Abstract: Calcium is an essential element for good growth and development of the organism, and its requirement is increased at school age. Low socio-economic populations of developing countries such as Colombia may have food deficiency of this mineral in schoolchildren that could be reflected in calcium biochemical indicators, bone alterations and anthropometric indicators. The objective of this investigation was to evaluate some calcium biochemical indicators in a group of schoolchildren of low socioeconomic level from Barranquilla city and to correlate with body mass index. 60 schoolchildren aged 7 to 15 years were selected from Jesus’s Heart Educational Institution in Barranquilla-Atlántico, apparently healthy, without suffering from infectious or gastrointestinal diseases, without habits of drinking alcohol or smoking another hallucinogenic substance and without taking supplementation with calcium in the last six months or another substance that compromises bone metabolism. The research was approved by the ethics committee at Universidad del Atlántico. The selected children were invited to donate a blood and urine sample in a fasting time of 12 hours, the serum was separated by centrifugation and frozen at ˗20 ℃ until analyzed and the same was done with the urine sample. On the day of the biological collections, the weight and height of the students were measured to determine the nutritional status by BMI using the WHO tables. Calcium concentrations in serum and urine (SCa, UCa), alkaline phosphatase activity total and of bone origin (SAPT, SBAP) and urinary creatinine (UCr) were determined by spectrophotometric methods using commercial kits. Osteocalcin and Cross-linked N-telopeptides of type I collagen (NTx-1) in serum were measured with an enzyme-linked inmunosorbent assay. For statistical analysis the Statgraphics software Centurium XVII was used. 63% (n = 38) and 37% (n = 22) of the participants were male and female, respectively. 78% (n = 47), 5% (n = 3) and 17% (n = 10) had a normal, malnutrition and high nutritional status, respectively. The averages of evaluated indicators levels were (mean ± SD): 9.50 ± 1.06 mg/dL for SCa; 181.3 ± 64.3 U/L for SAPT, 143.8 ± 73.9 U/L for SBAP; 9.0 ± 3.48 ng/mL for osteocalcin and 101.3 ± 12.8 ng/mL for NTx-1. UCa level was 12.8 ± 7.7 mg/dL that adjusted with creatinine ranged from 0.005 to 0.395 mg/mg. Considering serum calcium values, approximately 7% of school children were hypocalcemic, 16% hypercalcemic and 77% normocalcemic. The indicators evaluated did not correlate with the BMI. Low values were observed in calcium urinary excretion and high in NTx-1, suggesting that mechanisms such as increase in renal retention of calcium and in bone remodeling may be contributing to calcium homeostasis.
Abstract: Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA4404. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.
Abstract: Rv3873 is a relatively large size protein (371 amino acids in length) and its gene is located in the immunodominant genomic region of difference (RD)1 that is present in the genome of Mycobacterium tuberculosis but deleted from the genomes of all the vaccine strains of Bacillus Calmette Guerin (BCG) and most other mycobacteria. However, when tested for cellular immune responses using peripheral blood mononuclear cells from tuberculosis patients and BCG-vaccinated healthy subjects, this protein was found to be a major stimulator of cell mediated immune responses in both groups of subjects. In order to further identify the sequence of immunodominant epitopes and explore their Human Leukocyte Antigen (HLA)-restriction for epitope recognition, 24 peptides (25-mers overlapping with the neighboring peptides by 10 residues) covering the sequence of Rv3873 were synthesized chemically using fluorenylmethyloxycarbonyl chemistry and tested in cell mediated immune responses. The results of these experiments helped in the identification of an immunodominant peptide P9 that was recognized by people expressing varying HLA-DR types. Furthermore, it was also predicted to be a promiscuous binder with multiple epitopes for binding to HLA-DR, HLA-DP and HLA-DQ alleles of HLA-class II molecules that present antigens to T helper cells, and to HLA-class I molecules that present antigens to T cytotoxic cells. In addition, the evaluation of peptide P9 using an immunogenicity predictor server yielded a high score (0.94), which indicated a greater probability of this peptide to elicit a protective cellular immune response. In conclusion, P9, a peptide with multiple epitopes and ability to bind several HLA class I and class II molecules for presentation to cells of the cellular immune response, may be useful as a peptide-based vaccine against tuberculosis.
