Arginine side chain assignments in uniformly 15N-labeled proteins using the novel 2D HE(NE)HGHH experiment

A novel 2D NMR experiment, 2D HE(NE)HGHH, is presented for the assignment ofarginine side chain ¹H and ¹⁵N resonances inuniformly ¹⁵N-labeled proteins. Correlations between¹H, ¹Hand ¹H are established on the basis of³J(¹⁵N,¹H) heteronuclear scalarcoupling constants, and sequence-specific assignments are obtained by overlapof these fragments with ¹H chemical shiftsobtained by assignment procedures starting from the polypeptide backbone.Since guanidino protons exchange quite rapidly with the bulk water, the 2DHE(NE)HGHH pulse scheme has been optimized to avoid saturation and dephasingof the water magnetization during the course of the experiment. As anillustration, arginine side chain assignments are presented for two uniformly¹⁵N-labeled proteins of 7 and 23 kDa molecular weight.     

Psychosocial Concerns and Health-Compromising Behaviors among Overweight and Nonoverweight Adolescents

Objective: To compare weight-specific and global psychosocial concerns and health-compromising behaviors among overweight and nonoverweight youth across gender and ethnicity. Methods: A cross-sectional school-based survey of 31,122 adolescents in grades 7 to 12. Based on self-reported heights and weights, respondents were categorized as nonoverweight (body mass index (BMI) <85th percentile), moderately overweight (85th percenile <BMI <95th percentile), or severely overweight (BMI >95th percentile). Results: Global psychosocial concerns, such as emotional well-being, suicidal ideation, future job concerns, and peer concerns, did not differ greatly between nonoverweight, moderately overweight, and severely overweight adolescents. Substance abuse behaviors were equally or less prevalent among the overweight group. Overweight girls were significantly less likely to consume alcohol, whereas overweight boys were at lower risk for marijuana use. In contrast, overweight youth were more likely to perceive their health as only fair or poor and were more likely to express weight-specific concerns and engage in behaviors such as chronic dieting and binge eating than nonoverweight youth. Overweight American Indian girls perceived their physical health more positively than nonoverweight American Indian girls. Strong associations were found between overweight status and chronic dieting among African American boys and girls. Conclusions: Nutritional counseling and educational programs need to address the weight-specific concerns and behaviors of overweight adolescents. However, assumptions regarding global psychosocial concerns and health-compromising behaviors among overweight adolescents of different genders and ethnicities should be avoided. These broad issues need to be explored in more depth at both the research and intervention levels.     

A High-Resolution Interval Map of the q21 Region of the Human X Chromosome

In a previous study, we have developed a panel of chromosomal rearrangements for the physical mapping of the q13-q21 region of the human X chromosome (Philippe et al., Genomics 17: 147-152, 1993). Here, we report the physical localization of 36 additional polymorphic markers by polymerase chain reaction analysis. The high density of chromosomal breakpoints in Xq21 allows us to map 58 DNA loci in 22 intervals. As a result, this segment of the X chromosome is saturated with approximately three sequence tagged sites per megabase of DNA, which will facilitate the construction of a YAC contig of this region.     

A distance geometry program for determining the structures of small proteins and other macromolecules from nuclear magnetic resonance measurements of intramolecular1H−1H proximities in solution

DISGEO is a new implementation of a distance geometry algorithm which has been specialized for the calculation of macromolecular conformation from distance measurements obtained by two-dimensional nuclear Overhauser enhancement spectroscopy. The improvements include (1) a decomposition of the complete embedding process into two successive, more tractable calculations by the use of “substructures”, (2) a compact data structure for storing incomplete distance information on a structure, (3) a more efficient shortest-path algorithm for computing the triangle inequality limits on all distances from this information, (4) a new algorithm for selecting random metric spaces from within these limits, (5) the use of chirality constraints to obtain good covalent geometry without the use ofad hoc weights or excessive optimization. The utility of the resultant program with nuclear magnetic resonance data is demonstrated by embedding complete spatial structures for the protein basic pancreatic trypsin inhibitor vs all 508 intramolecular, interresidue proton-proton contacts shorter than 4.0 Å that were present in its crystal structure. The crystal structure could be reproduced from this data set to within 1.3 Å minimum root mean square coordinate difference between the backbone atoms. We conclude that the information potentially available from nuclear magnetic resonance experiments in solution is sufficient to define the spatial structure of small proteins.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

The binding of selenium to the lipids of two unicellular marine algae

Axenic cultures of the green algae $\text{Dunaliella}\text{primolecta}$ and red algae $\text{Porphyridium}\text{cruentum}$ were grown in the presence of sublethal quantities of selenite. All purified lipids from both algae were found to contain bound selenium, except for saturated hydrocarbons. Of the lipids which contain selenium, carotenoid pigments contain the greatest concentrations. Lipid-associated selenium is not metabolically incorporated. The selenium is probably non-covalently bound to the lipids.     

Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety

Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl α-d-mannoside, α-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for β-glucuronidase from human urine. The inhibition of α-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl α-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2026–2030]. Endocytosis of `low-uptake' forms of α-N-acetylglucosaminidase and α-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that `low-uptake' forms are either contaminated with `high-uptake' forms or are internalized via the same route as `high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver β-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.     

The paramedial neurosecretory cells of the suboesophageal ganglion of the cricket,Teleogryllus commodus (Walk.)

Summary Ovariectomy, performed immediately after the final hatch, caused a reduction of stainable (neurosecretory?) material in the paramedial neurosecretory cells (PNC) (A-type) of the suboesophageal ganglion in 10 day-old females of Teleogryllus commodus (Walk.). A concomitant increase in nuclear volume and in the incorporation of ³⁵S-cysteine indicates increased synthesis of neurosecretory material. From these findings it is concluded that more stainable material is secreted in the cerebral neurohaemal organ after Ovariectomy. A functional relationship between the PNC and the ovaries is suggested.     

The effect of monochromatic radiation in the range 350 to 750 nm on the carotenogenesis in Verticillium agaricinum

An action spectrum for carotenogenesis in V. agaricinum has maxima at 395, 433, 660 and 737 nm. In a previous study it had been shown that a light-minus-dark difference spectrum of a crude extract from V. agaricinum had maxima at 390 and 420 nm, and furthermore a red, far-red interaction suggesting phytochrome involvement has been proposed. All these data suggest that there may be at least two photoreceptor systems operating in the photoinduction process here; one for the near-ultraviolet (UV-A)-mediated carotenogenesis, presumably a novel pigment, and the other for the red, far-red region, most likely phytochrome.