Chronorhythms of the Circulating Erythrocytes and Leukocytes—Correlation with the E.S.R. Cycles

2750 healthy and fasting subjects, 20-30 years old, were studied over a half-year period in 1980. Considering the mean day value as a basic piece of information for statistics, the Erythrocyte Sedimentation Rate (E.S.R.) and the blood counts (erythrocytes, leukocytes, polymorphonuclears, lymphocytes, monocytes and eosinophils) were compared.

The timeless relation between E.S.R. and each cell type number or percentage is rectilinear. The stronger slope and relation apply to the polymorphonuclears (PMN) or to the overall leukocytes.

The chronological normalized variations of the E.S.R. and of the PMN or leukocyte number or percentage are highly correlated. E.S.R. is less correlated with monocytes, eosinophils and Lymphocytes. Contrary to what could have been expected, the erythrocyte situation is but an intermediate one.

The spectra derived from the time variations show that all the cell types, whatever they are, are to be taken into account to explain the E.S.R. value and variation with time, even if, for a given cell type, the correlation and timeless relation were but faint ones.

Each cell type has a specific spectrum. Erythrocytes are subject to low frequency variations (316-158 days). PMNs oscillate with time within the medium frequency range (90 days). Lymphocytes, monocytes and eosinophils fluctuate more quickly (53 days).     

Sensitivity of three thyrotropin receptor antibody assays in thyroid-associated orbitopathy

BackgroundThyrotropin receptor autoantibodies (TSH-RAb) are indispensable biomarkers in the laboratory assessment of thyroid-associated orbitopathy (TAO). Clinical sensitivity of three different assays for TSH-R-Ab determination was evaluated in patients with TAO.Methods87 consecutive TAO patients were enrolled and their serum samples analyzed in parallel with three assays. An ECLIA competitive binding and a chemiluminescent bridge immunoassay were used to measure total and binding TSH-R-Ab concentration, while their functional activity was determined using a stimulatory TSH-R-Ab (TSAb) cellbased bioassay.ResultsCompared to the two binding assays (ECLIA p<0.001, bridge p=0.003), the TSAb bioassay was more sensitive pertaining to the positive detection of TSH-R-Ab in TAO patients. No difference (p=0.057) was noted between the ECLIA and bridge assays regarding sensitivity rate. All patients with active and/or moderate-to-severe TAO tested positive in the TSAb bioassay (100% and 100%, respectively), while the positivity rates for bridge and ECLIA binding assays were 89.7% and 82.1% for active TAO, and 90.2% and 86.3% for severe TAO, respectively. Negative predictive values of the bioassay, bridge, and ECLIA assays were 100%, 75%, and 71%, respectively for active TAO, and 100%, 86%, and 71%, respectively for moderate-to-severe TAO. The superiority of the bioassay was most prominent in euthyroid (ET) TAO. Positivity rates of the TSAb bioassay, bridge and ECLIA binding assays were 89.6%, 75%, and 64.6%, respectively for inactive TAO; 86.1%, 69.4%, and 52.8%, respectively for mild TAO; 87.5%, 62.5%, and 12.5%, respectively for euthyroid TAO. The bridge assay correlated better with the ECLIA binding assay (r=0.893, p<0.001), compared to the bioassay (r=0.669, p<0.001).ConclusionsIn patients with TAO of various activity and severity, the TSAb bioassay demonstrates a superior clinical performance compared to both ECLIA and bridge binding assays.     

Lack of Correlation Between the Glucocorticoid Receptor Density and the in Vitro Growth-Inhibitory Effect of Dexamethasone in Human Leukemia Cell Lines

Abstract

We have developed a whole cell binding assay with [³H] dexamethasone as the ligand for the measurement of the glucocorticoid receptor (GR) content of normal and malignant human leukocytes. A panel of eleven phenotypically well-defined human leukemia cell lines were investigated for their GR expression and in vitro sensitivity to glucocorticoids.

There were great variations in the GR contents of different cell lines (2200–18100 sites/cell) while no marked differences in the binding affinities of the GRs were seen. No obvious correlation was found between the GR content and the phenotype of the cell line nor between the GR content and the in vitro growth inhibition by glucocorticoids.     

