Improving the PCR protocol to amplify a repetitive DNA sequence.

Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers. These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle. Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus. We describe the amplification of the MaSp1 gene through minor changes in the PCR program. The results show that a denaturation temperature of 98°C is the key determinant in the amplification of the MaSp1 partial gene sequence.

Molecular architecture and evolution of a modular spider silk protein gene.

Spider flagelliform silk is one of the most elastic natural materials known. Extensive sequencing of spider silk genes has shown that the exons and introns of the flagelliform gene underwent intragenic concerted evolution. The intron sequences are more homogenized within a species than are the exons. This pattern can be explained by extreme mutation and recombination pressures on the internally repetitive exons. The iterated sequences within exons encode protein structures that are critical to the function of silks. Therefore, attributes that make silks exceptional biomaterials may also hinder the fixation of optimally adapted protein sequences.

An about 50,000-dalton protein in adrenal medulla: a common precursor of [Met]- and [Leu]enkephalin.

A protein that may be an enkephalin precursor has been identified in extracts of bovine adrenal medulla. This protein (about 50,000 daltons) appears to contain seven copies of [Met]enkephalin and one copy of [Leu]enkephalin. Digestion with trypsin and carboxypeptidase B yields [Met]enkephalin and [Leu]enkephalin in a ratio of almost 7 to 1. The enkephalins were identified by chromatography and by their binding to opiate receptors. Some characteristics of several other adrenal peptides that may serve as intermediates in the biosynthesis of the enkephalins are presented.

Spidroins from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae).

Spiders produce up to six different kinds of silk, each one for a specific biological function. Spider silks are also known for their unique mechanical properties. The possibility of producing new materials with similar properties motivated research on these silk proteins (spidroins). Using expression sequence tags, we identified four spidroins produced by major ampullate, minor ampullate, flagelliform and tubuliform silk glands from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae). The new protein sequences showed substantial similarity to other spidroins previously described, with high content of alanine and glycine due to the presence of the highly repetitive motifs (polyAla, (GA)n, (GGX)n, (GPGGX)n). Similarities among sequences were also observed between the different spidroins with the exception of tubuliform spidroin, which presents a unique complex amino acid sequence with high amounts of serine and low amounts of glycine.

A novel methodology to explore the viscoelasticity of spider major ampullate silk.

Even before material science was a recognized discipline, the amazing mechanical properties of spider silk were documented and became the object of much study. In addition to the exceptional material properties of spider silk and the reported low immunogenicity, its concatenated amino acid motif arrangement facilitates a distinct possibility of manipulating the silk to create a designer biomaterial for medical applications. Crystalline protein regions imbedded in a mobile protein matrix give it a distinct set of viscoelastic abilities. Consequently, elasticity cannot be simply quantified by only measuring extensibility. To understand how the sequence of the major ampullate proteins affects elasticity, the hysteresis of single fibers from two different species, Argiope aurantia and Nephila clavipes, were examined using cyclic loading and unloading. The yield point that discriminates a transition from elastic extension to a plastic extension was analyzed by examining three different properties: Young's modulus, energy recovery and slack in the fiber after recovery. Young's modulus remained relatively constant regardless of the cycle. However, the energy recovered decreased as the slack and cycle number increased. Large standard deviations masked any quantitative differences between species and substantiated the necessity of developing synthetic silk to harness the amazing mechanical properties of spider silk.

Evidence from flagelliform silk cDNA for the structural basis of elasticity and modular nature of spider silks.

Orb-web weaving spiders rely on their aerial nets to entrap flying prey. A key mechanical feature of orb-web design is the high elasticity of the capture spiral. We report the cloning of substantial cDNA for flagelliform gland silk protein, which forms the core fiber of the catching spiral. Like all silks, the flagelliform protein is composed largely of iterated sequences. The dominant repeat of this protein is Gly-Pro-Gly-Gly-X, which can appear up to 63 times in tandem arrays. This motif likely forms Pro2-Gly3 type II beta-turns and the resulting series of concatenated beta-turns are thought to form a beta-spiral. We propose that this spring-like helix is the basis for the elasticity of silk. The variable fifth position of the motif (X) is occupied by a small subset of residues (Ala, Ser, Tyr, Val). Moreover, these X amino acids occur in specific patterns throughout the repeats. This ordered variation strongly suggests that with hydration, the beta-spirals form hydrogen-bonded networks that increase the elasticity of flagelliform silk. The self-assembly of flagelliform protein monomers into silk fibers may be promoted by beta-spiral/beta-spiral interactions. Additionally, the other two motifs in the flagelliform protein, Gly-Gly-X and a spacer that disrupts the glycine-rich regions, may contribute to the alignment of monomers into fibers. The flagelliform protein cDNA was compared to the other members of the spider silk gene family. We show that all spider silk proteins can be characterized as sets of shared structural modules. The occurrence of these modules among the proteins is inconsistent with the phylogenetic relationships inferred from the C-terminal regions. This observation, along with the high level of variation among individual flagelliform protein repeats, but striking lack of such variation in the other silk proteins, suggests that unusual homogenization processes are involved in silk protein evolution.

