In vitro and in vivo recombination-related reactions of Escherichia coli recA protein and glucosyl-hydroxymethyl-deoxycytidine DNA

Summary Recombination of T4 phage is not controlled by the host recA gene but by an analogous phage gene, uvsX. We have tested the hypothesis that recA protein is inactive in T4-infected cells because it is unable to catalyze reactions involving single stranded DNA containing glucosyl-hydroxylmethyl-deoxycytidine. We found, however, that with modified and unmodified deoxycytidine containing DNAs, uvsX protein and recA protein catalyze in vitro reactions related to DNA recombination, but in T4-infected cells recA protein fails to promote strand transfer of DNA which contains unmodified deoxycytidine.Abbreviations dC-DNA deoxycytidine containing DNA - dC-T4 T4 phage containing dC-DNA - dHMC-DNA glucosyl-hydroxymethyl-deoxycytidine containing DNA - dsDNA double stranded DNA - gp gene product - ssDNA single stranded DNA     

PLD4 Is Involved in Phagocytosis of Microglia: Expression and Localization Changes of PLD4 Are Correlated with Activation State of Microglia

Phospholipase D4 (PLD4) is a recently identified protein that is mainly expressed in the ionized calcium binding adapter molecule 1 (Iba1)-positive microglia in the early postnatal mouse cerebellar white matter. Unlike PLD1 and PLD2, PLD4 exhibits no enzymatic activity for conversion of phosphatidylcholine into choline and phosphatidic acid, and its function is completely unknown. In the present study, we examined the distribution of PLD4 in mouse cerebellar white matter during development and under pathological conditions. Immunohistochemical analysis revealed that PLD4 expression was associated with microglial activation under such two different circumstances. A primary cultured microglia and microglial cell line (MG6) showed that PLD4 was mainly present in the nucleus, except the nucleolus, and expression of PLD4 was upregulated by lipopolysaccharide (LPS) stimulation. In the analysis of phagocytosis of LPS-stimulated microglia, PLD4 was co-localized with phagosomes that contained BioParticles. Inhibition of PLD4 expression using PLD4 specific small interfering RNA (siRNA) in MG6 cells significantly reduced the ratio of phagocytotic cell numbers. These results suggest that the increased PLD4 in the activation process is involved in phagocytosis of activated microglia in the developmental stages and pathological conditions of white matter.     

Hollow spheroids in ascites of ovarian clear cell carcinoma: how are they formed and how do they behave?

N. Kato, K. Narutomi, M. Fukase and T. Motoyama Hollow spheroids in ascites of ovarian clear cell carcinoma: how are they formed and how do they behave? Objective: Although the multicellular aggregates (spheroids) in malignant ascites are usually solid throughout, they sometimes have acellular hollow spaces, especially in ascites of ovarian clear cell carcinoma. The purpose of this study is to analyse the origin and behaviour of hollow spheroids. Methods: Archival cytological and histological specimens of 32 ovarian carcinomas, including 12 clear cell carcinomas, were reviewed. HAC‐2, a clear cell carcinoma cell line, was injected into the abdominal cavity of nude mice for direct comparison of ascitic cytology and tumour histology. Spheroids that were collected from nude mice ascites were cultured in vitro to observe their behaviour. Results: Five of six clear cell carcinomas with hollow spheroids showed spherule‐like hyaluronan‐rich stroma in their tumour tissue, whereas those without hollow spheroids did not. After heterotransplantation, both ascites and tumour imprints showed small or large hollow spheroids. Hyaluronan was detected in the former but not in the latter. The abdominal tumours showed compact spherule‐like hyaluronan‐rich stroma, enlarged oedematous stroma or intermediate stroma. In both size and hyaluronan status, small and large hollow spheroids were approximately comparable to spherule‐like hyaluronan‐rich stroma and oedematous stroma, respectively. During culture in vitro, hollow spheroids were maintained as hollow spheroids in suspension, and produced daughter hollow spheroids. Conclusions: The hollow space in the spheroids originates from spherule‐like hyaluronan‐rich stroma, where water trapping by hyaluronan causes enlargement of the space. The matrix within the hollow space serves as a scaffold that regulates cell polarity and matrix production.     

The Chemical Constitution of Rubrofusarin,a Pigment from Fusarium graminearum

The zinc dust distillates of rubrofusarin, nor-rubrofusarin, and methylxanthones (1-, 2-, 3-, and 4-methylxanthones and l-methyl-3,6,8-trihydroxyxanthone) have been investigated.

On zinc dust distillation, rubrofusarin and nor-rubrofusarin afforded naphthalene and anthracene in low yield, but did not give methylxanthene expected from the formerly proposed structure of methylxanthone.

