Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification,properties, and amino acid sequences

Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is atpH 4.55 for LA-1 and atpH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 å. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0×10⁹ M^?1 sec^?1 for LA-1 and 0.8 × 10⁹ M^?1 sec^?1 for LA-2 and that of K₂HPO₄ quenching is 1.6×10¹¹ M^?1 sec^?1 for LA-1 and 1.2×10¹¹M^?1 sec^?1 for LA-2. Analysis of the circular dichroic spectra yields 40%α-helix and 60%Β-turn for La-1 and 45%α-helix and 55%Β-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzymeinhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Impact of rapid urbanization on the rates of infection by Vibrio cholerae O1 and enterotoxigenic Escherichia coli in Dhaka, Bangladesh

Background

In Bangladesh, increases in cholera epidemics are being documented with a greater incidence and severity. The aim of this prospective study was to identify the prevalence and importance of V. cholerae O1 and enterotoxigenic Escherichia coli (ETEC) as causal agents of severe diarrhea in a high diarrhea prone urban area in Dhaka city.

Methodology

Systematic surveillance was carried out on all diarrheal patients admitted from Mirpur between March 2008 to February 2010 at the ICDDR, B hospital. Stool or rectal swabs were collected from every third diarrheal patient for microbiological evaluation.

Principal Findings

Of diarrheal patients attending the hospital from Mirpur, 41% suffered from severe dehydration with 39% requiring intravenous rehydration therapy. More diarrheal patients were above five years of age (64%) than those below five years of age (36%). About 60% of the patients above five years of age had severe dehydration compared with only 9% of patients under five years of age. The most prevalent pathogen isolated was Vibrio cholerae O1 (23%) followed by ETEC (11%). About 8% of cholera infection was seen in infants with the youngest children being one month of age while in the case of ETEC the rate was 11%. Of the isolated ETEC strains, the enterotoxin type were almost equally distributed; ST accounted for 31% of strains; LT/ST for 38% and LT for 31%.

Conclusion

V. cholerae O1 is the major bacterial pathogen and a cause of severe cholera disease in 23% of patients from Mirpur. This represents a socioeconomic group that best reflects the major areas of high cholera burden in the country. Vaccines that can target such high risk groups in the country and the region will hopefully be able to reduce the disease morbidity and the transmission of pathogens that impact the life and health of people.     

PCR-based identification of Vibrio cholerae and the closely related species Vibrio mimicus using the large chromosomal ori sequence of Vibrio cholerae

The bacterial chromosomal replication origin (ori) sequences are a highly conserved essential genetic element. In this study, the large chromosomal replication origin sequence of Vibrio cholerae (oriCIVC) has been targeted for identification of the organism, including the biotypes of serogroup O1. The oriCIVC sequence-based PCR assay specifically amplified an 890 bp fragment from all the V. cholerae strains examined. A point mutation in the oriCIVC sequence of the classical biotype of O1 serogroup led to the loss of a BglII site, which was utilized for differentiation from El Tor vibrios. Interestingly, the PCR assay amplified a similarly sized ori segment, designated as oriCIVM, from V. mimicus strains, but failed to produce any amplicon with other strains. Cloning and sequencing of the oriCIVM revealed high sequence similarity (96%) with oriCIVC. The results indicate that V. mimicus is indeed very closely related to V. cholerae. In addition, the BglII restriction fragment length polymorphism (RFLP) between oriCIVM and oriCIVC sequences allowed us to differentiate the two species. The ori sequence-based PCR-RFLP assay developed in this study appears to be a useful method for rapid identification and differentiation of V. cholerae and V. mimicus strains, as well as for the delineation of classical and El Tor biotypes of V. cholerae O1.     

Colorimetric estimation of human glucose level using γ-Fe2O3 nanoparticles: An easily recoverable effective mimic peroxidase

This article reports simple, green and efficient synthesis of γ-Fe₂O₃ nanoparticles (NPs) (maghemite) through single-source precursor approach for colorimetric estimation of human glucose level. The γ-Fe₂O₃ NPs, having cubic morphology with an average particle size of 30 nm, exhibited effective peroxidase-like activity through the catalytic oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ producing a blue-colored solution. On the basis of this colored-reaction, we have developed a simple, cheap, highly sensitive and selective colorimetric method for estimation of glucose using γ-Fe₂O₃/TMB/glucose–glucose oxidase (GOx) system in the linear range from 1 to 80 μM with detection limit of 0.21 μM. The proposed glucose sensor displays faster response, good stability, reproducibility and anti-interference ability. Based on this simple reaction process, human blood and urine glucose level can be monitored conveniently.     

