Stage-specific keratins in Xenopus laevis embryos and tadpoles: the XK81 gene family.

This report describes the isolation and characterization of genomic and cDNA clones which define a subfamily of type I keratins in Xenopus laevis whose expression is restricted to embryonic and larval stages. The XK81 subfamily, named after the prototype cDNA clone DG81, contains four members arranged in two pairs of closely homologous loci; they were named 81A1, A2, B1, and B2. Genomic clones were obtained representing all of these regions. The A1 gene has been completely sequenced together with approximately 1 kb of flanking sequences at each end; this gene corresponds to the previously reported cDNA clone 8128 (Jonas, E., T. D. Sargent, and I. B. Dawid, 1985, Proc. Natl. Acad. Sci. USA, 82:5413-5417). The B2 gene is represented by a partial cDNA clone, DG118. Upstream sequences and about half of the coding regions have been sequenced for the B1 and B2 genes, whereas the A2 locus has been identified on the basis of hybridization data and could be a gene or pseudogene. Genomic Southern blotting indicates that all members of the subfamily have been isolated. The keratin proteins encoded by the B1 and B2 genes are 96% homologous in the central rod domain, whereas A/B gene homology in this region is 81%. During development mRNAs derived from A and B genes accumulate coordinately during gastrula and neurula stages; in the tadpole, 81A mRNA decays rapidly, whereas 81B mRNA shows a second abundance peak, persists for most of tadpole life, and decays by metamorphosis. RNAs derived from the XK81 keratin subfamily are undetectable in the adult, where different type I keratin genes are expressed.

Neural cadherin: role in selective cell-cell adhesion.

Cadherins are a family of Ca2+-dependent intercellular adhesion molecules. Complementary DNAs encoding mouse neural cadherin (N-cadherin) were cloned, and the cell binding specificity of this molecule was examined. Mouse N-cadherin shows 92 percent similarity in amino acid sequence to the chicken homolog, while it shows 49 percent and 43 percent similarity to epithelial cadherin and to placental cadherin of the same species, respectively. In cell binding assays, mouse N-cadherin did not cross-react with other mouse cadherins, but it did cross-react with chicken N-cadherin. The results indicate that each cadherin type confers distinct adhesive specificities on different cells, and also that the specificity of N-cadherin is conserved between mammalian and avian cells.

R-cadherin: a novel Ca(2+)-dependent cell-cell adhesion molecule expressed in the retina.

cDNAs encoding a novel member of the cadherin cell adhesion receptor family were cloned. This cadherin is expressed in the retina of the chicken and is termed R-cadherin. It is similar to other cadherins in its primary structure, but most resembles N-cadherin, showing 74% amino acid identity. Cells expressing R-cadherin can adhere to those expressing N-cadherin when mixed, but they form homotypic clusters within their chimeric aggregates. In the development of the neural retina, R-cadherin begins to be expressed around embryonic day 8 in both neuronal and glial cells, and this expression continues up to the hatching stage. The pattern of the expression of R-cadherin was different from that of N-cadherin, suggesting distinctive roles in retinal morphogenesis.

Xerl: a novel secretory protein expressed in eye and brain of Xenopus embryo.

A novel gene, Xerl (Xenopus EGF-like repeat with laminin-G domain protein) was isolated from a Xenopus head cDNA library prepared from tailbud. This gene encoded 779 amino acids including a potential signal sequence, twelve EGF-like repeats, a laminin-G domain, a RGD sequence and a VWF motif. In the EGF-like repeat and the laminin-G domain, Xerl showed similarity to those of Drosophila Crumbs, respectively. Zygotic expression of Xerl began at late gastrula, and increased through neurula up to the tailbud stage. In adult organs, Xerl was detected in brain and eye. Whole-mount in situ hybridization showed that Xerl expression occurred first in the anterior bilateral region of neurula and gradually localized to retina and forebrain and boundaries of midbrain and hindbrain.