Methylxanthones are considered to give the corresponding methylxanthenes in the distillates; and it is true for 2-, 3-, and 4-methylxanthones. However, in the 1-methyl derivatives the main product was not 1-methylxanthene but anthracene. It has been found also that on that condition all methylxanthones examined did not give naphthalene that was a main zinc dust distillate in rubrofusarin.

Thus, we conclude that it is appropriate to assign the structure of naphthalene derivative for rubrofusarin instead of the so far proposed methylxanthone structure.     

Drosophila evaluates and learns the nutritional value of sugars

Living organisms need to search for and ingest nutritional chemicals, and gustation plays a major role in detecting and discriminating between chemicals present in the environment. Using Drosophila as a model organism, we asked whether animals have the ability to evaluate the nutritional value of sugars. In flies, chemosensilla on the tarsi and labellum are the gustatory organs used to discriminate between edible and nonedible compounds [1, 2]. We noticed that Drosophila do not assign nutritional values to all sweet chemicals. D-arabinose is sweet to flies, but it provides them with no nutrition. By contrast, the sugar alcohol D-sorbitol is not sensed as sweet, but flies can live on it. We performed behavioral and electrophysiological measurements to confirm these gustatory and feeding responses. We found that Drosophila can learn the nutritional value of nonsweet D-sorbitol when it is associated with an odor cue. The learning process involved the synapsin molecule, suggesting that a neuronal mechanism is involved. We propose that Drosophila uses neural machinery to detect, evaluate, and learn the nutritional value of foods after ingestion.     

A Prognostic Score for Patients with Intermediate-Stage Hepatocellular Carcinoma Treated with Transarterial Chemoembolization

Background

Intermediate-stage hepatocellular carcinoma (HCC), defined according to the Barcelona Clinic Liver Cancer (BCLC) staging system, is a heterogeneous condition with variable clinical benefits from transarterial chemoembolization (TACE). This study aimed to develop a simple validated prognostic score based on the predictive factors for survival in patients with intermediate-stage HCC treated with TACE.

Methods

Three-hundred and fifty patients with intermediate-stage HCC undergoing initial TACE at Chiba University Hospital (training cohort; n = 187) and two affiliated hospitals (validation cohort; n = 163) were included. Following variables were entered into univariate and multivariate Cox regression models to develop a points-based clinical scoring system: gender, age, etiology, pretreatment, Child–Pugh score, aspartate aminotransferase, creatinine, C-reactive protein, alfa-fetoprotein, size of the largest lesion, and number and location of lesions.

Results

The number of lesions and the Child–Pugh score were identified as independent prognostic factors in the training cohort. The development of a 0–7-point prognostic score, named the Chiba HCC in intermediate-stage prognostic (CHIP) score, was based on the sum of three subscale scores (Child–Pugh score = 0, 1, 2, or 3, respectively, number of lesions = 0, 2, or 3, respectively, HCV-RNA positivity = 0 or 1, respectively). The generated scores were then differentiated into five groups (0–2 points, 3 points, 4 points, 5 points, and 6–7 points) by the median survival time (65.2, 29.2, 24.3, 13.1, and 8.4 months, respectively; p < 0.0001). These results were confirmed in the external validation cohort (p < 0.0001).

Conclusions

The CHIP score is easy-to-use and may assist in finding an appropriate treatment strategy for intermediate-stage HCC.     

Cyclodextrin, a novel therapeutic tool for suppressing amyloidogenic transthyretin misfolding in transthyretin-related amyloidosis

TTR (transthyretin), a β-sheet-rich protein, is the precursor protein of familial amyloidotic polyneuropathy and senile systemic amyloidosis. Although it has been widely accepted that protein misfolding of the monomeric form of TTR is a rate-limiting step for amyloid formation, no effective therapy targeting this misfolding step is available. In the present study, we focused on CyDs (cyclodextrins), cyclic oligosaccharides composed of glucose units, and reported the inhibitory effect of CyDs on TTR amyloid formation. Of various branched β-CyDs, GUG-β-CyD [6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-β-CyD] showed potent inhibition of TTR amyloid formation. Far-UV CD spectra analysis showed that GUG-β-CyD reduced the conformational change of TTR in the process of amyloid formation. In addition, tryptophan fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized the TTR conformation via interaction with the hydrophobic amino acids of TTR, especially tryptophan. Moreover, GUG-β-CyD exerted its inhibitory effect by reducing TTR deposition in transgenic rats possessing a human variant TTR gene in vivo. Collectively, these results indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation.