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Acute ataxia is not an uncommon childhood complaint. It most commonly occurs in young patients secondary to a postinfectious cerebellitis, which is typically associated with a very good prognosis and recovery. In adolescence, acute cerebellar ataxia is more often the product of an etiology likely to progress into a chronic disorder without recovery to preillness baseline. In the present case, the authors describe a 15-year-old girl with subacute cerebellar ataxia of presumed immune-mediated etiology that advanced into a chronic cerebellar ataxia. Due to a family history, celiac disease was suspected as the origin of the ataxia; biopsy ruled out enteropathy, and the severe, abrupt radiological changes to the patient's cerebellum are inconsistent with the reported sequelae of gluten ataxia. This case serves as a discussion for diagnostic challenges in adolescent patients with acute cerebellar ataxia with long-term sequelae as well as providing an adjunct discussion on the neurological complications of celiac disease.

Background: Bacterial DNA gyrase is a validated target for antibacterial chemotherapy. It consists of two subunits, GyrA and GyrB, which form an A₂B₂ complex in the active enzyme. Sequence alignment of Mycobacterium tuberculosis GyrB with other bacterial GyrBs predicts the presence of 40 potential additional amino acids at the GyrB N-terminus. There are discrepancies between the M. tuberculosis GyrB sequences retrieved from different databases, including sequences annotated with or without the additional 40 amino acids. This has resulted in differences in the GyrB sequence numbering that has led to the reporting of previously known fluoroquinolone-resistance mutations as novel mutations., Findings: We have expressed M. tuberculosis GyrB with and without the extra 40 amino acids in Escherichia coli and shown that both can be produced as soluble, active proteins. Supercoiling and other assays of the two proteins show no differences, suggesting that the additional 40 amino acids have no effect on the enzyme in vitro. RT-PCR analysis of M. tuberculosis mRNA shows that transcripts that could yield both the longer and shorter protein are present. However, promoter analysis showed that only the promoter elements leading to the shorter GyrB (lacking the additional 40 amino acids) had significant activity., Conclusion: We conclude that the most probable translational start codon for M. tuberculosis GyrB is GTG (Val) which results in translation of a protein of 674 amino acids (74 kDa).

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.

Pragmatic clinical trials (PCTs) seek to improve the generalizability and increase the statistical power of traditional explanatory trials. They are a major tenet of comparative effectiveness research. While a powerful study design, PCTs have been limited by high cost, modest efficiency, and limited ability to fill relevant evidence gaps. Based on an American Reinvestment and Recovery Act (ARRA) supported meeting of national stakeholders, we propose several innovations and future research that could improve the efficiency and effectiveness of such studies focused in the U.S. Innovations discussed include optimizing the use of community based practices through partnership with Practice Based Research Networks (PBRNs), using information technology to simplify PCT subject recruitment, consent and randomization processes, and utilizing linkages to large administrative databases, such as Medicare, as a mechanism to capture outcomes and other important PCT variables with lower subject and research team burden. Testing and adaptation of such innovations to PCT are anticipated to improve the public health value of these increasingly important studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca(2+)-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca(2+) cannot support topoisomerase reactions in the absence of Mg(2+), but partial removal of Ca(2+) from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca(2+). More extensive removal of Ca(2+) by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca(2+)-binding residues also leads to loss of activity. We propose that Ca(2+) has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca(2+) binding.

Systemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disorder commonly caused by the A3243G mutation. We report a patient who initially presented with visual hallucinations, headaches, and nonconvulsive status epilepticus originating in left occipital lobe who subsequently progressed to have multifocal seizures. His magnetic resonance imaging (MRI) showed subtle T2 hyperintensity at first presentation that subsequently fully resolved. He then had more typical diffusion restriction not conforming to vascular territories. Evolution of his neuroimaging and electroencephalogram (EEG) is discussed with a brief review of literature. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes should be suspected early with occipital lobe seizures.

MicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii, like diverse multicellular organisms, contains miRNAs. These RNAs resemble the miRNAs of land plants in that they direct site-specific cleavage of target mRNA with miRNA-complementary motifs and, presumably, act as regulatory molecules in growth and development. Utilizing these findings we have developed a novel artificial miRNA system based on ligation of DNA oligonucleotides that can be used for specific high-throughput gene silencing in green algae., (© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd.)