Novel microbiological system for antibiotic detection in ovine milk

This article presents a microbiological system composed of a “BT” bioassay (Beta-lactams and Tetracyclines) and a “QS” bioassay (Quinolones and Sulfonamides). The “BT” bioassay contains spores of Geobacillus stearothermophilus, bromocresol purple and cloramphenicol in a culture medium (incubation time: 2.45 h), while the “QS” bioassay uses spores of Bacillus subtilis, trifenyltetrazolium – toluidine blue and trimethoprim in a suitable culture medium (incubation time: 5.5 h). The detection capability (CC_β) of 27 antimicrobial agents in ovine milk were determined by logistic regression models. Thus, the “BT” bioassay detects amoxycillin, ampicillin, penicillin “G”, cloxacillin, oxacillin, cephalexin, cefoperazone, ceftiofur, chlortetracycline, oxytetracycline, tetracycline, neomycin, gentamycin and tylosin, while “QS” bioassay detects: ciprofloxacin, enrofloxacin, marbofloxacin, sulfadiazine, sulfadimethoxine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfathiazole, erythromycin, lincomycin and spiramycin at levels close to their respective Maximum Residue Limits. The simultaneous use of both bioassays detects a large number of antibiotics in milk given each method's adequate complementary sensitivity.     

Physiological effects of gibberellic Acid. X. The release of gibberellin-like substances by germinating barley embryos

Barley embryos, completely free from endosperm, were excised from germinating grain at various times and allowed to diffuse into an aqueous medium for varying lengths of time. At the end of this time, the embryos and ambient solutions were separately extracted. Gibberellin-like activity in the extracts was determined with the barley endosperm bioassay using seed from the same variety, harvest and treatment schedule as was employed for the embryo diffusion experiments. Gibberellin-like substances were released by embryos throughout the 60 hour germination period, though at no time during this period could sufficient activity be extracted from the embryos themselves to account for the observed release. Solvent partitioning and chromatography identified at least one major acidic component migrating at an Rf similar to that of GA₃.

It is concluded that the endogenous gibberellin-like substance(s) originates within the embryo during germination, and that the release of this substance(s) is temporally consistent with, and quantitatively sufficient to account for the in vivo endosperm mobilization response syndrome. A gibberellin-like substance is undoubtedly the endosperm mobilizing hormone.

     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

Effect of Test Leaf and Temperature on Mortality of Frankliniella occidentalis in Leaf Dip Bioassay of Insecticides

Effects of the kind of test leaf and temperature on leaf dip bioassay to the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), were examined with nine different insecticides. For the test of different leaves, cucumber, pepper and potato leaves were evaluated. The test leaves significantly influenced thrips mortalities on leaf dip bioassay. Generally the mortalities on pepper leaf were higher than on cucumber leaf or filter paper. Temperature effects were determined at 15, 20, 25 and 30°C. Regardless of the test leaf, the mortality was increased with increasing temperature except for the case of imidacloprid. Local variations in mortality of F. occidentalis populations were not observed in this study.     

Impiego delle sezioni di coleoptile di alcune varietà italiane di frumento nel dosaggio biologico di sostanze promotrici ed inibitrici della crescita

Abstract

Growth response of coleoptile sections from four Italian cultivars of wheat to some growth regulators.—The following Italian wheat varieties were used to obtain the coleoptile sections used throughout the experiments: Funo, S. Pastore, Abbondanza, Generoso.

The growth substances tested were Indolacetic acid (IAA), Abscisic acid (ABA), Kinetin and Gibberellic acid.

The colcoptile response of each variety was tested with three different concentrations for each growth regulator in combination with two sucrose concentrations and three different pH levels.

The experiments indicated that the coleoptiles from the variety « Funo » react linearly to incresing IAA and ABA concentration resulting the best one to be used in this bioassay.     

