Short communication: Fractional milking distribution of immunoglobulin G and other constituents in colostrum.

The provision of quality colostrum with a high concentration of immunoglobulins is critical for newborn calf health. Because first colostrum may be low in overall concentration to effectively reduce the risk of newborn infections, we tested equivalent milking fractions of colostrum for possible IgG differences. The objective of this study was to determine if the fractional composition of colostrum changes during the course of milking with a focus on immunoglobulins. Twenty-four Holstein and Simmental cows were milked (first colostrum) within 4h after calving. The colostrum of 1 gland per animal was assembled into 4 percentage fractions over the course of milking: 0 to 25%, 25 to 50%, 50 to 75%, and 75 to 100%. The IgG concentration among the various fractions did not change in any significant pattern. Concentration of protein, casein, lactose and somatic cell count remained the same or exhibited only minor changes during the course of fractional milking colostrum. We determined that no benefit exists in feeding any particular fraction of colostrum to the newborn.

Anti-Alpha-2-Macroglobulin-Like-1 Autoantibodies Are Detected Frequently and May Be Pathogenic in Paraneoplastic Pemphigus

Paraneoplastic pemphigus (PNP) shows autoantibodies mainly to plakin and desmosomal cadherin family proteins. We have recently identified alpha-2-macroglobulin-like-1 (A2ML1), a broad range protease inhibitor, as a unique PNP antigen. In this study, we tested a large number of PNP sera by various methods. Forty (69.0%) of 58 PNP sera recognized A2ML1 recombinant protein expressed in COS7 cells by immunofluorescence (IF) and/or immunoprecipitation (IP)/immunoblotting (IB). IP/IB showed higher sensitivity than IF. In addition, 22 (37.9%) PNP sera reacted with A2ML1 by IB of cultured normal human keratinocytes (NHKs) under non-reducing conditions. Statistical analyses using various clinical and immunological data showed that the presence of anti-A2ML1 autoantibodies was associated with early disease onset and absence of ocular lesions. Next, to investigate the pathogenic role of anti-A2ML1 antibody, we performed additional functional studies. Addition of anti-A2ML1 polyclonal antibody to culture media decreased NHK cell adhesion examined by dissociation assay, and increased plasmin activity detected by casein zymography, suggesting that anti-A2ML1 antibody may decrease NHK cell adhesion through plasmin activation by inhibition of A2ML1. This study demonstrates that autoantibodies to A2ML1 are frequently and specifically detected and may have a pathogenic role in PNP.

Protein deficiency and the growing rat lung. I. Nutritional findings and related lung volumes

We investigated the consequences of early malnutrition on milk production by dams and on body weight and structural lung growth of young rats using two models of protein restriction. Dams of the early restriction group were fed an 8% casein diet starting at parturition. Those of the delayed restriction group received a 12% casein diet from lactation d 8-14 and thereafter the 8% diet. After weaning, early restriction and delayed restriction group rats were maintained on low protein until d 49, then refed the control diet (18% casein) up to d 126. Milk was analyzed on d 12. Animals were killed at d 21, 49, and 126 for lung fixation in situ. In this report, we show that protein restriction lowered milk yield to 38% of normal. Milk lipid per gram of dry weight tended to be increased, whereas lactose and protein were significantly decreased. Pups from protein-restricted dams grew less and had lower lung volumes, effects being more serious at d 49. However, specific lung volumes (in milliliters per 100 g body weight) were constantly increased. This means that lung was either less affected than body mass or overdistended due to less connective tissue. After refeeding, both groups showed a remarkable catch-up in growth with restoration of the normal allometric relationship between lung volume and body weight. Thus, even after an early onset of protein restriction to total body, the lung is still capable to substantially recover from growth retardation.

