Single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk.

Single-domain antibody generation technology was applied to make new Sepharose-bound ligands for affinity separation of closely related proteins, such as human and goat lactoferrin. We generated recombinant antibodies that can selectively bind/recognize only lactoferrins having amino acid sequences identical to that of human natural lactoferrin (anti-hLF Ab). Selected and purified histidine-tagged single-domain antibodies were used as ligands, and different lactoferrins were used as analytes in the kinetics analysis of lactoferrin binding to captured anti-hLF Abs using the Bio-Rad ProteOn XPR36 protein interaction array system. The data obtained were consistent with a 1:1 binding model with very high affinity, practically equal in the case of hLF and rec-hLF (calculated KD varied from 0.43nM to 3.7nM). Interaction of captured fsdAbs with goat LF was significantly weaker and not detectable under the same analysis conditions. We demonstrated the high efficiency of the recombinant human lactoferrin purification from goat lactoferrin and other proteins using the obtained single domain antibody-based affinity ligands. We believe this approach can be used for the generation of single-domain antibody-based affinity media for the efficient separation/purification of a wide spectrum of other highly homologous proteins.

Decrease in pool of T lymphocytes with surface phenotypes of effector and central memory cells under influence of TCR transgenic ß-chain expression.

Peripheral T lymphocytes can be subdivided into naïve and antigen-experienced T cells. The latter, in turn, are represented by effector and central memory cells that are identified by different profiles of activation markers expression, such as CD44 and CD62L in mice. These markers determine different traffic of T lymphocytes in the organism, but hardly reproduce real antigenic experience of a T lymphocyte. Mechanisms of homeostasis maintenance of T lymphocytes with different activation phenotypes remain largely unknown. To investigate impact of T cell receptor (TCR) transgenic chains on formation of T lymphocytes, their peripheral survival and activation surface phenotypes, we have generated the transgenic mouse strain expressing transgenic ß-chain of TCR 1D1 (belonging to the Vß6 family) on the genetic background B10.D2(R101). Intrathymic development of T cells in these transgenic mice is not impaired. The repertoire of peripheral T lymphocytes in these mice contains 70-80% of T cells expressing transgenic ß-chain and 20-30% of T cells expressing endogenous ß-chains. The ratio of peripheral CD4⁺CD8⁻ and CD4⁻CD8⁺ T lymphocytes remained unchanged in the transgenic animals, but the percent of T lymphocytes with the "naïve" phenotype CD44⁻CD62L⁺ was significantly increased, whereas the levels of effector memory CD44⁺CD62L⁻ and central memory CD44⁺CD62L⁺ T lymphocytes were markedly decreased in both subpopulations. On the contrary, T lymphocytes expressing endogenous ß-chains had surface phenotype of activated T cells CD44⁺. Thus, for the first time we have shown that the pool of T lymphocytes with different activation phenotypes depends on the structure of T cell receptors.

Neolactoferrin as a stimulator of innate and adaptive immunity.

The effect of the innovative product Neolactoferrin, a natural combination of recombinant human lactoferrin (90%) and goat lactoferrin (10%) isolated from the milk of transgenic goats carrying the full-length human lactoferrin gene, on human immune system cells was studied. Neolactoferrin enhanced the production of IL-1ß. Neolactoferrin saturated with iron ions increased the synthesis of pro-inflammatory cytokine TNFα. It determined the direction of the differentiation of precursor dendrite cells. Under the action of T cells, Neolactoferrin amplified the expression of the transcription factors responsible for the differentiation of Th- and Treg-cells and stimulated the production of both IFNγ and IL-4. The results suggest that Neolactoferrin exhibits an immunotropic activity and hinders the development of immune inflammatory processes. Iron saturation of Neolactoferrin increases its pro-inflammatory activity.

Production of human lactoferrin in animal milk.

Genetic constructs containing the human lactoferrin (hLf) gene were created within a joint program of Russian and Belorussian scientists. Using these constructs, transgenic mice were bred (the maximum hLf concentration in their milk was 160 g/L), and transgenic goats were also generated (up to 10 g/L hLf in their milk). Experimental goatherds that produced hLf in their milk were also bred, and the recombinant hLf was found to be identical to the natural protein in its physical and chemical properties. These properties included electrophoretic mobility, isoelectric point, recognition by polyclonal and monoclonal antibodies, circular dichroic spectra, interaction with natural ligands (DNA, lipopolysaccharides, and heparin), the binding of iron ions, the sequence of the 7 terminal amino acids, and its biological activity. The latter was assessed by the agglutination of Micrococcus luteus protoplasts, bactericidal activity against Escherichia coli and Listeria monocytogenes , and fungicidal activity against Candida albicans . We also demonstrated a significant increase in the activity of antibiotics when used in combination with Lf.

