Manure nutrient excretion by lactating cows fed exogenous phytase and cellulase.

The effect of an exogenous phytase and cellulase-containing enzyme formulation on nutrient digestibility and excretion was evaluated in 24 Holstein cows. Cows were fed corn silage- and alfalfa silage-based diets with or without a cellulase-phytase blend for 31 d in a continuous random design. Treatment groups were balanced for parity, days in milk, and mature-equivalent projected milk yield. Diets contained 37% forage, 18.3% crude protein, 35.4% neutral detergent fiber, 18% acid detergent fiber, and 0.42% P (no supplemental P). Cows were fed once daily in Calan doors and milked 2 times daily. Body weight and milk yield were recorded at each milking. Milk samples were collected on d 28 to 31 at 8 consecutive milkings. On d 28 to 31, fecal grab samples were collected every 8 h, with sampling times advanced by 2 h each day. Feces samples were pooled by cow. Feed and feces samples were analyzed for acid detergent lignin (used as an internal marker) and for N, P, neutral detergent fiber, and acid detergent fiber. Days in milk were similar between treatments, and body weight and milk yield were unaffected by treatment. Cows fed the enzyme formulation had reduced fecal dry matter, neutral detergent fiber, and acid detergent fiber excretion and reduced fecal excretion of N and P. Apparent digestibility of dry matter, acid detergent fiber, neutral detergent fiber, and N tended to increase with the enzyme formulation. Addition of an exogenous phytase and cellulase enzyme formulation to diets for lactating cows reduced fecal nutrient excretion.

Cdc42 stimulates RNA splicing via the S6 kinase and a novel S6 kinase target, the nuclear cap-binding complex.

Cdc42 is a low molecular weight GTP-binding protein that plays a key regulatory role in a variety of cellular activities. The importance of the coordination of different cell functions by Cdc42 is underscored by the fact that a constitutively active Cdc42 mutant induces cellular transformation. In this study, we describe a novel function for Cdc42: its ability to stimulate pre-messenger RNA splicing. This activity is dependent on cysteine 37 in the effector loop of Cdc42 but is not dependent on cell growth. A likely candidate protein for mediating the Cdc42 effects on pre-mRNA splicing is the nuclear RNA cap-binding complex (CBC), which plays a key role in an early step of cap-dependent RNA splicing. Activation of the CBC by Cdc42 can be inhibited by rapamycin. Additionally, phosphatidylinositol 3-kinase and the Cdc42 effector, pp70 S6 kinase, stimulate the RNA cap-binding activity of the CBC. S6 kinase may directly target the CBC in vivo as it can phosphorylate the 80-kDa subunit of the CBC, CBP80, at residues that are subject to a growth factor-dependent and rapamycin-sensitive phosphorylation in vivo. Together these data suggest the involvement of a Cdc42-S6 kinase pathway in the regulation of RNA splicing, mediated by an increase in capped RNA binding by the CBC, as well as raise the possibility that the effects of Cdc42 on cell growth may be due in part to its regulation of RNA processing.

Signal transduction and post-transcriptional gene expression.

Traditionally, growth factor-coupled signaling to the nucleus has been thought to be primarily directed toward transcriptional regulation. However, there are now increasing indications from a diversity of experimental systems that other aspects of RNA processing, including translation, lifetime and stability, and splicing are under strict growth factor control. In this review, we present the emerging evidence for growth factor signaling pathways that impact on these different RNA processing events. Particularly noteworthy is the realization that growth factor signaling through Ras can effect the regulation of two RNA cap-binding proteins, the cytosolic eIF-4E complex, which is necessary for initiating translation, and the nuclear cap-binding complex, the CBC, which plays a role in cap-dependent pre-mRNA splicing, U snRNA export and 3'-end processing. This, taken together with other findings that demonstrate the ability of stress response pathways and the small G protein, Cdc42, to activate the CBC, raises some interesting possibilities regarding how signaling to the two cellular RNA cap-binding protein complexes may coordinate the growth-coupled regulation of gene expression at the level of RNA processing.

The nuclear cap-binding complex is a novel target of growth factor receptor-coupled signal transduction.

In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor control. Using a sensitive photoaffinity labeling assay that measured [alpha-32P]GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells. The CBC represents a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have demonstrated that the [alpha-32P]GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the [alpha-32P]GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors. We show that the CBC is activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation. Additionally, we show that cap-dependent splicing of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells. Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest novel mechanisms by which growth factors can influence gene expression and cell growth.

Long-term effects of insulin-like growth factor (IGF)-I treatment on serum IGFs and IGF binding proteins in adolescent patients with growth hormone receptor deficiency.

