Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH-activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure-function relationships, pH-rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA.

Size and shape separation of gold nanoparticles with preparative gel electrophoresis.

We employed agarose gel preparative electrophoresis to separate gold nanoparticles based on size, shape, and charge. The separating technique was first demonstrated by size separation of 5 nm, 15 nm, and 20 nm spherical gold nanoclusters; and further evidenced through the purification of crude 15 +/- 2.7 nm nanoclusters to nanoclusters that were 15 +/- 0.4 nm. The ability to separate gold nanoparticles by shape was also shown by the purification of a mixture of gold spheres, plates, and long rods.

Optical detection of thymine dinucleoside monophosphate and its cis-syn photodimer by inorganic nanoparticles.

The Watson-Crick DNA double helix is an averaged ideal of multitudinous natural sequence-directed local structural deviations. By effectively derailing normal cellular physiological processes, damaged bases can induce noncanonical irregularities in the local structure of DNA if not efficiently repaired. Pyrimidine bases, especially thymine, are prone to dimerization when exposed to ultraviolet light. A [2 + 2] photocyclo-addition between adjacent thymine bases predominantly produces the cis-syn photodimer. These lesions, implicated in skin cancer, bend DNA by approximately 30 degrees due to their structural and conformational changes. Such changes in molecular properties can be detected by differential quenching of CdS nanoparticle luminescence and by surface-enhanced Raman scattering spectroscopy on metal nanoparticle substrates.

Preferential end-to-end assembly of gold nanorods by biotin-streptavidin connectors.

A major challenge in nanoscience and nanotechnology is the rational assembly of nanoscale objects. Here we report that gold nanorods, aspect ratio 18, can be functionalized with a biotin disulfide, and subsequent addition of streptavidin links the rods together in an end-to-end manner much more often than expected.

Recognition of hypermethylated triplet repeats in vitro by cationic nanoparticles.

Genomic DNA contains many higher-order structural deviations from the Watson-Crick global average. The massive expansion and hypermethylation of the duplex triplet repeat (CCG)(n)(CGG)(n) has characteristic higher-order structures that are associated with the fragile X syndrome. We have used luminescent mineral nanoparticles of protein-sized cadmium sulfide in optical assays to detect anomalous DNA structures. The photoluminescence of these particles is sensitive to the presence and nature of adsorbates. We previously found that our nanoparticles bind the fragile X repeat well but do not bind to normal double-helical DNA. In this study, we have determined that these particles are also able to detect the hypermethylated forms of these triplet repeats. Therefore, these nanoparticles may form the basis for future optical assays of higher-order DNA structures, especially those associated with human disease.

The effect of different promoter-sequences on transient expression of gus reporter gene in cultured barley (Hordeum vulgare L.) cells.

The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcohol dehydrogenase (Adh1) intron1 or maize shrunken locus (sh1) intronl along with maize Adh1 and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli ß-glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoterintron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cultured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells.

Advantages of achiral h.p.l.c. as a preparative step for chiral analysis in biological samples and its use in toxicokinetic studies.

1. Achiral reverse-phase h.p.l.c. with semi-automated post-column fraction collection and solid-phase sample reconcentration, has been applied as the purification procedure during the enantiomeric quantification of two widely differing experimental drugs; an HMG-CoA reductase inhibitor (I) and an alpha 2-adrenoceptor antagonist (II). 2. The robust and specific achiral methodologies were available prior to the need for chiral analyses and recovery of drug from the fractions provided clean samples from a variety of biological matrices, without the need to develop compatible achiral/chiral mobile phases. 3. Compared with direct chiral chromatography of plasma extracts, this approach decreased the potential for metabolites and endogenous components to interfere or impair the performance of the chiral stationary phase. 4. The availability of quantitative data from achiral analysis of samples negated the need for internal standardization of the chiral analyses, helped confirm assay specificity and provided potential to determine enantiomeric ratios where only one isomer could be accurately measured. 5. Routine enantiomeric analyses were successfully carried out on samples taken from animals dosed orally with the racemic drugs, providing important data on the possible levels of exposure to individual enantiomers during toxicity testing.