Abstract: The comparisons of mycobacterial genomes have identified several Mycobacterium tuberculosis-specific genomic regions that are absent in other mycobacteria and are known as regions of differences. Due to M. tuberculosis-specificity, the peptides encoded by these regions could be useful in the specific diagnosis of tuberculosis. To explore this possibility, overlapping synthetic peptides corresponding to 39 proteins predicted to be encoded by genes present in regions of differences were tested for antibody-reactivity with sera from tuberculosis patients and healthy subjects. The results identified four immunodominant peptides corresponding to four different proteins, with three of the peptides showing significantly stronger antibody reactivity and rate of positivity with sera from tuberculosis patients than healthy subjects. The fourth peptide was recognized equally well by the sera of tuberculosis patients as well as healthy subjects. Predication of antibody epitopes by bioinformatics analyses using ABCpred server predicted multiple linear epitopes in each peptide. Furthermore, peptide sequence analysis for sequence identity using BLAST suggested M. tuberculosis-specificity for the three peptides that had preferential reactivity with sera from tuberculosis patients, but the peptide with equal reactivity with sera of TB patients and healthy subjects showed significant identity with sequences present in nob-tuberculous mycobacteria. The three identified M. tuberculosis-specific immunodominant peptides may be useful in the serological diagnosis of tuberculosis.
Abstract: In recent years, the consumption of functional foods, including foods containing probiotic bacteria, has come to notice. Milk proteins have been identified as a source of angiotensin-I-converting enzyme )ACE( inhibitory peptides and are currently the best-known class of bioactive peptides. In this study, the effects of adding prebiotic ingredients (inulin and wheat fiber) and fat percentage (0%, 2% and 3.5%) in yogurt containing probiotic Lactobacillus casei on physicochemical properties, degree of proteolysis, antioxidant and ACE-inhibitory activity within 21 days of storage at 5 ± 1 °C were evaluated. The results of statistical analysis showed that the application of prebiotic compounds led to a significant increase in water holding capacity, proteolysis and ACE-inhibitory of samples. The degree of proteolysis in yogurt increases as storage time elapses (P < 0.05) but when proteolysis exceeds a certain threshold, this trend begins to decline. Also, during storage time, water holding capacity reduced initially but increased thereafter. Moreover, based on our findings, the survival of Lactobacillus casei in samples treated with inulin and wheat fiber increased significantly in comparison to the control sample (P < 0.05) whereas the effect of fat percentage on the survival of probiotic bacteria was not significant (P = 0.095). Furthermore, the effect of prebiotic ingredients and the presence of probiotic cultures on the antioxidant activity of samples was significant (P < 0.05).
Abstract: T7 phage could be used as a perfect vector for peptides expression and haptens presentation. T7-3GnRH recombinant phage was constructed by inserting three copies of Gonadotrophin Releasing Hormone (GnRH) gene into the multiple cloning site of T7 Select 415-1b phage genome. The positive T7-3GnRH phage was selected by using polymerase chain reaction amplification, and the p10B-3GnRH fusion protein was verified by SDS-PAGE and Western-blotting assay. T7-3GnRH vaccine was made and immunized with 1010 pfu in 0.2 ml per dose in mice. Blood samples were collected at an interval in weeks, and anti-GnRH antibody and testosterone concentrations were detected by ELISA and radioimmunoassay, respectively. The results show that T7-3GnRH phage particles confer a high immunogenicity to the GnRH-derived epitope. Moreover, the T7-3GnRH vaccine induced higher level of anti-GnRH antibody than ImproVac®. However, the testosterone concentrations in both immunized groups were at a similar level, and the testis developments were significantly inhibited compared to controls. These findings demonstrated that the anti-GnRH antibody could neutralize the endogenous GnRH to down regulate testosterone level and limit testis development, highlighting the potential value of T7-3GnRH in the immunocastration vaccine research.
