Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

Stabilizing ER Ca2+ Channel Function as an Early Preventative Strategy for Alzheimer’s Disease

Alzheimer’s disease (AD) is a devastating neurodegenerative condition with no known cure. While current therapies target late-stage amyloid formation and cholinergic tone, to date, these strategies have proven ineffective at preventing disease progression. The reasons for this may be varied, and could reflect late intervention, or, that earlier pathogenic mechanisms have been overlooked and permitted to accelerate the disease process. One such example would include synaptic pathology, the disease component strongly associated with cognitive impairment. Dysregulated Ca²⁺ homeostasis may be one of the critical factors driving synaptic dysfunction. One of the earliest pathophysiological indicators in mutant presenilin (PS) AD mice is increased intracellular Ca²⁺ signaling, predominantly through the ER-localized inositol triphosphate (IP₃) and ryanodine receptors (RyR). In particular, the RyR-mediated Ca²⁺ upregulation within synaptic compartments is associated with altered synaptic homeostasis and network depression at early (presymptomatic) AD stages. Here, we offer an alternative approach to AD therapeutics by stabilizing early pathogenic mechanisms associated with synaptic abnormalities. We targeted the RyR as a means to prevent disease progression, and sub-chronically treated AD mouse models (4-weeks) with a novel formulation of the RyR inhibitor, dantrolene. Using 2-photon Ca²⁺ imaging and patch clamp recordings, we demonstrate that dantrolene treatment fully normalizes ER Ca²⁺ signaling within somatic and dendritic compartments in early and later-stage AD mice in hippocampal slices. Additionally, the elevated RyR2 levels in AD mice are restored to control levels with dantrolene treatment, as are synaptic transmission and synaptic plasticity. Aβ deposition within the cortex and hippocampus is also reduced in dantrolene-treated AD mice. In this study, we highlight the pivotal role of Ca²⁺ aberrations in AD, and propose a novel strategy to preserve synaptic function, and thereby cognitive function, in early AD patients.     

A mutant lymphoma cell line with a defectiveThy-1 glycoprotein gene

We characterized a mutant T -cell lymphoma line selected for the inability to express the Thy-1 glycoprotein. This cell line is a member of the D complementation class of Thy-1^– somatic cell mutants, and it lacks detectable cell-surface Thy-1.1 glycoprotein and detectable cytoplasmic Thy-1 mRNA. Southern blot analysis using a number of probes isolated from the clonedThy-1.2 gene demonstrated that, in the mutant, one copy of theThy-1 gene is absent from the genome and the other has undergone rearrangement. This rearrangement results from a deletion of the 5 portion of the gene removing the first two alternate exons and promoters and a portion of the second intron. The deletion breakpoint within the mutantThy-1 gene was localized to within 400 nucleotides by Southern blot analysis. The breakpoint is near two classes of mouse repetitive elements-a mouse B1-family repetitive element and a simple repetitive sequence-suggesting a mechanism of rearrangement leading to the mutation. Southern blot analysis demonstrated that two closely linked molecular markers on chromosome 9 are unaltered, demonstrating that the deletion in this mutant cell line is subchromosomal.     

Replacement of the invariant lysine 77 by arginine in yeast iso-1-cytochrome c results in enhanced and normal activities in vitro and in vivo

Oligonucleotide-directed mutagenesis of the yeast Saccharomyces cerevisiae was used to generate an abnormal iso-1-cytochrome c having an Arg-77 replacement of the normal Lys-77; this Lys-77 residue is evolutionarily conserved in most eukaryotic cytochromes c and is trimethylated in fungal and plant cytochromes c. Examination of strains having a single chromosomal copy of the gene encoding the Arg-77 protein indicated that the altered protein was synthesized at the normal rate and that it had normal or near normal activity in vivo. Examination of enzymatic activities in vitro with cytochrome b2, cytochrome c peroxidase, and cytochrome c oxidase indicated that the altered iso-1-cytochrome c has equal or enhanced catalytic efficiencies. Thus, replacement of the evolutionarily conserved residue Lys-77 produces no or only minor effects both in vivo and in vitro.     

Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.     

Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus

Summary Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12–13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.     

Identification of a Putative Structural Gene for Cathepsin D in Caenorhabditis Elegans

Mutants of Caenorhabditis elegans having about 10% of wild-type activity of the aspartyl protease cathepsin D have been isolated by screening. Mutant homozygotes have normal growth rates and no obvious morphological or developmental abnormalities. The mutant gene (cad-1) has been mapped to the right extremity of linkage group II. Heterozygous animals (cad-1/+) show intermediate enzyme levels and animals heterozygous for chromosomal deficiencies of the right extremity of linkage group II have 50% of wild-type activity. Cathepsin D purified from a mutant strain has a lower activity per unit mass of pure enzyme. These data suggest that cad-1 is a structural gene for cathepsin D.     

Blood vessel adaptation to gravity in a semi-arboreal snake

The effects of vasoactive agonists on systemic blood vessels were examined with respect to anatomical location and gravity acclimation in the semi-arboreal snake, Elaphe Obsoleta. Major blood vessels were reactive to putative neurotransmitters, hormones or local factors in vessel specific patterns. Catecholamines, adenosine triphosphate, histamine and high potassium (80 mM) stimulated significantly greater tension per unit vessel mass in posterior than anterior arteries. Anterior vessels were significantly more sensitive to catecholamines than midbody and posterior vessels. Angiotensin II stimulated significantly greater tension in carotid artery than in midbody and posterior dorsal aorta. Arginine vasotocin strongly contracted the left and right aortic arches and anterior dorsal aorta. Veins were strongly contracted by catecholamines, high potassium and angiotensin II, but less so by adenosine triphosphate, arginine vasotocin and histamine. Precontracted vessels were relaxed by acetylcholine and sodium nitroprusside, but not by atrial natriuretic peptide or bradykinin. Chronic exposure of snakes to intermittent hypergravity stress (+1.5 G_z at tail) did not affect the majority of vessel responses. These data demonstrate that in vitro tension correlates with known patterns of sympathetic innervation and suggest that catecholamines, as well as other agonists, are important in mediating vascular responses to gravitational stresses in snakes.Abbreviations ACH acetylcholine - ADA anterior dorsal aorta - ANG II salmon asn¹-val⁵-angiotensin II - ANP rat ile²⁶-atrial natriuretic peptide - ATP adenosine triphosphate - AVT arginine vasotocin - BK human bradykinin - BL total body length - CA carotid artery - CONT control - EC ₅₀ effective concentration producing 50% maximal response - EPI epinephrine - + G _z earth's gravity force - HI-G high gravity acclimation - HI K ⁺ 80 mM high potassium - JV jugular vein - LAA left aortic arch - MDA midbody dorsal aorta - MPV midbody portal vein - MS Mackenzie's solution - NEPI norepinephrine - pD ₂ log EC₅₀ - PDA posterior dorsal aorta - PPV posterior portal vein - RAA right aortic arch - SNP sodium nitroprusside     

Sampling dust from human dwellings to estimate the prevalence ofDermatophagoides mite and cat allergens

Summary Quantification of specific allergens in household dust samples may provide important information for selecting appropriate allergen control methods, and monitoring efficacy and compliance. The purpose of this study was to investigate the source of variation in mite and cat allergen measurements associated with dust sample collection. Discrete and composite dust samples were collected on a filter using a special vacuum sampling device. Aqueous extracts of the dust samples were prepared thenDer p I,Der f I, andFel d I were quantitated by enzyme immunoassays (EIA). Mite and cat allergens were frequently detected in dust samples from human dwellings, and the amounts of these allergens varied significantly (p<0.01) among dwellings. The differences of allergen measurements among duplicate samples taken immediately and up to three weeks later appear to be much smaller than differences among houses and between rooms. Variation among dust samples taken from living rooms and bedrooms of the same dwelling suggest differences in allergen reservoirs. Composite samples formed by sampling specific objects within a room may provide a reliable estimate of allergen exposure in that room. Dust samples from discrete objects are useful to find and monitor specific reservoirs of mite and cat allergens.