Responses of sodium balance, blood pressure, and other variables to sodium loading in Papua New Guinea highlanders.

For determination of the responses of sodium balance, blood pressure, and other relevant variables to Na loading in people with a low intake of Na, 10 male Papua New Guinea highland subjects were given additional Na at two levels (128 and 256 mmol/d) for 10 d after a 3-d control period of low-Na diet. Na loading caused a marked positive balance of Na, decreases of aldosterone concentration and renin activity in the plasma, and a decrease of urinary aldosterone excretion. The blood pressure, particularly that measured at noon, increased in the latter half of the Na-loading period, the increase being significant in the group given 256 mmol of sodium daily: the systolic and diastolic blood pressure increased from 92 +/- 8 over 56 +/- 7 mm Hg in the control period to 102 +/- 7 over 60 +/- 4 mm Hg in the latter half of the test period (p less than 0.05).

Molecular cloning of delta-4, a new mouse and human Notch ligand.

Complementary DNAs encoding a previously unidentified mouse Notch ligand and its human ortholog were isolated. The new Notch ligand contains a signal sequence, a DSL domain, eight epidermal growth factor-like repeats, a transmembrane domain, and an intracellular region, all of which are characteristics of members of the Delta protein family. The new protein was therefore designated Delta-4. Several previously unidentified sequences in both the extracellular and intracellular regions were shown to be conserved among vertebrate Delta proteins. The tissue distribution of Delta-4 mRNA resembles that previously described for Notch-4 (Int-3) transcripts. However, in situ hybridization with mouse lung revealed that Delta-4 mRNA is abundant in squamous alveolar cells that neighbor endothelial cells; Notch-4 expression is largely restricted to the latter cell type. Soluble forms of the extracellular portion of Delta-4 inhibit the apparent proliferation of human aortic endothelial cells, but not human pulmonary arterial endothelial cells.

Closed-circuit metabolic system with multiple applications.

A closed-circuit metabolic system has been designed and tested for multiple applications. Air pressure within a closed chamber is regulated electronically while allowing for respiratory gas exchange. Compared with a previously reported standard indirect calorimetry system, the new device had by virtue of longer duration of measurement improved precision (coefficient of variation 3% vs. 14%) during studies of O2 consumption both at room temperature and at 5 degrees C. In addition, a more physiological atmospheric environment is maintained. This system has also been utilized for simultaneously labeling groups of up to 20 weanling rats with 18O2 over a 2-day period and for exposure of rats to a hyperoxic (84% O2), normobaric environment for 4-day periods. Potential applications include maintenance of pressure (hypobaric through hyperbaric) and O2 (hypoxic through hyperoxic) controlled environments, exposure to toxic gases, study of diurnal variations in metabolic rate, measurement of metabolic expenditure with activity, and adaptation to other species including humans.

Determining double-bond positions in monoenoic 2-hydroxy fatty acids of glucosylceramides by gas chromatography-mass spectrometry.

We applied a gas chromatography-mass spectrometry (GC-MS) method using dimethyl disulfide (DMDS) adducts and were able to determine the double-bond positions in monounsaturated 2-hydroxy fatty acids (2-HFA). 2-HFA methyl esters, prepared from the hydrolysate of Arabidopsis thaliana leaf glucosylceramides, were acetylated and methylthiolated. GC-MS analysis of the resulting DMDS adducts showed simple mass spectra with recognizable molecular ions and a series of key fragment ions indicating the original double-bond positions in the aliphatic chain. Based on this GC-MS elucidation, we confirmed that Arabidopsis leaf glucosylceramides have C22, C23, C24, C25, and C26 chain length 2-HFA with monounsaturation, and all their double bonds are placed at the n-9 position. This procedure is simple, time efficient, and highly sensitive.

Effect of dietary protein levels on urea utilization in Papua New Guinea highlanders.