Protein deficiency and the growing rat lung. II. Morphometric analysis and morphology

Effects of protein deficiency during the whole period of postnatal development and intensive growth were studied in the rat lung parenchyma. Dams received a low protein diet as follows: early restriction, 8% casein diet from parturition, and delayed restriction, 12% then 8% casein diet from lactation d 8. After weaning (d 21), early restriction and delayed restriction group rats were maintained on the 8% casein diet until d 49, wherefore they were returned to normal food (18% casein) for 11 wk. Lungs were processed for light and electron microscopic morphometry on d 21, 49, and 126. The diffusion capacity of the lung for O2 (DLO2) was also determined from the morphologic parameters. Volume and surface densities of the parenchymal components of malnourished rats did not consistently differ from controls. Because of lower lung volumes, absolute values, including DLO2, were all significantly decreased. Further, although lung volume growth was less impaired than body growth and thus deviated from the normal allometric relationship, most morphometric parameters paralleled body weight changes. Visually, we detected minor morphologic alterations at d 21 and 49, not necessarily reflected by morphometric data. But, importantly, lung parenchyma appeared mature at weaning despite the growth retardation. Normal refeeding resulted in a striking regrowth of the lung parenchyma. Although early restriction rats did not fully catch up in lung volume, most parenchymal parameters and DLO2 were largely restored in both refed groups.

The role of post-translation regulation of Twist expression in the tumor progression of squamous cell carcinoma of head and neck

Squamous cell carcinoma of head and neck (SCCHN) is the tenth most common cancer in the world. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years. Therefore new targets for therapy are needed, and to this end we are studying signaling pathways activated by IL-6 which we have found stimulates cell migration and soft agar growth in SCCHN. Our data show that IL-6 increases TWIST expression in a transcription-independent mechanism in many SCCHN cell lines. Further investigation reveals TWIST can be phosphorylated upon IL-6 treatment. By computation prediction (http://scansite.mit.edu/motifscan_seq.phtml ), we found that TWIST has a putative phosphorylation site for casein kinase 2 (CK2) suggesting that this kinase could serve as a link between IL-6 stimulation and Twist stability. To test this hypothesis, we used a CK2 inhibitor and shRNA to CK2 and found that these interventions inhibited IL-6 stimulation of TWIST stability. In addition, mutation of the putative CK2 phosphorylation site (S18/S20A) in TWIST decreased the amount of phospho-ATP incorporated by TWIST in an in vitro kinase assay, and altered TWIST stability. In Boyd chamber migration assay and wound-healing assay, the CK2 inhibitor, DMAT, was found to decrease the motility of IL-6 stimulated SCCHN cells and over expression of either a wild-type or the hyperphosphorylated mimicking mutant S18/20D –Twist rather than the hypo-phosphorylated mimicking mutant S18/20A-Twist can promote SCCHN cell motility.To our knowledge, this is the first report to identify the importance of IL-6 stimulated CK2 phosphorylation of TWIST in SCCHN. As CK2 inhibitors are currently under phase I clinical trials, our findings indicate that CK2 may be a viable therapeutic target in SCCHN. Therefore, further pre-clinical studies of this inhibitor are underway.

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

Multisite phosphorylation of ornithine decarboxylase in transformed macrophages

Ornithine decarboxylase (ODC), the initial inducible enzyme in the polyamine biosynthetic pathway, exists in the transformed macrophage RAW264 cell line as a phosphoprotein following cell stimulation. The hypothesis that ODC is phosphorylated at multiple sites in stimulated RAW264 cells was investigated. ODC isolated from tetradecanoyl-phorbol-13-acetate (TPA)-stimulated cells metabolically radiolabeled in the presence of $\sp{32}$P$\sb{\rm i}$ was subjected to cyanogen bromide (CNBr) cleavage followed by phosphopeptide mapping and two dimensional phosphoamino acid analysis. These phosphorylation studies demonstrated six in situ phosphorylated CNBr-generated fragments having apparent molecular weights of 17, 14.3, 8, 6.5, 4, and 2.7 kDa and also revealed that ODC is phosphorylated in RAW264 cells on at least 5 serine and 2 threonine residues.^ In addition, the in vivo specific activity and phosphorylation pattern of ODC in response to various kinase cascade stimulants was studied. A differential response in ODC specific activity and a variation in the relative distribution of $\sp{32}$P-labeling of serine and threonine residues on the ODC molecule was noted in response to fetal bovine serum, cAMP and isobutylmethylxanthine, lipopolysaccharide, or TPA.^ Based on information derived from consensus sequence motifs, three protein kinases responsible for the phosphorylation of ODC in vitro were identified. Purified ODC was phosphorylated in vitro by casein kinase II (CK II), extracellular signal-regulated kinase 1 (ERK1), and its activator, extracellular signal-regulated kinase kinase (MEK). CK II phosphorylated ODC on serine residues contained on three CNBr-generated peptides with apparent molecular weights of 14.3, 6.5, and 2.7 kDa. Both ERK1 and MEK phosphorylated ODC on serine and threonine residues on a CNBr-generated peptide fragment with an apparent molecular weight of 6.5 kDa. The in vitro radiolabeled peptides corresponded in molecular mass with some of the CNBr fragments of ODC phosphorylated in situ in stimulated RAW264 cells.^ This study concludes that ODC is phosphorylated in the transformed macrophage RAW264 cell line at multiple sites in response to various kinase cascade stimulants. These stimulants also led to a differential response in specific activity and phosphorylation pattern of ODC in RAW264 cells. Three protein kinases have been identified which phosphorylate ODC in vitro on peptides and amino acid residues which correspond with those phosphorylated in situ. ^