On betaH-Leu5-endorphin and schizophrenia.

A previously unknown peptide, betaH-Leu5-endorphin, has been reported in the dialysates of schizophrenic patients. Accordingly, hemofiltrates from two schizophrenic and two control patients were examined for the presence of betaH-Leu5-endorphin. The opioid peptides were detected by a radioreceptor assay after separation and identification by gel filtration and high-performance liquid chromatography. With a detection limit of 30 pmole/L of hemofiltrate, no betaH-Leu5-endorphin or Met5-endorphin was found in controls or in patients. Whatever the possible involvement of endorphins in schizophrenic behavior, they are not present at detectable levels in the hemofiltrates of two well-characterized schizophrenic patients, thereby casting doubt on a general relationship of Leu-endorphin and schizophrenia.

Synthetic spider silk: a modular fiber.

Spiders make their webs and perform a wide range of tasks with up to seven different types of silk fiber. These different fibers allow a comparison of structure with function, because each silk has distinct mechanical properties and is composed of peptide modules that confer those properties. By using genetic engineering to mix the modules in specific proportions, proteins with defined strength and elasticity can be designed, which have many potential medical and engineering uses.

Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non-silk-like "spacer regions".

Spider minor ampullate silk is a strong non-elastic deformably stretchable silk used in web formation. This silk from Nephila clavipes is composed of two proteins, MiSp 1 and 2, whose transcripts are 9.5 and 7.5 kb, respectively, as determined by Northern blots. Both MiSp proteins are organized into a predominantly repetitive region and a small nonrepetitive carboxy terminal region. These highly repetitive regions are composed mainly of glycine and alanine, but also contain tyrosine, glutamine, and arginine. The sequences are mainly GGX and GA repeats. The repetitive regions are interrupted by nonrepetitive serine-rich spacer regions. Although the sequences of the spacer regions differ from the repetitive regions, sequences of the spacers from different regions of the proteins are nearly identical. The sequence differences between major and minor ampullate silks may explain the differing mechanical properties of the fibers.

ScreenMax plasmid mini-prep: super rapid plasmid DNA extraction method.

An ultra-quick method for plasmid DNA extraction using the ScreenMax Plasmid Mini-prep Kit was optimized. Since the method has fewer steps than current methods, the entire process takes only 12 min for two sets of plasmid DNA extractions. DNA quality was excellent for further analyses, including DNA sequencing. The kit's new medium, MMB, provided 5 times the viable cell count and 7 times the dry cell weight compared with the conventional LB medium. The yield of plasmid DNA was 5 times better than from LB medium. From 200 microL of culture, the maximum yield was 7.5 micrograms. The ScreenMax Plasmid Mini-Prep is not only simple and economical but also safe because of its mild reagents. This new procedure is both reliable and reproducible.

Atrial fibrillation. The therapeutic options.

Atrial fibrillation (AF) is a common cardiac arrhythmia which is particularly prevalent among the elderly. In patients with AF of recent onset, restoration of sinus rhythm may be feasible and this can be achieved by DC cardioversion, or by the use of one of a number of drugs including amiodarone, flecainide or propafenone. Neither digoxin nor the calcium antagonists facilitate the restoration of sinus rhythm. Recurrence of AF is common after successful cardioversion and, although long term antiarrhythmic drug therapy may help to maintain sinus rhythm, all such drugs are potentially toxic and can have important proarrhythmic actions. In patients with chronic AF, restoration of sinus rhythm is rarely possible and treatment is directed towards control of the ventricular response rate, which may be achieved with digoxin and/or a rate-limiting calcium antagonist such as verapamil or diltiazem; beta-blockers may also be used although they appear to impair effort tolerance. In addition, long term anticoagulation may be indicated to reduce the risks of systemic embolisation, even in patients with 'nonrheumatic' AF; antiplatelet drugs are of no apparent value in this context. A minority of patients present with AF associated with ventricular pre-excitation; in these individuals both digoxin and the calcium antagonists are contraindicated and the ventricular response rate should be controlled with flecainide, amiodarone or propafenone.