Translocation of radioactive kinetin

Kinetin has generally been thought to be immobile in plants. This was confirmed in the case of laminar applications in this study, but not in regard to petiole, vein, or root applications. Radioactivity from kinetin-8-¹⁴C (Kn^*) moved freely in the vascular system of several types of leaves. This movement was usually distal to the point of application and seemed to occur with the transpiration stream. Basipetal as well as acropetal translocation of radioactive kinetin was achieved in tobacco leaves. The translocated material was extracted from veinal tissue, shown to be radioactive, and to be able to retard senescence. Similar but less decisive results were obtained from agar blocks inserted into the vascular system of leaves receiving Kn^* by petiole uptake.

A bioassay employing disks from primary bean leaves was developed for the qualitative determination of substances like kinetin which possess the ability to retard chlorophyll breakdown and plant senescence. The use of radioactive kinetin provided a refinement in this bioassay because treated non-senescent areas could be correlated with exposed areas on radioautographs made from dried leaf disks.

Root treatments showed that cotton seedlings did not take up Kn^* but that similarly treated tobacco seedlings both absorbed and translocated the isotope readily.

     

Isolation, Purification, and Characterization of an Endogenous Root-promoting Factor Obtained from Basal Sections of Pear Hardwood Cuttings

Basal segments taken from Old Home and Bartlett pear hardwood cuttings collected at intervals during the rooting period in September were extracted with ethanol and fractionated by paper chromatography in different solvent systems. Different zones on the chromatograms were bioassayed by the mung bean rooting test, which showed high levels of promotion in Old Home basal extracts when the cuttings were obtained during the period of maximum rooting. Extracts from Bartlett cuttings, however, showed considerably less promotion activity in the bioassay but did show high levels of inhibitory activity.

After the easily-rooted Old Home cuttings had been in the rooting medium for 10 days, a highly active endogenous root-promoting material was found in extracts from basal segments of cuttings having buds and which had been treated with indolebutyric acid. Similar extracts obtained from disbudded cuttings, or from cuttings with buds but not treated with indolebutyric acid, lacked this rooting-factor. Extracts obtained from all types of the difficult-to-root Bartlett cuttings also lacked this rooting-factor. The latter is believed to be produced by physiologically active Old Home buds, and is very effective in the mung bean bioassay, even at extremely low concentrations.

From paper chromatographic studies, tests with spray reagents, solubility determinations, biological tests, UV spectrum analysis, and infrared spectroscopy, it is believed that this rooting factor could be a condensation product between exogenous auxin (indolebutyric acid) and a phenolic compound produced by physiologically active Old Home pear buds.

     

Elevated CO2 mediates the short-term drought recovery of ecosystem function in low-diversity grassland systems

Aims

Our study aimed to determine whether, and to what extent, stand characteristics and topography affected spatial variations in soil organic carbon (SOC), total nitrogen (TN) and total phosphorus (TP) concentrations in subtropical forests.

Methods

Soil samples were taken from a Choerospondias axillaris deciduous broadleaved forest and a Lithocarpus glaber–Cyclobalanopsis glauca evergreen broadleaved forest. Spatial variations in SOC, TN and TP concentrations and the factors affecting them were investigated using geostatistical analysis and stepwise linear regression, respectively.

Results

The L. glaber–C. glauca forest exhibited higher coefficients of variation (CVs) of SOC (35 %) and TN (34 %) concentrations than the C. axillaris forest (27 % for SOC and 21 % for TN), but the CV of TP concentration in the L. glaber–C. glauca forest (17 %) was lower than that in the C. axillaris forest (24 %). Stand characteristics contributed the most to spatial variations in SOC and TP, while soil texture made the greatest contribution to variations in TN. Topography contributed the least to variations in SOC, TN and TP.

Conclusions

Stand characteristics, together with topography and soil texture, contributed to spatial variations in SOC, TN and TP concentrations. The contributions of stand characteristics differed in SOC, TN and TP due to their different cycling characteristics.

     

Investigation of the relationship between GSTM1 gene variations and serum trace elements,plasma malondialdehyde levels in patients with colorectal cancer

Background

The aim of this study is to investigate of the relationship between GSTM1 gene variations and serum trace elements, plasma malondialdehyde levels in patient with colorectal cancer.

Mateials and Methods.

Genotype distributions of GSTM1 gene variations were determined using real-time polymerase chain reaction method. Serum trace element levels were determined using atomic absorption spectrophotometer method and plasma MDA levels were measurement by spectrophotometric method.

Results

Serum Cu levels, plasma MDA levels and Cu/Zn ratio were determined significantly higher in the group of CRC patient carrying the GA heterozygous genotype of the GSTM1 (rs 112,778,559) gene variation compared to healthy controls (p?<?0.05). Serum Cu, Zn levels, plasma MDA levels and Cu/Zn ratio were determined significantly higher in patients carrying GG homozygous genotype of the GSTM1 (rs 112778559) gene variation compared to healthy controls carrying same genotype (p?<?0.05). Serum Cu, Zn levels, plasma MDA levels and Cu/Zn ratio were determined significantly higher in the group of CRC patient carrying the GG homozygous genotype of the GSTM1 (rs 12068997) gene variation compared to healthy controls (p?<?0.05). On the other hand, serum Se levels were detected significantly lower in CRC patients carrying GA heterozygous and GG homozygous genotypes for GSTM1 (rs 112,778,559) and (rs 12,068,997) gene variations compared to healthy controls (p?<?0.05).

Conclusion

In our study, the evaluation of serum Cu, Zn and Se trace element levels and plasma MDA levels according to GSTM1 gene variations genotype distributions were enabled to obtain important biomarkers in terms of CRC development and progression.

     

Improved in vitro bioassay for Musa acuminata cv. Pisang ambon kuning (AAA group) based on quantitative analysis of necrosis area and biomass changes during Foc4 infection

Abstract

Efforts in improving banana plants that are resistant to the Fusarium wilt-causing Fusarium oxysporum f.sp. cubense tropical race 4 (Foc4) are indispensable. In this study, we developed rapid, space-efficient in vitro bioassay for assessing banana plant resistance to Foc4 using 35?×?150?mm glass test tubes, followed by quantitative and objective analysis of necrosis area and biomass changes as represented by fresh weight changes. Disease resistance screening was conducted based on the necrosis area as quantified using ImageJ software and on biomass gain during in vitro bioassay. In vitro banana plantlets showed age-related resistance during the development of necrosis (p?=?.034, Kruskal–Wallis test in root and shoot system and p?=?.027, one-way ANOVA in shoot system only), in which plantlets that were infected at the youngest age (24 weeks’ post-initiation) showed the largest necrosis area (up to 46.6%). In addition, plant fresh weight gain in this group (0.233?±?0.041?g) was higher compared to the gains in older plantlets (0.079?±?0.117 and 0.009?±?0.069?g, infected at 28 and 38?weeks’ post-initiation, respectively). Overall, for consistent and reliable result, the age of banana plantlet should be taken into consideration in interpreting the result of this in vitro bioassay.     

Photomorphogenesis in Sinningia speciosa, cv. Queen Victoria I. Characterization of Phytochrome Control

The morphological development of Sinningia speciosa plants that were exposed to supplementary far red light was very different from that of plants receiving dark nights. After several nights of such irradiation, stems and petioles were elongated, petioles were angulated, leaf blade expansion was inhibited, plants were chlorotic and the accumulation of shoot dry weight was retarded.

Red reversibility of the morphological changes potentiated by far red light indicated control by the phytochrome system. A high P_FR level during the last half of the night inhibited stem elongation and promoted leaf blade expansion, but both of these processes were hardly affected by the P_FR level during the first half of the night. Thus sensitivity to P_FR was cyclic.

The interpretation of our experiments was complicated by quantitative morphological differences resulting from long, as compared to short, far red irradiations.