Characterization of the Replication Initiator Orc1/Cdc6 from the Archaeon Picrophilus torridus

Eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at or very near origins in G₁ phase, which licenses origin firing in S phase. The archaeal DNA replication machinery broadly resembles the eukaryal apparatus, though simpler in form. The eukaryotic replication initiator origin recognition complex (ORC), which serially recruits Cdc6 and other pre-RC proteins, comprises six components, Orc1-6. In archaea, a single gene encodes a protein similar to both the eukaryotic Cdc6 and the Orc1 subunit of the eukaryotic ORC, with most archaea possessing one to three Orc1/Cdc6 orthologs. Genome sequence analysis of the extreme acidophile Picrophilus torridus revealed a single Orc1/Cdc6 (PtOrc1/Cdc6). Biochemical analyses show MBP-tagged PtOrc1/Cdc6 to preferentially bind ORB (origin recognition box) sequences. The protein hydrolyzes ATP in a DNA-independent manner, though DNA inhibits MBP-PtOrc1/Cdc6-mediated ATP hydrolysis. PtOrc1/Cdc6 exists in stable complex with PCNA in Picrophilus extracts, and MBP-PtOrc1/Cdc6 interacts directly with PCNA through a PIP box near its C terminus. Furthermore, PCNA stimulates MBP-PtOrc1/Cdc6-mediated ATP hydrolysis in a DNA-dependent manner. This is the first study reporting a direct interaction between Orc1/Cdc6 and PCNA in archaea. The bacterial initiator DnaA is converted from an active to an inactive form by ATP hydrolysis, a process greatly facilitated by the bacterial ortholog of PCNA, the β subunit of Pol III. The stimulation of PtOrc1/Cdc6-mediated ATP hydrolysis by PCNA and the conservation of PCNA-interacting protein motifs in several archaeal PCNAs suggest the possibility of a similar mechanism of regulation existing in archaea. This mechanism may involve other yet to be identified archaeal proteins.     

Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells

Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.     

The microbiology of metalworking fluids

Metalworking fluids (MWFs) are complex mixtures of chemicals and are indispensable materials in industry. They are used as cooling and lubricating agents in different machining process such as grinding, milling, and cutting. The quality of MWFs is affected by physical, chemical, and microbial contaminates. In particular, MWFs are highly vulnerable to microbial contamination, which may act both as potential pathogens and deteriorgens. Microbial contamination is of major concern due to potential health hazards such as skin dermatitis and hypersensitivity pneumonitis. The contaminated MWFs can exhibit high degrees of microbial loading, ranging from 10(4) to 10(10) colony-forming units (CFU)/ml. Wide varieties of microorganisms are reported to colonize MWFs. Traditional culturing techniques are not only laborious and time consuming but also underestimate the actual distribution of the microorganisms present in the contaminated MWFs. Therefore, rapid molecular methods such as real-time PCR and fluorescent in situ hybridization are implemented to monitor the microbial load. In industry, biocides are presently used to control microbial contamination. However, it has its own disadvantages and therefore, in recent years, alternative methods such as UV irradiation were evaluated to reduce microbial contamination in MWFs. Microbes inhabiting the MWF are also capable of forming biofilm which is detrimental to the MWF system. Biofilm is resistant to common disinfectant methods, and thus further research and development is required to effectively control its formation within MWF systems. This review is intended to discuss the overall microbiological aspects of MWF.     

Response properties of visual neurons in the turtle nucleus isthmi

The optic tectum holds a central position in the tectofugal pathway of non-mammalian species and is reciprocally connected with the nucleus isthmi. Here, we recorded from individual nucleus isthmi pars parvocellularis (Ipc) neurons in the turtle eye-attached whole-brain preparation in response to a range of computer-generated visual stimuli. Ipc neurons responded to a variety of moving or flashing stimuli as long as those stimuli were small. When mapped with a moving spot, the excitatory receptive field was of circular Gaussian shape with an average half-width of less than 3°. We found no evidence for directional sensitivity. For moving spots of varying sizes, the measured Ipc response-size profile was reproduced by the linear Difference-of-Gaussian model, which is consistent with the superposition of a narrow excitatory center and an inhibitory surround. Intracellular Ipc recordings revealed a strong inhibitory connection from the nucleus isthmi pars magnocellularis (Imc), which has the anatomical feature to provide a broad inhibitory projection. The recorded Ipc response properties, together with the modulatory role of the Ipc in tectal visual processing, suggest that the columns of Ipc axon terminals in turtle optic tectum bias tectal visual responses to small dark changing features in visual scenes.