Adverse reactions with beta-adrenoceptor blocking drugs. An update.

beta-Adrenoceptor blocking drugs are widely used throughout the world, and serious adverse reactions are relatively uncommon. Many of those that do occur, including bronchospasm and peripheral ischaemia, are due primarily to blockade of beta 2-adrenoceptors. Recently developed beta-blockers with enhanced beta 1-selectivity and partial beta 2-agonist activity appear, in general, to have lesser effects upon airways function and vascular resistance, but none are regarded as being entirely 'safe' in patients with asthma. In the treatment of hypertensive patients with co-existing airways disease there are now effective alternatives to the beta-blockers, including calcium antagonists, alpha-adrenoceptor antagonists and angiotensin converting enzyme (ACE) inhibitors. However, in the presence of ischaemic heart disease, beta-blockers have specific advantages and may still be considered necessary in patients with airways disease. In this situation, agents with beta 2-agonist activity are preferable to 'conventional' beta-blockers. However, there is still some risk that bronchospasm may occur in certain individuals, and the bronchodilator response to inhaled beta 2-agonists might be impaired. In patients with peripheral vascular disease, beta-blockers with beta 2-agonist activity are less likely to worsen the symptoms and signs of peripheral ischaemia, and may reduce the prevalence of peripheral coldness, a common adverse effect of beta-blockers. There is concern that beta-blockers may have significant central effects, including impairment of memory and concentration, although these are difficult to quantify. A number of pharmacologically unpredictable adverse reactions may occur rarely, including skin reactions, alopecia and arthropathy.

Enkephalin-containing polypeptide levels in normal tensive and SHR rat adrenal glands.

The levels of enkephalin-containing polypeptides (ECPs) in the adrenal glands of normal tensive rats (WKY) and spontaneously hypertensive rats (SHR) were compared. Innervated and denervated adrenals from both types of rats showed very similar levels of ECPs. The only difference observed was a small increase in the 18 kdal ECP and a concomitant decrease in the 12 kdal and 5.3 kdal ECPs in the innervated SHR rat adrenal gland. From these data it appears that the adrenal ECPs are not a major contributor to hypertension in the SHR rat nor does hypertension, at this age, affect the ECP levels.

Response of plasma proenkephalin peptide F to exercise.

Plasma proenkephalin Peptide F immunoreactivity was measured in the plasma of endurance trained and untrained males at various exercise intensities. Significantly different levels were found between the two groups at rest and at 54% maximum oxygen consumption (VO2 max). Maximum levels were at 5 min of recovery for the untrained group and at 54% VO2 max and at recovery 5 min for the trained group. These data suggest an adaptation in the trained group fro the release of this peptide and further suggest it may play a role in recovery since the highest levels are following the termination of the exercise period.

Purification and sequence of a non-opioid peptide derived from ovine proenkephalin: implications for possible species specific processing.

A non-enkephalin containing pentadeca peptide derived from ovine adrenal proenkephalin has been purified and sequenced. The sequence of the peptide is: Phe-Ala-Glu-Pro-Leu-Pro-Ser-Glu-Glu-Glu-Gly-Glu-Ser-Tyr-Ser (preproenkephalin 237-251) representing the amino portion of peptide B (preproenkephalin 237-268). The sequence is identical to bovine preproenkephalin 237-251, differing from the corresponding human sequence at positions 240 and 244. This peptide can be generated by a processing event common to other opioid peptides and is present in chromaffin granules in significant amounts. The presence of this peptide in substantial quantities suggests a possible difference in proenkephalin processing between the bovine and ovine adrenal medulla.

The effects of indomethacin and sulindac upon the blood pressures of individuals with untreated labile or mild hypertension.

