Chlamydia and mycoplasma infections during pregnancy and their relationships to orofacial cleft

Serum antibodies to Mycoplasma pneumoniae and Chlamydia trachomatis have been studied in a group of newborns with orofacial cleft (OC) and their mothers (n = 59) as compared to a control group of healthy newborns and their mothers (n = 40) assayed by ELISA and Western blot analysis. In the first group, IgG antibodies to M. pneumoniae were found by ELISA in 12 newborns with OC and 22 mothers, while IgA antibodies were detected only in 5 and 11 cases, respectively. IgM antibodies indicating an acute infection were found in 2 mothers only. IgG antibodies to C. trachomatis were found in 2 newborns with OC and 4 mothers. In the control group, IgG antibodies to M. pneumoniae were found in 3 newborns and 7 mothers. IgG antibodies to C trachomatis were observed in 1 newborn and 1 mother, while IgM antibodies to C trachomatis were present in 1 mother only. Immunoblot analysis revealed in newborns with OC and their mothers C. trachomatis-specific bands associated with MOMP 1, 29 kDa, 45 kDa, and heat shock proteins (HSP) 60 and 70. Based on these results we suggest that the risk associated with the exposure to M. pneumoniae and/or C. trachomatis is so far unknown and further study is needed for its elucidation.     

CD94/NKG2A inhibits NK cell activation by disrupting the actin network at the immunological synapse

An adequate immune response is the result of the fine balance between activation and inhibitory signals. The exact means by which inhibitory signals obviate activation signals in immune cells are not totally elucidated. Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and some CD8+ T cells that recognize HLA-E. We show that the engagement of this receptor prevents NK cell activation by disruption of the actin network and exclusion of lipid rafts at the point of contact with its ligand (inhibitory NK cell immunological synapse, iNKIS). CD94/NKG2A engagement leads to recruitment and activation of src homology 2 domain-bearing tyrosine phosphatase 1. This likely explains the observed dephosphorylation of guanine nucleotide exchange factor and regulator of actin, Vav1, as well as ezrin-radixin-moesin proteins that connect actin filaments to membrane structures. In contrast, NK cell activation by NKG2D induced Vav1 and ezrin-radixin-moesin phosphorylation. Thus, CD94/NKG2A prevents actin-dependent recruitment of raft-associated activation receptors complexes to the activating synapse. This was further substantiated by showing that inhibition of actin polymerization abolished lipid rafts exclusion at the iNKIS, whereas cholesterol depletion had no effect on actin disruption at the iNKIS. These data indicate that the lipid rafts exclusion at the iNKIS is an active process which requires an intact cytoskeleton to maintain lipid rafts outside the inhibitory synapse. The net effect is to maintain an inhibitory state in the proximity of the iNKIS, while allowing the formation of activation synapse at distal points within the same NK cell.     

Nitrergic Proprioceptive Afferents Originating from Quadriceps Femoris Muscle are Related to Monosynaptic Ia-Motoneuron Stretch Reflex Circuit in the Dog