Ability to utilize urea nitrogen for body protein synthesis was examined with Papua New Guinea (PNG) highlanders and Japanese (JPN). Eight male PNG highlanders and 8 male JPN were fed on a low protein diet containing 0.55 g protein/kg or an adequate protein diet containing 1.34 g protein/kg for 1 or 2 weeks. The fate of 15N was measured after oral administration of 15N-labelled urea. There was no difference in 15N incorporation into serum protein between PNG highlanders and JPN receiving low protein diets. On the other hand, on the adequate protein diet, 15N incorporation in PNG highlanders was similar to that on the low protein diet, in contrast to that in JPN which was hardly detected in the adequate protein diet. When PNG highlanders take more protein than protein in their usual diet, they effectively incorporate ingested protein into their body protein and urea nitrogen is utilized for synthesis of body protein.

Nutritive efficiencies of lactalbumin and wheat gluten at very low levels of intake in adult rats.

The nutritive values of proteins in relation to their intake levels were evaluated by feeding adult male rats weighing 250 g diets containing 0%, 0.39%, 0.78%, 1.56%, 2.34%, 3.90%, 7.79% and 15.58% lactalbumin or wheat gluten for three weeks. The biological values (BV) of both proteins were high at low levels of protein intake but decreased with increase in protein intake. The BV of wheat gluten was estimated to be about 100 at a level of intake of 1.56% but only 25 at a level of 15.58%. Similarly, the BV of lactalbumin decreased with increase in the protein level, being 67 at a level of 7.79%. The BVs of both proteins at low levels of dietary protein (below 2.34% of lactalbumin or 0.78% of wheat gluten) were apparently more than 100 because urinary N excretion was less than endogenous N. The BVs also decreased with time during the three-week test period. It is concluded that BV of a protein is not a fixed value but varies with the experimental conditions especially with changes in the amount of intake, and that differences in the qualities of various proteins cannot be compared quantitatively at a single level of protein. The results were briefly discussed in relation to protein requirements.

Effects of low protein intake on protein metabolism of Papua New Guinea highlanders studied with [15N]glycine.

The effects of low protein intake on protein metabolism, including the size of pools and the protein synthesis rates, were studied by use of [15N]glycine in Papua New Guinea highlanders. Studies were made on 9 men between October and December in 1982. In experiment 1, two subjects were given a protein-free diet (PFD) containing 49.1 kcal/kg of energy. In experiment 2, subjects were given a sweet-potato diet (SPD) containing 45.4 kcal/kg of energy and 0.507 g/kg of protein for 8 days, and then were given a low-protein sweet-potato diet (LPSPD) containing 50.0 kcal/kg of energy and 0.265 g/kg of protein. During the SPD period, the sizes of the metabolic and active protein pools (mean +/- SD) were 270 +/- 134 mgN/kg and 362 +/- 107 mgN/kg, respectively, and the rates of active and inactive protein synthesis were 463 +/- 161 mgN/kg/day and 299 +/- 38 mgN/kg/day, respectively. During the LPSPD period, the sizes of the metabolic pool and active protein pool were 131 +/- 64 mgN/kg and 378 +/- 106 mgN/kg, respectively, and the rates of active and inactive protein synthesis were 490 +/- 206 mgN/kg/day and 280 +/- 26 mgN/kg/day, respectively. The protein metabolism in the LPSPD showed no significant difference from the SPD. The results suggest that, when the energy levels were approximately the same, protein metabolism in Papua New Guinea highlanders was maintained in spite of the decrease in protein intake.

Blood status and serum free amino acids in Papua New Guinea highlanders.

