Irreversible thermoinactivation of ribonuclease‐A by soft‐hydrothermal processing

Ribonuclease (RNase), which often represents molecular biological contamination, is a thermostable enzyme. When RNase is heated at 121°C by autoclave sterilization for 20 min, it does not lose its activity. However, the nature of the molecular events by which the irreversible denaturation occurs remains unknown. The purpose of this study was to elucidate the molecular mechanisms of irreversible thermal denaturation of RNase A and to develop an advanced sterilization method using soft‐hydrothermal processing, which has the advantages of improved safety and cost‐efficiency. The enzymatic activity of RNase was measured using polyacrylamide gel electrophoresis with torula yeast RNA. We evaluated the temperature and time course of irreversible thermoinactivation of RNase by normal autoclaving, hot‐air sterilization, and soft‐hydrothermal processing that had been controlled to the desired steam saturation ratio. The results indicated that RNase A was deactivated by autoclave sterilization (121°C, 20 min) immediately after treatment, but was reactivated over time. Hot‐air sterilization (180°C, atmospheric pressure, 60 min) produced results similar to that of autoclave sterilization. In contrast, RNase A was irreversibly thermoinactivated by soft‐hydrothermal processing (110°C, 20 min) at 100% steam saturation ratio. We also determined that the mechanism of irreversible thermoinactivation of RNase A involved hydrolysis and deamidation under this condition at a steam saturation ratio of more than 100%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

PAPP‐A: a new anti‐aging target?

This article focuses on the role of PAPP‐A in mammalian aging. It introduces PAPP‐A and a little of its history, briefly discusses the function of PAPP‐A in the insulin‐like growth factor (IGF) system and the regulators of PAPP‐A expression, and then reviews data concerning PAPP‐A in aging and age‐related diseases especially in regard to the PAPP‐A knockout (KO) mouse. The PAPP‐A KO mouse is a valuable new model to test hypotheses concerning the control of the tissue availability of IGF, independent from systemic levels, on healthspan as well as lifespan.     

Identification and Biological Activities of Long‐Chain Peptaibols Produced by a Marine‐Derived Strain of Trichoderma longibrachiatum

Six long‐chain peptaibols, 1  –  6 , were identified from agar cultures of a marine‐derived Trichoderma longibrachiatum Rifai strain (MMS151) isolated from blue mussels. The structure elucidation was carried out using electrospray ionization ion trap mass spectrometry (ESI‐IT‐MS) and GC/EI‐MS. The long‐chain peptaibols exhibited the general building scheme Ac‐Aib‐Ala‐Aib‐Ala‐Aib‐XXX‐Gln‐Aib‐Vxx‐Aib‐Gly‐XXX‐Aib‐Pro‐Vxx‐Aib‐XXX‐Gln‐Gln‐Pheol and were similar or identical to recurrent 20‐residue peptaibols produced by Trichoderma spp. Three new sequences were identified and were called longibrachins A‐0, A‐II‐a, and A‐IV‐b. The isolated peptaibols were assayed for cytotoxic, antibacterial, and antifungal activities, and acute toxicity on Dipteran larvae.     

Human insulin A‐chain peptide analog(s) with in vitro biological activity

In a previous study, we showed that a synthetic human insulin 1‐chain analog, named analog ( 3 ) was capable of mimicking in vitro effects of native insulin, including stimulation of cell proliferation, glucose uptake and glycogen synthesis. Here, we have synthesized three new analogs ( 6, 9, 12 ) of the human A‐chain, bearing or not their N‐ or C‐terminal residue, to determine the structural features which are responsible for their biological properties. In vitro experiments clearly demonstrated that the N‐terminal part of the peptides is required for the biological activity of the molecules, suggesting its crucial role in the mechanism underlying the cellular effect. Our findings may help to better understand the mechanism of interaction between insulin and its receptor. In addition, the present data demonstrate that some mini‐insulin derived from the A‐chain can exert similar effects as native insulin. These small peptides may offer specific advantages over insulin in the definition of new strategies for diabetes treatment. Copyright © 2009 John Wiley & Sons, Ltd. This article was published online on 17 July 2009. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 4 August 2009.     

