Body mass index as an indicator of obstructive sleep apnea in pediatric Down syndrome.

OBJECTIVE: Our objective was to determine if higher body mass index (BMI) increases the likelihood of, obstructive sleep apnea (OSA) in pediatric Down syndrome (DS) patients. METHODS: We performed a, retrospective chart review of 63 DS patients evaluated by overnight polysomnography from December 1995 to February 2005. Patients aged less than 2 years were excluded. Remaining patients were grouped, according to presence (n=19) or absence (n=33) of OSA based on apnea hypopnea index (AHI). OSA, and non-OSA DS groups were age matched while blinded to patient attributes other than age and OSA, status. Patients without appropriate age matches were excluded. We recorded various patient information, including age, sex, height, weight, number of apneas, number of hypopneas, respiratory distress index (RDI), apnea-hypopnea index (AHI), lowest oxygen saturation during sleep, mean oxygen saturation, number of arousals per hour, and mean time spent in REM sleep. We calculated BMI using the, standard kg/m(2) formula and converted this into a Z-score. RESULTS: Fifty-two DS patients were analyzed with average age of 9.3+/-4.5 years (10.2+/-4.2 in 33 OSA patients, 7.8+/-4.3 in 19 non-OSA patients). There were 28 males and 24 females. The OSA group mean BMI Z-score was 2.09+/-0.94, and the non-OSA group Z-score was 1.4+/-1.40. The Z-scores for BMI were statistically significant between OSA and non-OSA patients with p=0.03 by t-test. CONCLUSIONS: When age and sex adjusted, BMI has a statistically significant association with the presence of OSA in Down syndrome patients. The incidence of OSA also increases with increasing age in this population.

Expression of tetanus toxin fragment C.

1. The yield of fragment C was only modestly affected by Mut phenotype, and the site and type of integration event. 2. Fragment C accumulation was closely correlated with gene dosage and maximal expression levels required high gene copy number. 3. Yields were greatly increased in controlled fermenters, compared to shake-flasks, owing to the high cell density achieved and to an increased efficiency of induction (2.5- to 10-fold). 4. In fermenter inductions of a 14-copy strain, fragment C accumulated to 27% of total protein, giving an estimated yield of 12 g/L. 5. Considerable clonal variation in the level of expression occurred with transplacement transformants, and this was owing to a diversity of different integration events and to differences in gene copy number. 6. These multicopy transplacement events occur by in vivo circularization of transforming DNA fragments followed by repeated single-crossover integration. This is presumably a general phenomenon, such that it should be possible to obtain multicopy integrants from all P. pastoris transplacement transformations.

Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.

Numerous heterologous proteins have been produced at greater than gram per liter levels in the methylotrophic yeast, Pichia pastoris, using the methanol oxidase promoter. The factors that drastically influence protein production in this system include: copy number of the expression cassette, site and mode of chromosomal integration of the expression cassette, mRNA 5'- and 3'-untranslated regions (UTR), translational start codon (AUG) context, A+T composition of cDNA, transcriptional and translational blocks, nature of secretion signal, endogenous protease activity, host strain physiology, media and growth conditions, and fermentation parameters. All these factors should be considered in designing an optimal production system. The inherent ability of P. pastoris to convert the zymogen (pro-enzyme) form of matrix metalloproteinases (MMP) into active mature forms (which tend to self-degrade, and in some instances also cause damage to cells), largely limits the use of this system for the production of MMP. However, this problem can be partly alleviated by co-expression of tissue inhibitor of MMP (TIMP-1).

Binding of the radiolabeled glycine site antagonist [3H]MDL 105,519 to homomeric NMDA-NR1a receptors.

We have characterized the binding of [3H]MDL 105,519 ((E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1 H-indole-2-carboxylic acid), a NMDA receptor glycine recognition site antagonist, to homomeric NMDA subunit 1a (NR 1a) receptors. Chinese hamster ovary cells (CHO-K1) were transfected with the rat NR 1a gene and cell lines stably expressing the receptor were isolated from amongst clones resistant to the neomycin analog G418. Saturation analysis indicated that the radioligand bound to the homomeric receptor with a similar high affinity (Kd = 1.8 nM) to that reported for the native receptor. The binding capacity (Bmax) was 370 fmol/mg protein reflecting approximately 110000 receptors per cell. The radioligand interacted with a single class of binding sites as indicated by linear Scatchard transformation of the saturation data and a unitary Hill slope in competition experiments. Thus, the MDL 105,519 recognition site is present on the NR 1a subunit and has similar radioligand binding properties to the native brain-derived receptor. However, pharmacologic characterization of [3H]MDL 105,519 binding indicated that agonists were weaker competitors at the homometric receptor relative to the native receptors. In contrast, representative of three distinct chemical classes of glycine site antagonists exhibited similar potencies at both types of binding sites.

Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeast Pichia pastoris.

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leech Haementeria ghilianii. In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an inframe fusion of the ghilanten-coding sequences with the region encoding the pre-pro alpha-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter. Pichia pastoris yeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5'-AOX1 locus via homologous recombination. Both strains yielded His+ transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level of r-ghilanten than KM 71. Significant clonal variation in the expression of r-ghilanten was found among the His+ transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales. r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.

Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression.

Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.

Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies.

We have constructed a synthetic secretion cassette encoding the alpha-factor prepro leader peptide from Saccharomyces cerevisiae fused to mouse epidermal growth factor (mEGF). This was used to compare the secretion of mEGF, a 53-amino acid polypeptide, in S. cerevisiae and Pichia pastoris. In both yeasts the leader sequence was accurately and efficiently cleaved showing that the S. cerevisiae-derived alpha-factor prepro region is correctly recognised and processed in P. pastoris. Of the total mEGF produced, over 90% was exported to the culture supernatant, although the final level of accumulation was dependent on the composition of the growth medium. With P. pastoris there was instability of the protein in minimal medium (yeast nitrogen base), probably caused by extracellular proteases. This was overcome by adding 1% Casamino acids and buffering the medium to pH 6.0. To increase the level of secreted mEGF we have developed a method for rapidly screening large numbers of P. pastoris transformants for the presence of many copies of a foreign gene. Using this procedure we isolated a strain containing 19 integrated copies of the mEGF gene which secreted 450 micrograms/ml of mEGF in high-density fermentations. Characterisation of the yeast-derived mEGF showed the presence of truncated forms, mEGF1-51 and mEGF1-52, as was found with S. cerevisiae-secreted human EGF [George-Nascimento et al., Biochemistry 27 (1988) 797-802]. In addition, the full-length protein, mEGF1-53, was secreted by P. pastoris.

High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene.

We have used the methylotrophic yeast, Pichia pastoris, to express high levels of tetanus toxin fragment C, a potential subunit vaccine against tetanus. In high biomass fermentations fragment C was induced to 27% of total cell protein or about 12 g/l of culture. The purified protein was as effective as native fragment C in immunizing mice. In order to optimize fragment C production, we have examined the parameters affecting foreign gene expression in Pichia. The level of expression was found to be largely independent of the site of chromosomal integration of the gene (AOX1 or HIS4), the type of integrant (insertion or transplacement), and the methanol utilisation phenotype of the host strain (Mut+ or Muts). The most important factor in obtaining high levels was the presence of multiple integrated copies of the fragment C expression cassette. Multicopy clones could be isolated from transformations using DNA fragments targeted for single-copy transplacement into the chromosome. These multicopy transformants were surprisingly stable over multiple generations during growth and induction in high cell density fermentations. Analysis of chromosomal DNA from these clones suggests that they arose by circularization of the transforming DNA fragment in vivo followed by multiple insertion into the chromosome via repeated single crossover recombination, in addition to the expected transplacement event. We have found this to be a general phenomenon and have used these multicopy "transplacement" clones to obtain high-level expression of several other foreign genes in Pichia.

High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris.