1. The aim of the present study was to examine the occurrence of the neuronal nitric oxide synthase immunoreactivity in the stretch reflex circuit pertaining to the quadriceps femoris muscle in the dog.2. Immunohistochemical processing for neuronal nitric oxide synthase and histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase were used to demonstrate the presence of neuronal nitric oxide synthase in the proprioceptive afferents issuing in the quadriceps femoris muscle. The retrograde tracer Fluorogold injected into the quadriceps femoris muscle was used to detect the proprioceptive afferents and their entry into the L5 and L6 dorsal root ganglia.3. A noticeable number of medium-sized intensely nitric oxide synthase immunolabelled somata (1000–2000 μm² square area) was found in control animals in the dorsolateral part of L5 and L6 dorsal root ganglia along with large-caliber intraganglionic nitric oxide synthase immunolabelled fibers, presumed to be Ia axons. Before entering the dorsal funiculus the large-caliber nitric oxide synthase immunolabelled fibers of the L5 and L6 dorsal roots formed a massive medial bundle, which upon entering the dorsal root entry zone reached the dorsolateral part of the dorsal funiculus and were distributed here in a funnel-shaped fashion. The largest nitric oxide synthase immunolabelled fibers, 8.0–9.2 μm in diameter, remained close to the dorsal horn, while medium-sized fibers were seen dispersed across the medial portion of the dorsal funiculus. Single, considerably tapered nitric oxide synthase immunolabelled fibers, 2.2–4.6 μm in diameter, were seen to proceed in ventrolateral direction until they reached the mediobasal portion of the dorsal horn and the medial part of lamina VII. In lamina IX, only short fragments of nitric oxide synthase immunoreactive fibers and their terminal ramifications could be seen. Nitric oxide synthase immunolabelled terminals varying greatly in size were identified in control material at the base of the dorsal horn, in the vicinity of motoneurons ventrally and ventrolaterally in L5 and L6 segments and in Clarke’s column of L3 and L4 segments. Injections of the retrograde tracer Fluorogold into the quadriceps femoris muscle and cut femoral nerve, combined with nitric oxide synthase immunohistochemistry of the L5 and L6 dorsal root ganglia, confirmed the existence of a number of medium-sized nitric oxide synthase immunoreactive and Fluorogold-fluorescent somata presumed to be proprioceptive Ia neurons (1000–2000 μm² square area) in the dorsolateral part of both dorsal root ganglia. L5 and L6 dorsal rhizotomy caused a marked depletion of nitric oxide synthase immunoreactivity in the medial bundle of the L5 and L6 dorsal roots and in the dorsal funiculus of L5 and L6 segments.4. The analysis of control material and the degeneration of the large- and medium-caliber nitric oxide synthase immunoreactive Ia fibers in the dorsal funiculus of L5 and L6 segments confirmed the presence of nitric oxide synthase in the afferent limb of the monosynaptic Ia-motoneuron stretch reflex circuit related to the quadriceps femoris muscle. Abbreviations ABC, avidin–biotin complex; bNOS, neuronal nitric oxide synthase; bNOS-IR, neuronal nitric oxide synthase immunoreactive; bNOS-IRB_s, neuronal nitric oxide synthase immunoreactive boutons; cNOS, catalytic nitric oxide synthase; DAB, diaminobenzidine; DF, dorsal funiculus; DH, dorsal horn; DREZ-one, dorsal root entry zone; DRG_s, dorsal root ganglia; eNOS, endothelial nitric oxide synthase; FG, Fluorogold; FN, femoral nerve; mNOS, macrophage nitric oxide synthase; NADPHd, nicotinamide adenine dinucleotide phosphate diaphorase; NBT, nitroblue tetrazolium; NO, nitric oxide; NOS, nitric oxide synthase; NOS-IR, nitric oxide synthase immunoreactive; PBS, phosphate-buffered saline; VGLUT1 and VGLUT2, vesicular glutamate transporters     

Apolipoprotein E polymorphism in relation to plasma lipid levels and other risk factors of atherosclerosis in two ethnic groups from Slovakia

The influence of apolipoprotein E (ApoE) genotypes on plasma lipid levels and interaction with other environmental factors was determined in two Slovakian population samples; 146 Romany and 351 Slovak individuals. The two samples differ significantly in the distribution of E3/3 genotypes (p<0.014) and E3/2 (p<0.035). Analysis of variance did not reveal any significant effect of the ApoE genotypes on any of the plasma lipid levels in the Romany individuals. In the Slovak sample the variation in plasma low-density lipoprotein cholesterol (LDL-C) levels was significantly associated with the ApoE genotypes (p=0.012). We detected decreased LDL-C concentrations in males with E2 genotype when compared with E3 and E4 carriers (p=0.008). Further, the E2 genotype was found to be associated with high triglycerides levels (p=0.009). The ethnic samples differ significantly in the prevalence of metabolic syndrome and in the case of males of diabetes. Both the Romany and the Slovak males can be considered as having a more atherogenic profile compared with the females.     