Effects of fish oil and endorphins on the cold pressor test in hypertension.

The effects of fish oil and naloxone on blood pressure, catecholamines, and endorphins during the cold pressor test were evaluated in a randomized, double-blind, placebo-controlled, two-way crossover trial of normotensive and medication-free hypertensive men (n = 13 each). Subjects were given 5 gm omega-3 fatty acids per day or placebo for 30 days with a 1-month washout between interventions. The cold pressor test (hand in ice water for 5 minutes) was done at the end of the treatment periods. Intravenous naloxone (10 mg) or placebo was given before the cold pressor test. Fish oil-treated, normotensive, or hypertensive groups had similar changes in blood pressure, plasma catecholamine levels, and beta-endorphins during the cold pressor test, but naloxone treatment was associated with fivefold and tenfold increases in plasma epinephrine and cortisol levels, respectively. Naloxone may modulate sympathomedullary discharge through blockade of endorphin activity. It is unlikely that endorphins are involved in the blood pressure increase during the cold pressor test or that fish oil alters this response.

Regeneration of Freezing-Tolerant Spring Wheat (Triticum aestivum L.) Plants from Cryoselected Callus.

A cryoselection protocol has been developed that provides freezing-tolerant callus that, in turn, can regenerate plants with enhanced cold hardiness. Tolerant calli were selected from spring wheat (Triticum aestivum L.) callus by immersion in liquid nitrogen without addition of cryoprotectants. Less than 15% of the calli survived the initial challenge, whereas 30 to 40% of previously selected calli survived subsequent exposure. Seed progeny from five of 11 regenerant (R2) lines tested exhibited significantly enhanced tolerance to freezing at -12 degrees C. Thus, cryoselection appears to involve at least in part, selection for genetic rather than epigenetic variants. Analysis of one callus line indicated that cryoselection did not induce significant alterations in lipid composition, adenylate energy charge, or freezing point. An increase in the soluble sugar component was detected. Changes were also detected in the protein complement of microsomal membrane and soluble protein extracts of cryoselected callus. In all, seven unique proteins ranging from 79 to 149 kilodaltons were identified. The results demonstrate that freezing tolerant callus can be isolated from a heterogeneous population by cryoselection, and factors that contribute to hardiness at the callus level are biologically stable and can contribute to tolerance at the whole plant level.

Transient expression of chloramphenicol acetyltransferase (CAT) gene in barley cell cultures and immature embryos through microprojectile bombardment.

Transient expression of chloramphenicol acetyl transferase gene has been detected in cultured barley (Hordeum vulgare L. cv. Heartland) cells and freshly isolated immature zygotic embryos (cv. Ellice) following the introduction of the gene by microprojectile bombardment. The DNA expression vector used to introduce the CAT gene, pCaMVI1CN, is a pUC8 derivative and consisted of a CaMV35S promoter, a fragment of alcohol dehydrogenase intron1, a CAT coding region and NOS polyadenylation region. The inclusion of the Adh1 intron1 was essential for the expression of CAT activity in cultured cells as well as immature zygotic embryos. Expression of CAT activity, which was dependent upon the DNA concentration used, could be detected as early as 20 h after bombardment. The results also suggested that the recipient cells have to be in an active state of cell division in order for the introduced gene to be expressed since mature zygotic as well as somatic embryos failed to reveal any gene expression. The effect of other parameters which influence the expression of the introduced gene as well as the potential of this novel technology for cereal transformation are also discussed.

Use of plaster casts in the management of diabetic neuropathic foot ulcers.