     

Disparate results between proliferation rates of surgically excised prostate tumors and an in vitro bioassay using sera from a positive randomized controlled trial

Abstract

In vitro bioassay has been used extensively to test the effects of culturing cancer cells in sera from humans participating in dietary interventions, i.e, studies of modified intake of nutrients for the purpose of reducing cancer risk or progression. It has been hypothesized that cell proliferation rates determined by the in vitro bioassay indicate whether modification of dietary intake could decrease cancer cell growth in vivo. It has been suggested, however, that the in vitro bioassay may not correlate with tumor cell proliferation rates in prostate cancer. We investigated the concordance of cell proliferation rates from surgically excised prostate tumor tissue with the in vitro bioassay using sera from matched patients. We used samples from an earlier randomized clinical trial that showed that supplementation with flaxseed significantly inhibited prostate cancer cell proliferation rates in vivo as indicated by Ki67 staining in tumor specimens. Proliferation rates of LNCaP, DU145 and PC3 cell lines cultured in 10% human sera from participants in the flaxseed trial were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Spearman's Rho correlation coefficients (ρ) indicated no association between Ki67 staining in prostate tumors and the in vitro bioassay for the three cell lines. These disparate findings suggest that the in vitro bioassay may not provide an accurate assessment of the environment in vivo.     

Improving the sensitivity of long read overlap detection using grouped short k-mer matches

Background

Single-molecule, real-time sequencing (SMRT) developed by Pacific BioSciences produces longer reads than second-generation sequencing technologies such as Illumina. The increased read length enables PacBio sequencing to close gaps in genome assembly, reveal structural variations, and characterize the intra-species variations. It also holds the promise to decipher the community structure in complex microbial communities because long reads help metagenomic assembly. One key step in genome assembly using long reads is to quickly identify reads forming overlaps. Because PacBio data has higher sequencing error rate and lower coverage than popular short read sequencing technologies (such as Illumina), efficient detection of true overlaps requires specially designed algorithms. In particular, there is still a need to improve the sensitivity of detecting small overlaps or overlaps with high error rates in both reads. Addressing this need will enable better assembly for metagenomic data produced by third-generation sequencing technologies.

Results

In this work, we designed and implemented an overlap detection program named GroupK, for third-generation sequencing reads based on grouped k-mer hits. While using k-mer hits for detecting reads’ overlaps has been adopted by several existing programs, our method uses a group of short k-mer hits satisfying statistically derived distance constraints to increase the sensitivity of small overlap detection. Grouped k-mer hit was originally designed for homology search. We are the first to apply group hit for long read overlap detection. The experimental results of applying our pipeline to both simulated and real third-generation sequencing data showed that GroupK enables more sensitive overlap detection, especially for datasets of low sequencing coverage.

Conclusions

GroupK is best used for detecting small overlaps for third-generation sequencing data. It provides a useful supplementary tool to existing ones for more sensitive and accurate overlap detection. The source code is freely available at https://github.com/Strideradu/GroupK.

     

A new model for sensitive detection of zoonotic prions by PrP transgenic Drosophila

Prions are transmissible protein pathogens most reliably detected by a bioassay in a suitable host, typically mice. However, the mouse bioassay is slow and cumbersome, and relatively insensitive to low titers of prion infectivity. Prions can be detected biochemically in vitro by the protein misfolding cyclic amplification (PMCA) technique, which amplifies disease-associated prion protein but does not detect bona fide prion infectivity. Here, we demonstrate that Drosophila transgenic for bovine prion protein (PrP) expression can serve as a model system for the detection of bovine prions significantly more efficiently than either the mouse prion bioassay or PMCA. Strikingly, bovine PrP transgenic Drosophila could detect bovine prion infectivity in the region of a 10⁻¹² dilution of classical bovine spongiform encephalopathy (BSE) inoculum, which is 10⁶-fold more sensitive than that achieved by the bovine PrP mouse bioassay. A similar level of sensitivity was observed in the detection of H-type and L-type atypical BSE and sheep-passaged BSE by bovine PrP transgenic Drosophila. Bioassays of bovine prions in Drosophila were performed within 7 weeks, whereas the mouse prion bioassay required at least a year to assess the same inoculum. In addition, bovine PrP transgenic Drosophila could detect classical BSE at a level 10⁵-fold lower than that achieved by PMCA. These data show that PrP transgenic Drosophila represent a new tractable prion bioassay for the efficient and sensitive detection of mammalian prions, including those of known zoonotic potential.     