Genetic and pharmacological inactivation of the adenosine A2A receptor attenuates 3-nitropropionic acid-induced striatal damage

Adenosine A2A receptor (A2AR) antagonism attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration and quinolinic acid-induced excitotoxicity in the neostriatum. As A2ARs are enriched in striatum, we investigated the effect of genetic and pharmacological A2A inactivation on striatal damage produced by the mitochondrial complex II inhibitor 3-nitropriopionic acid (3-NP). 3-NP was administered to A2AR knockout (KO) and wild-type (WT) littermate mice over 5 days. Bilateral striatal lesions were analyzed from serial brain tissue sections. Whereas all of the 3-NP-treated WT mice (C57BL/6 genetic background) had bilateral striatal lesions, only one of eight of the 3-NP-treated A2AR KO mice had detectable striatal lesions. Similar attenuation of 3-NP-induced striatal damage was observed in A2AR KO mice in a 129-Steel background. In addition, the effect of pharmacological antagonism on 3-NP-induced striatal neurotoxicity was tested by pre-treatment of C57Bl/6 mice with the A2AR antagonist 8-(3-chlorostyryl) caffeine (CSC). Although bilateral striatal lesions were observed in all mice treated either with 3-NP alone or 3-NP plus vehicle, there were no demonstrable striatal lesions in mice treated with CSC (5 mg/kg) plus 3-NP and in five of six mice treated with CSC (20 mg/kg) plus 3-NP. We conclude that both genetic and pharmacological inactivation of the A2AR attenuates striatal neurotoxicity produced by 3-NP. Since the clinical and neuropathological features of 3-NP-induced striatal damage resemble those observed in Huntington's disease, the results suggest that A2AR antagonism may be a potential therapeutic strategy in Huntington's disease patients.     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Role of carbohydrate moiety of bleomycin-A2 in caspase-3 activation and internucleosomal chromatin fragmentation in apoptosis of laryngeal carcinoma cells

Bleomycin (BLM), an antitumor antibiotic, is currently used during anticancer therapy. The therapeutic efficiency of BLM for the treatment of malignant tumors is related to its ability to cleave DNA. However, little is known about the biological activity of the glycannic moiety in BLM-induced cytotoxicity. In this study, cell death induced by BLM-A2 and deglycosylated BLM-A2 was studied in a laryngeal carcinoma cell line (HEp-2 cells). Our results indicate that HEp-2 cells showed morphological and biochemical changes associated with apoptosis in the presence of low concentrations of BLM-A2. In contrast, the same changes, except activation of caspase-3 and internucleosomal digestion of genomic DNA, were observed when cells were exposed to high concentrations of deglycosylated BLM-A2. These observations indicate that the glycannic moiety from the bleomycin molecule has important biological effects on the cytotoxicity of the drug.     

Hepatic stellate cell (vitamin A-storing cell) and its relative--past, present and future

HSCs (hepatic stellate cells) (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells) exist in the space between parenchymal cells and liver sinusoidal endothelial cells of the hepatic lobule and store 50-80% of vitamin A in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. In physiological conditions, these cells play pivotal roles in the regulation of vitamin A homoeostasis. In pathological conditions, such as hepatic fibrosis or liver cirrhosis, HSCs lose vitamin A and synthesize a large amount of extracellular matrix components including collagen, proteoglycan, glycosaminoglycan and adhesive glycoproteins. Morphology of these cells also changes from the star-shaped SCs (stellate cells) to that of fibroblasts or myofibroblasts. The hepatic SCs are now considered to be targets of therapy of hepatic fibrosis or liver cirrhosis. HSCs are activated by adhering to the parenchymal cells and lose stored vitamin A during hepatic regeneration. Vitamin A-storing cells exist in extrahepatic organs such as the pancreas, lungs, kidneys and intestines. Vitamin A-storing cells in the liver and extrahepatic organs form a cellular system. The research of the vitamin A-storing cells has developed and expanded vigorously. The past, present and future of the research of the vitamin A-storing cells (SCs) will be summarized and discussed in this review.     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.     

Isolation of Components from the Low-Sulphur Proteins of Wool by Fractional Precipitation

The two major components of the low-sulphur S-carboxymethylkerateine fraction from wool (components 7 and 8) have been isolated by methods involving fractional precipitation from n-propanol/water and from 4 M LiBr. The materials obtained in this way are similar in amino acid composition and in gel electrophoresis properties to fractions obtained by column chromatographic techniques and are of at least equal purity. The procedures are relatively quick and can be scaled up.     