Bioassay-guided fractionation of an ethanol extract of Roupellina (Strophanthus) boivinii from the rainforest of Madagascar afforded the six new cardenolide glycosides boivinides 1-6, as well as the four known cardenolide glycosides digitoxigenin 3-O-[beta-D-glucopyrananosyl-(1-->4)-alpha-L-acofriopyranoside], corotoxigenin 3-O-beta-D-boivinoside, 17alpha-corotoxigenin 3-O-beta-D-sarmentoside, and uzarigenin 3-O-alpha-L-rhamnoside. The structures of these compounds were elucidated by various 1D and 2D NMR techniques. All new compounds showed significant antiproliferative activity against the A2780 human ovarian cancer cell line, with boivinide A being the most active at IC50 = 0.17 microM.

Nucleic acid analogs and mimics are commonly the modifications of native nucleic acids at the nucleobase, the sugar ring, or the phosphodiester backbone. Many forms of promising nucleic acid analogs and mimics are available, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos. LNAs, PNAs, and morpholinos can form both duplexes and triplexes and have improved biostability. They have become a general and versatile tool for DNA and RNA recognition. LNA is a general and versatile tool for specific, high-affinity recognition of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA). LNA can be used for designing LNA oligoes for hybridization studies or as real time polymerase chain reaction probes in the form of Taqman probes. LNA also has therapeutic and diagnostic applications. PNA is another type of DNA analog with neutral charge. The extreme stability of PNA makes it an ideal candidate for the antisense and antigene application. PNA is used as probe for gene cloning, mutation detection, and in homologous recombination studies. It was also used to design transcription factor decoy molecules for target gene induction. Morpholino, another structural type, was devised to circumvent cost problems associated with DNA analogs. It has become the premier knockdown tool in developmental biology due to its cytosolic delivery in the embryos by microinjection. Thus, the nucleic acid analogs provide an advantage to design and implementation, therapies, and research assays, which were not implemented due to limitations associated with standard nucleic acids chemistry.

RNA interference (RNAi) is silencing of gene expression by double-stranded RNA (dsRNA) having complementary sequence to the target gene to be silenced. This phenomenon has transformed into a complete technology for functional genomic studies. Small interfering RNAs (siRNAs) are 21- to 23-nucleotide dsRNAs, in which the sense strand is the same as the target mRNA and the antisense strand is the complement of the target mRNA sequence. These are the effector molecules for inducing RNAi, leading to posttranscriptional gene silencing with RNA-induced silencing complex. Besides siRNA, which can be chemically synthesized, various other systems in the form of potential effector molecules for posttranscriptional gene silencing are available, such as short hairpin RNAs (shRNAs), long dsRNAs, short temporal RNAs, and micro RNAs (miRNAs). These effector molecules either are processed into siRNA such as in the case of shRNA or directly aid gene silencing as in the case of miRNA. RNAi for various unknown genes may facilitate to elucidate inherited genetic diseases and provide drug candidates for viral and oncogenic diseases. This can be achieved by targeting mRNA from oncogenic genes or mRNA for viral cellular receptor and viral structural proteins for RNAi. In this article, we evaluate various aspects and applications of RNAi technology and provide comprehensive information for the system currently available for inducing RNAi.

This report reviews previously published studies from our laboratory and shows some recent morphological data obtained with scanning and transmission electron microscopy regarding gallstone formation and alteration of the gallbladder epithelium in the Syrian hamster model. Both male and female hamsters were treated with female sex steroids (estradiol alone, estradiol and medroxyprogesterone, medroxyprogesterone alone) during one month. The results show that the Syrian hamster is a good model to study bile changes, gallbladder structure changes, including gallstone formation, and the regulation of cholesterol metabolism at the molecular level. Arguments in favor of this animal model are presented and, during gallstone formation, epithelial cell changes, anionic mucus secretion, and formation of gallbladder luminal deposits can be demonstrated. Recent molecular biology observations related to the effect of female sex steroids on liver cholesterol 7 alpha-hydroxylase (CYP7) gene suggest that progestin alone or primed by estrogen down regulates CYP7 transcription and activity. In addition, progesterone in cell culture systems has been shown to enhance intracellular accumulation of free cholesterol by increasing its uptake and synthesis and by decreasing its esterification by inhibiting the activity of acylcoenzyme A: cholesterol acyltransferase. Non-esterified cholesterol is free to migrate to the extracellular spaces and may contribute to nucleation within the bile. It is suggested that these effects of progesterone on cholesterol metabolism combined with the CYP7 gene down regulation, physical changes in the mucus and the hypomotility of the gallbladder and biliary ducts result in hypersaturation of cholesterol in the bile which favors gallstone formation.

We report here the expression of recombinant human prokallikrein and kallikrein in engineered Chinese hamster ovary cells transfected with a human genomic gene encoding preprokallikrein. At high expression levels, recombinant prokallikrein, an inactive proenzyme form, is predominantly secreted into the culture medium. Upon chromatographic separations, the inactive prokallikrein as well as the mature kallikrein after thermolysin activation of the proenzyme can be prepared to apparent purity. Both prokallikrein and kallikrein can be further separated into two distinct high- and low-molecular-weight isoforms. Kallikrein preparations are fully active in standard kallikrein activity assays such as esterase activity and kinin release from kininogen. Both kallikrein and prokallikrein display multiple molecular forms with differences in both molecular sizes and charges. The structural differences in high- and low-molecular-weight kallikreins or prokallikreins were found to be due to glycosylation, with the high-molecular-weight species glycosylated at three Asn-linked sites and the low-molecular-weight species at two of the three Asn-linked sites. The multiply charged kallikrein isoforms are derived from different numbers of sialic acids attached at the detected Asn-linked carbohydrates. In comparison with kallikrein, prokallikrein appears to show a significant decrease in the magnitude of near uv-circular dichroism bands, suggesting a change in local conformation. This conformational change correlates with the loss of activity in proenzyme due to the presence of propeptide.

The primary objective of this research project was to study the role of sex steroids in the pathogenesis of cholelithiasis using the Syrian hamster as a model. In addition to the morphological examination of the gallbladder epithelium, we thought it imperative to observe the changes induced in the biliary tract in response to the sex steroid treatment. This report focuses on the morphological changes induced in the liver. The hamsters were randomly divided into 4 groups, control (C), estrogen-treated (E), estrogen and medroxyprogesterone-treated (E+MP), and medroxyprogesterone-treated (MP) groups. The E group hepatocytes demonstrated proliferation of the smooth endoplasmic reticulum, lipofuscin-like granules, aggregates of glycogen rosettes, and dense bodies. Lipid droplets in the hepatocyte cytoplasm as well as the nuclei were detected in this group. E+MP combined treatment induced an exacerbation of all the changes observed in the E group, furthermore, there appeared to be a disruption of the hepatic parenchymal architecture. The MP-treated group also exhibited the architectural changes observed in the E+MP group, but also showed sinusoidal dilation. In response to MP alone, the fatty changes in the liver appeared to be accentuated. A striking feature induced in response to MP treatment, was a focal area suggestive of adenomatous changes.

Scanning (SEM), and transmission (TEM) electron microscopy were used and correlated to morphologically characterize changes induced in the gallbladder epithelium of female Syrian hamster in response to treatments with estradiol (E) alone, and estradiol with medroxyprogesterone (E + MP). Compared with control (C), the E- and E + MP-treated groups demonstrated alterations in the serum lipid profile as well as significantly decreased body weights. The liver with gallbladder weights, as well as the uterus weights, were significantly increased. Two-month E and E + MP treatment groups exhibited increased number of anionically charged apical granules, and luminal mucoid elements. Contrastingly, the three-month treatment groups demonstrated larger and more gallstone-like deposits as compared to the C and two-month E and E + MP groups. This report presents a comprehensive overview of our previous and current data, including that of other investigators in relation to the various factors and parameters involved in the cholelithiatic process.

Light (LM), transmission (TEM), and scanning (SEM) electron microscopy were used to characterize morphological changes induced in the gallbladder epithelium of female Syrian hamsters in response to one-month estradiol alone (E) and estradiol with medroxyprogesterone (E + MP) treatments. TEM data were correlated with the SEM observations. Compared with control (C), E- and E + MP-treated hamsters showed significant decreases in body weight, while the liver and gallbladder, and uterus weights increased. Moreover, E treatment induced some subcellular changes (microvilli, nucleus, mitochondria, RER, glycogen, abundant apical granules). The E + MP treatment appeared to exacerbate these similar changes and, in addition, induced apical excrescences and cell shedding. Both E and E + MP gallbladders showed luminal micelles, cellular debris and crystalliths associated with mucus. Simultaneously, an increased acidification of the mucoid content of the apical granules was noticed.