Hematological values and serum amino-acid concentrations were measured in 17 healthy male adult Papua New Guinea highlanders who live on a sweet-potato staple diet. Hematological values were within the normal range, except for a low serum urea concentration. The concentrations of serum threonine, valine, isoleucine, leucine and tyrosine were significantly lower, and those of arginine, glutamate, glycine and alanine were significantly higher, than in Japanese controls. These amino acid patterns in the serum of Papua New Guinea highlanders are an indication of low protein nutrition and adequate energy supply. Some essential amino acid and urea concentrations in the serum of nine Papua New Guinea subjects fed on an adequate protein diet (1.3 g/kg body weight, about twice their habitual diet) for 13 days were significantly increased but were still significantly lower than those of Japanese subjects. Serum alanine decreased on an adequate protein diet. These results show that amino acid uptake and utilization by peripheral tissues may be accelerated on an adequate protein diet. Blood status and serum amino acid concentrations did not show any change except for urea and some amino acids, when five Papua New Guinea highlanders were fed on a low protein diet (0.6 g/kg) consecutively for 13 days.

Antihypercholesterolemic effect of undigested fraction of soybean protein in young female volunteers.

The significant antihypercholesterolemic effect of the undigested high molecular fraction (HMF) of soybean protein is known in rats, but such an effect has not been shown in humans. The present two experiments were designed to elucidate it in humans. Subjects were female university students who had relatively high serum cholesterol levels for their age. In Experiment 1, subjects took 8% of their total energy from casein, soybean protein isolate (SPI), or HMF daily for 14 days. Five basic menus and snacks were cycled. Energy intakes and daily activities were kept constant and body weight was maintained. The HMF group showed decreased low-density lipoprotein cholesterol (LDL-C) as compared to other groups. In Experiment 2, subjects took 4% of total energy from casein or HMF daily for a menstruation period. Five basic menus and snacks which contained two egg yolks (about 500 mg cholesterol) were cycled. Energy intakes and daily activities were kept constant and body weight was maintained. A decrease in LDL-C and an increase in high-density lipoprotein cholesterol (HDL-C) were observed in the HMF group as compared to the casein group. Fecal acidic steroid excretion was greater in the HMF group than in the casein group (p < 0.05). The results confirmed that HMF increases fecal steroid excretion and reduces serum cholesterol levels in humans.

[A case of primary orbital chondrosarcoma].

The case of a twenty-year-old male with orbital chondrosarcoma is reported. He visited National Defense Medical College Hospital because of reduced vision in the right eye since two months previously. His corrected visual acuity was 8/20 in the right eye and 20/20 in the left eye. Fifteen degrees lateral displacement of the right globe and limitation of right ocular movement were recognized. Right fundus examination revealed optic disc edema and protuberant nasal fundus. Orbital computed tomography (CT) demonstrated a high density area between the inner part of the right orbit and the ethmoid sinus. Magnetic resonance imaging (MRI) showed a smoothly outlined and low intensity (T1) space occupying lesion. This lesion was irregularly enhanced by gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA). This orbital tumor was removed by an anterior approach. Histopathological examination revealed well-differentiated chondrosarcoma (grade 1) as determined by small prominent chondromatous cell projection into the collagen fibrous stroma, and existence of binucleate cells in the hypercellular region. After the operation the disc edema disappeared and his corrected right visual acuity improved to 20/20.

Ectopic expression of N-cadherin perturbs histogenesis in Xenopus embryos.

Xenopus embryos express N-cadherin in a pattern similar to that observed in other species, and cells expressing Xenopus N-cadherin can bind to cells expressing chicken N-cadherin in vitro. To investigate the developmental role of this molecule, we injected mRNA encoding chicken N-cadherin into one blastomere of 2-cell-stage Xenopus embryos and examined the effect of its expression on their development. The ectopic expression of N-cadherin occurred in various regions of the injected embryos and induced abnormal histogenesis, such as thickening, clumping or fusion of cell layers. These results suggest that the precise quantitative and qualitative regulation of the expression of cadherins is essential to embryonic morphogenesis.

Requirement and utilization of egg protein by Japanese young men with marginal intakes of energy.