New member of the protein disulfide isomerase (PDI) family identified in Amblyomma variegatum tick

Ticks belonging to arthropoda are blood feeding, geographically widespread ectoparasites of mammals, reptiles and birds. Their saliva contains active substances that protect them from host immune attack and allow for transmission of various pathogens during the feeding process. Characterization of tick saliva components can therefore contribute to the development of effective methods for the control of tick-borne diseases.

Here we describe the identification and basic characterization of a gene encoding a 55 kDa protein found in the salivary glands (SG) of Amblyomma variegatum tick. Based on the primary structure and homology to the family of protein disulfide isomerases (PDI; EC 5.3.4.1) the gene was named AvPDI. The 1461 nt long AvPDI open reading frame codes for a 487 amino acid protein. In vitro expressed AvPDI was exclusively localized in the endoplasmic reticulum. RT-PCR and Western blot analysis revealed that AvPDI expression is not restricted to the SG of the tick. More detailed analysis on tissue slides from SG detected an AvPDI specific signal in granular cells of the acini type II and III. Finally, reductase activity of AvPDI was confirmed in an insulin assay. The structural and functional characteristics suggest that AvPDI is another member of the PDI protein family and represents the first more closely characterized PDI in the ticks.     

Analysis of the tellurite resistance determinant on the pNT3B derivative of the pTE53 plasmid from uropathogenic Escherichia coli

We have found and sequenced a significant part of the previously described tellurite resistance determinant on mini-Mu derivative pPR46, named pNT3B, originally cloned from a large conjugative plasmid pTE53, found in Escherichia coli. This plasmid contains genes essential for tellurite resistance, together with the protective region bearing genes terX, Y, W, and the conserved spacing region bearing several ORFs of unknown function. Computer analysis of obtained sequence revealed a close similarity to the formerly described ter operons found on the Serratia marcescens plasmid R478 and the chromosome of Escherichia coli O157:H7. This finding confirms the presence of a whole region on the large conjugative plasmid that pTE53 originated from a uropathogenic E. coli strain, and suggests its possible role in horizontal gene transfer, resulting in the development of new pathogenic E. coli strains.     

Telomerase-associated Protein 1, HSP90, and Topoisomerase II�� Associate Directly with the BLM Helicase in Immortalized Cells Using ALT and Modulate Its Helicase Activity Using Telomeric DNA Substrates

