Alternative rib bone biopsy measurements to estimate changes in skeletal mineral reserves in cattle.

Rib bone biopsy samples are often used to estimate changes in skeletal mineral reserves in cattle but differences in sampling procedures and the bone measurements reported often make interpretation and comparisons among experiments difficult. 'Full-core' rib bone biopsy samples, which included the external cortical bone, internal cortical bone and trabecular bone (CBext, CBint and Trab, respectively), were obtained from cattle known to be in phosphorus (P) adequate (Padeq) or severely P-deficient (Pdefic) status. Experiments 1 and 2 examined growing steers and Experiment 3 mature breeder cows. The thickness of cortical bone, specific gravity (SG), and the amount and concentration of ash and P per unit fresh bone volume, differed among CBext, CBint and Trab bone. P concentration (mg/cc) was closely correlated with both SG and ash concentrations (pooled data, r=0.99). Thickness of external cortical bone (CBText) was correlated with full-core P concentration (FC-Pconc) (pooled data, r=0.87). However, an index, the amount of P in CBext per unit surface area of CBext (PSACB; mg P/mm2), was more closely correlated with the FC-Pconc (pooled data, FC-Pconc=37.0+146×PSACB; n=42, r=0.94, RSD=7.7). Results for measured or estimated FC-Pconc in 10 published studies with cattle in various physiological states and expected to be Padeq or in various degrees of Pdefic status were collated and the ranges of FC-Pconc indicative of P adequacy and P deficiency for various classes of cattle were evaluated. FC-Pconc was generally in the range 130 to 170 and 100 to 120 mg/cc fresh bone in Padeq mature cows and young growing cattle, respectively. In conclusion, the FC-Pconc could be estimated accurately from biopsy samples of CBext. This allows comparisons between studies where full-core or only CBext biopsy samples of rib bone have been obtained to estimate changes in the skeletal P status of cattle and facilitates evaluation of the P status of cattle.

Using gene transfer to circumvent off-target effects.

Many recombinant growth factors have failed in clinical trials due to off-target effects. We describe a method for circumventing off-target effects that involves equipping cells with a conditionally active signaling protein that can be specifically activated by an exogenously administered synthetic ligand. We believe that this approach will have many applications in gene and cell therapy.

Transcription of the rat glucagon gene by the cyclic AMP response element-binding protein CREB is modulated by adjacent CREB-associated proteins.

The cyclic AMP (cAMP) response element (CRE) of the rat glucagon gene (Glu-CRE, 5'-TGACGTCA-3') mediates transcriptional responses to 8-bromo-cAMP and protein kinase A (PKA) in a glucagon-producing hamster islet cell line (InR1G9). By several different DNA-protein binding assays, we show that the transcription factor CREB binds to the CRE octamer and that additional nuclear proteins bind to sequences adjacent to the CRE. Mutation of the Glu-CRE octamer attenuates both the binding of CREB and cAMP-dependent PKA-stimulated transcriptional activity in transient transfection experiments but does not affect the binding of adjacent CREB-associated proteins. Progressive deletions and clustered point mutations of the sequences flanking the Glu-CRE identify sequences (5'-TCATT-3') located both 5' and 3' to the core CRE octamer that bind several proteins. Two proteins with molecular masses of 80 and 100 kDa bind to each of the 5' and 3' TCATT sites. Formation of additional protein-DNA complexes containing 45- and 20-kDa proteins depends upon the integrity of both TCATT sequences. Deletion or point mutation of the TCATT motif located on the 3' side of the CRE octamer results in enhanced transcriptional responses to PKA, suggesting that the CREB-associated proteins decrease the ability of CREB to mediate PKA-stimulated transcription. Results from these studies demonstrate that nucleotides flanking the core CRE octamer can influence the activity of the CRE by serving as binding sites for proteins that modulate the function of CREB and suggest a mechanism to explain why some consensus palindromic CREs are less responsive to cAMP stimulation than others.

POU domain transcription factor brain 4 confers pancreatic alpha-cell-specific expression of the proglucagon gene through interaction with a novel proximal promoter G1 element.