Administration of raw onion inhibits platelet-mediated thrombosis in dogs.

A number of studies suggest that dietary intake of onions is of benefit to cardiovascular health. Onion juice inhibits in vitro human platelet aggregation. To study the in vivo effect of onion on platelet aggregation, 11 dogs were prepared with mechanically damaged and stenosed coronary arteries. Periodic platelet-mediated thrombus formation followed by embolization produced cyclic flow reductions (CFR). In five dogs, 0.09 +/- 0.01 mL/kg onion juice administered intravenously abolished CFR within 20 min. This was followed by a 60 +/- 14% (P = 0.002) reduction in collagen-induced ex vivo whole-blood platelet aggregation. Six dogs were given 2.0 g/kg raw onion homogenate intragastrically. CFR were eliminated within 2.5-3 h in five of the dogs. This was accompanied by a 44 +/- 24% (P = 0.04) reduction in ex vivo aggregation. These findings suggest that the consumption of raw onion may help prevent platelet-mediated cardiovascular disorders. However, in vitro incubations of onion juice demonstrated that the platelet inhibitory response was significantly greater in dog blood than in human blood.

Genetic analyses of correlated solids, flavor, and health-enhancing traits in onion (Allium cepa L.).

Onion possesses organosulfur compounds and carbohydrates that provide unique flavor and health-enhancing characteristics. Significant phenotypic correlations have been reported among soluble solids content (SSC), total dry matter, pungency, and onion-induced in vitro antiplatelet activity. A genetic map and segregating F3M families derived from a cross between two inbred populations were used to identify and estimate the effects of quantitative trait loci (QTLs) controlling these traits at 30 and 90 days postharvest. In vitro antiplatelet activities among different onion populations were consistent across six human blood donors. Most of the populations showed in vitro antiplatelet activities; however, for some donors, one of the parental lines and two F3M families had pro-aggregatory effects under our experimental conditions. SSC, dry matter, pungency, and in vitro antiplatelet activity showed significant positive phenotypic and genetic correlations. A chromosome region on linkage group E accounted for a significant amount of the phenotypic variation for all of these traits. The correlations among these traits may be due to linkage or pleiotropy of genes controlling solids content. Our results indicate that it will be difficult to develop onion populations with lower pungency and high in vitro antiplatelet activity; however, the strong genetic and phenotypic correlations between high in vitro antiplatelet activity and high SSC are beneficial for the health functionality of onion.

Differential inhibition of human platelet aggregation by selected Allium thiosulfinates.

Thiosulfinates (TSs) have been implicated as a principle source of the antiplatelet property of raw onion and garlic juice. The in vitro responses of human platelets to dosages of four TSs were measured using whole blood aggregometry and compared by regression analysis. Of the compounds evaluated, methyl methane-TS (MMTS), propyl propane-TS (PPTS), and 2-propenyl 2-propene-TS (allicin) are present in freshly cut Allium vegetables, whereas ethyl ethane-TS (EETS) has not been detected. All TSs were synthesized using a model reaction system. PPTS and allicin had the strongest antiplatelet activity at 0.4 mM, inhibiting aggregation by 90 and 89%, respectively. At the same concentration, EETS and MMTS were significantly weaker, inhibiting 74 and 26%, respectively. Combinations of TSs were not additive in their inhibition of aggregation, indicating that the antiplatelet potential of Allium extracts cannot be easily predicted by quantifying organosulfur components. EETS, PPTS, and allicin were significantly more potent platelet inhibitors than aspirin at nearly equivalent concentrations.

Influence of production, handling, and storage on phytonutrient content of foods.

