Determination of selected sulfonamide antibiotics and trimethoprim in manure by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry.

A method for the determination of sulfonamides and trimethoprim in the complex matrix liquid manure has been developed using reversed-phase liquid chromatography and atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. A comparison was made between electrospray and atmospheric pressure chemical ionization. APCI proved to be more robust and less sensitive to matrix effects.

Distribution and regulation of telencephalic aromatase expression in the zebra finch revealed with a specific antibody.

In songbirds, aromatase (estrogen synthase) activity and mRNA are readily detectable in the brain. This neural aromatization presumably provides estrogen to steroid-sensitive targets via autocrine, paracrine, and synaptic mechanisms. The location of immunoreactive protein, however, has been difficult to describe completely, particularly in distal dendrites, axons, and terminals of the forebrain.

Placental failure in mice lacking the mammalian homolog of glial cells missing, GCMa.

The GCM family of transcription factors consists of Drosophila melanogaster GCM, an important regulator of gliogenesis in the fly, and its two mammalian homologs, GCMa and GCMb. To clarify the function of these mammalian homologs, we deleted GCMa in mice. Genetic ablation of murine GCMa (mGCMa) is embryonic lethal, with mice dying between 9.

Protein stability and domain topology determine the transcriptional activity of the mammalian glial cells missing homolog, GCMb.

The glial cells missing (GCM) family of transcription factors consists of Drosophila GCM and the mammalian proteins GCMa and GCMb. They are expressed in a highly restricted manner during development and are known or assumed to be important regulators of developmental fate decisions. As the biochemical properties of GCMb have not been studied so far, we have undertaken a detailed structure-function analysis of the mouse GCMb (mGCMb) protein.

A comparative analysis of plant mitochondrial small subunit ribosomal RNA sequences.

The small subunit of the ribosomal RNA has long been used as a tool in determining phylogenetic relationships. This project explored a region of the mitochondrial small subunit ribosomal RNA (MSrRNA) that is found only in the plant mitochondrial genome referred to as Variable Region 7 (V7). The V7 region of cauliflower and radish, both members of the Brassicaceae family, was amplified with the polymerase chain reaction, cloned into the expression vector PBSSK +, and sequenced.

RNA ligands to human nerve growth factor.

High affinity RNA ligands to human nerve growth factor (NGF) were selected from pools of random RNA using SELEX [Tuerk, C. and Gold, L. (1990) Science, 249, 505-510].

Comprehensive chemical modification interference and nucleotide substitution analysis of an RNA pseudoknot inhibitor to HIV-1 reverse transcriptase.

We had previously used in vitro RNA selection techniques to describe a consensus RNA pseudoknot that binds and inhibits HIV-1 reverse transcriptase (HIV-RT). In this work we constructed variants of this consensus pseudoknot in order to evaluate the contributions of individual nucleotide identities and secondary structure to affinity for HIV-RT. We have also used chemical modification of ligand RNAs to corroborate the predicted structure of the pseudoknot, to discover which modifiable groups are protected from chemical attack when bound to HIV-RT, and to find which modifications interfere with binding to HIV-RT.

Characterization of an in vitro-selected RNA ligand to the HIV-1 Rev protein.

A small RNA ligand with high affinity for the HIV-1 Rev protein, generated by the SELEX in vitro evolution method, was used in a series of chemical modification studies to aid in determining the secondary structure of the ligand, to detect which modifications interfere with the binding of the ligand to Rev, and to find those modifiable groups that are protected from attack when bound to the Rev protein. This SELEX RNA ligand, like the high-affinity binding site of the Rev-responsive element, seems to bind the Rev protein within or along the major groove. There are two major regions of the RNA that interact with the Rev protein, and these regions appear to be close in space.

In vitro evolution of functional nucleic acids: high-affinity RNA ligands of HIV-1 proteins.

SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a protocol for isolating, from a pool of variant nucleic acid sequences, high-affinity ligands to a target protein [Tuerk and Gold, Science 249 (1990) 505-510]. This procedure involves cycles of affinity selection by a target molecule from a heterogeneous population of nucleic acids, replication of the bound species (the ligands), and in vitro transcription to generate an enriched pool of RNA. We have used the SELEX procedure to obtain high-affinity RNA ligands against the reverse transcriptase and the Rev and Tat proteins of human immunodeficiency virus 1 (HIV-1).

ESWL in situ or ureteroscopy for ureteric stones?