The proglucagon gene is expressed in a highly restricted tissue-specific manner in the alpha cells of the pancreatic islet, the hypothalamus, and the small and large intestines. Proglucagon is processed to glucagon and glucagon-like peptides GLP-1 and -2. Glucagon is expressed in alpha cells and regulates glucose homeostasis. GLP-1 is implicated in the control of insulin secretion, food intake, and satiety signaling, and GLP-2 is implicated in regulating small-bowel growth. Cell-specific expression of the proglucagon gene is mediated by proteins that interact with the proximal G1 promoter element which contains several AT-rich domains with binding sites for homeodomain transcription factors. In an attempt to identify major homeodomain proteins involved in pancreatic alpha-cell-specific proglucagon expression, we found that the POU domain transcription factor brain 4 is abundantly expressed in proglucagon-producing islet cell lines and rat pancreatic islets. In the latter, brain 4 and glucagon immunoreactivity colocalize in the outer mantle of islets. Electrophoretic mobility shift assays with specific antisera identify brain 4 as a major constituent of nuclear proteins of glucagon-producing cells that bind to the G1 element of the proglucagon gene proximal promoter. Transcriptional transactivation experiments reveal that brain 4 is a major regulator of proglucagon gene expression by its interaction with the G1 element. The finding that a neuronal transcription factor is involved in glucagon gene transcription may explain the presence of proglucagon in certain areas of the brain as well as in pancreatic alpha cells. Further, this finding supports the idea that the neuronal properties of endodermis-derived endocrine pancreatic cells may find their basis in regulation of gene expression by neuronal transcription factors.

Reduced insulin, GLUT2, and IDX-1 in beta-cells after partial pancreatectomy.

Reduction of GLUT2 is associated with loss of glucose-induced insulin secretion in genetic and chemical diabetes and in transplanted islets exposed to chronic hyperglycemia. To examine the mechanisms for this loss of GLUT2 in normal islets exposed to hyperglycemia, we performed studies on Sprague Dawley rats 4 weeks after a 90% partial pancreatectomy (Px), a well-characterized model of hyperglycemia. GLUT2 immunofluorescence in the beta-cell of Px rats was greatly reduced. Western blot analysis of homogenates of isolated Px islets also showed a reduction in GLUT2 protein; densitometry measurements were 36 +/- 3% of values from islets of sham-operated controls. Insulin protein levels were decreased to a similar extent. Islet GLUT2 and insulin mRNA were measured with quantitative reverse transcriptase-polymerase chain reaction. The level of GLUT2 mRNA from Px islets was 24 +/- 4% of that of islets from sham-operated controls; similar results were obtained for insulin. Because both these beta-cell-specific messages were reduced, we analyzed the Px islets for the pancreas-duodenum-specific transcription factor IDX-1(IPF-1, STF-1, PDX-1) protein. It was markedly reduced (approximately 80%) in islets from the Px rats. These data suggest that 1) the loss of GLUT2 protein associated with hyperglycemia is at least partially explained by reduced levels of the GLUT2 gene transcripts; 2) the reduction of beta-cell insulin content during chronic hyperglycemia may not be completely due to degranulation (reduced levels of gene transcripts may play a role); and 3) the reduction in the transcription factor IDX-1 raises the possibility that dysregulation of transcription factors may contribute to the abnormal beta-cell function found in states of chronic hyperglycemia.

The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration.

