Identification and in silico analysis of 14 novel GJB1, MPZ and PMP22 gene mutations

Duplication within the chromosome 17p11.2 (CMT1Adup), peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ) and gap junction β1-protein (GJB1) gene mutations are frequent causes of the Charcot-Marie-Tooth disease (CMT). A large number of mutations in these genes are listed in databases. Sequence variants identified in patients are frequently reported as mutations without further evaluation. We analyzed 250 consecutively recruited unrelated Austrian CMT patients for CMT1Adup by microsatellite marker typing, real-time PCR or MLPA, and found 79 duplications (31.6%). The coding regions of the PMP22, MPZ and GJB1 genes were analyzed by direct sequencing in the remaining patients; 28 patients showed mutations, 14 of which were novel. We scored the pathogenicity of novel missense mutations by segregation studies and by their exclusion in control samples. Our comprehensive literature study found that up to 60% of the reported mutations in these genes had not been evaluated regarding their pathogenicity, and the PANTHER bioinformatics tool was used to score novel and published missense variants. The PANTHER program scored known polymorphisms as such, but scored ∼82–88% only of the published and novel mutations as most likely deleterious. Mutations associated with axonal CMT were less likely to be classified as deleterious, and the PMP22 S72L mutation repeatedly associated with severe CMT was classified as a polymorphism using default parameters. Our data suggest that this in silico analysis tool could be useful for assessing the functional impact of DNA variations only as a complementary approach. The CMT1Adup, GJB1, MPZ and PMP22 mutation frequencies were in the range of those described in other CMT patient collectives with different ethnical backgrounds.     

Differential regulation of Th1-type and Th2-type cytokine profiles in pancreatic islets of C57BL/6 and BALB/c mice by multiple low doses of streptozotocin

In the nonobese diabetic (NOD) mouse, the T helper (Th)1-type inflammatory cytokines interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha play a critical role in the development of type 1 diabetes, whereas the Th2-type anti-inflammatory cytokines interleukin (IL)-4 and IL-10 operate counterregulatory. There are no comprehensive analyses on cytokine profiles in the mouse model of diabetes induced with multiple low doses of streptozotocin (MLD-STZ). Therefore, we used islets to study ex vivo effects of MLD-STZ and in vitro effects of STZ on IFN-gamma, TNF-alpha, IL-4, and IL-10 on both levels of protein-producing cells and the mRNA expression, as well as the mRNA expression of the Th3-type cytokine transforming growth factor TGF-beta1. C57BL/6 and BALB/c mice of both genders were injected intraperitoneally with 40 mg/kg body wt STZ on five consecutive days and islets were isolated on day I and 3 after the fifth STZ-injection. Control mice received the solvent of STZ. In islets of C57BL/6 mice of both genders MLD-STZ similarly stimulated production of IFN-gamma and TNF-alpha, but significantly reduced IL-4 and IL-10 levels in male mice only. Opposite results were obtained in islets of BALB/c mice of both genders. Here, MLD-STZ markedly decreased the levels of IFN-gamma and TNF-alpha, but significantly increased the levels of IL-4 and IL-10. The functional results were in line with MLD-STZ effects on the mRNA expression of the cytokines. Moreover, MLD-STZ effects on the TGF-beta1 mRNA expression were reversed to the effects on IFN-gamma and TNF-alpha. The in vitro effects of STZ in islets, in general, were similar to those exerted by MLD-STZ. Apparently, reduction and upregulation of Th2-type cytokines was more associated with susceptibility and resistance, respectively, to MLD-STZ-induced diabetes than upregulation of Th1-type cytokine levels.     

Cranial translation of the humeral head on radiographs in rotator cuff tear patients: the modified active abduction view