Human tumor necrosis factor (TNF) alpha/cachectin was expressed in the methylotrophic yeast Pichia pastoris at high levels (greater than 30% of the soluble protein) by placing the TNF cDNA under the control of regulatory sequences derived from the alcohol oxidase gene. Batch fermentor cultures at cell densities of 50 and 85 g dry cell weight/L contained approximately 6 X 10(10) and 10(11) units/L TNF bioactivity (6 and 10 g/L TNF), respectively. TNF productivity of 0.108 g L-1 h-1 was obtained in the continuous mode on glycerol- and methanol-mixed feed at 25 g dry cell weight/L cell density. TNF contained in the yeast cell lysate was soluble, displayed full cytotoxic activity, and was recognized by antibodies prepared against TNF derived from Escherichia coli. TNF was purified to greater than 95% purity with greater than 75% recovery by using three sequential chromatographic steps with a coordinated effluent-affluent buffer scheme which allowed one eluate to also serve as the loading buffer for the succeeding column. The amino acid composition, NH2-terminal amino acid sequence, isoelectric point, and minimal molecular weight determined for TNF corroborated those properties predicted from the nucleotide sequence. Sedimentation data indicated that TNF in the native form is a compact trimer held by noncovalent interactions. Circular dichroic spectra of TNF resemble those of proteins with high beta structure. TNF exhibited cachectic activity on mouse 3T3-L1 cells at about the same equivalence as the cytotoxic activity toward mouse L929 cells. In the criteria examined, TNF derived from P. pastoris closely resembles TNF derived from recombinant E. coli and human HL-60 cells.

High level expression of heterologous proteins in methylotrophic yeast Pichia pastoris.

Expression of human tumor necrosis factor-alpha (TNF) and four different TNF analogs has been studied in Pichia pastoris by utilizing the alcohol oxidase gene promoter. TNF expression level in certain transformants accounted for as much as 36% of the soluble protein. TNF expression was stably maintained during high cell density fermentation (100 g dry cell weight/liter) resulting in a TNF production level of 6-10 g/liter. TNF contained in P. pastoris cell lysates was biologically active as determined by its cytotoxic effect on murine L-929 fibroblast cells and the bioactivity was retained for at least 6 months in the lysates stored frozen at -20 degrees C in the presence of protease inhibitor PMSF. TNF expressed in P. pastoris was recognized by monoclonal antibodies prepared against recombinant Escherichia coli derived TNF. TNF purified from P. pastoris has the expected N-terminal amino acid sequence and specific activity of 10(7) units/mg protein. TNF analogs were also expressed at levels comparable to that of native TNF. Three of the four analogs were insoluble when produced in P. pastoris.

Isolation and characterization of mutants of Kluyveromyces lactis defective in lactose transport.

Mutants of Kluyveromyces lactis defective in lactose transport were identified among lactose-resistant revertants of lactose-sensitive strains. The mutations are closely linked to the beta-galactosidase gene, LAC4, and they are located in a previously identified gene, LAC12, which has been shown to code for a lactose permease. Our data establish that LAC12 is the only lactose permease gene in K. lactis. The lactose permease also transports galactose. LAC12 is transcribed in a direction opposite to that of LAC4, there being about 2.5 kb between their transcription start sites. Transcription of LAC12 is inducible as is that of all other structural genes in the lactose-galactose regulon of K. lactis.

Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation of Pichia pastoris.

A two-step method for the selection of transformants of prototrophic industrial strains of the methylotrophic yeast Pichia pastoris has been developed. This method is based on our observation that P. pastoris cannot use sucrose as the sole carbon source (Suc-) and that introduction of the invertase gene (SUC2) of Saccharomyces cerevisiae renders P. pastoris Suc+. P. pastoris was transformed with a plasmid which contains the SUC2 gene of S. cerevisiae and an autonomously replicating sequence PARS1 from P. pastoris. The transformants were initially allowed to regenerate on medium containing dextrose and the regenerated cells were pooled and plated on sucrose medium to screen for Suc+ transformants. It was shown that the Suc+ transformants of P. pastoris with the autonomously replicating plasmid were highly unstable with respect to the plasmid maintenance, even when grown on sucrose as the sole carbon and energy source. This high instability was attributed to an efficient cross-feeding by Suc- segregants on glucose and fructose generated due to hydrolysis of sucrose by the invertase enzyme secreted by Suc+ cells. Spontaneous integration of the plasmid DNA resulting in a stable Suc+ phenotype was also observed. However, stable Suc+ transformants were obtained more readily by integration of SUC2 into P. pastoris genome following transformation with a linearized plasmid with the ends homologous to P. pastoris HIS4 locus. All such integrants were completely stable for Suc+ phenotype after 20 generations of growth in a nonselective medium.

Construction of strains of Saccharomyces cerevisiae that grow on lactose.