Neuropathic foot ulceration is a major medical and economic problem among diabetic patients, and the traditional treatment involves bed rest with complete freedom from weight-bearing. We have investigated the use of walking plaster casts in the management of seven diabetic patients with long-standing, chronic plantar ulcers. Although all ulcers healed in a median time of 6 wk, this therapy was not without side effects, which are described in detail. We conclude that casting is a useful therapy for neuropathic ulcers, although several clinic visits, including cast removal and foot inspection, are necessary to avoid potential side effects caused by the casting of insensitive feet.

Absence of cardiac arrhythmias during a very-low-calorie diet with high biological quality protein.

Fourteen women who were at least 50 lb (22.7 kg) overweight entered the 15 week study which included 4 weeks of 1200 cal (5028 J) balanced diet followed by 7 weeks on a very-low-calorie diet (VLCD) and 4 weeks of refeeding. During the VLCD high biological quality protein (poultry, fish) and recommended supplements of vitamins, minerals and water were used. With the exception of week 5 and 14, 25-h Holter monitorings were done weekly. During the initial 4 weeks, 2 patients showed disturbances of cardiac rhythm and were discharged from the study. Twelve patients completed the trial without any clinically significant changes in cardiac rhythm. The 12 lead ECGs remained normal in all patients throughout the study. The average weight loss was 46 lb (20.9 kg) and the whole program was well tolerated. It is concluded that seven weeks of VLCD with high biological quality protein and recommended supplements appears to be a safe method for weight reduction for severely obese patients.

Open-heart surgery.

A randomized prospective study of the relative effectiveness of broad-spectrum versus specific antistaphylococcal antibiotic prophylaxis in open-heart surgery was done between May 1972 and June 1973. All patients undergoing open-heart surgery were assigned randomly (by hospital number) to receive either methicillin (M) or cephalothin (C) beginning the night before operation. There were 132 patients in the C group and 129 in the M group. There were no statistically significant differences in age or duration of hospitalization, cardiopulmonary bypass, urinary tract drainage, or postoperative fever. There was a significant difference in the ratio of male to total patients (0.67 C, 0.52 M, P less than .02) and duration of operation in hours (4.27 C, 3.87 M, P less than .02). A statistically significant higher rate of urinary tract infection (3 C, 22 M, P less than .05), pneumonia (0 C, 9 M, P less than .01), and episodes of sepsis and prosthetic endo carditis (0 C, 11 M, P less than .001) was found in the methicillin group. The incidence of wound infections and positive cultures from blood obtained immediately after termination of cardiopulmonary bypass was not significantly different. Cephalothin replaced methicillin as the routine antibiotic prophylaxis for open-heart surgery at our institution.

Antibiotic prophylaxis for open-heart surgery.

A randomized, prospective study of the relative effectiveness of broad-spectrum versus specific antistaphylococal antibiotic prophylaxis in patients having open-heart surgery was performed between May, 1972, and June, 1973. All patients undergoing open-heart surgery was assigned randomly (by hospital number) to receive either methicillin or cephalothin beginning the night before operation. There were 132 patients in the cephalothin group and 129 in the methicillin group. There was no statistically significant differences in age or duration of hospitalization, cardiopulmonary bypass, urinary tract drainage, or postoperative fever. There was a significant difference in the ratio of male to total patients (cephalothin group, 0.67; methicillin group, 0.52; p less than 0.02) and duration of operation (cephalothin group, 4.27 hours; methicillin group, 3.87 hours; p less than 0.05). The methicillin group had a statistically significant higher rate of urinary tract infection (cephalothin group, 3 cases; emthicillin group, 22 cases, p less than 0.05), pneumonia (cephalothin group, no cases; methicillin group, 9 cases; p less than 0.01), and episodes of sepsis and prosthetic valve endocarditis (cephalothin group, no cases; methicillin group, 11 cases, p less than 0.001). The incidence of wound infections and positive blood cultures from blood obtained immediately after termination of cardiopulmonary bypass was not significantly different between the two groups. Cephalothin has replaced methicillin as the routine prophylaxis for open-heart surgery at our institution.