OBJECTIVE: The aim of this investigation was to study the effect of relatively high dose IGF-I therapy given for several months, on serum levels of IGF-I, IGF-II and IGFBP-3, and on IGF-I pharmacokinetics in patients with growth hormone insensitivity due to GH receptor dysfunction. DESIGN AND PATIENTS: Two adolescent subjects from Ecuador were treated with recombinant IGF-I at a dosage of 120 micrograms/kg s.c. twice daily, in combination with a GnRH analogue for 8 months. MEASUREMENTS: Serum was sampled at baseline and at 3-8 months, for determination of IGF-I, IGF-II and IGFBP-3 by radioimmunoassay, and for evaluation of IGFBPs and IGFBP-3 protease activity by Western ligand blot and protease assay, respectively. RESULTS: Peak serum IGF-I levels ranged from 272 to 492 micrograms/l. Mean serum IGF-II levels were decreased concurrently with the increase in IGF-I. Serum IGFBP-3 levels failed to rise with prolonged IGF-I treatment. There was no apparent change in the half-life of IGF-I during the treatment period. CONCLUSIONS: IGF-I administration does not increase serum levels of IGFBP-3 or significantly alter IGF-I pharmacokinetics.

Insulin-like growth factor binding protein-3 concentrations and insulin-like growth factor binding protein-3 protease activity in sera of patients with malignant solid tumors or leukemia.

IGF binding proteins (IGFBP) regulate the bioavailability and bioactivity of IGF. The major IGFBP in serum is IGFBP-3. We investigated whether sera from children with malignancies show alterations in levels of IGFBP-3 as measured by Western ligand blot analysis (WLB) and RIA with alpha IGFBP-3gl, a specific rabbit polyclonal antibody. Furthermore, IGFBP-3 proteolysis was quantified by densitometric analysis of [125I]IGFBP-3 protease assays, and IGFBP-3 fragments were visualized by Western immunoblot with alpha IGFBP-3gl. We examined sera from 21 children with solid tumors, five patients with sarcoma who had reached complete remission, and 13 children with acute leukemia. Serum samples were collected at diagnosis, before initiation of therapy. Sera of 10 healthy children served as normal controls. Children with solid tumor or leukemia had significantly higher (p < 0.001) IGFBP-3 protease activity in serum than did normal controls or patients with sarcoma in complete remission. Corresponding to this finding, densitometry of WLB showed lower IGFBP-3 levels in sera of children with malignancies in comparison with normal controls. The negative correlation (p < 0.001, r = -0.80) between IGFBP-3 proteolysis, as measured by [125I]IGFBP-3 protease assay, and IGFBP-3 band density on WLB indicates that proteolysis is the probable reason for reduction of IGFBP-3 on WLB. IGFBP-3 concentrations measured by RIA were in the normal range for most patients, further indicating that differences in serum IGFBP-3 levels measured by WLB reflect protease activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of insulin-like growth factor I treatment on the molecular distribution of insulin-like growth factors among different binding proteins.

The molecular distribution of insulin-like growth factor I (IGF-I) and IGF-II among the IGF binding proteins (IGFBPs) was studied before and during IGF-I therapy in Ecuadorean adults with growth hormone receptor deficiency (GHRD). Of the total circulating IGF-I and IGF-II, 70% was carried by the 150 kDa complex in normal subjects, while in patients with GHRD, 50% of serum IGF-I, but only 30-35% of serum IGF-II, was measured within the 150 kDa IGFBP-3 region. Administration of IGF-I altered the concentration of IGF-I and IGF-II, although the percentage of total IGF measured within each IGFBP region was not affected, as the increase in IGF-I and the decrease in IGF-II were proportional. Similarly, serum concentrations of IGFBP-3 and the acid-labile subunit, measured by radioimmunoassay, were unaltered. Thus, administration of IGF-I to patients with GHRD was unable to correct the aberrant distribution of IGFs among the IGFBPs.

The composition and distribution of insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) in the serum of growth hormone receptor-deficient patients: effects of IGF-I therapy on IGFBP-3.