Abstract: In the present study, highly effective iTLC single strip method for the determination of radiochemical purity (RCP) of 68Ga-BCA-peptides was developed (with no double-developing, changing of eluents or other additional manipulation). In this method iTLC-SG strips and commonly used eluent TFAaq. (3-5 % (v/v)) are used. The method allows determining each of the key radiochemical forms of 68Ga (colloidal, bound, ionic) separately with the peaks separation being no less than 4 σ. Rf = 0.0-0.1 for 68Ga-colloid; Rf = 0.5-0.6 for 68Ga-BCA-peptides; Rf = 0.9-1.0 for ionic 68Ga. The method is simple and fast: For developing length of 75 mm only 4-6 min is required (versus 18-20 min for pharmacopoeial method). The method has been tested on various compounds (including 68Ga-DOTA-TOC, 68Ga-DOTA-TATE, 68Ga-NODAGA-RGD2 etc.). The cross-validation work for every specific form of 68Ga showed good correlation between method developed and control (pharmacopoeial) methods. The method can become convenient and much more informative replacement for pharmacopoeial methods, including HPLC.
Abstract: The main cause of several neurodegenerative diseases such as Alzhemier, Parkinson and spongiform encephalopathies is formation of amyloid fibrils and plaques in proteins. We have analyzed different sets of proteins and peptides to understand the influence of sequence based features on protein aggregation process. The comparison of 373 pairs of homologous mesophilic and thermophilic proteins showed that aggregation prone regions (APRs) are present in both. But, the thermophilic protein monomers show greater ability to ‘stow away’ the APRs in their hydrophobic cores and protect them from solvent exposure. The comparison of amyloid forming and amorphous b-aggregating hexapeptides suggested distinct preferences for specific residues at the six positions as well as all possible combinations of nine residue pairs. The compositions of residues at different positions and residue pairs have been converted into energy potentials and utilized for distinguishing between amyloid forming and amorphous b-aggregating peptides. Our method could correctly identify the amyloid forming peptides at an accuracy of 95-100% in different datasets of peptides.
Abstract: Amyloid aggregation of polypeptides is related to a
growing number of pathologic states known as amyloid disorders. In
recent years, blocking or reversing amyloid aggregation via the use of
small compounds are considered as two useful approaches in
hampering the development of these diseases. In this research, we
have compared the ability of several manganese-salen derivatives, as
synthetic compounds, and apigenin, as a natural flavonoid, to inhibit
of hen egg-white lysozyme (HEWL) aggregation, as an in vitro
model system.
Different spectroscopic analyses such as Thioflavin T (ThT) and
Anilinonaphthalene-8-sulfonic acid (ANS) fluorescence, Congo red
(CR) absorbance along with transmission electron microscopy were
used in this work to monitor the HEWL aggregation kinetic and
inhibition. Our results demonstrated that both type of compounds
were capable to prevent the formation of lysozyme amyloid
aggregation in vitro. In addition, our data indicated that synthetic
compounds had higher activity to inhibit of the β-sheet structures
relative to natural compound. Regarding the higher antioxidant
activities of the salen derivatives, it can be concluded that in addition
to aromatic rings of each of the compounds, the potent antioxidant
properties of salen derivatives contributes to lower lysozyme fibril
accumulation.
Abstract: Soy protein is a common ingredient added to processed meats to enhance its functional characteristics. In our study, soybean products (fermented soy Natto and protein hydrolysate) containing hydrolyzed peptides and amino acids, with or without ascorbic acid were added to burger in order to improve its quality characteristics. Results showed that soy additives significantly increased moisture and protein content and reduced (P < 0.05) fat values. Ash content did not affect with Natto additive. Color tools, lightness and yellowness were higher (P
Abstract: Soybean Natto powder was added to the burger in order to enhance the oxidative stability as well as decreases the microbial spoilage. The soybean bioactives compound (soybean Natto) as antioxidant and antimicrobial were added at level of 1, 2 and 3%. Chemical analysis and physical properties were affected by soybean Natto addition. All the tested soybean Natto additives showed strong antioxidant properties. The microbiological indicators were significantly (P < 0.05) affected by the addition of the soybean Natto. Decreasing trends of different extent were also observed in samples of the treatments for total viable counts, Coliform, Staphylococcus aureus, yeast and molds. Storage period was significantly (P < 0.05) affected on microbial counts in all samples Staphylococcus aureus were the most sensitive microbe followed by Coliform group of the sample containing soybean Natto. Sensory attributes were also performed, added soybean Natto exhibits beany flavor which was clear about samples of 3% soybean Natto.