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri

Species in the genus Naegleria are free-living amoebae of the soil and warm fresh water. Although around 30 species have been recognized, Naegleria fowleri is the only one that causes primary amoebic meningoencephalitis (PAM) in humans. PAM is an acute and fast progressing disease affecting the central nervous system. Most of the patients die within 1-2 weeks of exposure to the infectious water source. The fact that N. fowleri causes such fast progressing and highly lethal infections has opened many questions regarding the relevant pathogenicity factors of the amoeba. In order to investigate the pathogenesis of N. fowleri under defined experimental conditions, we developed a novel high- versus low-pathogenicity model for this pathogen. We showed that the composition of the axenic growth media influenced growth behaviour and morphology, as well as in vitro cytotoxicity and in vivo pathogenicity of N. fowleri. Trophozoites maintained in Nelson's medium were highly pathogenic for mice, demonstrated rapid in vitro proliferation, characteristic expression of surface membrane vesicles and a small cell diameter, and killed target mouse fibroblasts by both contact-dependent and -independent destruction. In contrast, N. fowleri cultured in PYNFH medium exhibited a low pathogenicity, slower growth, increased cell size and contact-dependent target cell destruction. However, cultivation of the amoeba in PYNFH medium supplemented with liver hydrolysate (LH) resulted in trophozoites that were highly pathogenic in mice, and demonstrated an intermediate proliferation rate in vitro, diminished cell diameter and contact-dependent target cell destruction. Thus, in this model, the presence of LH resulted in increased proliferation of trophozoites in vitro and enhanced pathogenicity of N. fowleri in mice. However, neither in vitro cytotoxicity mechanisms nor the presence of membrane vesicles on the surface correlated with the pathologic potential of the amoeba. This indicated that the pathogenicity of N. fowleri remains a complex interaction between as-yet-unidentified cellular mechanisms.

L-mimosine and dimethyloxaloylglycine decrease plasminogen activation in periodontal fibroblasts

BACKGROUND The use of prolyl hydroxylase inhibitors such as l-mimosine (L-MIM) and dimethyloxaloylglycine (DMOG) to improve angiogenesis is a new approach for periodontal regeneration. In addition to exhibiting pro-angiogenic effects, prolyl hydroxylase inhibitors can modulate the plasminogen activator system in cells from non-oral tissues. This study assesses the effect of prolyl hydroxylase inhibitors on plasminogen activation by fibroblasts from the periodontium. METHODS Gingival and periodontal ligament fibroblasts were incubated with L-MIM and DMOG. To investigate whether prolyl hydroxylase inhibitors modulate the net plasminogen activation, kinetic assays were performed with and without interleukin (IL)-1. Moreover, plasminogen activators and the respective inhibitors were analyzed by casein zymography, immune assays, and quantitative polymerase chain reaction. RESULTS The kinetic assay showed that L-MIM and DMOG reduced plasminogen activation under basal and IL-1-stimulated conditions. Casein zymography revealed that the effect of L-MIM involves a decrease in urokinase-type plasminogen activator activity. In agreement with these findings, reduced levels of urokinase-type plasminogen activator and elevated levels of plasminogen activator inhibitor 1 were observed. CONCLUSION L-MIM and DMOG can reduce plasminogen activation by fibroblasts from the gingiva and the periodontal ligament under basal conditions and in the presence of an inflammatory cytokine.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

KLIKK proteases of Tannerella forsythia: putative virulence factors with a unique domain structure.