Islet duodenal homeobox 1 (IDX-1/PF-1/STF-1/PDX-1), a homeodomain protein that transactivates the insulin promoter, has been shown by targeted gene ablation to be required for pancreatic development. After 90% pancreatectomy (Px), the adult pancreas regenerates in a process recapitulating embryonic development, starting with a burst of proliferation in the epithelium of the common pancreatic duct. In this model, IDX-1 mRNA was detected by semiquantitative reverse transcription-polymerase chain reaction in total RNA from isolated common pancreatic ducts at levels 10% of those of isolated islets. The IDX-1 mRNA levels were not significantly different for common pancreatic ducts of Px, sham Px, and unoperated rats and did not change with time after surgery. By immunoblot analysis, IDX-1 protein was only faintly detected in these ducts 1 and 7 days after Px or sham Px but was easily detected at 2 and 3 days after Px. Similarly, IDX-1 immunostaining was barely detectable in sham or unoperated ducts but was strong in ducts at 2-3 days after Px. The increase of IDX-1 immunostaining followed that of BrdU incorporation (proliferation). These results indicate a posttranscriptional regulation of the IDX-1 expression in ducts. In addition, islets isolated 3-7 d after Px showed higher IDX-1 protein expression than control islets. Thus, in pancreatic regeneration IDX-1 is upregulated in newly divided ductal cells as well as in islets. The timing of enhanced expression of IDX-1 implies that IDX-1 is not important in the initiation of regeneration but may be involved in the differentiation of ductal cells to beta-cells.

Improved glucose tolerance and acinar dysmorphogenesis by targeted expression of transcription factor PDX-1 to the exocrine pancreas.

The homeodomain protein PDX-1 is critical for pancreas development and is a key regulator of insulin gene expression. PDX-1 nullizygosity and haploinsufficiency in mice and humans results in pancreatic agenesis and diabetes, respectively. At embryonic day (e) 10.5, PDX-1 is expressed in all pluripotential gut-derived epithelial cells destined to differentiate into the exocrine and endocrine pancreas. At e15, PDX-1 expression is downregulated in exocrine cells, but remains high in endocrine cells. The aim of this study was to determine whether targeted overexpression of PDX-1 to the exocrine compartment of the developing pancreas at e15 would allow for respecification of the exocrine cells. Transgenic (TG) mice were generated in which PDX-1 was expressed in the exocrine pancreas using the exocrine-specific elastase-1 promoter. These mice exhibited a marked dysmorphogenesis of the exocrine pancreas, manifested by increased rates of replication and apoptosis in acinar cells and a progressive fatty infiltration of the exocrine pancreas with age. Interestingly, the TG mice exhibited improved glucose tolerance, but absolute beta-cell mass was not increased. These findings indicate that downregulation of PDX-1 is required for the proper maintenance of the exocrine cell phenotype and that upregulation of PDX-1 in acinar cells affects beta-cell function. The mechanisms underlying these observations remain to be elucidated.

A new antiestrogen, 2-(4-hydroxy-phenyl)-3-methyl-1-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-1H-indol-5-ol hydrochloride (ERA-923), inhibits the growth of tamoxifen-sensitive and -resistant tumors and is devoid of uterotropic effects in mice and rats.

PURPOSE: Tamoxifen is an antiestrogen used in women who have estrogen receptor (ER)-alpha-positive breast cancer. Unfortunately, resistance to tamoxifen is common in women with metastatic disease and side effects, including increased risk of endometrial cancer, exist. Here we describe the activity of a new selective ER modulator, ERA-923, in preclinical models focused on these limitations. EXPERIMENTAL DESIGN: The ability of ERA-923, 4-OH tamoxifen, or raloxifene to inhibit estrogen-stimulated growth was evaluated in cell-based and xenograft assays with tumor cells that are sensitive or resistant to tamoxifen. Uterine effects of selective ER modulators were compared in rodents. RESULTS: ERA-923 potently inhibits estrogen binding to ER-alpha (IC(50), 14 nM). In ER-alpha-positive human MCF-7 breast carcinoma cells, ERA-923 inhibits estrogen-stimulated growth (IC(50), 0.2 nM) associated with cytostasis. In vitro, a MCF-7 variant with inherent resistance to tamoxifen (10-fold) or 4-OH tamoxifen (>1000-fold) retains complete sensitivity to ERA-923. Partial sensitivity to ERA-923 exists in MCF-7 variants that have acquired profound tamoxifen resistance. In tumor-bearing animals, ERA-923 (10 mg/kg/day given p.o.) inhibits 17beta-estradiol-stimulated growth in human tumors derived from MCF-7, EnCa-101 endometrial, or BG-1 ovarian carcinoma cells, including a MCF-7-variant that is inherently resistant to tamoxifen. Raloxifene is inactive in the MCF-7 xenograft model. Unlike tamoxifen, droloxifene, or raloxifene, ERA-923 is not uterotropic in immature rats or ovariectomized mice. Consistent with this, tamoxifen, but not ERA-923, stimulates the growth of EnCa-101 tumors. CONCLUSIONS: In preclinical models, ERA-923 has an improved efficacy and safety compared with tamoxifen. Clinical trials with ERA-923 are in progress.

