Functional gene-discovery systems based on libraries of hammerhead and hairpin ribozymes and short hairpin RNAs

Abundant information about the nucleotide sequence of the human genome has become readily available and it is now necessary to develop methods for the identification of genes that are involved in important cellular, developmental and disease-related processes. Identification methods based on the activities of hammerhead and hairpin ribozymes and of short hairpin RNAs (shRNAs), whose target specificities are coupled with loss-of-function phenotypes, have received increasing attention as possible tools for the rapid identification of key genes involved in such processes. We describe here recent advances that have been made with libraries of ribozymes and shRNAs and compare the advantages of the different types of library. The use of such libraries has already revealed new details of several important physiological phenomena.     

Preclinical Cushing's syndrome resulting from black adrenal adenoma. A case report

A 59-year-old Japanese woman, admitted for the treatment of diabetes mellitus and hypertension, was incidentally discovered to have a solid mass of 1.4 cm in diameter by CT scan with the attenuation value of 38 Hounsfield units, relatively higher for ordinary adrenal adenomas. Magnetic resonance imaging revealed no reduction of signal intensity on opposite-phase image on T1-weighted sequence. Adrenal scintigraphy imaging with 131I-adosterol did not show any uptake of the isotope in the area corresponding to both adrenals. Although she had no characteristic feature of overt Cushing's syndrome, her serum cortisol level was not suppressed after an overnight dexamethasone administration. She was diagnosed as having preclinical Cushing's syndrome. Left adrenalectomy was performed, revealing the well-circumscribed black tumor, mainly consisted of compact cell, in which cytoplasm was filled with numerous granules pigmented with dark to golden brown colors on hematoxylin-eosin staining. These findings suggested that her incidentaloma was a black adrenal adenoma. Production of steroid hormones was confirmed by immunohistochemical analysis of steroidogenic enzymes and by measurement of the tissue contents of hormones, whose levels were comparable with those in adenomas of overt Cushing's syndrome. This is the first case report of preclinical Cushing's syndrome resulting from black adrenal adenoma.     

World of small RNAs: from ribozymes to siRNA and miRNA

RNAs, besides bridging genetic information to proteins, the major determinants of bio-structures and functions, serve as active regulators of gene expression. Initiated nearly 20 years ago with ribozymes (the small RNAs with catalytic activity providing fine tuning of gene expression and function, used as molecular scissors and tools for gene discovery), an era of more complex and coordinated gene regulation by small RNAs, siRNA, and miRNA has recently started. Simple nucleotide complementarity results in highly ordered and regulated events, such as assembly of RNA and proteins, resulting in gene silencing either by mRNA degradation or suppression of translation. This article reviews our contributions to the understanding of structure, the function of small RNAs, their use in biotechnology, and the understanding of phenotypes such as apoptosis, metastasis, and differentiation.     

Age-dependent changes of elements in human trachea

To elucidate compositional changes of human trachea by aging, element contents in tracheae were determined by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. The sulfur content of the tracheae decreased gradually with aging. In regard to calcium and phosphorus, both the contents increased to about three-fold amounts in their seventies compared with those in their sixties, and decreased thereafter. The contents of calcium and phosphorus were the highest in their seventies. Therefore, it is likely that surplus calcium released from bones is deposited temporally in the trachea, and the deposits are released from the trachea at older age. Based on our results of human cartilages, there are two types in regard to calcium accumulation: The first type is that calcium accumulation occurs progressively with aging; the second one is that calcium accumulation becomes the highest in the seventies or eighties, and decreases there-after. Therefore, the trachea belongs to the second type. Furthermore, the magnesium content remained constant through the age range.     

Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity

The cellular protein retinoic acid-inducible gene I (RIG-I) senses intracellular viral infection and triggers a signal for innate antiviral responses including the production of type I IFN. RIG-I contains a domain that belongs to a DExD/H-box helicase family and exhibits an N-terminal caspase recruitment domain (CARD) homology. There are three genes encoding RIG-I-related proteins in human and mouse genomes. Melanoma differentiation associated gene 5 (MDA5), which consists of CARD and a helicase domain, functions as a positive regulator, similarly to RIG-I. Both proteins sense viral RNA with a helicase domain and transmit a signal downstream by CARD; thus, these proteins share overlapping functions. Another protein, LGP2, lacks the CARD homology and functions as a negative regulator by interfering with the recognition of viral RNA by RIG-I and MDA5. The nonstructural protein 3/4A protein of hepatitis C virus blocks the signaling by RIG-I and MDA5; however, the V protein of the Sendai virus selectively abrogates the MDA5 function. These results highlight ingenious mechanisms for initiating antiviral innate immune responses and the action of virus-encoded inhibitors.     

Double-strand RNA dependent protein kinase (PKR) is involved in the extrastriatal degeneration in Parkinson's disease and Huntington's disease

Double-strand RNA dependent protein kinase (PKR) plays an important role in control of cell death. We previously reported that activation of PKR is associated with hippocampal neuronal loss in Alzheimer's disease (AD). Recent studies have reported that Parkinson's (PD) and Huntington's (HD) disease brains displayed progressive hippocampal neuronal loss in extrastriatal degeneration. However, association between PKR and hippocampal neuronal loss in PD and HD brains is not known. In this report, brain tissues from patients with PD and HD displayed strong induction of phosphorylated-PKR (p-PKR) in hippocampal neurons. Immunoblotting analysis also demonstrated that levels of nuclear p-PKR in the hippocampus affected by these diseases were increased compared with age-matched disease controls. These results suggest that a close association exists between PKR and extrastriatal degeneration in PD and HD pathology.     

Selective cell attachment to a biomimetic polymer surface through the recognition of cell-surface tags

Reactive phosphorylcholine polymers, which can recognize biosynthetic cell-surface tags, were synthesized to control cell attachment. Human promyelocytic leukemia cells (HL-60) with unnatural carbohydrates as cell-surface tags were harvested by treatment with N-levulinoylmannosamine (ManLev). The attachment of ManLev-treated HL-60 cells to 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers with hydrazide groups was studied. HL-60 cells, which are nonadhesive, did not attach to any polymer surface without ManLev treatment. In contrast, ManLev-treated HL-60 cells attached to a poly[MPC-co-n-butyl methacrylate (BMA)-co-methacryloyl hydrazide (MH)] (PMBH) surface following 15 min of incubation. The cells that attached to the PMBH surface retained their native morphology and viability for 24 h of incubation. On the other hand, approximately half of the HL-60 cells that attached to the poly(BMA-co-MH) (PBH) surface died. These results suggest that MH units in the polymer act as anchors for cell attachment and MPC units help to preserve cell viability on a polymer surface. The coculture of ManLev-treated HL-60 and fluorescence-stained human uterine cervical cancer cells (HeLa) was carried out on polymer surfaces. ManLev-treated HL-60 cells specifically attached to the PMBH surface. In contrast, both HL-60 and HeLa cells were observed on the PBH surface. The control of cellular interactions with synthetic polymers may be useful for the future development of cell-integrated biosensors and biomedical devices.     

Efficient immobilization of enzymes on microchannel surface through His-tag and application for microreactor

We developed a simple immobilisation method for His-tagged enzymes on a microchannel surface. It facilitates immobilisation of protein molecule on microchannel surface through Ni-complex, using crude or purified protein solutions. By this method, we could immobilize proteins on microcapillary constantly. This method might be useful for further development of microreactor with reversibly immobilized enzymes.     

Gene discovery by ribozyme and siRNA libraries

Catalytic RNAs, also known as ribozymes, can be engineered to optimize their activities in the intracellular environment. The introduction of a library of active ribozymes into cells, and the subsequent screening for phenotypic changes, allows the rapid identification of gene function. For the determination of gene function, ribozyme technology complements another RNA-based tool that is based on libraries of small interfering RNAs.