Comparative genomics of virulent Tannerella forsythia ATCC 43037 and a close health-associated relative, Tannerella BU063, revealed, in the latter, the absence of an entire array of genes encoding putative secretory proteases that possess a nearly identical C-terminal domain (CTD) that ends with a -Lys-Leu-Ile-Lys-Lys motif. This observation suggests that these proteins, referred to as KLIKK proteases, may function as virulence factors. Re-sequencing of the loci of the KLIKK proteases found only six genes grouped in two clusters. All six genes were expressed by T. forsythia in routine culture conditions, although at different levels. More importantly, a transcript of each gene was detected in gingival crevicular fluid (GCF) from periodontitis sites infected with T. forsythia indicating that the proteases are expressed in vivo. In each protein, a protease domain was flanked by a unique N-terminal profragment and a C-terminal extension ending with the CTD. Partially purified recombinant proteases showed variable levels of proteolytic activity in zymography gels and toward protein substrates, including collagen, gelatin, elastin, and casein. Taken together, these results indicate that the pathogenic strain of T. forsythia secretes active proteases capable of degrading an array of host proteins, which likely represents an important pathogenic feature of this bacterium.

Impact of Acquired Pellicle Modification on Adhesion of Early Colonizers

New preventive approaches against dental erosion caused by acidic drinks and beverages include fortification of beverages with natural polymers. We have shown that the mixture of casein and mucin significantly improved the erosion-inhibiting properties of the human pellicle layer. This study aimed to investigate the effect of pellicle modification by casein, mucin and a casein-mucin mixture on the adhesion of early bacterial colonizers. Test specimens of human tooth enamel were prepared, covered with saliva and coated with 0.5% aqueous (aq.) casein, 0.27% aq. mucin or with 0.5% aq. casein-0.27% aq. mucin, after which the adhesion of Streptococcus gordonii, Streptococcus oralis, and Actinomyces odontolyticus was measured after incubation for 30 min and 2 h. log10 colony-forming units were compared by nonparametric tests. All three bacterial strains adhered in higher number to pellicle-coated enamel than to native enamel. The protein modifications of pellicle all decreased the counts of adhering bacteria up to 0.34 log10/mm2, the most efficient being the casein-mucin mixture. In addition to the recently shown erosion-reducing effect by casein-mucin, modification of the pellicle may inhibit bacterial adherence compared to untreated human pellicle.