Industrial and international standardization of microbial pesticides — II. Insect viruses

Nuclear polyhedrosis virus (Baculovirus) (NPV) preparations, designed for biological control of insects, have to be standardized by bioassay in nearly the same way asBacillus thuringiensis preparations. Specific problems and bioassay techniques of NPV preparations are discussed. It seems necessary to use a standard reference sample in each bioassay. For each particular NPV virus an experimental standard reference preparation could be prepared by those laboratories already involved in biological control studies of a given virus. They should pay particular attention to the study of storage conditions.

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Résumé Les préparations à base de virus à polyèdres nucléaires (Baculovirus) destinées à la lutte biologique contre les insectes, doivent être étalonnées par des essais biologiques selon une méthodologie analogue à celle utilisée pour les préparations à base deBacillus thuringiensis. Les problèmes spécifiques aux virus et les techniques de titrage biologique qui leur sont adaptées sont discutés. Il semble nécessaire d'employer dans chaque cas une préparation de référence. Pour chaque virus à polyèdres nucléaires cette préparation de référence est à mettre au point par les laboratoires qui sont déjà concernés par les recherches sur l'utilisation en lutte biologique du virus considéré. Une attention particulière doit être portée à l'étude des conditions de conservation de ces étalons.

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In cooperation with the Texas Agricultural Experiment Station, Texas A & M University, College Station 77843. Paper read at the Symposium “Standardization and Safety of Microbial Pesticides” XVth. International Congress of Entomology, Washington 19–27 August 1976.     

TNFalpha production in whole blood cultures from healthy individuals

Tumor necrosis factor alpha (TNFalpha) is a major mediator of inflammatory responses and also plays a prominent role in bridging the innate and adaptive phases of immunity. In the present work we attempted to study TNFalpha production in endotoxin-stimulated blood of healthy individuals, and the inter-individual variability in TNFalpha production. For this study, we used diluted whole blood stimulated with lipopolysaccharide (LPS). The levels of the pro-inflammatory cytokine TNFalpha were measured by ELISA and by the L929 cytotoxicity bioassay in 16 and 18 healthy donors, respectively. There were highly significant inter-individual variations in the induced TNFalpha production. It is worth noting that there was no difference in sensitivity between ELISA and the cytotoxicity L929 bioassay. We concluded that whole blood culture is a sensitive method to determine the pro-inflammatory cytokine production in response to endotoxin stimuli in a relevant physiologic milieu. Our data indicate that this method provides appropriate information about the state of cellular immunity of the individual.     

Structural insights into the unique inhibitory mechanism of Kunitz type trypsin inhibitor from Cicer arietinum L.

Kunitz-type trypsin inhibitors bind to the active pocket of trypsin causing its inhibition. Plant Kunitz-type inhibitors are thought to be important in defense, especially against insect pests. From sequence analysis of various Kunitz-type inhibitors from plants, we identified CaTI2 from chickpea as a unique variant lacking the functionally important arginine residue corresponding to the soybean trypsin inhibitor (STI) and having a distinct and unique inhibitory loop organization. To further explore the implications of these sequence variations, we obtained the crystal structure of recombinant CaTI2 at 2.8Å resolution. It is evident from the structure that the variations in the inhibitory loop facilitates non-substrate like binding of CaTI2 to trypsin, while the canonical inhibitor STI binds to trypsin in substrate like manner. Our results establish the unique mechanism of trypsin inhibition by CaTI2, which warrant further research into its substrate spectrum. Abbreviations BApNA Nα-Benzoyl-L-arginine 4-nitroanilide

BPT bovine pancreatic trypsin

CaTI2 Cicer arietinum L trypsin inhibitor 2

DrTI Delonix regia Trypsin inhibitor

EcTI Enterolobium contortisiliquum trypsin inhibitor

ETI Erythrina caffra trypsin inhibitor

KTI Kunitz type inhibitor

STI soybean trypsin inhibitor

TKI Tamarindus indica Kunitz inhibitor

Communicated By Ramaswamy H. Sarma