DNA hybridization in nanostructural molecular assemblies enables detection of gene mutations without a fluorescent probe

We have developed a simple single nucleotide polymorphisms (SNPs) analysis utilizing DNA hybridization in nanostructural molecular assemblies. The novel technique enables the detection of a single-base mismatch in a DNA sequence without a fluorescent probe. This report describes for the first time that DNA hybridization occurs in the nanostructural molecular assemblies (termed reverse micelles) formed in an organic medium. The restricted nanospace in the reverse micelles amplifies the differences in the hybridization rate between mismatched and perfectly matched DNA probes. For a model system, we hybridized a 20-mer based on the p53 gene sequence to 20-mer complementary oligonucleotides with various types of mismatches. Without any DNA labeling or electrochemical apparatus, we successfully detected the various oligonucleotide mismatches by simply measuring the UV absorbance at 260 nm.     

Metabolism of daidzein by intestinal bacteria from rhesus monkeys (Macaca mulatta)

PURPOSE: To identify the metabolites produced from an isoflavonoid, daidzein, by colonic bacteria of rhesus monkeys. METHODS: The metabolism of daidzein by the fecal bacteria of nine monkeys was investigated. Daidzein was incubated anaerobically with fecal bacteria, and the metabolites were analyzed by use of liquid chromatography and mass spectrometry. RESULTS: The fecal bacteria of all of the monkeys metabolized daidzein to various extents. Dihydrodaidzein was found in cultures of fecal bacteria from two monkeys; dihydrodaidzein and equol were found in cultures from four monkeys; dihydrodaidzein, equol, and an unknown metabolite (MW = 244) were found in cultures from one monkey; and dihydrodaidzein and the unknown metabolite were found in cultures from two monkeys. CONCLUSIONS: Similar to that in humans, variation was evident in the metabolism of isoflavonoids by fecal bacteria from rhesus monkeys. Some metabolites produced by fecal bacteria from monkeys were the same as those produced by fecal bacteria from humans.     

Insulin amyloid fibrillation at above 100 degrees C: new insights into protein folding under extreme temperatures

To investigate the folding behavior of amyloidogenic proteins under extreme temperatures, the kinetics of fibrillation and accompanying secondary structure transitions of bovine insulin were studied for temperatures ranging up to 140 degrees C. The presence of extreme heat stress had traditionally been associated with irreversible denaturation of protein while the initial steps of such a denaturation process may be common with a fibril formation pathway of amyloidogenic proteins. The present work demonstrates the ability of insulin to form amyloid fibrils at above 100 degrees C. Amyloid formation was gradually replaced by random coil generation after approximately 80 degrees C until no amyloid was detected at 140 degrees C. The morphology of insulin amyloid fibrils underwent sharp changes with increasing the temperature. The dependence of amyloid formation rate on incubation temperature followed non-Arrhenius kinetics, which is explained by temperature-dependent enthalpy change for amyloid formation. The intermediate stage of amyloid formation and random coil generation consisted of a partially folded intermediate common to both pathways. The fully unfolded monomers in random coil conformation showed partial reversibility through this intermediate by reverting back to the amyloid pathway when formed at 140 degrees C and incubated at 100 degrees C. This study highlights the non-Arrhenius kinetics of amyloid fibrillation under extreme temperatures, and elucidates its intermediate stage common with random coil formation.     

Stem cells: cross-talk and developmental programs

The thesis advanced in this essay is that stem cells-particularly those in the nervous system-are components in a series of inborn 'programs' that not only ensure normal development, but persist throughout life so as to maintain homeostasis in the face of perturbations-both small and great. These programs encode what has come to be called 'plasticity'. The stem cell is one of the repositories of this plasticity. This review examines the evidence that interaction between the neural stem cell (as a prototypical somatic stem cell) and the developing or injured brain is a dynamic, complex, ongoing reciprocal set of interactions where both entities are constantly in flux. We suggest that this interaction can be viewed almost from a 'systems biology' vantage point. We further advance the notion that clones of exogenous stem cells in transplantation paradigms may not only be viewed for their therapeutic potential, but also as biological tools for 'interrogating' the normal or abnormal central nervous system environment, indicating what salient cues (among the many present) are actually guiding the expression of these 'programs'; in other words, using the stem cell as a 'reporter cell'. Based on this type of analysis, we suggest some of the relevant molecular pathways responsible for this 'cross-talk' which, in turn, lead to proliferation, migration, cell genesis, trophic support, protection, guidance, detoxification, rescue, etc. This type of developmental insight, we propose, is required for the development of therapeutic strategies for neurodegenerative disease and other nervous system afflictions in humans. Understanding the relevant molecular pathways of stem cell repair phenotype should be a priority, in our view, for the entire stem cell field.     

