Fresh and cryopreserved amniotic membrane secrete the trefoil factor family peptide 3 that is well known to promote wound healing

Amniotic membrane (AM) is often used for the treatment of ocular surface ulcerations and other corneal defects. Trefoil factor family (TFF) peptide 3 is produced by conjunctival goblet cells, participates in tear film physiology and has also been shown to be involved in ocular surface restitution after corneal injury. In the present study, we questioned whether AM also might be a source of TFF3 and if yes whether the secretion rate of TFF3 is changed by proinflammatory cytokines or by cryoconservation of AM. By means of RT-PCR, the mRNA expression of all three known TFF peptides could be detected in AM. Immunohistochemistry on paraffin-embedded sections localized TFF3 protein and also TFF2 in AM cells and Western blot analysis revealed TFF3 protein in AM. Stimulation experiments with proinflammatory cytokines and subsequent TFF3 ELISA measurements revealed that the secretion rate of fresh or cryoconserved AM was not significantly changed. The results indicate that TFF peptides are produced by AM. TFF3 may contribute to ocular surface wound healing after AM transplantation, but its production by AM is not further inducible by proinflammatory stimuli. Cryopreservation has no effect on the secretion rate of TFF3 supporting the use of cryopreserved AM for transplantation.     

Germline PTEN promoter mutations and deletions in Cowden/Bannayan-Riley-Ruvalcaba syndrome result in aberrant PTEN protein and dysregulation of the phosphoinositol-3-kinase/Akt pathway

Germline intragenic mutations in PTEN are associated with 80% of patients with Cowden syndrome (CS) and 60% of patients with Bannayan-Riley-Ruvalcaba syndrome (BRRS). The underlying genetic causes remain to be determined in a considerable proportion of classic CS and BRRS without a polymerase chain reaction (PCR)-detectable PTEN mutation. We hypothesized that gross gene deletions and mutations in the PTEN promoter might alternatively account for a subset of apparently mutation-negative patients with CS and BRRS. Using real time and multiplex PCR techniques, we identified three germline hemizygous PTEN deletions in 122 apparently mutation-negative patients with classic CS (N=95) or BRRS (N=27). Fine mapping suggested that one deletion encompassed the whole gene and the other two included exon 1 and encompassed exons 1-5 of PTEN, respectively. Two patients with the deletion were diagnosed with BRRS, and one patient with the deletion was diagnosed with BRRS/CS overlap (features of both). Thus 3 (11%) of 27 patients with BRRS or BRRS/CS-overlap had PTEN deletions. Analysis of the PTEN promoter revealed nine cases (7.4%) harboring heterozygous germline mutations. All nine had classic CS, representing almost 10% of all subjects with CS. Eight had breast cancers and/or benign breast tumors but, otherwise, oligo-organ involvement. PTEN protein analysis, from one deletion-positive and five PTEN-promoter-mutation-positive samples, revealed a 50% reduction in protein and multiple bands of immunoreactive protein, respectively. In contrast, control samples showed only the expected band. Further, an elevated level of phosphorylated Akt was detected in the five promoter-mutation-positive samples, compared with controls, indicating an absence of or marked reduction in functional PTEN. These data suggest that patients with BRRS and CS without PCR-detected intragenic PTEN mutations be offered clinical deletion analysis and promoter-mutation analysis, respectively.     

Targeting pancreatic cancer with a G-quadruplex ligand

The integrity of telomeres in most cancer cells is maintained by the action of the telomerase enzyme complex, which catalyzes the synthesis of telomeric DNA repeats in order to replace those lost during replication. Telomerase is especially up-regulated in metastatic cancer and is thus emerging as a major therapeutic target. One approach to telomerase inhibition involves the sequestration of the single-stranded 3' ends of telomeric DNA into higher-order quadruplex structures. We have recently shown that tetra-substituted naphthalene diimide compounds are potent quadruplex-stabilizing molecules with telomerase inhibitory activity in cells. We show here that one such compound, BMSG-SH-3, which has been optimized by computer modeling, has significant in vivo antitumor activity against a model for pancreatic cancer, a cancer that is especially resistant to current therapies. A large reduction in telomerase activity in treated tumors was observed and the naphthalene diimide compound was found to be selectively localized in the treated tumors. We find that the expression of the therapeutically important chaperone protein HSP90, a regulator of telomerase is also reduced in vivo by BMSG-SH-3 treatment. The compound is a potent stabilizer of two G-quadruplex sequences found in the promoter region of the HSP90 gene, as well as a G-quadruplex from human telomeric DNA. It is proposed that the simultaneous targeting of these quadruplexes may be an effective anti-tumor strategy.     