Investigation of the synthetic route to pepstatin analogues by SPPS using O‐protected and O‐unprotected statine as building blocks

The synthetic route to pepstatin derivatives by a solid phase peptide synthesis using either O‐protected or O‐unprotected statine as a building block has been investigated. Statine was prepared according to a modified literature procedure, whereas protection of its 3‐hydroxyl moiety using tert‐butyldimethylsilylchloride (TBSCl) provided the novel O‐TBS‐protected statine building block. The O‐tert‐butyldimethylsilyl (TBS)‐protected statine approach provides an improved synthetic strategy for the preparation of statine‐containing peptides as demonstrated by the synthesis of the pepstatin analogue iva‐Val‐Leu‐Sta‐Ala‐Sta. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Prediction of recurrence using exfoliative cytology and melanoma‐associated antigen‐A mRNA analysis following wide excision of oral squamous cell carcinoma: short report

N. Mollaoglu, P. Metzler, J. Zenk, E. Nkenke, F. W. Neukam and J. Ries
Prediction of recurrence using exfoliative cytology and melanoma‐associated antigen‐A mRNA analysis following wide excision of oral squamous cell carcinoma: short report Background: Oral squamous cell carcinoma (OSCC) is the sixth most common cancer. The local recurrence of OSSC might result from the existence of occult cancer cells around tumour margins. Exfoliative cytology has lately gained great importance as a method for obtaining RNA samples from suspicious oral mucosal lesions in order to carry out molecular diagnosis. In addition, melanoma associated‐A antigens (MAGE‐A) are expressed in various tumours and their detection is a highly accurate sign that cancer cells are present. Objective: The prediction of a recurrence using MAGE‐A mRNA expression analysis to follow‐up OSCC cases using a newly established molecular diagnostic technique applied to cytological materials. Methods: RNA was extracted from three recurrent OSCC cases and from 20 healthy volunteers as a control group using a cytobrush. The expression of MAGE‐A3, A4, A6, A10 and A12 was investigated in these specimens using quantitative real‐time (RT‐PCR). Results: There was no expression of MAGE‐A in the specimens of normal oral mucosa. However, the expression analysis of five different MAGE‐A genes indicated a high potential for malignant change in biopsy‐proven recurrent OSCC cases. Except for MAGE‐A10, the rest of the genes were expressed in different ratios by the three recurrent cases, which had been determined on histopathology to be OSCC or carcinoma in situ. Conclusion: It is suggested that analysis of MAGE‐A expression may be used as a risk prediction method in the diagnosis of recurrence after wide excision of OSCC to enhance the accuracy of exfoliative cytology, which has limitations due to false negative and false positive results.     

Potent and Selective Monoamine Oxidase‐B Inhibitory Activity: Fluoro‐ vs. Trifluoromethyl‐4‐hydroxylated Chalcone Derivatives

For various neurodegenerative disorders like Alzheimer's and Parkinson’s diseases, selective and reversible MAO‐B inhibitors have a great therapeutic value. In our previous study, we have shown that a series of methoxylated chalcones with F functional group exhibited high binding affinity toward human monoamine oxidase‐B (hMAO‐B). In continuation of our earlier study and to extend the understanding of the structure–activity relationships, a series of five new chalcones were studied for their inhibition of hMAO. The results demonstrated that these compounds are reversible and selective hMAO‐B inhibitors with a competitive mode of inhibition. The most active compound, (2E)‐1‐(4‐hydroxyphenyl)‐3‐[4‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one, exhibited a K_i value of 0.33 ± 0.01 μm toward hMAO‐B with a selectivity index of 26.36. A molecular docking study revealed that the presence of a H‐bond network in hydroxylated chalcone with the N(5) atom of FAD is crucial for MAO‐B selectivity and potency.     

The function of classical and alternative non‐homologous end‐joining pathways in the fusion of dysfunctional telomeres

Repair of DNA double‐stranded breaks (DSBs) is crucial for the maintenance of genome stability. DSBs are repaired by either error prone non‐homologous end‐joining (NHEJ) or error‐free homologous recombination. NHEJ precedes either by a classic, Lig4‐dependent process (C‐NHEJ) or an alternative, Lig4‐independent one (A‐NHEJ). Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by removal of telomere‐binding proteins are recognized as DSBs. In this report, we studied which end‐joining pathways are required to join dysfunctional telomeres. In agreement with earlier studies, depletion of Trf2 resulted in end‐to‐end chromosome fusions mediated by the C‐NHEJ pathway. In contrast, removal of Tpp1–Pot1a/b initiated robust chromosome fusions that are mediated by A‐NHEJ. C‐NHEJ is also dispensable for the fusion of naturally shortened telomeres. Our results reveal that telomeres engage distinct DNA repair pathways depending on how they are rendered dysfunctional, and that A‐NHEJ is a major pathway to process dysfunctional telomeres.     

MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation

Enterovirus A71 (EV‐A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV‐A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV‐A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV‐A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV‐A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV‐A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID₅₀) values calculated in viral proliferations (ranged from 10^7.6 to 10^7.8); the TCID₅₀ being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV‐A71Jinan1002 amplifies more efficiently in MA104, Hep‐2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV‐A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV‐A71.