Effects of transferring in vitro-cultured rabbit embryos to recipient oviducts on mucin coat deposition, implantation and development

A mucin coat is deposited on rabbit embryos during passage through the oviduct; rabbit blastocysts cultured from the 1-cell stage in vitro have no mucin coat. When cultured blastocysts are transferred to recipients, the lack of mucin coat might account in part for subsequent failure of pregnancy. We have investigated the possibility that mucin coat deposition is induced following transfer of in vitro 72 h-cultured blastocysts to oviducts of asynchronous or synchronous recipients. One-cell embryos were collected by flushing oviducts 19-20 h post-coitus and were cultured in vitro for 72 h until they reached the blastocyst stage. The blastocysts were transferred to the oviducts of recipients that were synchronized either with the donors (synchronous) or 1 day later than the donors (asynchronous). They were recovered after 24-48 h and the mucin coat thickness and embryo degeneration rate were measured. The degeneration rate of blastocysts recovered from uteri of synchronous recipients was higher than that from asynchronous recipients (72.2% vs 40.0%). The mucin coats around embryos recovered from oviducts of asynchronous recipients after 48 h were thicker than those from synchronous recipients. More asynchronous recipients were pregnant and gave birth to more pups than synchronous recipients. These results indicate that the oviducts of asynchronous recipients secreted more mucin around the transferred embryos, causing higher rates of implantation of the in vitro-cultured blastocysts.     

Sulfamide derivatives as transition state analogue inhibitors for carboxypeptidase A

3-Phenyl-2-sulfamoyloxypropionic acid (2), 2-benzyl-3-sulfamoylpropionic acid (3), and N-(N-hydroxysulfamoyl)phenylalanine (5) have been synthesized and evaluated as inhibitors for carboxypeptidase A (CPA) to find that they inhibit the enzyme competitively with the Ki values in the microM range, suggesting that their binding modes to CPA are analogous to each other, and resemble the binding mode of N-sulfamoylphenylalanine (1) that has been established by the X-ray crystallographic method to form a complex with CPA in a manner reminiscent of the binding of a transition state in the catalytic pathway. It was concluded thus that they are a new type of transition state analogue inhibitors for CPA. (R)-N-Hydroxy-N-sulfamoyl-beta-phenylalanine (8) was shown to be also a potent CPA inhibitor (Ki = 39 microM), the high potency of which may be ascribed to the involvement of the hydroxyl in the binding of CPA, most likely forming bidentate coordinative bonds to the zinc ion in CPA together with the sulfamoyl oxygen atom.     

Site-directed mutagenesis of human brain GABA transaminase: lysine-357 is involved in cofactor binding at the active site

gamma-Aminobutyrate transaminase (GABA-T), a key enzyme of the GABA shunt, converts the major inhibitory neurotransmitter, GABA, to succinic semialdehyde. Although GABA-T is a pivotal factor implicated in the pathogenesis of various neurological disorders, its function remains to be elucidated. In an effort to clarify the structural and functional roles of specific lysyl residue in human brain GABA-T, we constructed human brain GABA-T mutants, in which the lysyl residue at position 357 was mutated to various amino acids including asparagine (K357N). The purified mutant GABA-T enzymes displayed neither catalytic activity nor absorption bands at 330 and 415 nm that are characteristic of pyridoxal-5'-phosphate (PLP) covalently linked to the protein. The wild type apoenzyme reconstituted with exogenous PLP had catalytic activity, while the mutant apoenzymes did not. These results indicate that lysine 357 is essential for catalytic function, and is involved in binding PLP at the active site.     

Changes in parvalbumin immunoreactivity in the parietofrontal cortex after transient forebrain ischemia in the Mongolian gerbil

We investigated the changes in parvalbumin (PV)-immunoreactive (IR) neurons in the parietofrontal cortex after transient forebrain ischemia. In the sham-operated group, PV-IR neurons were present in all layers of the parietofrontal cortex except layer I. Shortly after ischemia the number of PV-IR neurons in layer II/III first increased, and then declined dramatically 12 h after ischemic insult, followed by a second increase after 2 days. At this time the PV immunoreactivity was very weak and only present in the peripheral neuronal cytoplasm. The reversible increase in the number of PV-IR neurons and in the level of their immunoreactivity could result from a transient ischemia-induced increase in intracellular calcium. This pattern of expression was particularly pronounced in layer II/III of the parietofrontal cortex, suggesting that these neurons are especially\susceptible to ischemic insult.