The goals of agricultural production have traditionally been to try to accommodate needs for: 1) adequate and reliable yields to provide a sufficient food supply in a growing world; 2) food safety; 3) taste; 4) convenience; 5) profit; and 6) variety. Alternative strategies to enhance any of these outcomes are typically evaluated as to their probable effects on the key outcome: yield. However, with the burgeoning consumer interest in foods that optimize health, attention is shifting from concerns over quantity alone to concerns over the constituents of foods that may promote health, and thus to the agricultural practices that will protect, and perhaps enhance these constituents of the food supply. This shift in focus requires new thinking and new strategies across all segments of the food production system. This paper summarizes selected aspects of crop production that are pivotal to the nutrient value of foods for human consumption and suggests some strategies for establishing a new research and production paradigm that will embrace nutrient quality among the priorities of agricultural research.

3-dimensional computerized tomographic reconstruction of colovesical fistulas.

PURPOSE: Use of 3-dimensional tomographic reconstruction in evaluating colovesical fistulas is discussed. MATERIALS AND METHODS: We compared 3-dimensional computerized tomographic (CT) images of colovesical fistulas to conventional CT images. RESULTS: Successful surgical repair was facilitated by preoperative radiographic data. CONCLUSIONS: Three-dimensional CT reconstruction provides superior spacial detail and can clarify complex anatomical relationships preoperatively.

Accumulation and distribution of free folic acid content in red beet (Beta vulgaris L.).

Among vegetable plants, red beet contains a relatively high level of the B vitamin folic acid. Although many leafy green vegetables contain high levels of folic acid, red beet is consumed primarily as a root vegetable. Folic acid levels have been quantified in various vegetable plants, but little information exists regarding the accumulation and distribution of this vitamin in plant tissues. The objective of this study was to characterize free folic acid content (FFAC) in shoot and root tissue during growth of two red beet inbreds. Experiments were conducted in a greenhouse during 1993, 1994 and 1995. Two inbreds, W384 and W357, were planted in randomized complete blocks and shoot and root tissues were separately harvested at 60, 80, and 100 days after planting (DAP). Significant differences between years, tissue portions, and among harvest dates were detected, however, similar patterns in FFAC accumulation and distribution were observed between inbreds and years. FFAC in shoot tissue was significantly greater than root tissue for both inbreds. Accumulation of FFAC was linear for both inbreds across harvest dates for root tissue but not for shoot tissue. FFAC accumulation in shoot tissue increased sharply from 60 to 80 DAP but decreased sharply from 80 to 100 DAP. These results demonstrate that FFAC accumulates differentially in root and shoot tissue in a red beet plant. Maximum folic acid levels in shoot tissue are achieved prior to those in root tissue.

Antiplatelet activity in onion (Allium cepa) is sulfur dependent.

Plants of the genus Allium such as onion and garlic are often consumed as a source of compounds which inhibit human platelet activity, with the goal of decreasing vascular disease. Antiplatelet activity in these plants is determined in part by native concentrations of organosulfur compounds. Evaluation of four onion genotypes grown in a field study at four US locations in 1994 demonstrated onions with mild flavor and low sulfur content exhibited significantly lower antiplatelet activity than those containing high levels of sulfur. Antiplatelet activity was significantly positively correlated with genotypically determined bulb sulfur content and dissolved solids, indicating these latter factors are good predictors of antiplatelet strength. These data demonstrate antiplatelet activity is genotype dependent and correlated with bulb sulfur content. Genotype and bulb sulfur content should be taken into account in studies assessing onion antiplatelet effects.

Quantitative trait locus analysis of a recombinant inbred line population derived from a Lycopersicon esculentum x Lycopersicon cheesmanii cross.

Quantitative trait loci influencing fruit traits were identified by restriction fragment length polymorphism (RFLP) analysis in a population of recombinant inbred lines (RIL) derived from a cross of the cultivated tomato, Lycopersicon esculentum with a related wild species Lycopersicon cheesmanii. One hundred thirty-two polymorphic RFLP loci spaced throughout the tomato genome were scored for 97 F8 RIL families. Fruit weight and soluble solids were measured in replicated trials during 1991 and 1992. Seed weight was measured in 1992. Significant (P<0.01 level) quantitative trait locus (QTL) associations of marker loci were identified for each trait. A total of 73 significant marker locus-trait associations were detected for the three traits measured. Fifty-three of these associations were for fruit weight and soluble solids, many of which involved marker loci signficantly associated with both traits. QTL with large effects on all three traits were detected on chromosome 6. Greater homozygosity at many loci in the RIL population as compared to F2 populations and greater genomic coverage resulted in increased precision in the estimation of QTL effects, and large proportions of the total phenotypic variance were explained by marker class variation at significant marker loci for many traits. The RIL population was effective in detecting and discriminating among QTL for these traits previously identified in other investigations despite skewed segregation ratios at many marker loci. Large additive effects were measured at significant marker loci. Lower fruit weight, higher soluble solids, and lower seed weight were generally associated with RFLP alleles from theL. cheesmanii parent.

