Safety assessment of the biotechnologically produced human-identical milk oligosaccharide 3-Fucosyllactose (3-FL).

3-Fucosyllactose (3-FL), a highly abundant complex carbohydrate in human breast milk, functions as a prebiotic promoting early microbial colonization of the gut, increasing pathogen resistance and modulating immune responses. To investigate potential health benefits, 3-FL was produced by fermentation using a genetically modified E. coli K12 strain. The safety assessment of 3-FL included acute oral toxicity, in vitro and in vivo assessment of genetic toxicity, and a subchronic rodent feeding study. 3-FL was not acutely toxic at 5000 mg/kg bw, and there was no evidence of genetic toxicity in the bacterial reverse mutation test and chromosomal aberration assay. There was a repeatable statistically-significant trend in the 4-h S9-activated test conditions in the in vitro micronucleus assay; the confirmatory in vivo mouse micronucleus study was negative at all doses. Dietary subchronic exposure of rats to 3-FL (5% and 10%) did not produce any statistical or biologically-relevant differences in growth, food intake or efficiency, clinical observations, or clinical or anatomic pathology changes at average daily intakes of 5.98 and 7.27 g/kg bw/day for males and females, respectively. The weight of evidence from these studies support the safe use of 3-FL produced using biotechnology as a nutritional ingredient in foods.

Effect of conjugated linoleic acid on fungal delta6-desaturase activity in a transformed yeast system.

Conjugated linoleic acid (CLA; 18:2), a group of positional and geometric isomers of linoleic acid (LA; 18:2n-6), has been shown to modulate immune function through its effect on eicosanoid synthesis. This effect has been attributed to a reduced production of n-6 polyunsaturated fatty acid (PUFA), the precursor of eicosanoids. Since delta6-desaturase is the rate-limiting enzyme of the n-6 PUFA production, it is our hypothesis that CLA, which has similar chemical structure to LA, interacts directly with delta6-desaturase. A unique and simple model, i.e., baker's yeast (Saccharomyces cerevisiae) transformed with fungal delta6-desaturase gene, previously established, was used to investigate the direct effect of CLA on delta6-desaturase. This model allows LA to be converted to y-linolenic acid (GLA; 18:3n-6) but not GLA to its metabolite(s). No metabolites of CLA were found in the lipids of the yeast transformed with delta6-desaturase. The inability to convert CLA to conjugated GLA was not due to the failure of yeast cells to take up the CLA isomers. CLA mixture and individual isomers significantly inhibited the activity of delta6-desaturase of the transformed yeast in vivo. Even though its uptake by the yeast was low, CLA c9,t11 isomer was found to be the most potent inhibitor of the four isomers tested, owing to its high inhibitory effect on delta6-desaturase. Since CLA did not cause significant changes in the level of delta6-desaturase mRNA, the inhibition of GLA production could not be attributed to suppression of delta6-desaturase gene expression at the transcriptional level.

Inhibitory effect of conjugated linoleic acid on linoleic acid elongation in transformed yeast with human elongase.

Conjugated linoleic acid (CLA; 18:2) refers to a group of positional and geometric isomers derived from linoleic acid (LA; delta9,12-18:2). Using a growing baker's yeast (Saccharomyces cerevisiae) transformed with human elongase gene, we examined the inhibitory effect of CLA at various concentrations (10, 25, 50, and 100 microM) on elongation of LA (25 microM) to eicosadienoic acid (EDA; delta11,14-20:2). Among four available individual CLA isomers, only c9,t11- and t10,c12-isomers inhibited elongation of LA to EDA. The extent of inhibition (ranging from 20 to 60%) was related to the concentration of CLA added to the medium. In the meantime, only these two isomers, when added at 50 microM to the media, were elongated to conjugated FDA (c11,t13- and t12,c14-20:2) by the same recombinant elongase at the rate of 28 and 24%, respectively. The inhibitory effect of CLA on LA elongation is possibly due to competition between CLA isomers and LA for the recombinant elongase. Thus, results from this study and a previous study suggest that the biological effect of CLA is exerted through its inhibitory effect on delta6-desaturation as well as elongation of LA which results in a decrease in long-chain n-6 fatty acids and consequently the eicosanoid synthesis.

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells.

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.

Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids.

The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.

Identification and characterization of an enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty acids.

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of gamma-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Delta6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Delta5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

cDNA cloning and characterization of human Delta5-desaturase involved in the biosynthesis of arachidonic acid.

Two human expressed sequence tag (EST) cDNA sequences with identity with Delta(5)- and Delta(6)-desaturases from a filamentous fungus, Mortierella alpina, were identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. (Palo Alto, CA, U.S.A.). An oligonucleotide complementary to the 3' EST cDNA sequences was used to screen human liver cDNA using rapid amplification of cDNA ends (RACE)-PCR. The amplified DNA fragment had 98% identity with a putative open reading frame (ORF) predicted from a human genomic sequence, and encoded 444 amino acids. Expression of this ORF in mouse fibroblast cells demonstrated that the encoded protein was a Delta(5)-desaturase, as determined by the conversion of dihomo-gamma-linolenic acid (C(20:3,n-6)) into arachidonic acid (C(20:4,n-6)). The human Delta(5)-desaturase contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. A tissue expression profile revealed that this gene is highly expressed in fetal liver, fetal brain, adult brain and adrenal gland. A search of the existing databases led to localization of this ORF within a 14 kb interval flanked by the flap endonuclease-1 (FEN1) and vitelliform macular dystrophy (Best's disease; VMD2) loci of chromosome 11q12.

Polyunsaturated fatty acid-specific elongation enzymes.

We have isolated a novel gene (GLELO) from Mortierella alpina and its homologue (CEELO1) from Caenorhabditis elegans and demonstrate the involvement of their encoded proteins in the elongation of C(18) polyunsaturated fatty acids.

Cloning of delta12- and delta6-desaturases from Mortierella alpina and recombinant production of gamma-linolenic acid in Saccharomyces cerevisiae.

Two cDNA clones with homology to known desaturase genes were isolated from the fungus Mortierella alpina. The open reading frame in one clone encoded 399 amino acids and exhibited delta12-desaturase activity when expressed in Saccharomyces cerevisiae in the presence of endogenous fatty acid substrate oleic acid. The insert in another clone contained an open reading frame encoding 457 amino acids and exhibited delta6-desaturase activity in S. cerevisiae in the presence of exogenous fatty acid substrate linoleic acid. Expression of the delta12-desaturase gene under appropriate media and temperature conditions led to the production of linoleic acid at levels up to 25% of the total fatty acids in yeast. When linoleic acid was provided as an exogenous substrate to the yeast cultures expressing the delta6-desaturase activity, the level of gamma-linolenic acid reached 10% of the total yeast fatty acids. Co-expression of both the delta6- and delta12-desaturase cDNA resulted in the endogenous production of gamma-linolenic acid. The yields of gamma-linolenic acid reached as high as 8% of total fatty acids in yeast.

Concentrations and anti-Haemophilus influenzae activities of beta-casein phosphoforms in human milk.

BACKGROUND: The distribution and concentrations of six phosphorylated forms of human beta-casein, a major source of nutrition among breast-fed infants, have not been examined in milk samples without prior fractionation. In this study, the levels of beta-casein phosphoforms in untreated human milk samples were analyzed and their antiadhesion activities determined against Haemophilus influenzae, a pathogen implicated in middle ear infection in infants. METHODS: Human milk samples were analyzed using urea-polyacrylamide gel electrophoresis of whole-milk samples and scanning densitometry to determine the concentrations of beta-casein and its phosphoforms. A nontypable H. influenzae strain was radiolabeled to monitor its attachment to human pharyngeal cells in microtiter plates. Purified phosphoforms of beta-casein were preincubated for 15 minutes with radiolabeled bacteria to determine their antiadhesion activities. RESULTS: The average beta-casein concentration in 151 human milk samples was 5.37+/-2.26 mg/ml. On average, the phosphoforms in untreated milk are present in the following order ranked by concentration: tetra- > di- > non- > mono- > tri- > pentaphosphorylated beta-casein. The tri-, tetra-, and pentaphosphorylated forms of human beta-casein exhibited more than 60% inhibition of H. influenzae in the antiadhesion assay when used at a concentration of 0.6 to 0.9 mg/ml. CONCLUSION: The beta-casein level in untreated human milk is significantly higher than previously reported. The phosphoform distribution of beta-casein in individual donors varies widely. Anti-H. influenzae activity was detected in vitro among human beta-casein molecules with three or more phosphate groups.

Identification of Delta5-desaturase from Mortierella alpina by heterologous expression in Bakers' yeast and canola.

A DNA fragment with homology to Delta6-desaturases from borage and cyanobacteria was isolated after polymerase chain reaction amplification of Mortierella alpina cDNA with oligonucleotide primers corresponding to the conserved regions of known Delta6-desaturase genes. This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 446 amino acids from a M. alpina library. Expression of this open reading frame from an inducible promoter in Saccharomyces cerevisiae in the presence of various substrates revealed that the recombinant product had Delta5-desaturase activity. The effects of growth and induction conditions as well as host strain on activity of the recombinant Delta5-desaturase in S. cerevisiae were evaluated. Expression of the M. alpina Delta5-desaturase cDNA in transgenic canola seeds resulted in the production of taxoleic acid (Delta5,9-18:2) and pinolenic acid (Delta5,9,12-18:3), which are the Delta5-desaturation products of oleic and linoleic acids, respectively.

A controlled family history study of childhood-onset depressive disorder.

BACKGROUND: We studied the family psychiatric history of 125 youths with childhood-onset depressive disorder (a portion of whom developed bipolar disorder) and 55 psychiatric controls with nonaffective disorder. METHODS: Probands were classified according to prospectively observed clinical course in childhood. Family psychiatric history was determined by interviewers blind to probands' diagnosis, with mothers typically informing about themselves and about remaining first- and a all second-degree adult relatives. RESULTS: Families of affectively ill juveniles had 5-fold greater odds of lifetime depressive disorder and 2-fold greater odds of recurrent unipolar depressive disorder than did families of psychiatric controls. The higher risk of depression was most evident in first-degree and female relatives. Mothers of affectively ill youths were younger at onset of depression than were mothers of controls. Alcoholism and substance use disorders were more prevalent in relatives of affectively ill probands than in controls and cosegregated with familial depression. However, other covariates were more important at predicting patterns of familial depression. Familial illness patterns also varied somewhat with proband characteristics. CONCLUSIONS: Child probands with affective disorder identify families enriched with affective disorder (even compared with families of psychiatric controls), suggesting that juvenile- and adult-onset forms of this condition share the same diathesis. Rates of affective illness in the families of depressed youngsters also are notably higher than population-based estimates. The findings therefore indicate that very-early-onset affective disorder is familial and that pedigrees ascertained through affectively ill children are good candidates for family and genetic studies.

Expression and characterization of phosphorylated recombinant human beta-casein in Escherichia coli.

Specific serine and threonine residues of recombinant human beta-casein produced in Escherichia coli were shown to be phosphorylated in vivo when human casein kinase II was coexpressed in the same plasmid. All of the phosphorylated forms found in the native protein were also detected in the recombinant protein. The phosphorylation of recombinant human beta-casein was confirmed by immunoblots, fast protein liquid chromatography, urea-polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and liquid chromatography-mass spectrometry. The results indicate that the substrate specificity of casein kinase II in vivo was unaffected in its recombinant form. This is the first demonstration of in vivo phosphorylation of specific residues of a multiphosphorylated protein produced in E. coli with a single plasmid.

Relationship of depressive, conduct, and comorbid disorders and social functioning in childhood.

