Construction of a fast, inexpensive rapid-scanning diode-array detector and spectrometer.

A 512-element diode-array spectroscopic detection system capable of acquiring multiple spectra at a rate of 5 ms per spectrum with an effective scan rate of 102.9 kHz has been constructed. Spectra with fewer diode elements can also be acquired at scan rates up to 128 kHz. The detector utilizes a Hamamatsu silicon photodiode-array sensor that is interfaced to Hamamatsu driver/amplifier and clock generator boards and a DRA laboratories 12-bit 160-kHz analog-to-digital converter. These are standard, commercially available devices which cost approximately $3500. The system is interfaced to and controlled by an IBM XT microcomputer. Detailed descriptions of the home-built detector housing and control/interface circuitry are presented and its application to the study of the reaction of horseradish peroxidase with hydrogen peroxide is demonstrated.

Photon-gated spectral hole burning by donor-acceptor electron transfer.

We have observed a new mechanism for photon-gated spectral hole burning, donor-acceptor electron transfer, in a material composed of a zinc-tetrabenzoporphyrin derivative (donor) with chloroform acceptors in poly(methyl methacrylate) thin films. Gated holes form when we simultaneously excite the donor 0-0 singlet absorption (630 nm) and the donor triplet-triplet absorption (350-550 nm), with the largest gating enhancement (>30) occurring for gating light near 480 nm. The gating action spectrum and the photoproduct spectrum confirm that the mechanism is electron transfer from an excited triplet of the porphyrin donor to the chloroform acceptor. This result opens up a new class of materials for photon gating that should be of interest for frequency-domain optical storage applications as well as high-resolution spectroscopy of electron transfer processes in solids.

Measurement of serum alpha-fetoprotein in early infancy: utilization of dried blood specimens.

Serum alpha-fetoprotein (AFP) concentrations were measured in dried blood spots and the biologic half-life of AFP was determined during the first month after parturition. AFP blood levels, in babies at all birthweight groups during the first wk, were partitioned according to birthweight and sex. A synthetic surge of serum AFP at postnatal day 3 was observed in babies of the low birthweight category (less than 6 lb 8 oz), whereas a concomitant surge could not be demonstrated in babies of medium, (6 lb 8 oz to 8 lb 8 oz), and high birthweight, (greater than 8 lb 8 oz), categories. All babies weighing less than 6 lb 8 oz, regardless of prematurity likewise displayed the synthetic surge. The sex of the infant appeared to have no influence on AFP serum concentrations. In a population of infants ranging in age from 1-30 days, the biologic apparent half-life of AFP was calculated as 5.7 days; however, the half-life of AFP was found to differ among the three groups of infants when subdivided by birthweight and was higher (7.7 days) in babies of low birthweight. Our determination of 2 S.D. cutoff values for AFP in the first wk of life should prompt further studies in which the relationship of AFP to newborn disorders should be more fully explored.

Commercial radioimmunoassay kit for measurement of alpha-fetoprotein adapted for use with dried blood specimens from newborns.

We adapted a commercial RIA kit to measure alpha-fetoprotein (AFP) in 0.785-microliters portions of 60-microliters spots of dried blood from newborns. We evaluated sample elution, temperature and time stability, between- and within-assay variability, sensitivity, and use of liquid vs dried specimens of blood. Also, we present normal AFP concentrations for healthy neonates during the first postnatal week. Our procedure permits measurement of AFP concentrations both in maternal liquid plasma and in spots of dried blood from the infant with the same RIA kit reagents and standards. The sensitivity, precision, stability, and simplicity of this procedure makes more practical the routine measurement of AFP in dried blood specimens from newborns than measurement in liquid plasma or serum. Blood-sample collection by heel stick suffices for this simple, efficient, inexpensive determination of AFP concentration in the newborn.

Newborn screening for hemoglobinopathies in New York State: experience of physicians and parents of affected children.

Responses of physicians and parents to New York State-mandated newborn screening for sickle cell disease were solicited and evaluated. The index group comprised 25 infants born in western upstate New York. Each was found to have either sickle cell disease, hemoglobin SC disease, sickle beta-thalassemia, or hemoglobin C disease. In nondirective interviews the following factors were assessed: clinical course, the physician's policies of disease treatment and family counseling, the parents' reactions to the diagnosis and their level of understanding and compliance with medical recommendations, and the physicians' and parents' views on newborn screening. Newborn screening for sickle hemoglobin makes early prophylaxis and prompt treatment possible. Some morbidity may have been averted, judging from parental understanding of medical needs. Parents and physicians agreed that newborn screening for hemoglobinopathies is a valuable public health program. Suggestions for improving the New York state program included a need to increase communication among the screening laboratory, the hospital, and the physician; encouraging physicians to educate parents more fully, provide genetic counseling, and test parents and siblings of the identified neonate; and, most important, provide a well-delineated mechanism for follow-up of every infant with a potentially symptomatic hemoglobinopathy.

Thymidine kinase activity is reduced in the developing staggerer cerebellum.

