Role of SH3b binding domain in a natural deletion mutant of <Emphasis Type="Italic">Kayvirus</Emphasis> endolysin LysF1 with a broad range of lytic activity

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.     

Gender differences in behavioral changes elicited by prenatal methamphetamine exposure and application of the same drug in adulthood

The aim of the present study was to compare the response to sub‐chronic application of methamphetamine (MA) in adulthood in male and female rats prenatally exposed to the same drug. The spontaneous locomotor activity and exploratory behavior of adult male and female rats prenatally exposed to 5 mg/kg MA or saline (SAL) were tested in a Laboras apparatus (Metris B.V., Netherlands) for five consecutive days, 1 hr daily. MA 1 mg/kg or SAL were used as a challenge prior to testing. Our results showed that rats prenatally exposed to MA were more sensitive to sub‐chronic administration of MA in adulthood than prenatally SAL‐exposed rats. However, this sensitizing effect of prenatal MA exposure was manifested differently in males and females. In contrast, prenatal MA exposure decreased baseline locomotion in females. This study indicates that gender plays an important role in the sensitivity to MA during prenatal development and in adulthood. © 2012 Wiley Periodicals, Inc. Dev Psychobiol 55: 232–242, 2013     

Palatal growth changes in newborns with unilateral and bilateral cleft lip and palate from birth until 12 months after early neonatal cheiloplasty using morphometric assessment

Clinical Oral Investigations - To compare palatal growth changes in infants with complete unilateral (UCLP) or bilateral (BCLP) cleft lip and palate during the first year of life. Upper dental...     

High- and low-affinity sites for sodium in δ-OR-Gi1α (Cys351-Ile351) fusion protein stably expressed in HEK293 cells; functional significance and correlation with biophysical state of plasma membrane

The effect of sodium, potassium, and lithium on δ-opioid receptor ligand binding parameters and coupling with the cognate G proteins was compared in model HEK293 cell line stably expressing PTX-insensitive δ-OR-G_i1α (Cys³⁵¹-Ile³⁵¹) fusion protein. Agonist [³H]DADLE binding was decreased in the order Na⁺???Li⁺?>?K⁺?>?(+)NMDG. When plotted as a function of increasing NaCl concentrations, the binding was best-fitted with a two-phase exponential decay considering two Na⁺-responsive sites (r ²?=?0.99). High-affinity Na⁺-sites were characterized by K_d?=?7.9 mM and represented 25 % of the basal level determined in the absence of ions. The remaining 75 % represented the low-affinity sites (K_d?=?463 mM). Inhibition of [³H]DADLE binding by lithium, potassium, and (+)-NMDG proceeded in low-affinity manner only. Surprisingly, the affinity/potency of DADLE-stimulated [³⁵S]GTPγS binding was increased in a reverse order: Na⁺?<?K⁺?<?Li⁺. This result was demonstrated in PTX-treated as well as PTX-untreated cells. Therefore, it is not restricted to G_i1α(Cys³⁵¹-Ile³⁵¹) within the δ-OR-G_i1α fusion protein, but is also valid for stimulation of endogenous G proteins of G_i/G_o family in HEK293 cells. Biophysical studies of interaction of ions with polar head-group region of lipids using Laurdan generalized polarization indicated the low-affinity type of interaction only proceeding in the order: Cs⁺?<?K⁺?<?Na⁺?<?Li⁺. The results are discussed in terms of interaction of Na⁺, K⁺ and Li⁺ with the high- and low-affinity sites located in water-accessible part of δ-OR binding pocket. We also consider the role of negatively charged Cl^?, Br^?, and I^? counter anions in inhibition of both [³H]DADLE and [³⁵S]GTPγS binding.     

A novel fibrinogen variant--Liberec: dysfibrinogenaemia associated with gamma Tyr262Cys substitution

Objective:  A 22-yr-old woman had abnormal preoperative coagulation test results and congenital dysfibrinogenaemia was suspected.
Patients and methods:  The patient from Liberec (Czech Republic) had a low fibrinogen plasma level as determined by Clauss method, normal fibrinogen level as determined by immunoturbidimetrical method, and prolonged thrombin time. To identify the genetic mutation responsible for this dysfibrinogen, genomic DNA extracted from the blood was analysed. Fibrin polymerisation measurement, kinetics of fibrinopeptide release, fibrinogen clottability measurement and scanning electron microscopy were performed.
Results:  DNA sequencing showed the heterozygous fibrinogen γ Y262C mutation. Kinetics of fibrinopeptide release was normal, however fibrin polymerisation was impaired. Fibrinogen clottability measurement showed that only about 45% molecules of fibrinogen are involved in the clot formation. Scanning electron microscopy revealed thicker fibres, which were significantly different from the normal control.
Conclusion:  A case of dysfibrinogenaemia, found by routine coagulation testing, was genetically identified as a novel fibrinogen variant (γ Y262C) that has been named Liberec.