Chitosan particles agglomerated scaffolds for cartilage and osteochondral tissue engineering approaches with adipose tissue derived stem cells

In the XML file of the original article, H. Redl’s affiliation is incorrect. It is listed correctly in both the paper and PDF versions of the article, and can be found below: The online version of the original article can be found at     

Protein phosphorylation: technologies for the identification of phosphoamino acids

Protein phosphorylation plays a central role in many biological and biomedical phenomena. In this review, while a brief overview of the occurrence and function of protein phosphorylation is given, the primary focus is on studies related to the detection and analysis of phosphorylation both in vivo and in vitro. We focus on phosphorylation of serine, threonine and tyrosine, the most commonly phosphorylated amino acids in eukaryotes. Technologies such as radiolabelling, antibody recognition, chromatographic methods (HPLC, TLC), electrophoresis, Edman sequencing and mass spectrometry are reviewed. We consider the speed, simplicity and sensitivity of tools for detection and identification of protein phosphorylation, as well as quantitation and site characterisation. The limitations of currently available methods are summarised.     

Fluorescence in situ hybridization (FISH): DNA probe production and hybridization criteria

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.     

Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

The fusion of intracellular transport vesicles with their target membranes requires the assembly of SNARE proteins anchored in the apposed membranes. Here we use recombinant cytoplasmic domains of the yeast SNAREs involved in Golgi to plasma membrane trafficking to examine this assembly process in vitro. Binary complexes form between the target membrane SNAREs Sso1p and Sec9p; these binary complexes can subsequently bind to the vesicle SNARE Snc2p to form ternary complexes. Binary and ternary complex assembly are accompanied by large increases in alpha-helical structure, indicating that folding and complex formation are linked. Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of approximately 3 M(-1) s(-1). An N-terminal regulatory domain of Sso1p accounts for slow assembly, since in its absence complexes assemble 2,000-fold more rapidly. Once binary complexes form, ternary complex formation is rapid and is not affected by the presence of the regulatory domain. Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhibition by this regulatory domain.     

Is insurance for children enough? The link between parents'' and children''s health care use revisited

The gutless hydrothermal tubeworm Riftia pachyptila Jones relies mainly on its chemoautotrophic bacterial symbionts to supply nutrients in the form of secreted organic compounds resulting from fixation and incorporation of CO2. In this study, symbionts were purified, tested for viability, and incubated in the presence of labeled CO2. We demonstrated that purified symbionts can be used as a viable alternative to experiments with bacterial cultures. Several organic acids, sugars, and amino acids were labeled, but their fraction of the total label stayed generally constant during the incubation times used. However, increasing fractions of succinate and, to a lesser degree, glutamate were excreted into the incubation medium, indicating that these are probably the main carbon-containing compounds transferred from the symbionts to the host. Glutamate could also account for the transport of nitrogen from the symbionts to the host.     

Double blind, randomised, placebo controlled study of oral vancomycin in prevention of necrotising enterocolitis in preterm, very low birthweight infants

AIMS: To evaluate the effectiveness of oral vancomycin in the prophylaxis of necrotising enterocolitis in preterm, very low birthweight infants. METHODS: A prospective, double blind, randomised, placebo controlled study in a tertiary referral centre of a university teaching hospital was conducted on 140 very low birthweight infants consecutively admitted to the neonatal unit. The babies were randomly allocated to receive oral vancomycin (15 mg/kg every 8 hours for 7 days) or an equivalent volume of placebo solution. Prophylaxis was started 24 hours before the start of oral feeds. All suspected cases of necrotising enterocolitis were investigated with a full sepsis screen and serial abdominal radiographs. Necrotising enterocolitis was diagnosed and staged according to modified Bell's criteria. RESULTS: Nine of 71 infants receiving oral vancomycin and 19 of 69 infants receiving the placebo solution developed necrotising enterocolitis (p = 0.035). Infants with necrotising enterocolitis were associated with a significant increase in mortality (p = 0.026) and longer duration of hospital stay (p = 0.002). CONCLUSIONS: Prophylactic oral vancomycin conferred protection against necrotising enterocolitis in preterm, very low birthweight infants and was associated with a 50% reduction in the incidence. However, widespread implementation of this preventive measure is not recommended, as it would only be effective in necrotising enterocolitis caused by Gram positive organisms and could increase the danger of the emergence of vancomycin resistant or dependent organisms. Its use should be restricted to a high prevalence nursery for a short and well defined period in a selected group of high risk patients.     

Aorto-iliac thrombosis in a foal

A six-day-old Missouri foxtrotter colt was examined because it had had diarrhoea since it was 24 hours old. A diagnosis of colitis, septicaemia, and disruption of the arterial blood flow to the pelvic limbs was made on the basis of clinical and laboratory findings. Despite intensive medical therapy, the foal died 13 hours after being examined. Postmortem examination revealed diffuse fibrinous enteritis with lymphoid necrosis, multifocal fibrinonecrotic typhlocolitis, disseminated intravascular coagulation, and a large occluding thrombus at the aortic termination. The results of bacteriological culturing supported the diagnosis of septicaemia leading to activation of the clotting cascade, disseminated intravascular coagulation, aorto-iliac thrombosis and infarction of the pelvic limbs.     

Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation

The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR-chloromethyl ketone). Thermal unfolding monitored by differential scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.