In the mouse cerebellar mutant staggerer, thymidine kinase levels do not increase developmentally at ages when the wild-type level is high. Mixing experiments show that this effect is not due to an endogenous inhibitor of the enzyme. Both the Km and the susceptibility of the thymidine kinase to nucleotide inhibitors are unaltered in the mutant animals, suggesting that the enzyme is not induced in the mutant.

Human brain cerebroside beta-galactosidase: deficiency of transgalactosidic activity in Krabbe's disease.

Under experimental conditions optimal for the assay of D-galactosyl-N-acylsphingosine galactohydrolase (EC 3.2.1.46) activity, homogenates of neurologically normal human brain tissue could transfer galactose from galactosyl ceramide (gal-cer), lactosyl ceramide (lac-cer), 4-methylumbelliferyl-beta-galactoside (4-MU-gal), or p-nitrophenyl-beta-galactoside (PNP-gal) to [1-14C]oleoyl sphingosine, but homogenates of brain tissue from patients with Krabbe's disease lacked this ability. The rate of hydrolysis of ganglioside GM1 and, to a lesser extent, of PNP-gal by homogenates of Krabbe's brain tissue was also decreased. Activity of PNP-beta-galactosidase in normal brain tissue, like that of cerebroside beta-galactosidase from the same source, was considerably more heat-stable than the activity of either 4-MU-beta-galactosidase or the predominant GM1 beta-D-galactosidase (EC 3.2.1.23). Lac-cer and GM1, as well as 4-MU-gal and PNP-gal, were competitive inhibitors of human-brain cerebroside beta-galactosidase. These findings confirm the ability of mammalian cerebroside beta-galactosidase to catalyze a transgalactosylation reaction and provide additional information on the substrate specificity of human brain cerebroside beta-galactosidase.

Lipolytic action of cholera toxin on fat cells. Re-examination of the concept implicating GM1 ganglioside as the native membrane receptor.

The possible role of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM1) ganglioside in the lipolytic activity of cholera toxin on isolated fat cells has been examined. Analyses of the ganglioside content and composition of intact fat cells, their membranous ghosts, and the total particulate fraction of these cells indicate that N-acetylneuraminylgalactosylglucosylceramide (GM3) represents the major ganglioside, with substantial amounts of N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM2) and smaller amounts of other higher homologues also present. Native GM1 was not detected in any of these preparations. Examination of the relative capacities of various exogenously added radiolabeled sphingolipids to bind to the cells indicated that GM2 and glucosylsphingosine were accumulated by the cells to extents comparable to GM1. Galactosylsphingosine and sulfatide also exhibited significant, although lesser, binding affinities for the cells. The adipocytes appeared to nonspecifically bind exogenously added GM1; saturation of binding sites for GM1 could not be observed up to the highest concentration tested (2 X 10(-4) M), wherein about 7 X 10(9) molecules were associated with the cells. Essentially all of this exogenously added GM1 was found bound to the plasma membrane "ghost" fraction. Investigation of the biological responses of the cells confirmed their sensitivities to both cholera toxin and epinephrine-stimulated lipolysis, as well as the lag period displayed during the toxin's action. While we could confirm that the toxin's lipolytic activity can be enhanced by prior treatment of the fat cells with GM1, several of the observed characteristics of this phenomenon differ from earlier reported findings. Accordingly, added GM1 was able to enhance only the subsequent rate, but not the extent, of toxin-stimulated glycerol release (lipolysis) from the cells. We also were unable to confirm the ability of GM1 to enhance the toxin's activity at either saturating or at low toxin concentrations. The limited ability of added GM1 to enhance the toxin's activity appeared in a unique bell-shaped dose-response manner. The inability of high levels of GM1 to stimulate a dose of toxin that was ineffective on native cells suggests that the earlier reported ability of crude brain gangliosides to accomplish this was due to some component other than GM1 in the crude extract. While several glycosphingolipids and some other carbohydrate-containing substances that were tested lacked the ability to mimic the enhancing effect of GM1, 4-methylumbelliferyl-beta-D-galactoside exhibited an effect similar to, although less pronounced than, that of GM1. The findings in these studies are unable to lend support to the earlier hypothesis that (a) GM1 is cholera toxin's naturally occurring membrane receptor on native fat cells, and (b) the ability of exogenously added GM1 to enhance the toxin's lipolytic activity represents the specific creation of additional natural receptors on adipocytes...

Comparison of lipids in total brain tissue from five mouse genotypes.

Brain tissue from adult male and female mice of the C57BL/6J, C57BL/6J-AW-J, BALB/cJ, SJL/J, and DBA/2J genotypes was examined for brain weight, total protein, total lipid, cholesterol, phospholipid, plasmalogen, sulfatide, nonganglioside-glycolipid sphingosine, and ganglioside N-acetyl neuraminic acid, fatty acid, and sphingosine. No significant differences were found between sexes for any of these constituents. When compared to the overall average obtained for other animals, the DBA/2J, C57BL/6J-AW-J, and BALB/cJ mice contained lower quantities of plasmalogen and sulfatide compared to the overall averages obtained for the other genotypes. In addition, the sterol content in DBA/2J mice was significantly higher than the overall average value obtained for the other animals.