The NMR structure of the 47-kDa dimeric enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase and ligand binding studies reveal the location of the active site

Recent developments in NMR have extended the size range of proteins amenable to structural and functional characterization to include many larger proteins involved in important cellular processes. By applying a combination of residue-specific isotope labeling and protein deuteration strategies tailored to yield specific information, we were able to determine the solution structure and study structure-activity relationships of 3,4-dihydroxy-2-butanone-4-phosphate synthase, a 47-kDa enzyme from the Escherichia coli riboflavin biosynthesis pathway and an attractive target for novel antibiotics. Our investigations of the enzyme's ligand binding by NMR and site-directed mutagenesis yields a conclusive picture of the location and identity of residues directly involved in substrate binding and catalysis. Our studies illustrate the power of state-of-the-art NMR techniques for the structural characterization and investigation of ligand binding in protein complexes approaching the 50-kDa range in solution.     

Sphingosine kinase 2 deficient tumor xenografts show impaired growth and fail to polarize macrophages towards an anti‐inflammatory phenotype

A challenging task of the immune system is to fight cancer cells. However, a variety of human cancers educate immune cells to become tumor supportive. This is exemplified for tumor‐associated macrophages (TAMs), which are polarized towards an anti‐inflammatory and cancer promoting phenotype. Mechanistic explanations, how cancer cells influence the macrophage phenotype are urgently needed to address potential anti‐cancer strategies along this line. One potential immune modulating compound, sphingosine‐1‐phosphate (S1P), was recently highlighted in both tumor growth and immune modulation. Using a xenograft model in nude mice, we demonstrate a supportive role of sphingosine kinase 2 (SphK2), one of the S1P‐producing enzymes for tumor progression. The growth of SphK2‐deficient MCF‐7 breast tumor xenografts was markedly delayed when compared with controls. Infiltration of macrophages in SphK2‐deficient and control tumors was comparable. However, TAMs from SphK2‐deficient tumors displayed a pronounced anti‐tumor phenotype, showing an increased expression of pro‐inflammatory markers/mediators such as NO, TNF‐α, IL‐12 and MHCII and a low expression of anti‐inflammatory IL‐10 and CD206. These data suggest a role for S1P, generated by SphK2, in early tumor development by affecting macrophage polarization. © 2009 UICC     

Fabry disease and vascular risk factors: future strategies for patient-based studies and the knockout murine model

Fabry disease is secondary to deficiency of the lysosomal enzyme alpha-galactosidase A, leading to altered glycosphingolipid metabolism and accumulation that is often associated with endothelial dysfunction. Current evidence suggests that there is impairment of the vascular nitric oxide pathway, with abnormalities evident in the cerebral circulation and in the dermal vasculature of patients with Fabry disease. Some of these findings have been confirmed in a mouse model of Fabry disease. The murine model, however, allows investigation of Fabry disease at a non-clinical level and a near complete investigation of biological processes within an affected tissue. This is of particular utility in allowing gene expression analysis of clinically inaccessible tissues such as the aorta.Conclusion: Future developments in array technology for proteins and DNA single nucleotide polymorphism analysis, together with gene expression microarray analysis, may open a new chapter in our understanding of the biology of lysosomal storage disorders.     

Ablation of striatal somatostatin interneurons affects MSN morphology and electrophysiological properties,and increases cocaine‐induced hyperlocomotion in mice

The striatum is mainly composed by medium spiny neurons (95 %) (MSNs). Although outnumbered, in other brain regions such as the hippocampus and the cortex, somatostatin interneurons (SSTi) are known to control and fine‐tune the activity of principal cells. This information is still fragmented for the striatum. Here, we questioned the striatal functional consequences of the selective ablation of SSTi in the striatum at the behavioural and cellular levels. We identified increased excitability coupled with decreased distal spine density in MSNs from SSTi‐ablated mice. Although the ethological behavioural analysis did not reveal differences between the groups, SSTi‐ablated mice were significantly more sensitive to the locomotor effects of cocaine without changes in motivation. This was accompanied by increased expression of the dopamine transporter (DAT) in the ventral striatum. Altogether, we show that SSTi are important players in the maintenance of MSN excitability and spine density impacting on mechanisms towards hyperdopaminergic states.     

Rocaglates as Antivirals: Comparing the Effects on Viral Resistance,Anti-Coronaviral Activity,RNA-Clamping on eIF4A and Immune Cell Toxicity

Rocaglates are potent broad-spectrum antiviral compounds with a promising safety profile. They inhibit viral protein synthesis for different RNA viruses by clamping the 5′-UTRs of mRNAs onto the surface of the RNA helicase eIF4A. Apart from the natural rocaglate silvestrol, synthetic rocaglates like zotatifin or CR-1-31-B have been developed. Here, we compared the effects of rocaglates on viral 5′-UTR-mediated reporter gene expression and binding to an eIF4A-polypurine complex. Furthermore, we analyzed the cytotoxicity of rocaglates on several human immune cells and compared their antiviral activities in coronavirus-infected cells. Finally, the potential for developing viral resistance was evaluated by passaging human coronavirus 229E (HCoV-229E) in the presence of increasing concentrations of rocaglates in MRC-5 cells. Importantly, no decrease in rocaglate-sensitivity was observed, suggesting that virus escape mutants are unlikely to emerge if the host factor eIF4A is targeted. In summary, all three rocaglates are promising antivirals with differences in cytotoxicity against human immune cells, RNA-clamping efficiency, and antiviral activity. In detail, zotatifin showed reduced RNA-clamping efficiency and antiviral activity compared to silvestrol and CR-1-31-B, but was less cytotoxic for immune cells. Our results underline the potential of rocaglates as broad-spectrum antivirals with no indications for the emergence of escape mutations in HCoV-229E.     