We have previously reported that adult GH receptor-deficient (GHRD) patients treated subcutaneously with recombinant human insulin-like growth factor (IGF)-I have increased serum IGF-I levels and decreased IGF-II levels, whereas IGF-binding protein-3 (IGFBP-3) levels were unchanged. To further investigate the effects of IGF-I administration upon the IGF-IGFBP axis in GHRD, we have examined: 1) the molecular distribution of IGF-I and IGF-II among the IGFBPs; 2) the composition and distribution of the IGFBPs, in particular IGFBP-3; and 3) the acid labile subunit (ALS). Serum samples from adult GHRD patients who were treated sc with recombinant human IGF-I (40 micrograms/kg, sc, twice a day) or from normal Ecuadorian adults were incubated with [125I]IGF-II and subjected to neutral size-exclusion chromatography. The fractions were then subjected to Western ligand blot, Western immunoblot, IGFBP-3 RIA, and IGF RIAs. Serum of healthy adults incorporated [125I]IGF-II into the 150- and 44-kilodalton (kDa) IGFBP region. The 150-kDa IGFBP region contained most of the circulating IGFBP-3, whereas the 44-kDa IGFBP region contained mainly IGFBP-1, 2, and 4. The 150-kDa region also contained a unique 28-kDa immunoreactive form of IGFBP-3, which was not detectable by Western ligand blot. Endogenous IGF-I and IGF-II were distributed equally in the 150- and 44-kDa IGFBP regions. Sera from GHRD patients mainly incorporated [125I]IGF-II into the 44-kDa IGFBP region. Similar to control sera, the 150-kDa IGFBP region contained IGFBP-3, albeit at lower concentrations. The 44-kDa IGFBP region contained all IGFBPs including 50% of the total immunoreactive IGFBP-3. The two immunoreactive forms of IGFBP 3 (40- to 45-kDa doublet and 28-kDa band) were present in both IGFBP regions. The IGF size-distribution study revealed that the 150-kDa IGFBP region carried half of the circulating endogenous IGF-I, but only 30% of the IGF-II. Concentrations of the ALS were consistently low. Administration of IGF-I to GHRD patients was unable to increase concentrations of the molecular forms of IGFBP-3, correct the aberrant distribution of IGFs among the IGFBPs, or increase serum concentrations of ALS. In conclusion, we have found two forms of IGFBP-3 associated with IGF and ALS, which are capable of forming the ternary 150-kDa complex in healthy adult serum. The ratio of these two forms of IGFBP-3 and their distribution in serum was different between GHRD and control patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factors (IGFs) stimulate and dexamethasone inhibits IGF binding protein (BP)-5 expression in a mouse pituitary cell line.

The mouse pituitary cell line AtT-20 was found to secrete two low MW IGFBPs into conditioned medium (CM). The major IGFBP migrated at approximately 29 kDa and a minor IGFBP of 24 kDa was also present on western ligand blots (WLB). Both IGFBPs were purified from CM by IGF-affinity chromatography followed by reverse phase-FPLC. N-terminal analysis revealed that the first 10 amino acids of the 29 kDa and the 24 kDa IGFBPs were homologous to corresponding sequences of both human and rat IGFBP-5 and IGFBP-4, respectively. The 24 kDa IGFBP also crossreacted with a new antiserum specific for rodent IGFBP-4. The concentrations of both IGFBPs were increased by the addition of IGF-I, IGF-II, or insulin to the cell cultures, with IGFBP-5 demonstrating the greatest hormonal stimulation. The effects of IGF-I on IGFBP-5 expression were both time and dose dependent, with IGF-I being more potent than IGF-II, and IGF-II more potent than insulin. The relative potencies of these hormones in stimulating IGFBP-5 production were consistent with the peptides acting through the type-I IGF receptor. Similarly, the IGF-II analog [Leu 27]-IGF-II, which has very low affinity for the type-I receptor, only slightly stimulated an increase in IGFBP-5. Addition of dexamethasone to the cultures decreased both basal and IGF-stimulated IGFBP-5 production. Northern blotting demonstrated that IGF-I increased the expression of the mRNA for IGFBP-5, whereas dexamethasone decreased it. Together, these data suggest that the IGFs can increase IGFBP-5 production at both the protein and mRNA level.

Elevated levels of insulin-like growth factor-binding protein-2 in the serum of prostate cancer patients.

We have previously documented the presence of specific insulin-like growth factor (IGF)-binding protein (IGFBPs) in seminal plasma and prostate epithelial cell-conditioned medium IGFBP-2 is the prevalent IGFBP in both fluids. To assess whether patients with prostate carcinoma have alterations in serum IGFP levels related to the production of IGFBPs by their tumors, we performed Western ligand blots (WLB) and IGFBP-2 RIA on serum samples from 32 patients with prostate carcinoma of various degrees of clinical severity and compared them to results in 16 healthy age-matched controls. We have also measured serum IGF-I and -II by RIA. The mean level of IGFBP-2 in the prostate cancer patients was 170% of control levels by WLB analysis and 195% of control levels by RIA (P < 0.01). The degree of elevation of IGFBP-2 was related to the stage of the tumor and the levels of the serum tumor marker, prostate-specific antigen. Serum IGFBP-3 levels determined by WLB and serum IGF-I and IGF-II levels measured by RIA after acid chromatography were not different among the subjects with cancer and the normal controls. We conclude that IGFBP-2, which is the main IGFBP produced by prostate epithelial cells, is elevated in the serum of patients with prostate carcinoma, and that the degree of this elevation is related to serum prostate-specific antigen levels and the stage of the tumor. We speculate that prostate-derived IGFBPs may be secreted by prostate tumors and could e of value in understanding the pathophysiology of prostatic tumor growth as well as provide potential diagnostic markers.

Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: discrepancies between radioimmunoassay and ligand blotting.

The inability to detect insulin-like growth factor binding protein-3 (IGFBP-3) in some circumstances by Western ligand blot analysis has emphasized the need to characterize IGFBPs by both ligand binding and immunological techniques. In this study, we have: 1) characterized and quantified IGFBP-3 in nonpregnancy, pregnancy, and fetal cord serum, follicular, peritoneal, and amniotic fluid, seminal plasma, cerebrospinal fluid (CSF), and urine; 2) established a new IGFBP-3 RIA that detects both intact and fragments of IGFBP-3; 3) identified both intact and fragments of IGFBP-3 by Western immunoblot techniques; and 4) addressed the discordance between Western ligand blot analysis and RIA by assessing fluids for IGFBP proteolytic activity. All fluids examined, except pregnancy serum, CSF, and amniotic fluid, displayed a 44-34-kilodalton (kDa) IGFBP-3 doublet by Western ligand blot analysis. Western immunoblot analysis using specific IGFBP-3 antiserum showed a 44-34-kDa IGFBP-3 doublet and a 28-kDa fragment in nonpregnancy serum, fetal cord serum, follicular fluid, and peritoneal fluid. The immunoreactive 42-38-kDa doublet was faint in urine and seminal plasma. IGFBPs in CSF did not cross-react with IGFBP-3 antiserum. Pregnancy serum and amniotic fluid contained only the 28-kDa fragment when compared against equal volumes of nonpregnancy serum. With the development of an IGFBP-3 RIA, IGFBP-3 could be accurately measured; urine, CSF, and seminal plasma contained the lowest levels of IGFBP-3 at 27 +/- 3 ng/ml (mean +/- SEM), 110 +/- 26 ng/ml, and 209 +/- 56 ng/ml, respectively. In increasing concentration: fetal cord serum contained 753 +/- 101 ng/ml; peritoneal fluid, 1124 +/- 130 ng/ml; follicular fluid, 2356 +/- 211 ng/ml; nonpregnancy serum, 3556 +/- 508 ng/ml; pregnancy serum, 3718 +/- 842 ng/ml; and amniotic fluid, 5150 +/- 688 ng/ml. The measurable concentrations of IGFBP-3 in CSF and the high concentrations measured in pregnancy serum and amniotic fluid conflicted with Western blot analysis. Thus, fluids were assessed for IGFBP proteolytic activity by incubation with a source of IGFBP-3, either nonpregnancy serum or purified IGFBP-3. All fluids displayed some proteolytic activity with either assay. Fluids with little protease activity (nonpregnancy serum, follicular fluid, and urine) showed a close relationship between immunoassayable IGFBP-3 by RIA and IGFBP-3 band intensity by Western ligand blot. Fluids with high proteolytic activity (pregnancy serum, CSF, seminal plasma, peritoneal fluid, and amniotic fluid) gave discrepant IGFBP-3 values between RIA and Western ligand blot.(ABSTRACT TRUNCATED AT 400 WORDS)

Dendritic organization of class II (inter)neurons in the dorsal lateral geniculate nucleus of the tree shrew: observations based on Golgi, immunocytochemical, and biocytin methods.

In this report we examine the dendritic organization of putative interneurons (class II cells) in different layers of the dorsal lateral geniculate nucleus of the tree shrew. The results show that there is considerable morphological diversity within this class, but that two broad groups can be identified: neurons whose dendrites remain within a layer or its adjacent interlaminar zones (intralaminar class II cells); and neurons whose dendrites cross into an adjacent layer(s) (interlaminar class II cells). The majority of class II cells in every layer have intralaminar dendrites, some of which are oriented along a particular axis, and others that are organized radially. The paired layers (1 and 2, 4 and 5) contain a particular group of intralaminar class II cells that have radially organized dendrites and elaborate claw-like appendages. The dendrites of interlaminar class II cells are organized along lines of projection and extend across as many as four layers. These cells often reside close to or within the interlaminar zones. Overall, the organization of class II cells seems to follow a pattern similar to the class I (relay) cells identified previously. Most have intralaminar dendrites, which presumably underlie the fidelity of signals transmitted from the retina to a particular layer. However, there are also a number of other cells whose processes cross laminar borders, presumably to affect integrative functions within the nucleus.