Three unique base pair changes in a family with Gaucher disease

Summary Single-stranded cDNA was prepared from RNA obtained from a patient with type 1 Gaucher disease. The cDNA was amplified in vitro and analyzed by sequencing. Three base-pair changes were identified which included a G to C transversion at nucleotide 3119 of the active gene (Asp¹⁴⁰His), an A to C transversion at nucleotide 3170 (Lys¹⁵⁷Gln) and a G to A change at nucleotide 5309 (Glu³²⁶Lys). To study the mode of inheritance of the three different base-pair changes, genomic DNA was prepared from blood or skin fibroblasts of several family members. Genomic glucocerebrosidase DNA sequences were amplified and subjected to hybridization with allele-specific oligonucleotides (ASOs). The hybridization profiles demonstrated that two of the basepair changes originated from the mother and were transmitted to her two affected sons and to a grandchild, while the third base-pair change, originating from the father, was transmitted to his two affected sons, a carrier daughter and a second grandchild. Tests of other patients with Gaucher disease failed to disclose the presence of the three base-changes. This is a unique family with three base-pair changes tightly linked to Gaucher disease.     

Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes

Repeated DNAs from the constitutive heterochromatin of human chromosomes 1 and 18 were used as probes in nonradioactive in situ hybridization experiments to define specific numerical and structural chromosome aberrations in three human glioma cell lines and one neuroblastoma cell line. The number of spots detected in interphase nuclei of these tumor cell lines and in normal diploid nuclei correlated well with metaphase counts of chromosomes specifically labeled by in situ hybridization. Rapid and reliable assessments of aneuploid chromosome numbers in tumor lines in double hybridization experiments were achieved, and rare cells with bizarre phenotype and chromosome constitution could be evaluated in a given tumor cell population. Even with suboptimal or rare chromosome spreads specific chromosome aberrations were delineated. As more extensive probe sets become available this approach will become increasingly powerful for uncovering various genetic alterations and their progression in tumor cells.     

Mechanism for Cd2+ inhibition of (K++ Mg2+)ATPase activity and K+ (86Rb+) uptake join roots of sugar beet (Beta vulgaris)

Steady state kinetics were used to examine the influence of Cd²⁺ both on K⁺ stimulation of a membrane-bound ATPase from sugar beet roots (Beta vulgaris L. cv. Monohill) and on K⁺(⁸⁶Rb⁺) uptake in intact or excised beet roots. The in vitro effect of Cd²⁺ was studied both on a 12000–25000 g root fraction of the (Na⁺+K⁺+Mg²⁺)ATPase and on the ATPase when further purified by an aqueous polymer two-phase system. The observed data can be summarized as follows: 1) Cd²⁺ at high concentrations (>100 μM) inhibits the MgATPase activity in a competitive way, probably by forming a complex with ATP. 2) Cd²⁺ at concentrations <100 μM inhibits the specific K⁺ activation at both high and low affinity sites for K⁺. The inhibition pattern appears to be the same in the two ATPase preparations of different purity. In the presence of the substrate MgATP, and at K⁺ <5 mM, the inhibition by Cd²⁺ with respect to K⁺ is uncompetitive. In the presence of MgATP and K⁺ >10 μM, the inhibition by Cd²⁺ is competitive. 3) At the low concentrations of K⁺, Cd²⁺ also inhibits the 2,4-dinitrophenol(DNP)-sensitive (metabolic) K⁺(⁸⁶Rb⁺) uptake uncompetitively both in excised roots and in roots of intact plants. 4) The DNP-insensitive (non metabolic) K⁺(⁸⁶Rb⁺) uptake is little influenced by Cd²⁺. As Cd²⁺ inhibits the metabolic uptake of K⁺(⁸⁶Rb⁺) and the K⁺ activation of the ATPase in the same way at low concentrations of K⁺, the same binding site is probably involved. Therefore, under field conditions, when the concentration of K⁺ is low, the presence of Cd²⁺ could be disadvantageous.     

1-Methylguanine and 7-methylguanine increase cell agglutinability

Summary 1-Methylguanine and 7-methylguanine, both metabolic products of tRNA degradation, are known to induce transformation of Chinese hamster fibroblasts in culture. The effects of these compounds on the cell membrane have been studied by the method of Concanavalin A-mediated hemadsorption. 1-Methylguanine or 7-methylguanine induced a 50% increase of Con A-mediated hemadsorption within 20 hours of exposure of the cells to the agent at a concentration of 10^-5 M. This alteration was reversed within 13 days when the cells were grown in the control medium. Prolonged treatment with 1-methylguanine or 7-methylguanine resulted in changes which were only slowly reversed during growth of the cells in the control medium. The effect of the methylated purines on the cell membrane could be completely inhibited by simultaneous addition of dibutyryl-cAMP at a concentration of 10^-5 M. The possible mechanism of cell membrane alteration by methylated purines and its relevance to transformation in vitro are discussed.     

