Intrafetal insulin-like growth factor-I infusion stimulates adrenal growth but not steroidogenesis in the sheep fetus during late gestation.

We investigated the effects of an intrafetal infusion of IGF-I on adrenal growth and expression of the adrenal steroidogenic and catecholamine-synthetic enzyme mRNAs in the sheep fetus during late gestation. Fetal sheep were infused for 10 d with either IGF-I (26 microg/kg.h; n = 14) or saline (n = 10) between 120 and 130 d gestation, and adrenal glands were collected for morphological analysis and determination of the mRNA expression of steroidogenic and catecholamine-synthetic enzymes. Fetal body weight was not altered by IGF-I infusion; however, adrenal weight was significantly increased by 145% after IGF-I infusion. The density of cell nuclei within the fetal adrenal cortex (the zona glomerulosa and zona fasciculata), and within the adrenaline synthesizing zone of the adrenal medulla, was significantly less in the IGF-I-infused fetuses compared with the saline-infused group. Thus, based on cell-density measurements, there was a significant increase in cell size in the zona glomerulosa and zona fasciculata of the adrenal cortex and in the adrenaline-synthesizing zone of the adrenal medulla. There was no effect of IGF-I infusion on the adrenal mRNA expression of the steroidogenic or catecholamine-synthetic enzymes or on fetal plasma cortisol concentrations. In summary, infusion of IGF-I in late gestation resulted in a marked hypertrophy of the steroidogenic and adrenaline-containing cells of the fetal adrenal in the absence of changes in the mRNA levels of adrenal steroidogenic or catecholamine-synthetic enzymes or in fetal plasma cortisol concentrations. Thus, IGF-I infusion results in a dissociation of adrenal growth and function during late gestation.

Successful treatment of empty follicle syndrome by triggering endogenous LH surge using GnRH agonist in an antagonist down-regulated IVF cycle.

To date, empty follicle syndrome (EFS) has only been reported in GnRH agonist down-regulated IVF cycles. Some cases have been successfully treated by changing the batch, or by repeating the dose of hCG. A case of EFS was observed in both GnRH antagonist and GnRH agonist down-regulated IVF cycles when final oocyte maturation was triggered using urinary hCG (u-hCG). Failure to retrieve oocytes occurred, despite administration of a further dose of u-hCG from a different batch and a delayed repeated oocyte recovery performed in the second GnRH agonist down-regulated cycle. A successful oocyte recovery cycle was achieved after triggering of an endogenous gonadotrophin surge using GnRH agonist in an antagonist down-regulated cycle. Nine oocytes were readily retrieved from 10 follicles, at 36 h after GnRH agonist administration, and eight of these fertilized normally. Two good quality embryos were used for fresh transfer and four were cryopreserved for future use. EFS can occur in GnRH antagonist down-regulated IVF cycles, and can be successfully treated by triggering a natural gonadotrophin surge using GnRH agonist in the absence of any response to previous treatment methods. This represents a novel therapeutic modality for this uncommon but frustrating condition.

Risk factors for hypoxemia and respiratory failure in respiratory syncytial virus bronchiolitis.

Respiratory syncytial virus (RSV) bronchiolitis is a common infection in young children and may result in hospitalization. We examined the incidence of, and risk factors associated with, hypoxemia and respiratory failure in 216 children aged < 24 months admitted consecutively for proven RSV bronchiolitis. Hypoxemia was defined as SpO2 < 90% in room air and severe RSV bronchiolitis requiring intubation and ventilation was categorized as respiratory failure. Corrected age at admission was used for premature children (gestation < 37 weeks). Hypoxemia was suffered by 31 (14.3%) children. It was more likely to occur in children who were Malay (OR 2.56, 95%CI 1.05-6.23, p=0.03) or premature (OR 6.72, 95%CI 2.69-16.78, p<0.01). Hypoxemia was also more likely to develop in children with failure to thrive (OR 2.96, 95%CI 1.28-6.82, p<0.01). The seven (3.2%) children who were both premature (OR 11.94, 95%CI 2.50-56.99, p<0.01) and failure to thrive (OR 6.41, 95%CI 1.37-29.87, p=0.02) were more likely to develop respiratory failure. Prematurity was the only significant risk factor for hypoxemia and respiratory failure by logistic regression analysis (OR 1.17, 95%CI 1.06-1.55, p<0.01 and OR 1.14 95%CI 1.02-2.07, p=0.02 respectively). Prematurity was the single most important risk factor for both hypoxemia and respiratory failure in RSV bronchiolitis.

Synthesis, processing and export of cytoplasmic endo-beta-1,4-xylanase from barley aleurone during germination.

We have identified the major endo-beta-1,4-xylanase (XYN-1) in the aleurone of germinating barley grain, and show that it is expressed as a precursor of Mr 61 500 with both N- and C-terminal propeptides. XYN-1 is synthesized as an inactive enzyme in the cytoplasm, and only becomes active at a late stage of germination when the aleurone ceases to secrete hydrolases. A series of processing steps, mediated in part by aleurone cysteine endoproteases, yields a mature active enzyme of Mr 34 000. Processing and extracellular release of the mature enzyme coincide with the programmed cell death (PCD)-regulated disintegration of aleurone cells. We discuss the significance of delayed aleurone cell-wall degradation by endoxylanases in relation to the secretory capacity of the aleurone, and propose a novel role for aleurone PCD in facilitating the export of hydrolases.

Purification, enzymatic characterization, and nucleotide sequence of a high-isoelectric-point alpha-glucosidase from barley malt.

High-isoelectric-point (pI) alpha-glucosidase was purified 7, 300-fold from an extract of barley (Hordeum vulgare) malt by ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose chromatography. The enzyme had high activity toward maltose (k(cat) = 25 s(-1)), with an optimum at pH 4.5, and catalyzed the hydrolysis by a retaining mechanism, as shown by nuclear magnetic resonance. Acarbose was a strong inhibitor (K(i) = 1.5 microM). Molecular recognition revealed that all OH-groups in the non-reducing ring and OH-3 in the reducing ring of maltose formed important hydrogen bonds to the enzyme in the transition state complex. Mass spectrometry of tryptic fragments assigned the 92-kD protein to a barley cDNA (GenBank accession no. U22450) that appears to encode an alpha-glucosidase. A corresponding sequence (HvAgl97; GenBank accession no. AF118226) was isolated from a genomic phage library using a cDNA fragment from a barley cDNA library. HvAgl97 encodes a putative 96.6-kD protein of 879 amino acids with 93.8% identity to the protein deduced from U22450. The sequence contains two active site motifs of glycoside hydrolase family 31. Three introns of 86 to 4,286 bp interrupt the coding region. The four exons vary from 218 to 1,529 bp. Gene expression analysis showed that transcription reached a maximum 48 h after the start of germination.

Isolation and characterization of the gene encoding the starch debranching enzyme limit dextrinase from germinating barley.

The gene encoding the starch debranching enzyme limit dextrinase, LD, from barley (Hordeum vulgare), was isolated from a genomic phage library using a barley cDNA clone as probe. The gene encodes a protein of 904 amino acid residues with a calculated molecular mass of 98.6 kDa. This is in agreement with a value of 105 kDa estimated by SDS-PAGE. The coding sequence is interrupted by 26 introns varying in length from 93 bp to 825 bp. The 27 exons vary in length from 53 bp to 197 bp. Southern blot analysis shows that the limit dextrinase gene is present as a single copy in the barley genome. Gene expression is high during germination and the steady state transcription level reaches a maximum at day 5 of germination. The deduced amino acid sequence corresponds to the protein sequence of limit dextrinase purified from germinating malt, as determined by automated N-terminal sequencing of tryptic fragments coupled with matrix assisted laser desorption mass spectrometry. The sequenced peptide fragments cover 70% of the entire protein sequence, which shows 62% and 77% identity to that of starch debranching enzymes from spinach and rice and 37% identity to Klebsiella pullulanase. Sequence alignment supports the multidomain architecture and identifies both secondary structure elements of the catalytic (beta/alpha)8-barrel substrate, catalytic residues, and specificity associated motifs characteristic of members of the glycoside hydrolase family 13 which cleave alpha-1,6-glucosidic bonds. A remarkable distribution of the secondary structure elements to individual exons is observed.

