Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombin-cleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors.     

Activities of several classes of acyclic nucleoside phosphonates against camelpox virus replication in different cell culture models

Camelpox virus (CMLV) is the closest known virus to variola virus. Here we report on the anti-CMLV activities of several acyclic nucleoside phosphonates (ANPs) related to cidofovir [(S)-1-(3-hydroxy-2-phosphonomethoxypropyl)cytosine (HPMPC; Vistide)] against two CMLV strains, CML1 and CML14. Cytopathic effect (CPE) reduction assays performed with human embryonic lung fibroblast monolayers revealed the selectivities of the first two classes of ANPs (cHPMPA, HPMPDAP, and HPMPO-DAPy) and of the hexadecyloxyethyl ester of 1-{[(5S)-2-hydroxy-2-oxido-1,4,2-dioxaphosphinan-5-yl]methyl}-5-azacytosine (HDE-cHPMP-5-azaC), belonging to the newly synthesized ANPs, which are HPMP derivatives containing a 5-azacytosine moiety. The inhibitory activities of ANPs against both strains were also confirmed with primary human keratinocyte (PHK) monolayers, despite the higher toxicity of those molecules on growing PHKs. Virus yield assays confirmed the anti-CML1 and anti-CML14 efficacies of the compounds selected for the highest potencies in CPE reduction experiments. Ex vivo studies were performed with a 3-dimensional model of human skin, i.e., organotypic epithelial raft cultures of PHKs. It was ascertained by histological evaluation, as well as by virus yield assays, that CMLV replicated in the human skin equivalent. HPMPC and the newly synthesized ANPs proved to be effective at protecting the epithelial cells against CMLV-induced CPE. Moreover, in contrast to the toxicity on PHK monolayers, signs of toxicity in the differentiated epithelium were seen only at high ANP concentrations. Our results demonstrate that compounds belonging to the newly synthesized ANPs, in addition to cidofovir, represent promising candidates for the treatment of poxvirus infections.     

Psoriasis risk genes of the late cornified envelope-3 group are distinctly expressed compared with genes of other LCE groups

Deletion of the late cornified envelope (LCE) genes LCE3B and LCE3C has recently been identified as a risk factor for psoriasis. Expression of 16 LCE genes of LCE groups 1, 2, 3, 5, and 6 was examined in vivo and in vitro. Quantitative PCR demonstrated that moderate to high LCE expression was largely confined to skin and a few oropharyngeal tissues. Genes of the LCE3 group demonstrated increased expression in lesional psoriatic epidermis and were induced after superficial injury of normal skin, whereas expression of members of other LCE groups was down-regulated under these conditions. Immunohistochemistry and immunoelectron microscopy demonstrated that LCE2 protein expression was restricted to the uppermost granular layer and the stratum corneum. Stimulation of in vitro reconstructed skin by several psoriasis-associated cytokines resulted in induction of LCE3 members. The data suggest that LCE proteins of groups 1, 2, 5, and 6 are involved in normal skin barrier function, whereas LCE3 genes encode proteins involved in barrier repair after injury or inflammation. These findings may provide clues to the mechanistic role of LCE3B/C deletion in psoriasis.     

EB病毒感染与中线恶性网织细胞增多症

使用一个针对ＥＢ病毒编码的小分子ＲＮＡ（ＥＢＥＲ）的寡核苷酸探针对１９例中线恶性网织细胞增多症病变组织进行了原位杂交检测，并配合免疫表型分析。结果为：（１）本组病例中１８／１９例（９４．７％）的病变组织中的异形淋巴样细胞表达Ｔ细胞分化抗原。ＣＤ３阳性１５例，ＵＣＨＬ１阳性９例，其中二者均阳性６例，但各例中的异形淋巴样细胞均未表达Ｂ细胞及组织细胞分化抗原；（２）ＥＢＥＲ原位杂交检测的阳性数高（１５／     

Influence of polymer molecular weight in osteoinductive composites for bone tissue regeneration

