiliensis that remain soluble in trichloroacetic acid (TCA) can be precipitated by phosphotungstic acid (PTA). A combination of 5% TCA and 0.2% PTA precipitates a wide range of proteins effectively even when they are present in low concentrations (below 1 microgram ml-1). In addition to its protein purification function, acid precipitation also increases the sensitivity of the subsequent protein assay by allowing the test sample to be concentrated. Another advantage of protein precipitation by TCA and PTA is that very small amounts of protein (of the order of 10 micrograms) can be repeatably recovered without the use of precipitate-bulking agents such as sodium deoxycholate. This general procedure of protein purification and concentration is simple and rapid, but the use of PTA may not be fully compatible with the Bradford protein assay. A modified Lowry microassay is described which enables about 3 micrograms ml-1 to be quantitated at the photometric absorbance of 0.05. When used in conjunction with protein concentration by precipitating with TCA/PTA, approximately 0.4 microgram ml-1 protein present in 6 ml of solution can be assayed.
We have investigated the expression of a strain-specific malarial antigen on the surface of erythrocytes infected with knobless (K-) variants of knob-positive (K+) strains of Plasmodium falciparum. Aotus blood infected with K+ or K- parasites derived from two independent geographical isolates (Malayan camp and Santa Lucia) was surface iodinated by the lactoperoxidase method. Infected and uninfected erythrocytes were then separated by a new procedure involving equilibrium density sedimentation on a Percoll gradient containing sorbitol. Strain-specific antigens were readily identified on the surface of erythrocytes infected with either of the K+ strains by their characteristic size and detergent solubility. These proteins were not detected on the surface of erythrocytes infected with either of the K- variants nor on uninfected erythrocytes isolated from K+- or K- -infected blood. These results are consistent with a role for the strain-specific surface antigen in cytoadherence of P. falciparum-infected erythrocytes. Our findings represent the second biochemical difference (with the knob-associated histidine-rich protein) between K+ and K- P. falciparum.
Infants, one to 56-weeks-old, presenting with persistent diarrhoea were placed on a diet free of cow's milk protein which improved their clinical condition. Six weeks later, 67 infants were challenged with a low-lactose cow's milk formula and jejunal biopsy was taken before and 24-hours after challenge. On the basis of histological changes in the intestinal mucosa and development of clinical symptoms the infants were categorised into three groups: Group 1 (n = 16) with no clinical or mucosal abnormality, Group 2 (n = 20) with mucosal abnormality but lacking clinical symptoms, and Group 3 (n 31) with manifestation of mucosal abnormality and clinical symptoms. In addition to the total IgE the radioallergosorbent test (RAST) was performed on sera from the infants taken before and after milk provocation. The mean total serum IgE level ranged from 288 to 560 IU/ml. In Groups 2 and 3 the prechallenge serum IgE levels were significantly higher than the postchallenge levels but in Group 1 the levels remained unchanged on challenge. A positive RAST to milk proteins was observed in five infants (7.4%), that is, one in Group 2 and four in Group 3, of 67 infants studied. In a survey of 405 consecutive paediatric-age patients admitted for a variety of symptoms, 90 were positive for RAST specific for milk proteins. Interestingly the majority of the patients positive for RAST presented with gastrointestinal ailments. The measurement of specific IgE appears not to be a useful adjunct in the diagnosis of CMPSE in Malaysian children.
SIB1 FKBP22 is a peptidyl prolyl cis-trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC50 of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.
Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant-fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp.