NFĸB Targeting in Bone Marrow Mesenchymal Stem Cell-Mediated Support of Age-Linked Hematological Malignancies.

Mesenchymal stem cells (MSCs) can become dysfunctional in patients with hematological disorders. An unanswered question is whether age-linked disruption of the bone marrow (BM) microenvironment is secondary to hematological dysfunction or vice versa. We therefore studied MSC function in patients with different hematological disorders and found decreased MHC-II except from one sample with acute myeloid leukemia (AML). The patients' MSCs were able to exert veto properties except for AML MSCs. While the expression of MHC-II appeared to be irrelevant to the immune licensing of MSCs, AML MSCs lost their ability to differentiate upon contact and rather, continued to proliferate, forming foci-like structures. We performed a retrospective study that indicated a significant increase in MSCs, based on phenotype, for patients with BM fibrosis. This suggests a role for MSCs in patients transitioning to leukemia. NFĸB was important to MSC function and was shown to be a potential target to sensitize leukemic CD34+/CD38- cells to azacitidine. This correlated with their lack of allogeneic stimulation. This study identified NFĸB as a potential target for combination therapy to treat leukemia stem cells and showed that understanding MSC biology and immune response could be key in determining how the aging BM might support leukemia. More importantly, we show how MSCs might be involved in transitioning the high risk patient with hematological disorder to AML.

The validity of nasal endoscopy in patients with chronic rhinosinusitis-An inter-rater agreement study.

OBJECTIVES: Nasal endoscopy is a cornerstone in diagnosing sinonasal disease, but different raters might generate different results using the technique. Our study aims to evaluate the agreement between multiple raters to assess the validity of nasal endoscopy. DESIGN/PARTICIPANTS: Three independent and blinded raters evaluated 28 patients (56 nasal cavities) diagnosed with chronic rhinosinusitis according to the European Position Paper on Rhinosinusitis and Nasal Polyps. The ratings were compared using unweighted Fleiss' kappa coefficients (Kf ) for each objective parameter. SETTING: The department of Otorhinolaryngology, Odense University Hospital, Denmark. MAIN OUTCOME MEASURES: The ratings were quantified in a modified Lund-Kennedy endoscopy score and focused on the objective parameters specified in the diagnostic criteria: polyps, oedema and discharge. RESULTS: The raters agreed on the findings concerning polyps and discharge but not regarding oedema with the inter-rater agreement for the different parameters being: polyps Kf =.66 (SE .07, P<.001), oedema Kf =.05 (SE .07, P=.21), discharge Kf =.35 (SE .08, P<.001), oedema exclusively in middle meatus Kf =-.07 (SE .04, P=.8) and discharge exclusively in middle meatus Kf =.16 (SE .07, P=.01). CONCLUSION: Using nasal endoscopy, the evaluation of polyps by multiple raters showed sufficient reliability indicating an acceptable objective evaluation. The evaluation of discharge achieved a fair level of agreement while the assessment of oedema could not achieve a sufficient reliability questioning the inclusion of oedema in the criteria for diagnosing sinonasal disease.

Dietary fiber effects in chronic kidney disease: a systematic review and meta-analysis of controlled feeding trials.

BACKGROUND/OBJECTIVES: Chronic kidney disease (CKD) is a major health concern associated with increased risk of cardiovascular disease, morbidity and mortality. Current CKD practice guidelines overlook dietary fiber, which is chronically low in the renal diet. However, increasing dietary fiber has been proposed to ameliorate the progress of CKD. We therefore conducted a systematic review and meta-analysis on the effect of dietary fiber intake on serum urea and creatinine as classical markers of renal health in individuals with CKD. SUBJECTS/METHODS: We searched MEDLINE, EMBASE, CINHAL and the Cochrane Library for relevant clinical trials with a follow-up ⩾7 days. Data were pooled by the generic inverse variance method using random-effects models and expressed as mean difference (MD) with 95% confidence intervals (95% CIs). Heterogeneity was assessed by the Cochran Q statistic and quantified by I(2). RESULTS: A total of 14 trials involving 143 participants met the eligibility criteria. Dietary fiber supplementation significantly reduced serum urea and creatinine levels in the primary pooled analyses (MD, -1.76 mmol/l (95% CI, -3.00, -0.51), P<0.01 and MD, -22.83 mmol/l (95% CI, -42.63, -3.02), P=0.02, respectively) with significant evidence of interstudy heterogeneity only in the analysis of serum urea. CONCLUSIONS: This is the first study to summarize the potential beneficial effects of dietary fiber in the CKD population demonstrating a reduction in serum urea and creatinine, as well as highlighting the lack of clinical trials on harder end points. Larger, longer, higher-quality clinical trials measuring a greater variety of uremic toxins in CKD are required (NCT01844882).

Paraoxonase 1 arylesterase activity and mass are reduced and inversely related to C-reactive protein in patients on either standard or home nocturnal hemodialysis.

UNLABELLED: Human serum paraoxonase (PON1) activity is reduced in standard hemodialysis (SHD) (4 hours, 3 days/week) patients. Home nocturnal hemodialysis (HNHD) (8 hours, 6 days/week), provides a greater dialysis dose resulting in a greater clearance of metabolites. Whether improvements in the metabolic milieu of HNHD patients results in different PON1 activity levels compared to SHD patients is unclear. We determined serum PON1 mass and arylesterase activities in a group of HNHD patients and compared them to SHD patients and a group of healthy controls (HC). PATIENTS AND METHODS: We measured PON1 arylesterase activity and mass, C-reactive protein (CRP), cystatin C, total and high-density lipoprotein (HDL) cholesterol, triglycerides, apolipoproteins A-I and B in 15 HNHD, 15 SHD and 15 HC participants. RESULTS: PON1 arylesterase activity (p < 0.001) and mass (p < 0.05) were significantly higher in HC participants compared to SHD and HNHD participants, although no significant differences were noted between HD groups. CRP (p < 0.05) was significantly higher in SHD compared to HC participants and there were no significant differences noted between HD groups. Cystatin C (p < 0.001) was significantly different among the 3 groups. There were no significant differences noted in any lipoprotein parameters among the groups. PON1 activity (r = -0.636, p < 0.001) and mass (r = -0.425, p = 0.019) were inversely correlated with CRP in HD patients. CONCLUSION: PON1 is reduced in HNHD patients compared to HC subjects, independent of the concentration of HDL cholesterol. Within subjects on HD, the combination of increased CRP and reduced PON1 may identify subjects at a high risk for cardiovascular complications.

Identification of a siderophore receptor required for ferric ornibactin uptake in Burkholderia cepacia.

Ornibactins are linear hydroxamate siderophores produced by Burkholderia cepacia with peptide structures similar to that of pyoverdines produced by the fluorescent pseudomonads. The gene encoding the outer membrane receptor (orbA) was identified, sequenced, and demonstrated to have significant homology with hydroxamate receptors produced by other organisms. The orbA precursor was predicted to be a protein with a molecular mass of 81 kDa. An orbA mutant was constructed and demonstrated to be unable to take up (59)Fe-ornibactins or to grow in medium supplemented with ornibactins. Outer membrane protein profiles from the parent strain, K56-2, revealed an iron-regulated outer membrane protein of 78 kDa that was not detectable in the K56orbA::tp mutant. When this mutant harbored a plasmid containing the orbA gene, the 78-kDa protein was present in the outer membrane protein profiles and the mutant was able to utilize ornibactin to acquire iron. The orbA mutant was less virulent in a chronic respiratory infection model than the parent strain, indicating that ornibactin uptake and utilization are important in the pathogenesis of B. cepacia respiratory infections.

Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers.

The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined. The dimerization constant for cro has been measured as a prelude to these binding studies. Here, the energetics of cro binding to O(R) are evaluated using quantitative DNaseI footprint titration techniques. Binding data for wild-type and modified O(R) site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O(R) sites. Binding of cro dimers is strongest to O(R)3, then O(R)1 and lastly, O(R)2. Adjacently bound repressors exhibit positive cooperativity ranging from -0.6 to -1.0 kcal/mol. Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation. This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI-cro cooperativity.

Extracellular domains drive homo- but not hetero-dimerization of erbB receptors.

Many different growth factor ligands, including epidermal growth factor (EGF) and the neuregulins (NRGs), regulate members of the erbB/HER family of receptor tyrosine kinases. These growth factors induce erbB receptor oligomerization, and their biological specificity is thought to be defined by the combination of homo- and hetero-oligomers that they stabilize upon binding. One model proposed for ligand-induced erbB receptor hetero-oligomerization involves simple heterodimerization; another suggests that higher order hetero-oligomers are 'nucleated' by ligand-induced homodimers. To distinguish between these possibilities, we compared the abilities of EGF and NRG1-beta1 to induce homo- and hetero-oligomerization of purified erbB receptor extracellular domains. EGF and NRG1-beta1 induced efficient homo-oligomerization of the erbB1 and erbB4 extracellular domains, respectively. In contrast, ligand-induced erbB receptor extracellular domain hetero-oligomers did not form (except for s-erbB2-s-erbB4 hetero-oligomers). Our findings argue that erbB receptor extracellular domains do not recapitulate most heteromeric interactions of the erbB receptors, yet reproduce their ligand-induced homo-oligomerization properties very well. This suggests that mechanisms for homo- and hetero-oligomerization of erbB receptors are different, and contradicts the simple heterodimerization hypothesis prevailing in the literature.

Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding I: analysis of cro dimerization from nanomolar to micromolar concentrations.

The cro repressor from bacteriophage lambda is an important and classical transcription regulatory protein that binds DNA operator sites as a dimer. Therefore, a complete understanding of gene regulation by cro requires knowledge of the coupled energetics of its protein dimerization and site-specific DNA binding. A method is described by which cro repressor can be labeled in vivo with [(35)S]methionine to a specific activity of 2 x 10(15) cpm/mol. As a prelude to binding studies, the association equilibrium of cro was determined over the range 10(-)(9)-10(-)(3) M using large-zone analytical gel chromatography with radiolabeled repressor. The data are best described by a monomer-dimer stoichiometry with an equilibrium constant of 3.07 (+/-1.08) x 10(6) M(-)(1) total cro monomer. Stokes radii for monomers and dimers were evaluated from the resolved gel partition coefficients. Under the conditions employed in this study (10 mM Bis-Tris, 200 mM KCl, 2.5 mM MgCl(2), 1 mM CaCl(2), 100 microg/mL BSA, pH 7.0, 20 degrees C), self-association of cro to species with assembly states greater than dimers is not observed.

Threonine dehydrogenase is a minor degradative pathway of threonine catabolism in adult humans.

The threonine dehydrogenase (TDG) pathway is a significant route of threonine degradation, yielding glycine in experimental animals, but has not been accurately quantitated in humans. Therefore, the effect of a large excess of dietary threonine, given either as free amino acid (+Thr) or as a constituent of protein (+P-Thr), on threonine catabolism to CO(2) and to glycine was studied in six healthy adult males using a 4-h constant infusion of L-[1-(13)C]threonine and [(15)N]glycine. Gas chromatography-combustion isotope ratio mass spectrometry was used to determine [(13)C]glycine produced from labeled threonine. Threonine intakes were higher on +Thr and +P-Thr diets compared with control (126, 126, and 50 micromol x kg(-1) x h(-1), SD 8, P < 0.0001). Threonine oxidation to CO(2) increased threefold in subjects on +Thr and +P-Thr vs. control (49, 45, and 15 micromol x kg(-1) x h(-1), SD 6, P < 0.0001). Threonine conversion to glycine tended to be higher on +Thr and +P-Thr vs. control (3.5, 3.4, and 1.6 micromol x kg(-1) x h(-1), SD 1.3, P = 0.06). The TDG pathway accounted for only 7-11% of total threonine catabolism and therefore is a minor pathway in the human adult.

Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase.

Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients. B. cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity. These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins. These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of Pseudomonas aeruginosa. I117 contained an insertion in a pvdA homolog, the gene for the enzyme L-ornithine N(5)-oxygenase, which catalyzes the hydroxylation of L-ornithine. Ornibactin synthesis in this mutant was partially restored when the precursor L-N(5)-OH-Orn was added to the culture medium. T10 contained an insertion in a pvdD homolog, which is a peptide synthetase involved in pyoverdine synthesis. beta-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter. Tn5-OT182 contains a lacZ gene that is expressed when inserted downstream of an active promoter. Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism. The B. cepacia pvdA homolog was approximately 47% identical and 59% similar to L-ornithine N(5)-oxygenase from P. aeruginosa. Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of (59)Fe-ornibactin uptake in I117. A chromosomal pvdA deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA. The pvdA mutants were less virulent than the parent strain in chronic and acute models of respiratory infection. A functional pvdA gene appears to be required for effective colonization and persistence in B. cepacia lung infections.

Isotopic enrichment of amino acids in urine following oral infusions of L-[1-(13)C]phenylalanine and L-[1-(13)C]lysine in humans: confounding effect of D-[13C]amino acids.

Urine sampling of the free amino acid pool serves to reflect plasma enrichment and is used as a noninvasive means to determine isotope enrichment in studies of amino acid metabolism. We determined the effect of D-[13C]phenylalanine and D-[13C]lysine content of tracers on urinary amino acid enrichment following oral infusion of L-[13C]phenylalanine in 18 preterm infants and L-[1-(13)C]lysine in seven healthy adult females. Urinary [13C]phenylalanine enrichment was higher (P < .0001) for L-[13C]phenylalanine containing 0.4% D-[13C]phenylalanine (28.6 +/- 7.1) versus L-[1-(13)C]phenylalanine that contained undetectable D-[13C]phenylalanine (10.2 +/- 1.5). D-[13C]phenylalanine, measured by chiral column gas chromatography-mass spectrometry (GC-MS), accounted for 10% to 30% (20.5% +/- 7%) of total phenylalanine in the urine of infants who received 0.4% D-[13C]phenylalanine, and was absent from the urine of infants receiving tracer with undetectable [13C]phenylalanine. Urinary L-[13C]phenylalanine enrichment did not differ between tracer groups (9.8 +/- 1.5 and 9.8 +/- 2.5). In adult females, the use of L-[1-(13)C]lysine (1.6% D-lysine) resulted in a higher (P < .02) urine total L,D-[13C]lysine enrichment compared with plasma enrichment (40.8 +/- 4.1 v 11.1 +/- 0.7). This study demonstrates the significant presence of D-[13C]amino acids in urine that originate as contaminants from commercially manufactured tracers, as a result of renal tubular discrimination of D-amino acids. A tracer containing detectable amounts of D-[13C]isomer cannot be recommended for any study in which urine will be used to reflect enrichment in the arterial plasma pool.

Threonine kinetics in preterm infants fed their mothers' milk or formula with various ratios of whey to casein.

BACKGROUND: Plasma threonine concentrations are elevated in infants fed formula containing a whey-to-casein protein ratio of 60:40 compared with concentrations in infants fed formula containing a ratio of 20:80 or human milk (60:40). OBJECTIVE: We studied whether degradation of excess threonine was lower in formula-fed infants than in infants fed their mothers' milk. DESIGN: Threonine kinetics were examined in 17 preterm infants (gestational age: 31+/-2 wk: birth weight: 1720+/-330 g) by using an 18-h oral infusion of [1-13C]threonine at a postnatal age of 21+/-11 d and weight of 1971+/-270 g. Five infants received breast milk. Formula-fed infants (n = 12) were randomly assigned to receive 1 of 3 formulas (5.3 g protein/MJ) that differed only in the whey-to-casein ratio (20:80, 40:60, and 60:40). RESULTS: Threonine intake increased significantly in formula-fed infants with increasing whey content of the formula (48.5, 56.4, and 63.2 micromol.kg(-1).h(-1), respectively; pooled SD: 2.2; P = 0.0001), as did plasma threonine concentrations (228, 344, and 419 micromol/L, respectively; pooled SD: 75; P = 0.03). Despite a generous threonine intake by infants fed breast milk (58.0+/-16.0 micromol.kg(-1).h(-1), plasma threonine concentrations remained low (208+/-41 micromol/L). Fecal threonine excretion and net threonine tissue gain, estimated by nitrogen balance, did not differ significantly among groups. Threonine oxidation did not differ significantly among formula-fed infants but was significantly lower in formula-fed infants fed than in infants fed breast milk (17.1% compared with 24.3% of threonine intake, respectively). CONCLUSION: Formula-fed infants have a lower capacity to oxidize threonine than do infants fed breast milk.

Free amino acids in milks of human subjects, other primates and non-primates.

Preterm and term transitional milks of human subjects and mature milks of human subjects, non-human primates and non-primates were analysed for free amino acids (AA) using precolumn phenylisothiocyanate derivatization and liquid chromatography. Differences in free AA between three types of human milk were small. Milks of pinnipeds (seals and sea lions) contained the highest levels of total free AA (8634-20,862 mumol/l), while the milks of cows and sheep had the lowest levels of total free AA (1061-1357 mumol/l). The milks of human subjects, chimpanzees (Pan troglodytes), gorillas (Gorilla gorilla), elephants (Elephas maximus), horses and pigs had intermediate levels of total free AA (3069-7381 mumol/l). Glutamic acid was the most abundant free AA in milks of human subjects (1339-2157 mumol/l), non-human primates (423-2528 mumol/l), elephants (1332 mumol/l), horses (1119 mumol/l), and cows (349 mumol/l). Taurine was the most abundant free AA in milks of pinnipeds (5776-13,643 mumol/l), pigs (1238 mumol/l), goats (1150 mumol/l) and sheep (341 mumol/l). Taurine was the second most abundant free AA in milks of human subjects and non-human primates, while histidine was the second most abundant free AA in milks of pinnipeds. Milks of each species had a distinctive free AA pattern which may reflect the relative importance of the free AA during early postnatal development.

Siderophore production by cystic fibrosis isolates of Burkholderia cepacia.

Sixty-one Burkholderia cepacia isolates from patients with cystic fibrosis (CF) and four plant isolates were screened for production of the siderophores salicylic acid (SA), pyochelin, cepabactin, and ornibactins and fingerprinted by a PCR-based randomly amplified polymorphic DNA (RAPD) method. Of the 24 RAPD types determined, 22 (92%) were associated with isolates that produced SA, 21 (87%) were associated with isolates that produced ornibactins, 15 (60%) were associated with isolates that produced pyochelin, and 3 (12%) were associated with isolates that produced cepabactin. Of the 24 RAPD types plus 2 phenotypic variants of types 1 and 9, 3 were associated with isolates that produced all four siderophores, 8 were associated with isolates that produced three siderophores, 12 were associated with isolates that produced two siderophores, and 3 were associated with isolates that produced only one siderophore. These results suggest that the numbers and types of siderophores produced by CF isolates of B. cepacia correlate with RAPD type and that SA and ornibactins are the most prevalent siderophores produced.

Deposition of collagen VI in the extracellular matrix during mouse embryogenesis correlates with expression of the alpha 3(VI) subunit gene.

Collagen VI is a microfibrillar component of the extracellular matrix that is predicted to have an important structural role in matrix organization and a biological function in mediating cell-matrix interactions. Secreted collagen VI molecules are composed of three distinct subunits, the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. To determine when, and in which tissues, collagen VI is likely to have a role in embryonic processes, we have analyzed the expression patterns of the three subunit chains during postimplantation mouse development by reverse transcriptase-PCR (RT-PCR), in situ hybridization, and immunofluorescence. No collagen VI protein could be detected in the mouse embryo until Day 11.5 of gestation, when low levels were localized within the mesoderm layer of the visceral yolk sac, the subepidermal matrix of branchial arches, and the vessel wall of the dorsal aorta. Levels of collagen VI mRNA and protein increased during the period from Days 12.5 to 14.5 in the visceral yolk sac, subepidermal mesenchyme, lung, gut, meninges, muscle, perichondrium, and vertebral column. The cartilage matrix of ribs and developing long bones was not stained with collagen VI antisera, but pericellular staining of chondrocytes was seen in both tissues. Low levels of collagen VI mRNA and protein were seen in the fetal liver except for the connective tissue of the liver capsule, which was highly stained. Collagen VI was first detected at significant levels in the developing heart on Day 14.5. These data demonstrate a tissue-specific onset of collagen VI synthesis and deposition in the extracellular matrix of developing mouse embryos at a much later stage of development than that reported for fibronectin or collagen I. Sensitive RT-PCR assays showed that alpha 1(VI) and alpha 2(VI) mRNAs were amplified from extracts of embryonic tissues as early as Day 7.5, while alpha 3(VI) mRNA was not detected until Day 10.5. Expression of the alpha 3(VI) gene immediately preceded the appearance of collagen VI protein in embryonic tissues. This correlation is consistent with the proposal that expression of alpha 3(VI) chains regulates the formation and secretion of collagen VI trimers and collagen VI matrix deposition during development.

Use of amino acid profiles of preterm and term human milks in evaluating scoring patterns for routine protein quality assessment of infant formulas.

Reports on the amino acid composition of human milk vary considerably with respect to concentrations of sulfur amino acids. Often, analyses forego tryptophan determination. A complete analysis of protein and amino acid concentrations was performed on human milk samples (5-10 days postpartum) collected from mothers of preterm (gestations of 25-32 weeks) and term (gestations of > 36 weeks) infants. Careful attention was given to quantitate amino acids such as cysteine and tryptophan, which are vulnerable to acidic hydrolysis conditions. Differences in concentrations of total amino acids (expressed on protein basis) between preterm and term milks were small, despite the higher true protein content of preterm milk versus term milk (19.20 versus 12.60 g/L). The methionine + cyst(e)ine contents of term and preterm milks (3.72-3.84 g/100 g protein) were comparable with those reported in 1991 by the Food and Agricultural Organization/World Health Organization (FAO/WHO) for mature human milk (4.20 g/100 g protein) but higher than those reported in 1991 by the European Commission (2.9 g/100 g protein). The amino acid pattern of human milk obtained in this study confirms that the 1991 FAO/WHO amino acid scoring pattern for predicting protein quality of infant formulas is representative of the amino acid quality of both preterm and term human milks.

Expression of collagen alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains in the pregnant mouse uterus.

The alpha 1, alpha 2, and alpha 3 chains of collagen VI and mRNAs for these chains were localized in the female mouse reproductive tract by immunofluorescence and in situ hybridization. High levels of collagen VI protein and mRNAs were present in the endometrium and myometrium of the uterus up to Day 4.5 of pregnancy. After embryo implantation, reduction in collagen VI protein within the decidualizing endometrium correlated with significantly reduced steady-state levels of alpha 1(VI), alpha 2(VI), and alpha 3(VI) mRNAs, indicating either transcriptional down-regulation of collagen VI gene expression or decreased stability of transcripts. High levels of alpha 1(V1) and alpha 2(VI) mRNAs, but not alpha 3(VI) mRNA, in cells surrounding the uterine epithelium in the mesometrial region did not correlate with deposition of collagen VI protein in this region. These data are consistent with an important role of alpha 3(VI) in assembly of collagen VI heterotrimers. However, distinct immunostaining with antiserum to alpha 2(VI) chains in the extracellular matrix immediately beneath the uterine epithelium may indicate that alpha 2(VI) chains are deposited without the alpha 1(VI) or alpha 3(VI) collagen chains. No collagen VI protein or mRNAs were detected in any tissue layers of the embryo on Days 5.5 or 6.5 of gestation.

Neutralizing monoclonal antibodies to an extracellular Pseudomonas cepacia protease.

Pseudomonas cepacia produces at least two extracellular proteases with apparent molecular masses of 36,000 and 40,000 Da. The 36-kDa protease has high proteolytic activity and the 40-kDa protease has low proteolytic activity with hide powder azure as a substrate. Monoclonal antibodies (MAbs) were raised against the purified 36- and 40-kDa proteases. Several MAbs directed against the 36-kDa protease were found to recognize the 40-kDa protease by Western immunoblot analysis. Similarly, a MAb directed against the 40-kDa protease recognized the 36-kDa protease, suggesting that these two proteases may be immunologically related. A MAb directed against the 36-kDa protease, designated 36-6-8, and a MAb directed against the 40-kDa protease (MAb G-11) cross-reacted with other extracellular proteases, such as Pseudomonas aeruginosa elastase and alkaline protease, Pseudomonas pseudomallei protease, and the Vibrio cholerae hemagglutinin/protease. MAb 36-6-8 neutralized the P. cepacia 36-kDa protease, P. aeruginosa elastase, P. pseudomallei protease, and V. cholerae hemagglutinin/protease but did not affect P. aeruginosa alkaline protease activity. In contrast, MAb G-11 to the 40-kDa protease neutralized only the P. cepacia 36-kDa protease. This evidence suggests that the neutralizing MAb, 36-6-8, recognizes an epitope conserved among some metalloproteases. This epitope may lie at or near the active site of the P. cepacia 36-kDa protease and P. aeruginosa elastase.

Utilization of non-protein nitrogen in whey-dominant formulae by low-birthweight infants.

1. The effects of increasing non-protein nitrogen intake on nitrogen balance and alpha-amino nitrogen flux rate using [15N]glycine were examined in 30 low-birthweight appropriate-for-gestational-age infants (birthweight 1.5-2.0 kg). The compositions of the three whey-dominant formulae were similar except for the ratios of non-protein nitrogen/protein nitrogen, which were 6.5:93.5, 11.4:88.6 and 17.5:82.5. 2. Infants in the three diet groups each received similar total nitrogen intakes (395 mg of N day-1 kg-1, SD 2.6; n = 3). Protein nitrogen and non-protein nitrogen intakes were different as expected. Energy absorption (449 kJ day-1 kg-1, SD 13; n = 3) did not differ significantly between groups. A similar weight gain was observed in all groups. 3. Nitrogen absorption (76%, SD 4; n = 3) was not significantly different between groups. Apparent urea balance was significantly increased and became positive in the group receiving the formula with the higher proportion of non-protein nitrogen and urea nitrogen. Nitrogen retention, however, was significantly depressed in this group, indicating decreased efficiency of nitrogen utilization at this level of non-protein nitrogen despite an enhanced urea salvage. 4. The enrichment of the 15N label in urinary urea at isotopic steady state was significantly reduced in infants receiving the highest urea-containing formula, presumably due to the dilution of 15N-labelled urea by dietary urea. No difference, however, was found in the enrichment of the 15N label in urinary ammonia. Rates of alpha-amino nitrogen flux, protein synthesis and protein breakdown calculated from the ammonia labelling did not differ significantly between groups.(ABSTRACT TRUNCATED AT 250 WORDS)