In bone tissue regeneration, certain polymer and calcium-phosphate-based composites have been reported to enhance some biological surface phenomena, facilitating osteoinduction. Although the crucial role of inorganic fillers in heterotopic bone formation by such materials has been shown, no reports have been published on the potential effects the polymer phase may have. The present work starts from the assumption that the polymer molecular weight regulates the fluid uptake, which determines the hydrolysis rate and the occurrence of biological surface processes. Here, two composites were prepared by extruding two different molecular weight l/d,l-lactide copolymers with calcium phosphate apatite. The lower molecular weight copolymer allowed larger fluid uptake in the composite thereof, which was correlated with a higher capacity to adsorb proteins in vitro. Further, the large fluid absorption led to a quicker composite degradation that generated rougher surfaces and enhanced ion release. Following intramuscular implantation in sheep, only the composite with the lower molecular weight polymer could induce heterotopic bone formation. Besides influencing the biological potential of composites, the molecular weight also regulated their viscoelastic behaviour under cyclic stresses. The results lead to the conclusion that designing biomaterials with appropriate physico-chemical characteristics is crucial for bone tissue regeneration in mechanical load-bearing sites.     

4D-CT angiography differentiating arteriovenous fistula subtypes

Objective and methods

In the diagnostic work-up of patients suspected of a dural arteriovenous fistula (dAVF), imaging has a key role in order to diagnose the dAVF, assess its bleeding risk and choose optimal treatment strategy. Digital subtraction angiography (DSA) is the gold standard for the most detailed image of a dAVF. Nowadays four-dimensional CT angiography (4D-CTA) could possibly be an additional first-line tool in the work-up of a patient suspected of a dAVF. We describe three cases clinically suspected of a dAVF which had a diagnostic work-up with 4D-CTA as well as DSA. We evaluated the angioarchitecture of the dAVF both on 4D-CTA and DSA, with emphasis on the patterns of venous drainage as this is important in assessing the bleeding risk of a dAVF.

Results and conclusion

4D-CTA identified the dAVF, revealed its angioarchitecture and correctly differentiated different patterns of venous drainage (Borden type I, II and III) as confirmed on DSA. Although DSA has the advantage of higher spatial and temporal resolution, 4D-CTA seems to be a new useful non-invasive tool in the diagnostic work-up of a patient suspected of a dAVF.     

Pericardial Effusions in Adolescent Girls With Anorexia Nervosa: Clinical Course and Risk Factors

The aim of this study was to evaluate cardiac, biochemical and endocrine differences between female adolescents with anorexia nervosa (AN) with and without pericardial effusions. We studied 128 female adolescents (9.8–17.7 years) with anorexia nervosa (AN) diagnosed according to DSM-IV (American Psychiatric Association, 1994 American Psychiatric Association. 1994. Diagnostic and statistical manual of mental disorders, 4th, Washington, DC: Author.  [Google Scholar]) criteria. They all underwent an echocardiographic evaluation. In 29 patients (22.2 %) a pericardial effusion (ranging between ≥ 0.35–2.5 cm) was noted. None of the patients were clinically symptomatic. After 3 months of refeeding, the effusions disappeared in 18/29 patients while in 7/29 patients a pericardial effusion > 0.3 cm persisted. Risk factors for development of effusions were a BMI ≤ 13,5 kg/m², weight loss ≥ 25% and IGF-1-level ≤100 ng/ml. Pericardial effusions are common in adolescent AN patients. They are mostly asymptomatic not requiring any intervention and spontaneously regress with refeeding. They are more common in the patients with the most significant weight loss.     

Adeno-associated viral vector-mediated gene transfer of brain-derived neurotrophic factor reverses atrophy of rubrospinal neurons following both acute and chronic spinal cord injury

Rubrospinal neurons (RSNs) undergo marked atrophy after cervical axotomy. This progressive atrophy may impair the regenerative capacity of RSNs in response to repair strategies that are targeted to promote rubrospinal tract regeneration. Here, we investigated whether we could achieve long-term rescue of RSNs from lesion-induced atrophy by adeno-associated viral (AAV) vector-mediated gene transfer of brain-derived neurotrophic factor (BDNF). We show for the first time that AAV vectors can be used for the persistent transduction of highly atrophic neurons in the red nucleus (RN) for up to 18 months after injury. Furthermore, BDNF gene transfer into the RN following spinal axotomy resulted in counteraction of atrophy in both the acute and chronic stage after injury. These novel findings demonstrate that a gene therapeutic approach can be used to reverse atrophy of lesioned CNS neurons for an extended period of time.