Differential regulation of rat insulin I and II messenger RNA synthesis: effects of fasting and cyproheptadine.

Rats and mice retain a duplicated insulin (I) gene. Because the duplicated gene shares only incomplete homology with the ancestral insulin (II) gene it may be regulated differently. In the studies presented here we measured changes in abundance of these distinct insulin mRNAs and their precursors in response to fasting and fasting plus a single dose of cyproheptadine, two experimental manipulations that cause changes in the level of total insulin mRNA in rats. Both diminished rat insulin II mRNA to a greater extent than rat insulin I mRNA. Rat insulin II mRNA comprised 41% of the total insulin mRNA in 0 h controls and decreased to 33% of the total insulin mRNA after a 10-h fast. Insulin II mRNA decreased to 26% of the total insulin mRNA 10 h after treatment with cyproheptadine. To determine whether these manipulations had effects on insulin mRNA synthesis, precursors for each of the two mRNAs were quantified. Fasting for 24 h had only small effects on insulin I mRNA precursor, but diminished rat insulin II pre-mRNA to 32% of the 0 h control values. One and a half hours after fasting plus cyproheptadine administration, pre-mRNA for rat insulin II levels had decreased to 38%, while rat insulin I pre-mRNA remained at levels present in 0 h controls. Levels of rat insulin I and II pre-mRNAs were both maximally depressed at 10 h, but rat insulin II pre-mRNA decreased to 3%, while rat insulin I pre-mRNA diminished to only 49% of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

An optimized system for studies of EPO-dependent murine pro-erythroblast development.

OBJECTIVE: Objectives were to develop new means to isolate useful numbers of primary progenitor cells and to quantitatively assay the stepwise maturation of erythroblasts. METHODS: Approaches involved dosing mice with thiamphenicol (TAP) to yield staged cohorts of pro-erythroid cells; optimizing conditions for their EPO-dependent in vitro growth and survival; developing assays for CFU-E maturation; analyzing stage-specific transcript expression; and expressing a heterologous, erythroid-specific tag (EE372) in transgenic mice. RESULTS: Per TAP-treated mouse, 3 x 10(7) highly EPO-responsive erythroid progenitor cells were generated that represented up to 30% of total splenocytes and showed strict dependence on EPO for survival, growth, and immediate response gene expression. In this developing cohort, a tightly programmed sequence of gene expression was observed, and maximal expression of c-kit, EPO receptor, and beta-globin transcripts occurred at 72, 96, and 120 hours post-TAP withdrawal, respectively. Also, the newly discovered erythroid-specific dual-specificity kinase, DYRK3, was revealed to be expressed at a late CFU-E stage. In vitro, these progenitor cells matured stepwise from high FALS Ter119- cells (24-hour culture) to high FALS Ter119+ cells (24-36 hours) to low FALS Ter119+ maturing erythroblasts (40-48 hours) and sharp differences in their morphologies were observed. Finally, a MACS-based procedure for the purification of erythroid progenitor cells from TAP-treated EE372 transgenic mice also was developed. CONCLUSIONS: A comprehensive new system for isolating large numbers of primary murine erythroid progenitor cells and quantitatively monitoring their development is established that should serve well in investigations of endogenous and pharmacological regulators of red blood cell development.

Rapid alteration of pancreatic proinsulin and preproinsulin messenger ribonucleic acid in rats treated with cyproheptadine.