OBJECTIVE: To examine the relationship of depressive, conduct, and comorbid disorders and social functioning in psychiatrically referred youths. METHOD: Subjects were 94 boys and 67 girls (mean age at initial assessment = 11.5 years) who were repeatedly evaluated with standardized instruments during a mean interval of 4.4 years. On the basis of their diagnoses during the follow-up, children were designated as having had depressive, conduct, or both (comorbid) disorders or other conditions. Two domains of social functioning were assessed: social competence and self-esteem. RESULTS: Longitudinal analyses revealed that at any given point in time, depressive, conduct, and comorbid disorders were associated with low social competence and depressive disorder also was associated with low self-esteem. At the approximate age of 15 years, on average, children with a history of conduct or comorbid disorders had lower social competence than did children with a history of depressive disorder, but these groups endorsed similar levels of self-esteem. CONCLUSION: Some areas of social dysfunction associated with comorbid depressive and conduct disorders appear to reflect mostly the effects of conduct disorder. The latter condition has a more severe and longer-term impact on children's social competence than does depression. In addition, whereas depression has an adverse effect on self-esteem, this effect appears to be temporary.

Non-Hodgkin's lymphoma masquerading as empyema thoracis.

Non-hodgkin's lymphoma has varied presentations. Malignant lymphoma arising in chronic pyothorax is very rare and has been reported from Japan. We report a case of non-hodgkin's lymphoma presenting as empyema thoracis.

Psychiatric disorder and metabolic control among youths with IDDM. A longitudinal study.

OBJECTIVE: To investigate the longitudinal relationship between psychiatric diagnostic variables and metabolic control among youths with IDDM. RESEARCH DESIGN AND METHODS: A group of 88 youths, 8 to 13 years old at onset of IDDM, were evaluated repeatedly during a 9-year follow-up period, on average, using a standardized psychiatric protocol. Levels of HbA1 were also assessed repeatedly. Psychiatric diagnoses were derived independently of HbA1 values. RESULTS: In univariate longitudinal analyses, the psychiatric diagnosis of noncompliance with medical treatment was significantly related to HbA1 level. There was a trend of an association between any major psychiatric disorder, as well as nondepressive disorder, and HbA1. Interaction terms between IDDM duration (or age) and psychiatric variables were also significantly related to metabolic control. According to the final multivariate model of repeatedly assessed HbA1, noncompliance with medical treatment (irrespective of IDDM duration) and the interaction between nondepressive psychiatric disorder and IDDM duration contributed to worse metabolic control. CONCLUSIONS: We found some support for the hypothesis that psychiatric morbidity negatively affects blood glucose regulation and that its consequences are more marked the longer young patients have had IDDM. We did not confirm the hypothesis that depressive illness has particularly deleterious consequences on metabolic control. Noncompliance with medical treatment and having had nondepressive psychiatric illness in interaction with IDDM duration account for a statistically significant but clinically modest amount of variability in HbA1 over time. The weak relationship among these variables may explain the inconsistent findings in the literature regarding psychiatric morbidity and metabolic control.

Biomedical and psychiatric risk factors for retinopathy among children with IDDM.

OBJECTIVE: Illness duration and glycemic control influence the development of retinopathy in childhood-onset insulin-dependent diabetes mellitus (IDDM). Psychiatric disorders and sociodemographic factors also affect diabetes-related outcomes. However, biomedical and psychosocial factors have not been examined together in modeling the risk of retinopathy. RESEARCH DESIGN AND METHODS: We conducted a single-site prospective longitudinal study of 66 children (aged 8-13 years) newly diagnosed with IDDM. Repeated assessments served to derive psychiatric diagnoses. Poor glycemic control was defined as the upper 15th percentile of all HbA1 values. After a median follow-up of 10 years, severity of retinopathy was determined. It was modeled with a stepwise polychotomous regression procedure using antecedent biomedical and psychosocial variables. RESULTS: Young adults with childhood-onset IDDM were found to be at increased risk of retinopathy the longer they had IDDM, the more persistently they evidenced poor antecedent glycemic control, and the longer they suffered from depressive illness. These three factors operated individually and additively, with duration of IDDM conferring a baseline level of risk. In depressed patients (27%), depression onset antedated the detection of retinopathy generally by 7 years. CONCLUSIONS: Duration of childhood-onset IDDM confers a baseline level of risk of retinopathy irrespective of glycemic control; antecedent clinical depression is also a risk factor. Depression therefore may serve as a marker of vulnerability and help to identify a subgroup of patients at risk for complications. The findings raise the question whether timely treatment of depression could forestall diabetic retinopathy.