Intravenous infusion of insulin-like growth factor I in fetal sheep reduces hepatic IGF-I and IGF-II mRNAs.

Liver contains the highest concentrations of insulin-like growth factor (IGF) I mRNA in adult rats and sheep and is a major source of circulating IGF-I. In rats, inhibition of hepatic IGF-I production by exogenous IGF-I has been reported. In fetal sheep, skeletal muscle and liver are major sites of IGF-I synthesis and potential sources of circulating IGF-I. To determine whether feedback inhibition of IGF gene expression in fetal liver or muscle by IGF-I occurs, IGF-I and IGF-II mRNAs were measured in these tissues after intravenous infusion of recombinant human IGF-I into fetal sheep. Infusion of IGF-I (26 +/- 4 micrograms.h-1.kg-1; n = 6) or saline (n = 6) commenced on day 120 of pregnancy (term = 150 days) and continued for 10 days. Plasma concentrations of IGF-I were threefold higher in infused fetuses at 130 days of gestation (P < 0.0003), whereas those of IGF-II were unchanged. IGF-I infusion reduced the relative abundance of IGF-I mRNA (P < 0.0002) and IGF-II mRNA (P < 0.01) in fetal liver by approximately 50% but did not alter IGF-I or IGF-II mRNA in skeletal muscle. These results indicate that IGF-I inhibits the expression of both IGF-I and IGF-II genes in fetal liver and that IGF gene expression in fetal liver and muscle is differentially regulated by IGF-I.

Insulin-like growth factor I promotes growth selectively in fetal sheep in late gestation.

Insulin-like growth factor I (IGF-I) is required for normal fetal growth and skeletal maturation in late gestation, because null mutations of the IGF-I gene in mice reduce fetal weight and retard ossification of bones. To determine if, conversely, increased abundance of IGF-I promotes fetal growth and skeletal maturation, fetal sheep were infused intravascularly with recombinant human IGF-I (n = 7) (26 +/- 3 micrograms. h-1.kg-1) from 120 to 130 days gestation and compared with controls (n = 15). IGF-I infusion increased plasma IGF-I concentrations by 140% (P = 0.002) and weights of fetal liver, lungs, heart, kidneys, spleen, pituitary, and adrenal glands by 16-50% (P < 0.05). Weights and/or lengths of the fetus, placenta, gastrointestinal tract, individual skeletal muscles, and long bones were unchanged by IGF-I. However, IGF-I increased the percentage of proximal epiphyses of long bones present (P < 0.05) and their cross-sectional areas by 15 to 38% (P < 0.05). These results show that IGF-I promotes growth of major fetal organs, endocrine glands, and skeletal maturation in vivo, consistent with IGF-I actively controlling and not merely facilitating fetal growth. The variable response of different tissues may partly reflect tissue specificity in growth requirements for additional factors.

Placental control of fetal growth.

The placenta exerts its effects on the growth of the fetus from the beginning of pregnancy via metabolic and endocrine mechanisms. To achieve this, the placenta exchanges a wide array of nutrients, endocrine signals, cytokines and growth factors with the mother and the fetus. These exchanges modulate or programme fetal growth and development. This review concentrates on the function and structure of the placenta in humans and in animals, and the effects of experimental perturbation of placental size and function on fetal growth. The consequences for fetal growth of varying the abundance of peptides or, by deleting genes, insulin-like growth factors or cytokines, are also described. Maternal nutritional and hormonal state from as early as the first few days after fertilization, can influence the growth rate of the placenta and the fetus and also the length of gestation. Influences on placental development and their consequences will clearly have an impact on the placental control of fetal growth. Variations in the maternal environment and consequent perturbation of the metabolic and endocrine environment of the placenta and fetus are implicated as being responsible for the associations between prenatal growth of the placenta and its fetus and the subsequent risk of adult disease. The next challenge will be to determine the dominant influences at each stage of fetal and placental growth.