The effect of marginal intakes of energy on the requirement and utilization of egg protein was evaluated in 46 Japanese young men. The subjects were given a standard diet for 1 week and then low protein diets for 2 weeks. These diets contained about 32, 64, and 80 mgN/kg with whole eggs as the protein source. In the first experiment with excess energy, the energy intakes of 31 subjects were kept constant during the 3 week experiment, the mean intakes being 48.2 +/- 1.5 kcal/kg. The body weight was affected by changing protein intakes while maintaining energy intakes at 48 kcal/kg. From regression analysis, the N requirement for apparent N equilibrium was estimated to be 82.0 +/- 8.0 mgN/kg, where NPU was calculated as 56. In the second experiment with submaintenance energy, 15 subjects received 40 kcal/kg. The N requirement was 124 +/- 21 mgN/kg, where NPU was calculated as 37. From these results and those of previous studies, it was concluded as follows: 1) N balance and NPU were remarkably affected by energy intake changed around maintenance level; and 2) the NPU for egg protein in young men for maintenance intakes of energy and N is about 50 to 55. For estimation of the protein requirement for Japanese adults, a correction factor of 100/55 (about 1.8) was used instead of 1.3 adopted by the 1973 FAO/WHO.

Notch signaling suppresses IgH gene expression in chicken B cells: implication in spatially restricted expression of Serrate2/Notch1 in the bursa of Fabricius.

The bursa of Fabricius is a central organ for chicken B cell development and provides an essential microenvironment for expansion of the B cell pool and for generation of a diversified B cell repertoire. We report here that genes encoding the Notch family of transmembrane proteins, key regulators of cell fate determination in development, are differentially expressed in the bursa of Fabricius: Notch1 is expressed in medullary B cells located close to the basement membrane-associated epithelium (BMAE). In contrast, a Notch ligand, Serrate2, is expressed exclusively in the BMAE, which surrounds bursal medulla. A basic helix-loop-helix-type transcription factor, Hairy1, a downstream target of Notch signaling, is expressed in the bursa coordinately with Notch1 and Serrate2 and an immature B cell line, TLT1, which expresses both Notch1 and Serrate2. Furthermore, stable expression of a constitutively active form of chicken Notch1 or Notch2 in a B cell line results in a down-regulation of surface IgM expression, which is accompanied by the reduction of IgH gene transcripts. Transient reporter assay with the human IgH gene intronic enhancer reveals that an active form of Notch1 inhibits the IgH enhancer activity in chicken B cells, suggesting that Notch-mediated signals suppress the IgH gene expression via influencing the IgH intronic enhancer. These findings raise the possibility that the local activation of Notch1 in a subset of B cells by Serrate2 expressed in BMAE may influence the cell fate decision that is involved in B cell differentiation and selection inside the bursa.

Genomic structure and chromosomal mapping of the mouse N-cadherin gene.

N-cadherin is a member of the cadherin cell-cell adhesion receptor family that includes P-, E-, and R-cadherin and liver cell adhesion molecule (L-CAM). In this study, we determined the structure of the mouse N-cadherin gene by analyzing overlapping genomic clones obtained from a mouse genomic library. This gene consists of 16 exons that disperse over greater than 200 kilobases of genomic DNA. This large size of the N-cadherin gene, compared with its cDNA (4.3 kilobases), is ascribed to the fact that the first and second introns are 34.2 kilobases and greater than 100 kilobases long, respectively. When the N-cadherin gene was compared with that of L-CAM and P-cadherin, the exon-intron boundaries were found to be fully conserved between them, except that the P-cadherin first exon includes the first and second exons of the other two genes. Also, the second intron, which is equivalent to the first intron in P-cadherin, is exceptionally large and this structural feature is conserved in all of these genes. An interesting feature of the N-cadherin gene is that this gene has an extra 16th exon that is almost identical to the other exon, 100% in the coding region and 99% in the 3' untranslated region in the nucleotide level. We also determined the chromosomal localization of the N-cadherin gene by interspecific backcross analysis and found that this gene is localized in the proximal region of mouse chromosome 18. The E- and P-cadherin genes are tightly linked and located on chromosome 8 in this species. Thus, N-cadherin is unlinked to these other cadherin loci.