Twelve patients (mean age 48.8 years, 5 females) with untreated labile or mild essential hypertension completed a randomised, double-blind crossover study comparing the effects of two-week courses of indomethacin, sulindac and matching placebo upon sitting and standing blood pressure. Both indomethacin and sulindac significantly increased systolic BP when compared with placebo; indomethacin tended to have a greater pressor effect than sulindac. During treatment with indomethacin, the mean BP rose from 136/86 to 149/92 (sitting), and from 136/93 to 150/99 standing. The extent of the pressor response was unrelated to baseline BP. The results indicate that both non-steroidal anti-inflammatory drugs (NSAIDs) have a significant pressor effect in individuals with untreated labile or mild hypertension. The fact that sulindac had a pressor effect implies that this response may be unrelated to inhibition of renal prostaglandins, or that the renal-sparing effect of sulindac is only relative. Extra-renal prostaglandins may play a role in the control of BP in this population. Individuals with labile or mild hypertension do not appear to exhibit exaggerated pressor responses during treatment with these NSAIDs, as similar increases in BP have been reported in normal subjects.

Kinetics of chromium transformations in the environment.

Kinetics of chromium transformations under typical environmental conditions were systematically investigated using batch, microcosm and column experiments. Oxidation and reduction rates were evaluated in single phase water systems and in two phase water-solid systems. Natural and reference waters, sediments and soils representing a broad range of characteristics were used. Some of the investigated reactions, such as the reduction of Cr(VI) by S2- or Fe2+ ions under anaerobic conditions, were instantaneous and the experimental results exactly followed the stoichiometric and equilibrium calculations. Other reactions, such as the reduction of Cr(VI) by organics in sediments and soils, or the oxidation of Cr(III) by MnO2, were much slower and clearly exhibited kinetic controls. Reduction of Cr(VI) by organics was dependent on both the type and amount of organic material. Dissolved oxygen by itself did not induce measurable oxidation of Cr(III), spiked to the experimental waters, after 128 days. Slow Cr(III) oxidation to Cr(VI) was only noted in one of the natural waters and sediments with half-lives ranging from 2 to 9 years. In all cases the extent of Cr(III) oxidation did not exceed 15% of the initial Cr(III) present. The overall results indicate that the transport and transformation of chromium can be predicted using kinetics data derived from a simplified laboratory experiment, the Eh-pH data and the aquatic system characteristics. Results of the microcosm and laboratory experiments were in good agreement with the predicted concentrations.

Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins.

Several types of silks and silk protein coding genes have been characterized from orb-web weaving spiders. When the protein sequences of major ampullate, minor ampullate, and flagelliform silks from Nephila clavipes are compared, they can be summarized as sets of shared amino acid motifs. Four of these motifs and their likely secondary structures are described. Each structural element, termed a module, is then associated with its impact on the mechanical properties of a silk fiber. In particular, correlations are drawn between an alanine-rich 'crystalline module' and tensile strength and between a proline-containing 'elasticity module' and extensibility.

The absence of detectable fetal microchimerism in nontransgenic goats (Capra aegagrus hircus) bearing transgenic offspring.

Regulations for the disposal of genetically engineered animals are strict due to concern for their inappropriate introduction into the food chain, and of the possible public health and environmental impacts of these organisms. Nontransgenic animals that give birth to transgenic offspring are treated as if they are transgenic due to concern of fetal cells crossing the placental barrier and residing in the mother (fetal-maternal microchimerism). Determining whether or not fetal-fetal or fetal-maternal transfer of DNA or cells occurs during caprine gestation is critical to effectively protect the public without culling animals that pose no risk. Additionally, fetal-maternal transfer, should it exist in the goat, could contraindicate the rebreeding of nontransgenic dams due to the possible transfer of fetal cells from 1 pregnancy to the fetus of subsequent pregnancies. Fetal-maternal transfer in Capra hircus has not been reported in the literature, although it has been reported in another ruminant, Bos taurus. We examined blood from nontransgenic dams that carried transgenic offspring using a PCR method sensitive enough to detect the presence of a spider silk transgene to a 1:100,000 dilution. At this sensitivity, we did not detect the occurrence of fetal-maternal transfer in 5 nontransgenic dams. Likewise, fetal-fetal transfer was not observed from a transgenic to a nontransgenic twin in utero. To test tissue-specific expression of the silk transgene, proteins purified from standard necropsy tissue from a lactating transgenic dam were examined by Western blot analysis. Silk protein expression was only observed in mammary tissue consistent with the tissue specificity of the ß-casein promoter used in the transgenic construct. We report evidence collected from a limited caprine breeding pool against transfer of transgenes in utero from fetus to dam and fetus to fetus. In addition, we show evidence that the ß-casein promoter in our expression construct is not expressed ectopically as previously suggested. These results suggest that transgene transfer in utero does not occur, but further study is warranted with a larger sample group to confirm these results.