Glycosylation heterogeneity in recombinant human tissue kallikrein (r-HuTK) produced by Chinese hamster ovary (CHO) cells from microcarrier culture and from a serum-free suspension cell recycle process has been compared. Significant differences in the degree of sialylation were observed in glycoform distribution and oligosaccharide heterogeneity. High-performance liquid chromatography with a pellicular anion-exchange column under low pH eluant conditions was used to characterize the number and types of N-linked complex type oligosaccharides present. The oligosaccharides were released by N-glycanase and, after reduction, were resolved into a number of peaks containing one, two, three, and four sialic acids with an additional subfractionation based on the nature of the antennary structure. The microcarrier process resulted in a reduced amount of sialylated oligosaccharide species as compared to the suspension cell process. Removal of sialic acid followed by chromatography of the asialooligosaccharides under high pH anion-exchange conditions indicated that the same antennary structures were present but in slightly different relative amounts. The oligosaccharide profiles are indicative of a highly complex array of microheterogeneity present, encompassing mono-, di-, tri-, and tetrasialylated complex type oligosaccharides.

This report is a cytometric study of the female Syrian hamster gallbladder epithelium following 1-, 2-, and 3-month administration of female sex steroids. Nulliparous, multiparous, young, old and pregnant hamsters were used in this study. A 1 month treatment with estrogen alone significantly increases the nuclear volume of the gallbladder epithelial cells, while E + P treatment significantly affects the nuclear volume only after a 2 month treatment. On the other hand, E + P and P treatments significantly increase the cell volumes as compared to the E-treated groups, this effect is most striking following the 1 month period. Prolonged sex steroid treatment (2 and 3 month) does not appear to influence the gallbladder epithelial cell and nuclear volumes as dramatically as that observed following the 1 month treatment. The nulliparous, progesterone-treated hamsters appear to have a greater cytoplasmic volume than the multiparous group and this is substantiated by the bulging apices and the luminal cellular excrescences observed with scanning and transmission electron microscopy. These observations are similar to those reported in ovariectomized hamsters (Gilloteaux et al., 1992). Further, the gallbladder epithelial cells and nuclei of the older female hamsters demonstrate an accentuated response to a 1 month sex steroid treatment as compared to the younger hamsters for the same treatment duration. These results enable us to hypothesize that changes induced by a short term sex steroid treatment participate in the gallstone nucleation process, while longer duration of the treatments contribute to progressive enlargement and accumulation of gallbladder calculi.

All the intact female Syrian hamsters treated with medroxyprogesterone (MP) for a one-month period, without dietary manipulation, display gallbladder surface epithelial changes, and intraluminal deposits. These changes include excrescences in various stages, bulging, and extrusion of material from the epithelial cells. The most striking scanning electron microscopic observations are the dramatic events, comparable to apocrine-like secretory events observed in another related study using oophorectomized hamsters. Since the hamster gallbladder does not possess mucous goblet cells, it appears that this phenomenon could be a response to the MP treatment, thus providing a larger amount of mucous product than usual with cellular material, in addition to the possible alteration in the quality of the bile following this treatment. As a result of MP treatment, intraluminal deposits were also confirmed by using light and transmission electron microscopy. In control hamsters these events were not observed, however, small blebs outlining surface epithelial cells are seen. The results in this report complement the previous studies using the male and oophorectomized Syrian hamster model subjected to similar experimental conditions.

The effect of cloned gene copy number on growth and product formation has been studied in sufficient detail using a Chinese hamster ovary (CHO) cell line producing recombinant hepatitis B surface antigen (HbsAg). Batch culture experiments were carried out in T flasks in order to characterize cell growth and HbsAg secretion in various clones carrying different numbers of HbsAg gene copies integrated into CHO cell chromosomes. Specific growth rates were found to decrease with increasing gene copy number. Secreted HbsAg concentration and specific HbsAg secretion rates were found to increase with increase in gene copy number. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (dhfr) content using a flow cytometric assay. The mRNA levels quantitated using Northern hybridization followed by autoradiography and densitometry also gave the same trends. The flow cytometry experiments show that while parental cells were quite homogeneous with respect to intracellular dhfr content, the amplified clones exhibit a great deal of heterogeneity in dhfr content. Pulse-chase experiments show that the efficiency of HbsAg secretion (defined here as the fraction of initially labeled HbsAg that is secreted into the extracellular medium at the end of a 23.5-h chase) decreases and also that the intracellular HbsAg degradation increases with increasing gene copy number.