Quantitative trait loci influencing protein and starch concentration in the Illinois Long Term Selection maize strains.

A study was initiated to determine the number, chromosomal location, and magnitude of effect of QTL (quantitative trait loci or locus depending on context) controlling protein and starch concentration in the maize (Zea mays L.) kernel. Restriction fragment length polymorphism (RFLP) analysis was performed on 100 F3 families derived from a cross of two strains, Illinois High Protein (IHP), X Illinois Low Protein (ILP), which had been divergently selected for protein concentration for 76 generations as part of the Illinois Long Term Selection Experiment. These families were analyzed for kernel protein and starch in replicated field trials during 1990 and 1991. A series of 90 genomic and cDNA clones distributed throughout the maize genome were chosen for their ability to detect RFLP between IHP and ILP. These clones were hybridized with DNA extracted from the 100 F3 families, revealing 100 polymorphic loci. Single factor analysis of variance revealed significant QTL associations of many loci with both protein and starch concentration (P < 0.05 level). Twenty-two loci distributed on 10 chromosome arms were significantly associated with protein concentration, 19 loci on 9 chromosome arms were significantly associated with starch concentration. Sixteen of these loci were significant for both protein and starch concentration. Clusters of 3 or more significant loci were detected on chromosome arms 3L, 5S, and 7L for protein concentration, suggesting the presence of QTL with large effects at these locations. A QTL with large additive effects on protein and starch concentration was detected on chromosome arm 3L. RFLP alleles at this QTL were found to be linked with RFLP alleles at the Shrunken-2 (Sh2) locus, a structural gene encoding the major subunit of the starch synthetic enzyme ADP-glucose pyrophosphorylase. A multiple linear regression model consisting of 6 significant RFLP loci on different chromosomes explained over 64 % of the total variation for kernel protein concentration. Similar results were detected for starch concentration. Thus, several chromosomal regions with large effects may be responsible for a significant portion of the changes in kernel protein and starch concentration in the Illinois Long Term Selection Experiment.

Electromagnetic induced shock wave therapy of ureteral calculi.

Between March 1989 and July 1990, 86 patients with ureteral stones were treated with electromagnetic induced extracorporeal shock wave lithotripsy at our facility, of which 77 were available for follow-up. Sixty-one of the patients were male, 16 were female, and the average age was 53.8 +/- 16.0 (11-84). Seventy-four of the patients were treated with intravenous sedation. One patient required general anesthesia and two other patients requested no sedation. Sixty-four of 77 (83%) patients were stone free after therapy, 59 of 77 (77%) after a single treatment, and 5 of 9 (56%) after a second treatment. Thirteen patients were hospitalized post therapy, however, 6 had been hospitalized pretreatment because of pain or obstruction. Four major complications were encountered: hematoma (1), ureteral stricture (1), urinary extravasation (1), and proximal stent migration (1). There were no statistically significant differences between stone free and non-stone free patients when comparing for sex, age, stone size, number of shocks, maximum kilovoltage, or duration of symptoms pretreatment. The patients who had unsuccessful treatment, however, were noted to have a significantly greater incidence of distal stones (69% v 39%) (P less than .05).

Infected urachal cysts: a review of 10 cases.

Disorders of urachal remnants are uncommon. While a urachal cyst usually is asymptomatic, infection may mimic a variety of acute intra-abdominal or pelvic processes. We describe 10 patients in 2 distinct age groups (the young child and the young adult) with an infected urachal cyst. The presenting symptoms and signs in most patients included dysuria, severe lower abdominal pain and fever. In 7 patients the correct preoperative diagnosis was made. Diagnoses at referral included Crohn's disease, bladder carcinoma and pelvic inflammatory disease. A single procedure was performed in 7 cases and a staged technique was used in 3. The differential diagnosis of acute abdominal and pelvic pain or a midline lower abdominal mass in the pediatric or young adult age group should include infection of a urachal remnant.