Cranial translation of the humeral head is related to massive rotator cuff tears; however, it may be unapparent in early-stage tears. The goal of this study was to investigate whether active abduction leads to increased active cranial humeral translation in early-stage tears. We assessed 20 consecutive patients (9 full-thickness supraspinatus tears, 11 posterosuperior tears) using the newly introduced modified active abduction view: acromiohumeral (AH) distance was measured on radiographs acquired during rest and active isometric abduction and adduction tasks with the arm alongside the body. Rest AH was 7.5 mm (SD = 1.53); during abduction and adduction, it decreased to 2.1 mm (95 % CI 1.28–3.01, p < 0.001) and 1.1 mm (95 % CI 0.46–1.65, p = 0.001), respectively. Cranial translation during abduction was more severe in shoulders with posterosuperior cuff tears (?AH = 3 mm, SD = 1.5) compared to supraspinatus tears (?AH = 1 mm, SD = 1.6), with a mean difference of 2 mm (95 % CI 0.64–3.58, p = 0.007). Both active isometric abduction and adduction leads to active cranial translation in cuff tear patients. Cranial translation is largest during active abduction. Furthermore, there is significant more cranial translation in posterosuperior cuff tear patients compared to supraspinatus cuff tear patients. Possibly, radiographs combined with active tasks offer new possibilities in diagnosing early-stage rotator cuff tears.     

Reliability of histological malignancy grade,ER and HER2 status on core needle biopsy vs surgical specimen in breast cancer

Early determination of malignancy grade and biomarkers in primary breast cancer is important when planning the treatment with neoadjuvant therapy and breast‐conserving surgery. The aim of this study was to assess the reliability of malignancy grade on core needle biopsies, and to compare the concordance of ER and HER2 status on core needle biopsies vs surgical specimens. Eighty‐nine patients with invasive ductal or lobular carcinoma were included. Malignancy grade was upgraded by one degree in 23.0%. The mitotic count had most impact on the malignancy grade, and may be underestimated on core needle biopsies compared with surgical specimens. ER status was concordant in core needle biopsies and surgical specimens in 98.0%, but should be repeated when doubtfully negative. HER2 status was concordant in 84.0% prior to FISH‐HER2. Including FISH‐HER2 data, the core needle biopsies showed a final total concordance of 95.4%. In conclusion, ER and HER2 are useful markers in core needle biopsies, providing a reliable base for preoperative decisions in neoadjuvant chemotherapy, and may be omitted on surgical specimens with certain precautions and exceptions. Determination of malignancy grade should be repeated on surgical specimens, and one should be aware of underestimation on core needle biopsies.     

On the evolution of attachment-disordered behaviour

Using an example of a goose which grew up in complete isolation, this article shows how escape behaviour increases through all its developmental steps by lack of a sufficient secure primary attachment object. If a gosling has no mother, its fear influences wide parts of its behaviour and blocks the capacity to approach another animal in order to form a new attachment. But the urge for attachment is still alive and provokes strong conflicts between this urge and fear. In these situations displacement activities and apathy occur – attachment-disordered behaviour. Under partly artificial circumstances, a change to ‘normal’ attachment behaviour took place by reduction of escape motivation, which enabled the goose to accept her brood. From this moment on the attachment-disordered behaviour, the displacement activity and the apathy disappeared. Implications to human behaviour are drawn – a comparison between displacement activity and neurotic symptoms, the state of apathy and personal disorder. A synthesis of ethological and psychoanalytical models concerning etiological and therapeutic aspects is discussed. Fear and attachment, not aggression and sexuality – our ‘animal heritage’ – are the primary factors which influence our development. The balance of fear and attachment is the basis for a healthy or unhealthy development of social relationships. This applies to social living animals as well as to humans.     

Increased CD11b and Decreased CD62L in Blood and Airway Neutrophils from Long-Term Smokers with and without COPD

There is incomplete mechanistic understanding of the mobilization of neutrophils in the systemic and local compartment in smokers with chronic obstructive pulmonary disease (COPD). In this pilot study, we characterized how the adhesion molecules CD11b and CD62L, surface markers indicative of priming, are altered as neutrophils extravasate, and whether surface density of CD11b and CD62L differs between long-term tobacco smokers (LTS) with and without COPD compared with healthy never-smokers (HNS). Unstimulated blood neutrophils from LTS with (n = 5) and without (n = 9) COPD displayed lower surface density of CD62L compared with HNS (n = 8). In addition, surface density of CD11b was higher in bronchoalveolar lavage (BAL) neutrophils from LTS without COPD compared with those with COPD and HNS. Moreover, in BAL neutrophils from all study groups, CD62L was lower compared with matched blood neutrophils. In addition, BAL neutrophils responded with a further decrease in CD62L to ex vivo TNF stimulation. Thus, neutrophils in the airway lumen display a higher state of priming than systemic neutrophils and bear the potential to be further primed by local cytokines even with no smoking or the presence of COPD, findings that may represent a universal host defense mechanism against local bacteria. Moreover, systemic neutrophils are primed in LTS regardless of COPD. Further studies in larger materials are warranted to determine whether the priming of neutrophils is protective against COPD or merely preceding it.     

