Characterization of amine oxidases from pisum, lens, Lathyrus and Cicer

Amine oxidases have been purified to homogeneity from Pisum sativum, Lens esculenta, Lathyrus sativus and Cicer arietinum. The enzymes have a M_r. of 150 000 and are composed of two identical subunits of 72 000. The amine oxidases showed an isoelectrophoretic heterogeneity.     

Making more microtubules by severing: a common theme of noncentrosomal microtubule arrays?

Two related enzymes, katanin and spastin, use the energy from ATP hydrolysis to sever microtubules. Two new studies (one in this issue; see McNally et al., p. 881) show that microtubule severing by katanin provides a means for increasing microtubule density in meiotic spindles. Interestingly, loss of spastin leads to a sparser microtubule array in axons and synaptic boutons. Together, these studies hint at a wider role for microtubule-severing enzymes in the formation and organization of noncentrosomal microtubule arrays by generating new seeds for microtubule growth.     

Imidazo[1,2-a]pyrazine diaryl ureas: Inhibitors of the receptor tyrosine kinase EphB4

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent^® and Nexavar^®, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.     

Upper Limb Evaluation and One-Year Follow Up of Non-Ambulant Patients with Spinal Muscular Atrophy: An Observational Multicenter Trial

Assessment of the upper limb strength in non-ambulant neuromuscular patients remains challenging. Although potential outcome measures have been reported, longitudinal data demonstrating sensitivity to clinical evolution in spinal muscular atrophy patients are critically lacking. Our study recruited 23 non-ambulant patients, 16 patients (males/females = 6/10; median age 15.4 years with a range from 10.7 to 31.1 years) with spinal muscular atrophy type II and 7 patients (males/females = 2/5; median age 19.9 years with a range from 8.3 to 29.9 years) with type III. The Brooke functional score was on median 3 with a range from 2 to 6. The average total vital capacity was 46%, and seven patients required non-invasive ventilation at night. Patients were assessed at baseline, 6 months, and 1 year using the Motor Function Measure and innovative devices MyoGrip, MyoPinch, and MoviPlate, which assess handgrip strength, key pinch strength, and hand/finger extension-flexion function, respectively. The study demonstrated the feasibility and reliability of these measures for all patients, and sensitivity to negative changes after the age of 14 years. The younger patients showed an increase of the distal force in the follow-up period. The distal force measurements and function were correlated to different functional scales. These data represent an important step in the process of validating these devices as potential outcome measures for future clinical trials.

Trial Registration

ClinicalTrials.gov NCT00993161     

Parasitophorous vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem)

We present a short insight into the problem of parasitophorous vacuole (PV) formation as a most peculiar kind of cell vacuolization occurring in the course of intracellular development of coccidian pathogens of the genera Eimeria, Isospora, Toxoplasma, Sarcocystis, Cryptosporidium, Epieimeria, and Karyolysus. The review focuses on the morpho-functional diversity of PVs in these parasites. By the present time, the PVs containing different parasite genera and species have been examined to different extent. The membrane of the PV (PVM) obviously derives from the host cell plasmalemma. But soon after parasite penetration, the morphofunctional organization and biochemical composition of the PVM drastically changes: its proteins are selectively excluded and those of the parasite are incorporated. As the result, the PV becomes not fusigenic for lysosomes or any other vacuoles or vesicles, because host cell surface markers necessary for membrane fusion are eliminated from the PVM during parasite invasion.The pattern of the PVs is parasite specific and demonstrates a broad diversity within the same genera and species and even at different stages of the endogenous development. The PV is far from being an indifferent membrane vesicle containing the parasite. Instead, it represents a dynamic system that reflects the innermost events of host-parasite relationships, thus promoting the accomplishing of the parasite life cycle, which, in its turn, is a necessary prerequisite of the parasite eventual survival as a species.     

Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane

Huntingtin is a large membrane-associated scaffolding protein that associates with endocytic and exocytic vesicles and modulates their trafficking along cytoskeletal tracks. Although the progression of Huntington’s disease is linked to toxic accumulation of mutant huntingtin protein, loss of wild-type huntingtin function might also contribute to neuronal cell death, but its precise function is not well understood. Therefore, we investigated the molecular role of huntingtin in exocytosis and observed that huntingtin knockdown in HeLa cells causes a delay in endoplasmic reticulum (ER)-to-Golgi transport and a reduction in the number of cargo vesicles leaving the trans-Golgi network. In addition, we found that huntingtin is required for secretory vesicle fusion at the plasma membrane. Similar defects in the early exocytic pathway were observed in primary fibroblasts from homozygous Htt^140Q/140Q knock-in mice, which have the expansion inserted into the mouse huntingtin gene so lack wild-type huntingtin expression. Interestingly, heterozygous fibroblasts from a Huntington’s disease patient with a 180Q expansion displayed no obvious defects in the early secretory pathway. Thus, our results highlight the requirement for wild-type huntingtin at distinct steps along the secretory pathway.KEY WORDS: Exocytosis, Huntingtin, ER, Golgi, Vesicle fusion     

Tools,techniques, and future opportunities for characterizing the mechanobiology of uterine myometrium

The myometrium is the smooth muscle layer of the uterus that generates the contractions that drive processes such as menstruation and childbirth. Aberrant contractions of the myometrium can result in preterm birth, insufficient progression of labor, or other difficulties that can lead to maternal or fetal complications or even death. To investigate the underlying mechanisms of these conditions, the most common model systems have conventionally been animal models and human tissue strips, which have limitations mostly related to relevance and scalability, respectively. Myometrial smooth muscle cells have also been isolated from patient biopsies and cultured in vitro as a more controlled experimental system. However, in vitro approaches have focused primarily on measuring the effects of biochemical stimuli and neglected biomechanical stimuli, despite the extensive evidence indicating that remodeling of tissue rigidity or excessive strain is associated with uterine disorders. In this review, we first describe the existing approaches for modeling human myometrium with animal models and human tissue strips and compare their advantages and disadvantages. Next, we introduce existing in vitro techniques and assays for assessing contractility and summarize their applications in elucidating the role of biochemical or biomechanical stimuli on human myometrium. Finally, we conclude by proposing the translation of “organ on chip” approaches to myometrial smooth muscle cells as new paradigms for establishing their fundamental mechanobiology and to serve as next-generation platforms for drug development.     