Abstract: Using an enzyme of known specificity the hydrolysis of protein was carried out in a controlled manner. The aim was to obtain oligopeptides being the so-called active peptides or their direct precursors. An original way of expression of the protein hydrolysis kinetics was introduced. Peptide bonds contained in the protein were recognized as a diverse-quality substrate for hydrolysis by the applied protease. This assumption was positively verified taking as an example the hydrolysis of albumin by thermolysin. Peptide linkages for this system should be divided into at least four groups. One of them is a group of bonds non-hydrolyzable by this enzyme. These that are broken are hydrolyzed at a rate that differs even by tens of thousands of times. Designated kinetic constants were k'F = 10991.4 L/g.h, k'M = 14.83L/g.h, k'S about 10-1 L/g.h for fast, medium and slow bonds, respectively. Moreover, a procedure for unfolding of the protein, conducive to the improved susceptibility to enzymatic hydrolysis (approximately three-fold increase in the rate) was proposed.
Abstract: Protein hydrolysates prepared from a number of medicinal plants are promising sources of various bioactive peptides. In this work, proteins from dried whole plant of Euphorbia hirta Linn. were extracted and digested with pepsin for 12h. The hydrolysates of lesser than 3 KDa were fractionated by a cut-off membrane. The peptide hydrolysate was then purified by an anion-exchange chromatography on DEAE-Sephacel™ column and reverse-phase chromatography on Sep-pak C18 column, respectively. The cytotoxic effect of each peptide fraction against a gastric carcinoma cell line (KATO-III, ATCC No. HTB103) was investigated using colorimetric MTT viability assay. A human liver cell line (Chang Liver, CLS No. 300139) was used as a control normal cell line. Two purified peptide peaks, peak l and peak ll at 100µg peptides mL-1 affected cell viability of the gastric cancer cell lines to 63.85±4.94 and 66.92±6.46%, respectively. Our result showed for the first time that the peptide fractions derived from protein hydrolysate of Euphorbia hirta Linn. have anti-gastric cancer activity, which offers a potential novel and natural anti-gastric cancer remedy.
Abstract: The aim of the study was to determine how different
ripening processes (traditional vs. industrial) influenced the
proteolysis in traditional Serbian dry-fermented sausage Petrovská
klobása. The obtained results indicated more intensive pH decline
(0.7 units after 9 days) in industrially ripened products (I), what had a
positive impact on drying process and proteolytic changes in these
samples. Thus, moisture content in I sausages was lower at each
sampling time, amounting 24.7% at the end of production period
(90 days). Likewise, the process of proteolysis was more pronounced
in I samples, resulting in higher contents of non-protein nitrogen
(NPN) and free amino acids nitrogen (FAAN), as well as in faster
and more intensive degradation of myosin (≈220 kDa), actin (≈45
kDa) and other polypeptides during processing. Consequently, the
appearance and accumulation of several protein fragments were
registered.
Abstract: Recent research has shown that milk proteins can
yield bioactive peptides with opioid, mineral binding,
cytomodulatory, antihypertensive, immunostimulating, antimicrobial
and antioxidative activity in the human body. Bioactive peptides are
encrypted in milk proteins and are only released by enzymatic
hydrolysis in vivo during gastrointestinal digestion, food processing
or by microbial enzymes in fermented products. At present
significant research is being undertaken on the health effects of
bioactive peptides. A variety of naturally formed bioactive peptides
have been found in fermented dairy products, such as yoghurt, sour
milk and cheese. In particular, antihypertensive peptides have been
identified in fermented milks, whey and ripened cheese. Some of
these peptides have been commercialized in the form of fermented
milks. Bioactive peptides have the potential to be used in the
formulation of health-enhancing nutraceuticals, and as potent drugs
with well defined pharmacological effects.
Abstract: Bacterial molecular chaperone DnaK plays an essential role in protein folding, stress response and transmembrane targeting of proteins. DnaKs from many bacterial species, including Escherichia coli, Salmonella typhimurium and Haemophilus infleunzae are the molecular targets for the insect-derived antimicrobial peptide pyrrhocoricin. Pyrrhocoricin-like peptides bind in the substrate recognition tunnel. Despite the high degree of crossspecies sequence conservation in the substrate-binding tunnel, some bacteria are not sensitive to pyrrhocoricin. This work addresses the molecular mechanism of resistance of Helicobacter pylori DnaK to pyrrhocoricin. Homology modelling, structural and sequence analysis identify a single aminoacid substitution at the interface between the lid and the β-sandwich subdomains of the DnaK substrate-binding domain as the major determinant for its resistance.