Carbon monoxide-cytochrome interactions in the brain of the fluorocarbon-perfused rat

Reflectance spectrophotometry through the skull was used to investigate carbon monoxide (CO) binding by tissue hemoproteins in the brains of barbiturate-anesthetized Sprague-Dawley rats. After splenectomy and extensive perfluorotributylamine exchange transfusion, steady-state spectral scans were obtained in Soret and visible wave-length regions during O2 ventilation, during subsequent exposure to O2-enriched gases containing 1, 3, or 5% CO, and finally after N2 anoxia. These CO exposures were well-tolerated and electroencephalograph (EEG) activity continued to be present. Initial difference spectra were influenced by CO binding to residual hemoglobin, but spectral evidence of CO-mediated b-type cytochrome reduction was obtained in the visible region as CO concentration was increased to 3 or 5%. This was associated with Soret spectra compatible with formation of the reduced cytochrome a3-CO complex. Reduction of cytochrome a at 605 nm and cytochrome c + c1 at 550 nm was absent. These findings may indicate respiratory chain branching through b cytochromes, either to a separate a3-like oxidase independent of the classical cytochrome aa3 or to an unidentified alternative CO-sensitive oxidase.     

The possession of coli surface antigen CS6 by enterotoxigenicEscherichia coli of serogroups O25, O27, O148, and O159: a possible colonization factor?

A total of 134 enterotoxigenicEscherichia coli (ETEC) of serogroups O25, O27, O148, and O159 were tested in the enzyme-linked immunosorbent assays for the colonization factor antigens I (CFA/I), CFA/II (coli surface antigens CS1, 2 and 3) and putative colonization factor (PCF) 8775 (CS4, 5 and 6). CS6 was detected without CS4 or CS5 in 94% of the strains of serogroup O25, 86% of strains of serogroup O27, 87% of strains of serogroup O148, and 29% of strains of serogroup, O159. The frequency with which CS6 occurs in ETEC of common serotypes without the antigens CS4 or CS5 suggests that it might be a colonization factor.     

The effects of potassium, 5-hydroxytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3′:5′-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells

Dispersed rat adrenal cells prepared from both the capsule and the decapsulated gland were used to investigate the effects on cyclic AMP accumulation of known stimuli of steroidogenesis [ACTH (adrenocorticotrophin), angiotensin II, K(+) ions and 5-hydroxytryptamine]. Since glomerulosa-cell preparations from capsular strippings are normally contaminated with a proportion of fasciculata cells, cells purified by fractionation on a bovine serum albumin gradient were also used. The results showed that: (1) ACTH and angiotensin II stimulated cyclic AMP accumulation in both fractionated and unfractionated zona fasciculata cells; (2) 5-hydroxytryptamine and an increased extracellular K(+) concentration (from 3.6 to 8.4mm) had no effect on cyclic AMP concentrations in fasciculata cell preparations; (3) the addition of ACTH, angiotensin II, 5-hydroxytryptamine or K(+) to the incubation medium resulted in increased cyclic AMP concentrations in unpurified zona glomerulosa cell preparations; (4) fractionation and hence the virtual elimination of fasciculata contamination, did not affect the response to 5-hydroxytryptamine and increased K(+) concentration. However, the responses to ACTH and angiotensin II were markedly lowered but not abolished. These results strongly suggest a link between cyclic AMP production and steroidogenesis in the zone of the adrenal gland that specifically secretes aldosterone. All four agents used stimulated both steroid output and cyclic AMP accumulation. However, at certain doses of 5-hydroxytryptamine, K(+) and angiotensin II the significant increases in corticosterone output were not accompanied by measurable increases in cyclic AMP accumulation.     

A New Plating Medium for the Isolation of Enteric Pathogens: II. Comparison of Hektoen Enteric Agar with S S and E M B Agar

During this study, 2,855 stool specimens from patients at Cook County Hospital were cultured for enteric pathogens. Hektoen Enteric Agar (HE) was compared with E M B and S S Agars by replicate samplings with both direct and indirect methods. Shigella species were recovered more than twice as often on HE Agar as on S S Agar by both methods. With the direct method only, out of 98 Shigella isolated, 97 were isolated from HE Agar, 74 were recovered from E M B Agar, and 40 were found on S S Agar. In addition, HE yielded better isolation of Salmonella strains than did S S or E M B by either direct or indirect methods. The greater efficiency of HE medium is discussed with respect to colonial recognition of enteric pathogens.     

Purification and Chemistry of Bacteriophage ?

From a stock of varkappa phage grown on Salmonella, a host-range mutant which attacks Escherichia coli was isolated. As in the case of Salmonella, only motile strains of E. coli are sensitive to varkappa. The phage shows an eclipse period of 35 min and a minimal latent period of 52 min. The adsorption rate constant is 3 x 10(-9) ml/min. Adsorption shows a marked dependence on temperature. Bacteriophage varkappa was purified by differential centrifugation and CsCl density gradient centrifugation. It contains deoxyribonucleic acid (DNA) which is double-stranded. The DNA has a molecular weight of 42 million and a guanine plus cytosine content of 57%. Of 68 molecules of DNA inspected, 7 were circular. The phage particle weight is about 90 million.