We have constructed strains of Saccharomyces cerevisiae that grow on lactose (Lac+). S. cerevisiae strain YNN27, which, like all S. cerevisiae, is unable to grow on lactose, was transformed with pKR1B-LAC4-1. This plasmid has a selectable marker gene conferring resistance to the antibiotic G418 and carries a 13-kilobase region of the Kluyveromyces lactis genome including LAC4, a beta-galactosidase gene. Transformants were selected first for G418 resistance and then for growth on lactose. Southern hybridization experiments showed that Lac+ transformants had integrated 15-25 tandem copies of the vector into a host chromosome. Several lines of evidence indicate that the Lac+ phenotype in pKR1B-LAC4-1-transformed S. cerevisiae is due to expression of a K. lactis lactose permease gene that lies between 2 and 8.6 kilobase upstream of LAC4 and also to expression of LAC4. The permease gene has been designated LAC12.

Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903.

Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS). To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (L-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol. Several ARS-containing plasmids were selected from a K. lactis recombinant DNA library in K. lactis and in Saccharomyces cerevisiae. Two of four ARS clones selected in K. lactis promoted transformation frequencies of 5-10 X 10(2) G418-resistant cells/micrograms of plasmid DNA. This frequency of transformation was at least twice as high as with ARS clones selected in S. cerevisiae. The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug. In the absence of selection pressure less than 5% of the cells retained the drug-resistance phenotype. Plasmids containing the ARS1 or 2 mu replicon of S. cerevisiae failed to transform K. lactis for G418 resistance. Inclusion of S. cerevisiae centromere, CEN4, in a K. lactis ARS recombinant plasmid did not increase the stability of the plasmid in K. lactis, and marker genes on the vector segregated predominantly 4-:0+ through meiosis. We conclude that neither the ARS sequences or the centromere of S. cerevisiae was functioning in K. lactis.

Inhibition of the microsomal stearoyl coenyme A desaturation by divalent copper and its chelates.

Divalent copper and copper complexes of tyrosine, histidine and lysine inhibited at low concentrations the stearoyl-CoA desaturation reaction in both chicken liver microsomes and in a purified system consisting of chicken liver delta 9 terminal desaturase, cytochrome b5, ascorbate and liposome. Although the copper chelates lowered the steady-state level of ferrocytochrome b5 by 20%, and partially inhibited the NADH-ferricyanide reductase activity, the availability of the ferrocytochrome b5 during the time course of desaturation was not affected, indicating that the site of inhibition of desaturation was at the terminal step, i.e., on the delta 9 terminal desaturase. The presence of chalates during catalysis was essential for the observed inhibition. Based on the observation that O2 is involved in the desaturation and that there is an initial electron reduction of desaturase iron, it is plausible that the copper chelates are inhibiting by acting as superoxide scavengers.

Interaction of the fluorescent analogue stearoyl-(1,N6)-etheno coenzyme A with chicken liver acetyl coenzyme A carboxylase.

The interaction of stearoyl-(1,N6)-etheno coenzyme A (stearoyl-epsilon-CoA) with acetyl coenzyme A carboxylase was investigated by using fluorescence spectroscopy. The fluorescence emission of stearoyl-epsilon-CoA was partially quenched by acetyl coenzyme A carboxylase. Analysis of the data for dissociation constant (KD) and the stoichiometry of the interaction (n) gave values of 5.06 nM and 1.2, respectively, at pH 7.6 in 50 mM Tris-HCl and 25 degrees C. The KD value is comparable to the inhibition constant (Ki) obtained previously by others for the inhibition of rat liver acetyl coenzyme A carboxylase by long chain fatty acyl-CoAs. Citrate (which is known to polymerize and thus activate carboxylase) caused a partial quenching of the protein fluorescence of carboxylase, presumably due to polymerization of the enzyme. The quenching of the stearoyl-epsilon-CoA fluorescence caused by carboxylase as well as the inhibition of carboxylase activity by stearoyl-epsilon-CoA was reversed by citrate, but only in the presence of 6-O-methylglucose polysaccharide which forms a stable complex with fatty acyl-CoA. This shows that the stearoyl-epsilon-CoA bound to the enzyme is displaced by citrate only in the presence of an acceptor of fatty acyl-CoA. These results support the reciprocal relationship of citrate and fatty acyl-CoA in the regulation of acetyl coenzyme A carboxylase.