Proceedings of the Seventeenth Annual Biochemical Engineering Symposium

This is the seventeenth of a series of symposia devoted to talks by students about their biochemical engineering research. The first, third, fifth, ninth, twelfth, and sixteenth were at Kansas State University, the second and fourth were at the University of Nebraska-Lincoln, the sixth was in Kansas City and was hosted by Iowa State University, the seventh, tenth, thirteenth, and seventeenth were at Iowa State University, the eighth and fourteenth were at the University of Missouri–Columbia, and the eleventh and fifteenth were at Colorado State University. Next year's symposium will be at the University of Colorado. Symposium proceedings are edited by faculty of the host institution. Because final publication usually takes place elsewhere, papers here are brief, and often cover work in progress. ContentsThe Effect of Polymer Dosage Conditions on the Properties of ProteinPolyelectrolyte Precipitates, K. H. Clark and C. E. Glatz, Iowa State University An Immobilized Enzyme Reactor/Separator for the Hydrolysis of Casein by Subtilisin Carlsberg, A. J. Bream, R. A. Yoshisato, and G. R. Carmichael, University of Iowa Cell Density Measurements in Hollow Fiber Bioreactors, Thomas Blute, Colorado State University The Hydrodynamics in an Air-Lift Reactor, Peter Sohn, George Y. Preckshot, and Rakesh K. Bajpai, University of Missouri–Columbia Local Liquid Velocity Measurements in a Split Cylinder Airlift Column, G. Travis Jones, Kansas State University Fluidized Bed Solid Substrate Trichoderma reesei Fermentation, S. Adisasmito, H. N. Karim, and R. P. Tengerdy, Colorado State University The Effect of 2,4-D Concentration on the Growth of Streptanthus tortuosis Cells in Shake Flask and Air-Lift Permenter Culture, I. C. Kong, R. D. Sjolund, and R. A. Yoshisato, University of Iowa Protein Engineering of Aspergillus niger Glucoamylase, Michael R. Sierks, Iowa State University Structured Kinetic Modeling of Hybidoma Growth and Monoclonal Antibody Production in Suspension Cultures, Brian C. Batt and Dhinakar S. Kampala, University of Colorado Modelling and Control of a Zymomonas mobilis Fermentation, John F. Kramer, M. N. Karim, and J. Linden, Colorado State University Modeling of Brettanomyces clausenii Fermentation on Mixtures of Glucose and Cellobiose, Max T. Bynum and Dhinakar S. Kampala, University of Colorado, Karel Grohmann and Charles E. Yyman, Solar Energy Research Institute Master Equation Modeling and Monte Carlo Simulation of Predator-Prey Interactions, R. 0. Fox, Y. Y. Huang, and L. T. Fan, Kansas State University Kinetics and Equilibria of Condensation Reactions Between Two Different Monosaccharides Catalyzed by Aspergillus niger Glucoamylase, Sabine Pestlin, Iowa State University Biodegradation of Metalworking Fluids, S. M. Lee, Ayush Gupta, L. E. Erickson, and L. T. Fan, Kansas State University Redox Potential, Toxicity and Oscillations in Solvent Fermentations, Kim Joong, Rakesh Bajpai, and Eugene L. Iannotti, University of Missouri–Columbia Using Structured Kinetic Models for Analyzing Instability in Recombinant Bacterial Cultures, William E. Bentley and Dhinakar S. Kompala, University of Colorado

Dominance of facultative and obligate oligotrophic bacteria in surface waters above Gunnerus and Astrid Ridge, Antarctic Ocean

Facultative and obligate oligotrophs have been enumerated in March/April 1990 by the MPN-method with 14C-protein hydrolysate as tracer substrate. Obligate (10-3360 cells/ml) and facultative (110-9000 cells/ml) oligotrophs revealed to be the dominant population above Gunnerus Ridge (65°30'-68°S; 31-35°E) at a depth of 25 m compared with eutrophic bacteria (5 to 260 CFU/ml). Above Astrid Ridge (65-68°S; 8-18°E), obligate (0-1100 cells/ml) and facultative oligotrophs (300-9000 cells/ml) were also abundant but not always dominant. Bacterial biomass above Gunnerus Ridge was only between 7.3 and 43.6% of particulate biomass, but biomass of bacteria above Astrid Ridge amounted from 56.9 to >100% of particulate biomass; an exception was station no. PS16/552 with only 22.2% of bacterial biomass. Ratio of bacterial biomass to particulate biomass was negatively correlated with maximal primary production, complementing the view that phytoplankton was the dominant population above Gunnerus Ridge, whereas bacteria predominated above Astrid Ridge. Eutrophic bacteria were also more abundant above Astrid Ridge, with 3 to 6380 CFU/ml. Total bacteria by acridine orange direct counts amounted from 1 x 10**4 to 34.2 x 10**4 cells/ml. Bacterial biomass above Gunnerus Ridge was 1.8 to 10.7, and above Astrid Ridge 5.7 to 13.6 mg C/m*3. Maximal primary production above Gunnerus Ridge was 4.5 to 11.0, and above Astrid Ridge 2.3 to 3.5 mg C/m**3/d.

Trace element contents in different types of recent bottom sediments from the Indian Ocean

Trace element contents in different types of recent botoom sediments of the Indian Ocean are given. Sediment samples were obtained during cruises of the P.P. Shirshov Institute of Oceanology, Moscow.