Ribosomal proteins of Thermus thermophilus fused to beta-galactosidase are imported into the nucleus of eukaryotic cells

Archaea, Bacteria, and Eukarya have 34 homologous ribosomal protein (RP) families in common. Comparisons of published amino acid sequences prompted us to question whether RPs of the prokaryote Thermus thermophilus contain nuclear localization signals (NLSs), which are recognized by the nuclear import machinery of eukaryotic cells and are thereby translocated into the nucleoplasm ultimately accumulating in the nucleolus. Several RPs of T. thermophilus - specifically S12, S17, and L2 - were selected for this study since their three-dimensional structures as well as rRNA interaction patterns are precisely known at the molecular level. Fusion proteins of these RPs were constructed and subsequently expressed in COS cells. N-terminally tagged fusions with dimeric EGFP and C-terminally tagged hybrids with beta-galactosidase of prokaryotic RP S17 (S17p) were targeted to the nucleoplasm where they were visualized by direct fluorescence and by indirect immune staining, respectively. A region containing the classical monopartite NLS KRKR, which is known to physically interact with karyopherin alpha2, was delineated by tagging specific S17p fragments with beta-galactosidase. Unexpectedly, S12p and L2p hybrids accumulated in the nucleolus. Due to their size, RPs tagged with beta-galactosidase can only be imported into the nucleus when NLS-recognition is mediated by karyopherins since they are otherwise excluded from entry into the nucleoplasm of eukaryotic cells. Our results indicate that after the formation of the nuclear compartment during evolution, the newly established eukaryotic cell relied on the pre-existing basic amino acid clusters of the prokaryotic RPs for use as NLSs.     

Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization

We have previously reported (Hinescu & Popescu, 2005) the existence of interstitial Cajal-like cells (ICLC), by transmission electron microscopy, in human atrial myocardium. In the present study, ICLC were identified with non-conventional light microscopy (NCLM) on semi-thin sections stained with toluidine blue and immunohistochemistry (IHC) for CD117/c-kit, CD34, vimentin and other additional antigens for differential diagnosis. Quantitatively, on semi-thin sections, ICLC represent about 1-1.5% of the atrial myocardial volume (vs. approximately 45% working myocytes, approximately 2% endothelial cells, 3-4% for other interstitial cells, and the remaining percentage: extracellular matrix). Roughly, there is one ICLC for 8-10 working atrial myocytes in the intercellular space, beneath the epicardium, with a characteristic (pyriform, spindle or triangular) shape. These ICLC usually have 2-3 definitory processes, emerging from cell body, which usually embrace atrial myocytes (260 nm average distance plasmalemma/sarcolemma) or establish close contact with nerve fibers or capillaries (approximately 420 nm average distance to endothelial cells). Cell prolongations are characteristic: very thin (mean thickness = 0.15+/-0.1 microm), very long for a non-nervous cell (several tens of microm) and moniliform (uneven caliber). Stromal synapses between ICLC and other interstitial cells (macrophages) were found (e.g. in a multicontact type synapse, the average synaptic cleft was approximately 65 nm). Naturally, the usual cell organelles (mitochondria, smooth and rough endoplasmic reticulum, intermediate filaments) are relatively well developed. Caveolae were also visible on cell prolongations. No thick filaments were detected. IHC showed that ICLC were slightly and inconsistently positive for CD117/c-kit, variously co-expressed CD34 and EGF receptor, but appeared strongly positive for vimentin, along their prolongations. Some ICLC seemed positive for a-smooth muscle actin and tau protein, but were negative for nestin, desmin, CD13 and S-100. In conclusion, we provide further evidence of the existence of ICLC in human atrial myocardium, supporting the possible ICLC role in pacemaking, secretion (juxta- and/or paracrine), intercellular signaling (neurons and myocytes). For pathology, ICLC might as well be 'players' in arrhythmogenesis and atrial remodeling.     

Controlled degradation by ClpXP protease tunes the levels of the excision repair protein UvrA to the extent of DNA damage

UV irradiation damages DNA and activates expression of genes encoding proteins helpful for survival under DNA stress. These proteins are often deleterious in the absence of DNA damage. Here, we investigate mechanisms used to regulate the levels of DNA-repair proteins during recovery by studying control of the nucleotide excision repair (NER) protein UvrA. We show that UvrA is induced after UV irradiation and reaches maximum levels between ∼20 and 120 min post UV. During post-UV recovery, UvrA levels decrease principally as a result of ClpXP-dependent protein degradation. The rate of UvrA degradation depends on the amount of unrepaired pyrimidine dimers present; this degradation rate is initially slow shortly after UV, but increases as damage is repaired. This increase in UvrA degradation as repair progresses is also influenced by protein–protein interactions. Genetic and in vitro experiments support the conclusion that UvrA–UvrB interactions antagonize degradation. In contrast, Mfd appears to act as an enhancer of UvrA turnover. Thus, our results reveal that a complex network of interactions contribute to tuning the level of UvrA in the cell in response to the extent of DNA damage and nicely mirror findings with excision repair proteins from eukaryotes, which are controlled by proteolysis in a similar manner.