Abstract: In order to characterize the soy protein hydrolysate obtained in this study, gel chromatography on Sephadex G-25 was used to perform the separation of the peptide mixture and electrophoresis in SDS-polyacrylamide gel has been employed. Protein hydrolysate gave high antioxidant activities, but didn't give any antimicrobial activities. The antioxidant activities of protein hydrolysate was in the same trend of peptide content which gave high antioxidant activities and high peptide content between fractions 15 to 50. With increasing peptide concentrations, the scavenging effect on DPPH radical increased until about 70%, thereafter reaching a plateau. In compare to different concentrations of BHA, which exhibited higher activity (90%), soybean protein hydrolysate exhibited high antioxidant activities (70%) at a concentration of 1.45 mg/ml at fraction 25. Electrophoresis analysis indicated that, low- MW hydrolysate fractions (F1) appeared, on average, to have higher DPPH scavenging activities than high-MW fractions. These results revealed that soybean peptides probably contain substances that were proton donors and could react with free radicals to convert them to stable diamagnetic molecules.
Abstract: Dried soy protein hydrolysate powder was added to
the burger in order to enhance the oxidative stability as well as
decreases the microbial spoilage. The soybean bioactive compounds
(soy protein hydrolysate) as antioxidant and antimicrobial were added
at level of 1, 2 and 3 %.Chemical analysis and physical properties
were affected by protein hydrolysate addition. The TBA values were
significantly affected (P < 0.05) by the storage period and the level of
soy protein hydrolysate. All the tested soybean protein hydrolysate
additives showed strong antioxidant properties. Samples of soybean
protein hydrolysate showed the lowest (P < 0.05) TBA values at each
time of storage.
The counts of all determined microbiological indicators were
significantly (P < 0.05) affected by the addition of the soybean
protein hydrolysate. Decreasing trends of different extent were also
observed in samples of the treatments for total viable counts,
Coliform, Staphylococcus aureus, yeast and molds. Storage period
was being significantly (P < 0.05) affected on microbial counts in all
samples Staphylococcus aureus were the most sensitive microbe
followed by Coliform group of the sample containing protein
hydrolysate, while molds and yeast count showed a decreasing trend
but not significant (P < 0.05) until the end of the storage period
compared with control sample. Sensory attributes were also
performed, added protein hydrolysate exhibits beany flavor which
was clear about samples of 3% protein hydrolysate.
Abstract: Acute disseminated encephalomyelitis (ADEM) has
been reported to develop after a hymenoptera sting, but its
pathogenesis is not known in detail. Myelin basic protein (MBP)-
specific T cells have been detected in the blood of patients with
ADEM, and a proportion of these patients develop multiple sclerosis
(MS). In an attempt to understand the mechanisms underlying
ADEM, molecular mimicry between hymenoptera venom peptides
and the human immunodominant MBP peptide was scrutinized,
based on the sequence and structural similarities, whether it was the
root of the disease. The results suggest that the three wasp venom
peptides have low sequence homology with the human
immunodominant MBP residues 85-99. Structural similarity analysis
among the three venom peptides and the MS-related HLA-DR2b
(DRA, DRB1*1501)-associated immunodominant MHC
binding/TCR contact residues 88-93, VVHFFK showed that
hyaluronidase residues 7-12, phospholipase A1 residues 98-103, and
antigen 5 residues 109-114 showed a high degree of similarity
83.3%, 100%, and 83.3% respectively. In conclusion, some wasp
venom peptides, particularly phospholipase A1, may potentially act
as the molecular motifs of the human 3HLA-DR2b-associated
immunodominant MBP88-93, and possibly present a mechanism for
induction of wasp sting-associated ADEM.
Abstract: Radiolabeled cyclic RGD peptides targeting integrin αvβ3 are reported as promising agents for the early diagnosis of metastatic tumors. With an aim to improve tumor uptake and retention of the peptide, cyclic RGD peptide dimer E[c (RGDfK)] 2 (E = Glutamic acid, f = phenyl alanine, K = lysine) coupled to the bifunctional chelator DOTA was custom synthesized and radiolabelled with 68Ga. Radiolabelling of cyclic RGD peptide dimer with 68Ga was carried out using HEPES buffer and biological evaluation of the complex was done in nude mice bearing HT29 tumors.