As documented by follow-up data on ureteric stones in 1259 ureteric units treated, ESWL in situ on advanced lithotriptors with stone location by ultrasonography and fluoroscopy was successful without any retrograde ureteric manipulation in 98% of stones in the upper, 71% in the iliac, and 84% in the distal ureter; 85% of the units were stone-free within 3 months: ancillary measures were needed in 11% and the stone-free state was reached after a median of 39 days. The results obtained with treatment after manipulation of the stone from the upper and mid-ureter by retrograde instrumentation were similar, but ancillary measures were needed in 20% of cases. Endoscopic management with rod-lens ureteroscopes was highly efficient in the distal and mid-ureter, but involved a complication rate of about 11% and required general anaesthesia.

Selection of high affinity RNA ligands to the bacteriophage R17 coat protein.

RNA ligands with high affinity for the bacteriophage R17 coat protein were isolated from a pool of random RNA molecules using SELEX. Of the 38 ligands isolated, 36 were found to contain a hairpin very similar to the naturally occurring coat protein binding site in the R17 genome. The common features of these 36 sequences provide a consensus binding site and predict components of a hairpin that promote favorable interaction with the coat protein.

Extracorporeal shock wave lithotripsy retreatment ("stir-up") promotes discharge of persistent caliceal stone fragments after primary extracorporeal shock wave lithotripsy.

A prospective randomized study was performed to compare the results of piezoelectric extracorporeal shock wave lithotripsy (ESWL) retreatment versus surveillance only in 50 patients with persistent caliceal stone fragments after primary ESWL for renal calculi. After a 3-month followup significant decreases in residual debris were observed in the retreated group, while changes in the control group were negligible. Considering the low morbidity of outpatient ESWL with a pain-free, second generation lithotriptor, ESWL retreatment of completely fragmented but persistent stone debris appears to be justified to render the kidney stone-free.

RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase.

High-affinity ligands of the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) were isolated by the SELEX procedure (systematic evolution of ligands by exponential enrichment) from RNA populations randomized at 32 positions. Analysis of these ligands revealed a pseudoknot consensus with primary sequence bias at some positions. We demonstrated that at least one of the ligands inhibits cDNA synthesis by HIV reverse transcriptase but fails to inhibit other reverse transcriptases.

SELEXION. Systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis.

Recently, novel technologies for isolation of nucleic acid molecules with specific biological activities have been reported. In each case, the enrichment process involves repeated rounds of selection from complex mixtures of nucleic acid sequences, followed by polymerase chain reaction (PCR) amplification of ligand sequences that function in the desired manner. Particular variations in experimental conditions can dramatically alter the outcome of these processes.

Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.

High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized.

Autogenous translational operator recognized by bacteriophage T4 DNA polymerase.

The synthesis of the DNA polymerase of bacteriophage T4 is autogenously regulated. This protein (gp43), the product of gene 43, binds to a segment of its mRNA that overlaps its ribosome binding site, and thereby blocks translation. We have determined the Kd of the gp43-operator interaction to be 1.

Identification of a T4 gene required for bacteriophage mRNA processing.

A ribonucleolytic activity that cleaves within the Shine/Dalgarno sequences of the bacteriophage T4 motA and ORF2 mRNAs was recently described. We have identified additional sites of processing within several other ribosome binding sites, including two sites in the polycistronic frd transcript. Deletion mutants (farP) that overproduce the product of frd are defective in this mRNA processing.

DNA polymerase of bacteriophage T4 is an autogenous translational repressor.

In bacteriophage T4 the protein product of gene 43 (gp43) is a multifunctional DNA polymerase that is essential for replication of the phage genome. The protein harbors DNA-binding, deoxyribonucleotide-binding, DNA-synthesizing (polymerase) and 3'-exonucleolytic (editing) activities as well as a capacity to interact with several other T4-induced replication enzymes. In addition, the T4 gp43 is a repressor of its own synthesis in vivo.

CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes.

The mRNA of bacteriophage T4 contains a strikingly abundant intercistronic hairpin. Within the 55 kilobases of known T4 sequence, the hexanucleotide sequence CTTCGG is found 13 times in the DNA strand equivalent to mRNA sequences. In 12 of those occurrences, the sequence is flanked by inverted repeats predictive of RNA hairpins with UUCG in the loop.

A technique for draping of the infant for surgery of the head and neck.

A method of draping a young infant for head and neck surgery exposing the entire patient and still maintaining a sterile field is presented. This provides an additional aid in helping to monitor the young patient during surgery.

Single-dose ceftriaxone kinetics in liver insufficiency.

The disposition profile of ceftriaxone was studied in eight normal subjects and in 15 subjects with various degrees of chronic liver damage (alcoholic fatty liver [FL] and cirrhosis without [C] and with [CA] ascites) who received bolus injections of ceftriaxone, 1 gm iv. Plasma protein binding fell in all. As a result, mean free fraction in plasma rose between 140% (FL) and 320% (CA).