RNA aptamers that specifically bind to a 16S ribosomal RNA decoding region construct.

RNA-RNA recognition is a critical process in controlling many key biological events, such as translation and ribozyme functions. The recognition process governing RNA-RNA interactions can involve complementary Watson-Crick (WC) base pair binding, or can involve binding through tertiary structural interaction. Hence, it is of interest to determine which of the RNA-RNA binding events might emerge through an in vitro selection process. The A-site of the 16S rRNA decoding region was chosen as the target, both because it possesses several different RNA structural motifs, and because it is the rRNA site where codon/anticodon recognition occurs requiring recognition of both mRNA and tRNA. It is shown here that a single family of RNA molecules can be readily selected from two different sizes of RNA library. The tightest binding aptamer to the A-site 16S rRNA construct, 109.2-3, has its consensus sequences confined to a stem-loop region, which contains three nucleotides complementary to three of the four nucleotides in the stem-loop region of the A-site 16S rRNA. Point mutations on each of the three nucleotides on the stem-loop of the aptamer abolish its binding capacity. These studies suggest that the RNA aptamer 109.2-3 interacts with the simple 27 nt A-site decoding region of 16S rRNA through their respective stem-loops. The most probable mode of interaction is through complementary WC base pairing, commonly referred to as a loop-loop 'kissing' motif. High affinity binding to the other structural motifs in the decoding region were not observed.

Xenognosin sensing in virulence: is there a phenol receptor in Agrobacterium tumefaciens?

BACKGROUND: The mechanisms of signal perception and transmission in the 'two-component' autokinase transmitters/response regulators are poorly understood, especially considering the vast number of such systems now known. Virulence induction from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens represents one of the best understood systems with regard to the chemistry of the activating signal, and yet the existing data does not support a receptor-mediated perception event for the xenognostic phenols. RESULTS: Here we provide the first conclusive evidence that a specific receptor must be involved in xenognostic phenol perception, detail structural requirements of the xenognosins necessary for perception by this receptor, and develop a genetic strategy that demonstrates critical components of the phenol recognition system are not encoded on the Ti plasmid. CONCLUSIONS: Although the basic elements of the two-component system required for phenol-mediated induction of virulence gene expression are encoded on the Ti plasmid, they are dependent on the chromosomal background for even the very first stage of signal perception. This discovery suggests a curious evolutionary history, and also provides functional insight into the mechanisms of two-component signal detection and transmission in general.

Unilateral nevoid telangiectasia syndrome.

Unilateral nevoid telangiectasia syndrome can be classified into two major categories: congenital and acquired. There have been reports showing an increase in skin estrogen and progesterone receptors in patients with this syndrome. We report a case of acquired unilateral nevoid telangiectasia syndrome related to pregnancy with no estrogen and progesterone receptors. Results of more studies are needed to demonstrate the correlation of systemic estrogen levels with the presence of estrogen and progesterone receptors in the skin.

Novel synthesis and RNA-binding properties of aminoglycoside dimers conjugated via a naphthalene diimide-based intercalator.

The synthesis and RNA-binding properties of naphthalene-based diimide conjugated bis-aminoglycoside antibiotics are reported. Compared to the monomeric aminoglycoside, the conjugated ligands were observed to attain up to 35-fold enhancement in binding affinity towards a novel RNA construct that contained two 16S rRNA A-sites.

Enhanced binding of aminoglycoside dimers to a "dimerized" A-site 16S rRNA construct.

In this work, we investigated the binding of a series of dimeric aminoglycoside molecules to (i) a 27 nt A-site 16S rRNA construct, and (ii) an artificially grafted 46 nt 'dimerized' A-site 16S rRNA construct. It was observed that the dissociation constants of dimeric aminoglycosides to the dimerized A-site 16S rRNA construct can achieve up to approximately 19-fold enhancement compared to the monomeric aminoglycoside molecules.

Binding of a cyclic BIV beta-Tat peptide with its TAR RNA construct.

The ability of RNA structures to adopt diverse yet complex tertiary structures has resulted in numerous fascinating RNA-protein recognition events. It was recently reported that a close relative of the HIV Rev peptide, namely a 17 residue Tat peptide from bovine immuno-deficiency virus (BIV), is able to bind to the 28 nucleotide BIV TAR RNA construct. Here we report that by simply converting the 17 residue beta-ribbon peptide structure to a 19 residue cyclopeptide, the binding affinity (Kd) of the resulting cyclopeptide to the TAR RNA target, observed by fluorescence binding study, was enhanced approximately 5-fold.

Aminoglycoside antibiotics are able to specifically bind the 5'-untranslated region of thymidylate synthase messenger RNA.

The translational initiation codon for thymidylate synthase (TS) mRNA is located in a unique stem-loop structure which contains an internal cytosine-cytosine (CC) bubble. This stem-loop structure is thought to be important in the regulation of TS translation, which is itself an important target for anticancer drugs, such as 5-fluorouracil. Internal bubble or bulge structures are candidate receptors for the aminoglycoside antibiotics. It is shown here that aminoglycosides bind in a specific and saturable fashion with dissociation constants of approximately 1 microM to a TS mRNA site 1 construct and that the binding site for the aminoglycosides is located in the CC bubble region. In fact, the CC bubble, when grafted into other stem-loop structures, confers aminoglycoside binding on them. These studies reveal an additional binding domain for aminoglycosides and also suggest how novel anti-cancer drugs might be designed that affect TS mRNA translation rather than enzyme function.