Childhood ataxia with diffuse central nervous system hypomyelination

A significant number of patients with progressive leukodystrophy do not have a definitive diagnosis. This report describes the clinical, morphological, and biochemical characteristics of 4 unrelated girls with progressive ataxic diplegia of unknown etiology. These patients had normal development until the ages of 1.5 to 5 years. A diffuse confluent abnormality of the white matter of the central nervous system was present on computed tomography and magnetic resonance scans obtained early in the course of the illness. Dementia was not present and peripheral nerves were normal. All patients were evaluated for known metabolic and degenerative diseases and no abnormalities were observed. Light and electron microscopy of open-brain biopsy specimens from 2 girls showed selective white matter abnormalities including hypomyelination, demyelination, and gliosis. Myelin-specific proteins in the subcortical white matter were examined immunocytochemically and by Western blot analysis. The were of normal molecular size but were markedly reduced in quantity in both patients compared to control subjects. Lipid analysis revealed decreased levels of characteristic myelin lipids. When examined by magnetic resonance spectroscopic imaging, all patients showed a marked decrease of N-acetylaspartic acid, choline, and creatine in white matter only. The magnetic resonance spectroscopic imaging profile is a unique diagnostic feature of this clinicopathological syndrome.     

Immunocytochemical detection of GABAergic nerve cells in the human temporal cortex using a direct gamma-aminobutyric acid antiserum

Recently, an immunocytochemical method using glutaraldehyde fixation and an antiserum developed against a GABA--glutaraldehyde--protein conjugate has permitted direct visualization of GABAergic structures in the brains of perfused animals. This paper reports a successful use of this technique on human temporal cortex fixed by immersion. The cerebral tissue was obtained from patients operated for focal epilepsy. GABA-positive somata, fibres and terminals are observed in all layers of the temporal cortex. Terminals are particularly abundant in the superficial portion of layer I and in layers II, III and IV. Dense plexuses of fibres are located in layers II, III, IV and VI and in the underlying white matter. Somata are found in all cortical layers and in the underlying white matter; they are round, oval, fusiform or triangular and exhibit a multipolar, bitufted or bipolar dendritic pattern. This technique for the visualization of GABAergic structures in the human brain may allow a better understanding of the pathogeny of epilepsy in which the GABAergic transmission has been implicated.     

Kinetochore proteins, peripheral location of chromosomes and nuclear budding: another look at the genesis of aneuploidy.

Some chromosomes in the rat do not have detectable levels of kinetochore proteins as determined by antikinetochore antibody, which may constitute a mechanism for the genesis of aneuploidy. An analysis of three mouse cell lines for the presence of kinetochore proteins showed that some chromosomes lacked these proteins, i.e. are akinetochoric by this criterion. This phenomenon may contribute to the variability of chromosome number that is characteristic of such lines. In some cells there appears to be en masse detachment of kinetochores. Several kinetochore-bearing chromosomes were also observed to be located peripherally to the spindle. These might be associated with the genesis of aneuploidy. In one cell line an apparently new phenomenon of nuclear budding was observed. In this case, one to several chromosomes appear to be pushed out of the nucleus in bud-like structures. These were surrounded by a nuclear membrane and appeared to detach from the main nucleus. Perhaps these structures eventually break off as micronuclei and, hence, would also be associated with the genesis of aneuploidy.     

Detection of hyperdiploidy and chromosome breakage affecting the 1 (1cen-q12) region in lentigo malignant melanoma (LMM), superficial spreading melanoma (SSM) and congenital nevus (CN) cells in vitro by the multicolor FISH technique

The centric/pericentric region of chromosome 1 (cen-q12) of human melanoma cells of different stages of carcinogenicity (superficial spreading melanoma (SSM), lentigo malignant melanoma (LMM)) and premalignant precursor lesions (congenital nevus (CN)) were investigated by fluorescence in situ hybridization (FISH) with tandem DNA probes. The pericentric heterochromatin region 1(q12) is large and highly prone to breakage in contrast to the adjacent centromeric region which is much smaller and less prone to such events. All samples of melanoma cells were obtained from patients and cultivated in vitro. LMM cells showed the highest number of breakage events within the 1q12 region (90% of cells). The number of hyperdiploid cells was not increased in comparison to CN cells. In contrast to LMM cells, SSM cells showed a significant increased number of hyperdiploid cells which were mainly tetrasomic for chromosome 1 (P≤0.05). The number of chromosome breaks was not significantly increased in this type of melanoma cells. The spontaneous rates of chromosomal breakage and hyperdiploidy is relatively low in CN cells (1.5–2.5% and 3.2–5.8%, respectively) but these frequencies also differ between CN samples from different patients. These results show that the multicolor FISH technique represents a fast and reliable detection method, distinguishing structural and numerical chromosomal alterations in interphase nuclei. This technique is useful as a histological marker to differentiate between specific tumor subtypes and to investigate the relationship between genomic instability and clinopathological parameters (tumor grading and staging).