The BLM helicase associates with the telomere structural proteins TRF1 and TRF2 in immortalized cells using the alternative lengthening of telomere (ALT) pathways. This work focuses on identifying protein partners of BLM in cells using ALT. Mass spectrometry and immunoprecipitation techniques have identified three proteins that bind directly to BLM and TRF2 in ALT cells: telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα (TOPOIIα). BLM predominantly co-localizes with these proteins in foci actively synthesizing DNA during late S and G₂/M phases of the cell cycle when ALT is thought to occur. Immunoprecipitation studies also indicate that only HSP90 and TOPOIIα are components of a specific complex containing BLM, TRF1, and TRF2 but that this complex does not include TEP1. TEP1, TOPOIIα, and HSP90 interact directly with BLM in vitro and modulate its helicase activity on telomere-like DNA substrates but not on non-telomeric substrates. Initial studies suggest that knockdown of BLM in ALT cells reduces average telomere length but does not do so in cells using telomerase.Bloom syndrome (BS)⁴ is a genetic disease caused by mutation of both copies of the human BLM gene. It is characterized by sun sensitivity, small stature, immunodeficiency, male infertility, and an increased susceptibility to cancer of all sites and types. The high incidence of spontaneous chromosome breakage and other unique chromosomal anomalies in cells from BS patients indicate an increase in homologous recombination in somatic cells (1). Another notable feature of non-immortalized and immortalized cells from BS individuals is the presence of telomeric associations (TAs) between homologous chromosomes (2). Work from our group and others have suggested a role for BLM in recombination-mediated mechanisms of telomere elongation or ALT (alternative lengthening of telomeres), processes that maintain/elongate telomeres in the absence of telomerase (3–5). However, the exact mechanism by which BLM contributes to telomere stability is unknown.Several proteins interact with and regulate BLM helicase activity, including two telomere-specific proteins, TRF1 and TRF2 (6, 7). Although TRF2 stimulates BLM unwinding of telomeric and non-telomeric 3′-overhang substrates, TRF1 inhibits BLM unwinding of telomeric substrates. TRF2-mediated stimulation of BLM helicase activity on a telomeric substrate is observed when TRF2 is present in excess or with equimolar amount of TRF1 but not when TRF1 is present in molar excess. Both proteins associate with BLM specifically in ALT cells in vivo, suggesting their involvement in the ALT pathways. In addition to TRF1 and TRF2, the telomere single-strand DNA-binding protein POT1 strongly stimulates BLM helicase activity on long telomeric forked duplexes and D-loop structures (8). Other proteins also play an important role in telomere maintenance in telomerase-negative cells, including RAD50, NBS1, and MRE11, which co-localize with TRF1 and TRF2 in specialized ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) (9–11). Thus, we hypothesize that BLM complex formation may be essential for the ALT mechanism, and its modification may occur dynamically during the specific nucleic acid transactions required to protect the telomere in cells using the ALT pathways.This study has identified previously unknown protein partners of BLM and TRF2 in ALT cells using double immunoprecipitation and mass spectrometry (MS). These include telomerase-associated protein 1 (TEP1), heat shock protein 90 (HSP90), and topoisomerase IIα (TOPOIIα). These proteins associate with BLM and TRF2 in cells using ALT but not in cells using telomerase and directly interact with BLM in vitro. This complex of proteins localizes to sites of new DNA synthesis in vivo in ALT cells, suggesting a role in telomere maintenance. We also identified HSP90 and TOPOIIα in another ALT-specific complex consisting of BLM, TRF1, and TRF2 but not TEP1. In vitro analyses demonstrate that HSP90 inhibits BLM helicase activity using both telomeric and non-telomeric substrates, whereas TEP1 and TOPOIIα initially slow the kinetics of BLM unwinding only using telomeric substrates. These findings suggest the presence of dynamic BLM-associated ALT complexes that include previously unidentified interacting proteins. The function of TEP1 in the BLM·TRF2 complex remains unclear, although its previously described interaction with the RNA subunit of telomerase (12) suggests an interesting hypothesis of cross-talk between mechanisms of telomere elongation.     

Application of wound closure Molndal technique after laparoscopic cholecystectomy--initial comparative study

Because of a possible delayed wound healing, critical colonization and infection of wounds present a problem for surgeons. Colonized and infected wounds are a potential source for cross-infection. Molndal technique of wound dressing has proven to be effective in prevention of infection. Also the wound heal better and faster. In our study we wanted to describe the benefits of the Molndal technique wound dressing after laparoscopic cholecistectomy compared to traditional wound dressing technique. Molndal technique consisted of wound dressing with Aquacel Ag--Hydrofiber (ConvaTec, Dublin, Ireland). Traditional technique was performed using gauze compresses and hypoallergic adhesives. We analyzed the results of 100 patients after laparoscopic cholecystectomy. 50 patients were treated by Molndal technique and 50 patients by the traditional technique of wound dressing. In the group treated by Molndal technique only 1 (2%) patient has revealed a wound infection, proven by positive microbiological examination and suppuration, mostly in the subumbilical incision. In the traditional technique group 7 (14%) patients developed wound infection also predominantly in the subumbilical incision. The difference was statistically significant (p < 0.01). Our results are clearly showing that Molndal technique is effective in preventing the infection of subumbilical incision wound and is to by recommend for regular use at designated site after laparoscopic cholecistectomy.