Detection of clonal T-cell receptor gamma chain gene rearrangements by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE) in archival specimens from patients with early cutaneous T-cell lymphoma: correlation of histologic findings with PCR/DGGE.

BACKGROUND: Early stages of cutaneous T-cell lymphoma (CTCL) may be difficult to distinguish from benign inflammatory dermatoses by routine histologic examination. OBJECTIVE: Our purpose was to determine whether clonal rearrangements of the T-cell receptor (TCR) gamma gene by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE) could be detected in the early stages of CTCL and to correlate these findings with conventional histopathology. METHODS: A total of 39 specimens from 12 patients with CTCL were obtained. The slides were evaluated independently by three dermatopathologists, and categorized into three groups: nondiagnostic, suggestive of CTCL, and diagnostic of CTCL. Of the 39 specimens, 33 were tested by PCR/DGGE by means of GC-clamped primers for clonal rearrangement of the TCR gamma gene. RESULTS: The histologic evaluation of the 12 cases showed a significant variation among the three dermatopathologists. The correlation of PCR/DGGE with routine histology was as follows: Clonal TCR gamma gene rearrangements were demonstrated in 73% of the specimens nondiagnostic for CTCL, 71% of those suggestive of CTCL, and 74% of those diagnostic of CTCL. CONCLUSION: Clonal TCR gamma gene rearrangements may be detected in patients with early CTCL, even when the histologic findings are not unequivocally diagnostic. In patients with multiple biopsy specimens, identical clones were demonstrated in all rearranged samples, indicating the same neoplastic clone was present in the earliest stages of disease.

Binding of dimeric aminoglycosides to the HIV-1 rev responsive element (RRE) RNA construct.

Through a series of elegant fluorescence measurements, particularly through stopped-flow kinetic measurements, it was recently demonstrated that aminoglycoside antibiotics are able to bind to the HIV-1 Rev responsive element (RRE) RNA construct in more than a 1:1 stoichiometry (Lacourciere, K. A.; Stivers, J. T.; Marino, J. P. Biocheminstry 2000, 39, 5630). Here, we present the binding study results of dimeric neomycin ligands through fluorescence anisotropy studies, to the HIV-1 RRE RNA construct. The dimeric neomycin molecules are observed to be able to bind the HIV-1 RRE RNA construct approximately 17-fold higher when compared to the monomeric neomycin, lending evidence that there are indeed two or more neomycin binding sites within the HIV-1 RRE construct.

Cutaneous metastasis from a parotid adenocarcinoma. Report of a case with immunohistochemical findings and review of the literature.

We present a rare case of metastatic adenocarcinoma of the parotid gland to the skin. Reviewing the histologic features of the primary parotid gland and comparing the microscopic sections and immunohistochemical studies, we concluded the skin tumor to be metastases from the parotid adenocarcinoma. By histologic examination alone, it is difficult to distinguish an eccrine sweat gland carcinoma from a metastatic carcinoma of the salivary gland. Immunohistochemical analysis may not be conclusive. Therefore, clinical history and clinicopathologic correlation are essential in arriving at an accurate diagnosis in these cases.

A comparison of frozen and paraffin sections in dermatofibrosarcoma protuberans.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a tumor with a high local reoccurrence rate. Mohs micrographic surgery offers the highest cure rate. However, differentiating minimal residual tumor from normal skin can be difficult during Mohs surgery. OBJECTIVE: To clarify the problem of determining when a tumor-free plane had been achieved during Mohs surgery for a DFSP. METHODS: In two patients with DFSPs, we compared frozen and paraffin-embedded sections extending from tumor to normal skin, using both H&E and CD34 stains. RESULTS: On frozen, but not paraffin-embedded, sections scattered dermal spindle cells were seen in normal skin. CONCLUSIONS: Scattered dermal spindle cells in the dermis of normal skin make it difficult to differentiate minimal residual tumor from normal dermis during Mohs surgery. A biopsy of normal skin can be useful as a control in this setting.

Identification of mutations in the transglutaminase 1 gene in lamellar ichthyosis.

Lamellar ichthyosis (LI) is an autosomal recessive disorder of cornification. Mutations in the transglutaminase 1 gene (TGM1) have been identified in several families with this disorder. We analyzed two unrelated families with offspring affected with LI. Family 1 included affected monozygotic twins, in which a homozygous G-to-T transversion was identified in exon 6 at amino acid residue R315L. This mutation was also identified in the unaffected mother. In family 2, which consisted of one affected infant, a T-to-G transversion in exon 8 resulted in a change of phenylalanine to valine, F400V, and a C-to-T transition in exon 4 resulted in a change of proline to leucine, P248L. In this family, the mutation F400V was found in the unaffected father, and the mutation P248L was identified in the unaffected mother. These findings extend the growing body of literature documenting mutations in the TGM1 gene as the molecular basis of certain cases of lamellar ichthyosis.