Zur Katalyse der stereospezifischen Butadienpolymerisation mit den Katalysatorsystemen aus Nd(η3-C3H5)3 · dioxan und methylaluminoxan (MAO) sowie Hexaisobutylaluminoxan (HIBAO)

The activation of the tris(allyl)neodymium complex Nd(η³-C₃H₅)₃ · dioxane with alkylaluminoxanes (MAO or HIBAO) results in highly selective catalysts for the 1,4-cis-polymerization of butadiene (cis-selectivity up to 80%). Under standard conditions (50°C, toluene), the turnover frequency (TOF) of the catalyst/MAO system amounts to 10–15000 mol butadiene/(mol Nd · h). Molecular weight determinations indicate the formation of only one polymer chain per neodymium center as in a living polymerization reaction, and for the catalyst/HIBAO system the rate law r_p = k_p [Nd][C₄H₆] with k_p = 8,7 · 10^?2 mol/(L · s) (at 25°C) has been derived. As the catalytically active species, a cationic monobutenyl neodymium(III) complex is discussed, which is stabilized through coordinative interaction with the counter anion as well as the growing polybutadiene chain. This cationic complex reacts under insertion with butadiene in a bimolecular fashion.     

Postantibiotic effects with Bacteroides fragilis determined by viable counts and CO2 generation

Objective: To study the postantibiotic effect (PAE) for Bacteroides fragilis after exposure to common anaerobic antimicrobials with two different methods, by viable counting and by measuring CO₂ generation in a BACTEC® blood culture system.
Method: Four strains of B. fragilis were exposed for 1,2 and 4 h to cefoxitin, chloramphenicol, clindamycin, imipenem or metronidazole at concentrations from 1 to 16 X MIC. The drugs were removed by dilution into BACTEC 7A® vials and growth determined with viability counts and CO₂ production.
Results: The durations of the PAEs determined by the two methods correlated well ( r =0.913, p <0.005). PAEs of up to 4–5 h were induced by imipenem and metronidazole with achievable concentrations and exposure durations. Chloramphenicol induced short or no PAEs, but cefoxitin and clindamycin induced PAEs up to 2 h with high AUC values. The imipenem PAEs and the short cefoxitin and clindamycin PAEs were dependent on AUC.
Conclusions: Significant PAEs against B. fragilis were induced by imipenem and metronidazole. Determining PAE by measuring CO₂ production is an accurate and less time-consuming alternative to the conventional method of viable counts.     

Sulfur amino acids in methionine-restricted rats: Hyperhomocysteinemia

ObjectiveDietary methionine restriction in Fischer-344 rats favorably influences visceral fat mass, insulin sensitivity, metabolic parameters, and longevity. However, little is known about the effects of methionine restriction on serum methionine and its downstream sulfur amino acids. We investigated the serum sulfur amino acid profile of male Fischer-344 rats fed a methionine-restricted diet for 3 mo.Methods and resultsUsing tandem mass spectrometry, we observed marked reduction in serum concentrations of methionine, cystathionine, cysteine, and taurine in methionine-restricted rats compared with control (P < 0.001) and a 2.5-fold elevation of homocysteine (P < 0.001).ConclusionThis suggests that homocysteine trans-sulfuration may be inhibited by methionine restriction, and that some of the effects of methionine restriction may be mediated by changes in sulfur amino acids downstream of methionine.     

CTLA-4 trafficking and surface expression

The T-cell co-receptor cytotoxic T-cell antigen 4 (CTLA-4) has a strong inhibitory role as shown by the lymphoproliferative phenotype of CTLA-4-deficient mice. Despite its potent effects on T-cell function, CTLA-4 is primarily an intracellular antigen whose surface expression is tightly regulated by restricted trafficking to the cell surface and rapid internalisation. Recently, several signalling molecules such as Trim, PLD, ARF-1 and TIRC7 have been described to be involved in the transport of CTLA-4 to the cell surface. Minor changes in surface expression levels have major effects on the outcome of T-cell activation. Optimal regulation of CTLA-4 surface expression is crucial for the balance of stimulatory and inhibitory signals to maximize protective immune responses while maintaining immunological tolerance and preventing autoimmunity.