Blood-based protein biomarkers for diagnosis and classification of neurodegenerative diseases: current progress and clinical potential

Biomarker research is a rapidly advancing field in medicine. Recent advances in genomic, genetic, epigenetic, neuroscientific, proteomic, and metabolomic knowledge and technologies have opened the way to thriving research. In the most general sense, a biomarker refers to any useful characteristic that can be measured and used as an indicator of a normal biologic process, a pathogenic process, or a pharmacologic response to a therapeutic agent. Despite the extensive resources concentrated on this area, there are very few biomarkers currently available that qualify and are satisfactorily validated for mental disorders, and there is still a major lack of biomarkers for typifying neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. This article provides an overview of this field of research and focuses on recent advances in biomarker research in Alzheimer's disease and Parkinson's disease.     

Indirect estimation of biomass by rapid ribonucleic acid determination

Summary Microbial ribonucleic acid (RNA) was estimated by determinations of nucleotides formed by complete hydrolysis. The steps involved in this procedure comprise the alkaline hydrolysis of RNA (5 N NaOH; 100°C; 5 min) to a mixture of 2-and 3-mononucleotides and successive separation and quantification by means of high performance liquid chromatography (HPLC). Since guanylic acid (GMP) shows adequate stability during RNA degradation and may be separated well and rapidly from other compunds, it is used as an equivalent measure of RNA. The assay is performed within 15 min having a lowest detection limit of 4 mg RNA/l. It was applied to isolated yeast cells and fermentation broths of mycelial organisms. Linear correlation to mycelial dry weight was established for unlimited growth (trophophase), while in idiophase determination of RNA may serve as a measure of active biomass.     

Value of emergency toxicological investigations in differential diagnosis of coma.

Out of 208 cases of coma of unknown aetiology referred to the poisons unit of this hospital during 1978 for emergency toxicological investigations, 108 were found to be due to self-poisoning medical conditions, mainly neurological, accounted for coma in 90 patients; the cause was not ascertained in the remaining 10 cases. More than one preparation had been ingested by 58 (54%) of the poisoned patients, although barbiturates were the drugs most commonly encountered in the severe cases. Toxicological investigations should be considered in the differential diagnosis of coma when history, physical examination, and emergency biochemical measurements yield little diagnostic information.     

Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry.

The exocyclic amine protecting groups in oligonucleotide synthesis which require 8-16 hours at 55 degrees C for deprotection in ammonia have been replaced with more labile base protecting groups (dimethylformamidine for adenine and guanine and isobutyryl for cytosine). Using these fast oligonucleotide deprotecting groups which require 2-3 hours at 55 degrees C for complete deprotection, a new set of cyanoethyl phosphoramidite ribonucleoside monomers and supports has been developed. Ribozymes and substrate RNAs which were synthesized with these phosphoramidites were assayed and were found to have full catalytic (biological) activity.     

RNA catalytic properties of the minimum (-)sTRSV sequence

We have identified an RNA catalytic domain within the sequence of the 359 base long negative-strand satellite RNA of tobacco ringspot virus. The catalytic domain contains two minimal sequences of satellite RNA, a 50-base catalytic RNA sequence, and a 14-base substrate RNA sequence. The catalytic complex of catalytic RNA/substrate RNA represents a structure not previously found in any RNA catalytic reaction described to date. The reaction is truly catalytic since the catalytic RNA has multiple substrate cleavage events and is not consumed during the course of the reaction. A linear relationship is seen between reaction rate and catalytic RNA concentration. The reaction has a Km of 0.03 microM, a kcat of 2.1/min, a temperature optimum of near 37 degrees C, and an energy of activation of 19 kcal/mol.