Biochemical characterization of human enteropeptidase light chain

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.     

Evidence for contact stimulation of growth of homologous cells in superinoculated cultures of chick embryo cells

The secondary cultures of chick embryo cells were suspended and transferred to homologous cell cultures. Cell adhesion and proliferation were studied in these superinoculated cultures. It was shown that added cells soon adhered to the underlying cell layer which results in a prompt increase in culture density followed by the activation of DNA synthesis and cell division. Stimulation of cell proliferation involved both cell subpopulations composing the superinoculated culture: cells seeded on the built-up cell layer and cells of the layer. The contact nature of added cell mitogenic action on overlaid cell proliferation was evidenced. The cell system described can be used to investigate the adhesive properties of the cell layer apical surface, the relationship between cell growth rate and culture density, and the contact stimulation of cell proliferation.     

Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.     

Plasmid characteristics of naphthalene and salicylate biodegradation in Pseudomonas putida

The object of this work was to study the physico-chemical and biological properties of DNAs of the biodegradation plasmids NAH and SAL. A comparative analysis of the physico-chemical parameters for these DNAs made it possible to detect a number of identical properties in them: the same sedimentation profile for covalently-closed circular DNA forms, 68--70 S; the molecular weight of ca. 50 MD; a roughly equal number of fragments (up to 23) was found when the DNAs of NAH and SAL were restricted by EcoRI endonuclease. The transformation of the plasmidless strain PpGI was done.     

Localization of tryptophan residues in NADPH-adrenodoxin reductase with respect to the NADPH-binding center

It was demonstrated that the NADPH-adrenodoxin reductase molecule contains ten tryptophan residues titrated by N-bromosuccinimide. The effectiveness of the non-radiant energy transfer was used to calculate the average distance between the NADPH-binding site of the enzyme and tryptophan residues at different steps of N-bromosuccinimide-induced modification.     

Hovk 1 and the Middle and Upper Paleolithic of Armenia: a preliminary framework

The territory of present day Armenia is a geographic contact zone between the Near East and the northern Caucasus. Armenian Middle and Upper Paleolithic records are both few and patchy as a result of the historical paucity of systematic archaeological research in the country. Consequently, it is currently difficult to correlate the Armenian Middle and Upper Paleolithic records with those from other neighboring regions. We present new archaeological and chronometric data (luminescence, U-Th, and ¹⁴C) from our ongoing research at Hovk 1 Cave in northeast Armenia. We discuss in particular two activity phases in Hovk 1 Cave for which we have outline chronometric data: (1) an early Middle Paleolithic occupational phase, dated by optically stimulated luminescence (OSL) to 104 ± 9.8 ka BP_OSL; and (2) a Paleolithic occupational phase characterized by microlithic flakes dated by AMS ¹⁴C to 39,109 ± 1,324 calibrated years BP_Hulu. The two phases are separated by a hiatus in hominin occupation corresponding to MIS 4 and an episode in early MIS 3. These chronometric data, taken together with the preliminary paleoenvironmental reconstruction of the Hovk 1 Cave and environment, suggest that these activity phases represent short-lived and seasonal use of the cave presumably by small groups of hunters during episodes of mild climate. Neither tool manufacture nor butchery appears to have taken place within the cave, and consequently, the archaeological record included, for the most part, finished tools and blanks. We address the chronology and techno-typological aspects of Hovk 1 lithics in relation to: (1) the Paleolithic records of Armenia, and (2) the broader interregional context of early Middle Paleolithic hominin occupation and the Middle-Upper Paleolithic transition in the Caucasus.     

Use of inert gas jets to measure the forces required for mechanical gene transfection

Background

Abnormal blood glucose (BG) concentrations have been associated with increased morbidity and mortality in both critically ill adults and infants. Furthermore, hypoglycaemia and glycaemic variability have both been independently linked to mortality in these patients. Continuous Glucose Monitoring (CGM) devices have the potential to improve detection and diagnosis of these glycaemic abnormalities. However, sensor noise is a trade-off of the high measurement rate and must be managed effectively if CGMs are going to be used to monitor, diagnose and potentially help treat glycaemic abnormalities.

Aim

To develop a tool that will aid clinicians in identifying unusual CGM behaviour and highlight CGM data that potentially need to be interpreted with care.

Methods

CGM data and BG measurements from 50 infants at risk of hypoglycaemia were used. Unusual CGM measurements were classified using a stochastic model based on the kernel density method and historical CGM measurements from the cohort. CGM traces were colour coded with very unusual measurements coloured red, highlighting areas to be interpreted with care. A 5-fold validation of the model was Monte Carlo simulated 25 times to ensure an adequate model fit.

Results

The stochastic model was generated using ~67,000 CGM measurements, spread across the glycaemic range ~2-10?mmol/L. A 5-fold validation showed a good model fit: the model 80% confidence interval (CI) captured 83% of clinical CGM data, the model 90% CI captured 91% of clinical CGM data, and the model 99% CI captured 99% of clinical CGM data. Three patient examples show the stochastic classification method in use with 1) A stable, low variability patient which shows no unusual CGM measurements, 2) A patient with a very sudden, short hypoglycaemic event (classified as unusual), and, 3) A patient with very high, potentially un-physiological, glycaemic variability after day 3 of monitoring (classified as very unusual).

Conclusions

This study has produced a stochastic model and classification method capable of highlighting unusual CGM behaviour. This method has the potential to classify important glycaemic events (e.g. hypoglycaemia) as true clinical events or sensor noise, and to help identify possible sensor degradation. Colour coded CGM traces convey the information quickly and efficiently, while remaining computationally light enough to be used retrospectively or in real-time.     

Flora Europaea Notulae Systematicae ad Floram Europaeam spectantes No. 20 Corrigenda et Addenda

Following the publication of the last of the series of Flora Europaea Notulae, No. 20 in the Botanical Journal of the Linnean Society , 76: 297–384 (1978), a number of additions or alterations have been drawn to our attention. These are published in continuation.