Cyproheptadine (CPH) appears to be unique among islet beta-cell toxicants by virtue of its ability to rapidly and reversibly inhibit insulin biosynthesis in rats. These studies examined the mechanism of CPH-induced insulin depletion by determining the time course for CPH-induced changes in pancreatic preproinsulin mRNA, proinsulin, and insulin levels. A single oral dose of CPH decreased proinsulin levels to 35% of the control value by 3 h. Proinsulin stayed depressed for up to 24 h. Preproinsulin mRNA declined more slowly, reaching 35% of the control value at 6 h, and increased more rapidly, returning to the control value by 24 h. Pancreatic insulin levels did not decrease significantly until 24 h. Exposure of isolated rat islets to CPH for 30 min in vitro selectively inhibited proinsulin synthesis by 62%, without affecting preproinsulin mRNA levels. The dissociation between changes in proinsulin and preproinsulin mRNA levels suggests that the decrease in preproinsulin mRNA in vivo is associated with but does not cause CPH-induced changes in insulin biosynthesis.

Developmental expression of the homeodomain protein IDX-1 in mice transgenic for an IDX-1 promoter/lacZ transcriptional reporter.

Expression of the homeodomain transcription factor IDX-1 (also known as IPF-1, STF-1, and PDX-1) is required for pancreas development, because disruption of the gene in mice and humans results in pancreatic agenesis. During embryonic development the idx-1 gene is first expressed in a localized region of foregut endoderm from which the duodenum and pancreas later develop. To more fully understand the role of IDX-1 in pancreas development, transgenic mice expressing the Escherichia coli lacZ gene under control of the 5'-proximal 4.6 kb of the idx-1 promoter were created as a reporter for the developmental expression of IDX-1. Here we show that the determinants for the developmental and tissue-specific expression of the endogenous idx-1 gene are faithfully reproduced by the 4.6-kb region of the idx-1 promoter. Expression of lacZ is detected in the development of the exocrine and endocrine pancreas in pancreatic ducts, common bile and cystic ducts, pyloric glands of the distal stomach, Brunner's glands, the intestinal epithelium of the duodenum, and the spleen. The observed spatial and temporal pattern of lacZ expression directed by the IDX-1 promoter further supports an important role of IDX-1 in specifying the development of several endodermal structures within the midsegment of the body. An unexpected finding is that IDX-1 promoter-driven (transcriptional) lacZ activity does not always coincide with the localization of IDX-1 messenger RNA by in situ hybridization and IDX-1 protein by immunocytochemistry in adult rat duodenum, suggesting the existence of regulation of IDX-1 expression at the posttranscriptional level of expression of the idx-1 gene.

Cyproheptadine-induced alterations in clonal insulin-producing cell lines.

Cyproheptadine (CPH) inhibits glucose-stimulated insulin synthesis and secretion, and reversibly depletes pancreatic insulin content in the rat. To examine whether the inhibitory actions of CPH on insulin cell function are linked to the ability of glucose to stimulate insulin synthesis and secretion, studies were performed in two different insulin-producing cell lines. CPH effects were compared in HIT-T15 cells, which respond to glucose with increased insulin synthesis and secretion, and in glucose-unresponsive RINm5F cells. CPH produced similar alterations in both cells lines. After a 48-hr culture period in the presence of 0, 0.1, 1.0 or 10.0 microM CPH, cellular insulin stores and media insulin levels were decreased in a concentration-dependent manner. At 10.0 microM CPH, RIN and HIT cell insulin content declined to 34 and 33% of controls respectively. Cellular insulin returned to control levels 48 hr after removal of CPH. In experiments designed to test a direct inhibitory effect on stimulated insulin secretion, 1 and 10.0 microM concentrations of CPH were found to inhibit glucose-stimulated insulin release from HIT cells, and K+, alanine and glyceraldehyde-stimulated release from RIN cells. CPH was also shown to inhibit insulin biosynthesis in both cell lines at concentrations that did not alter the synthesis of total cellular proteins. All of these alterations in cellular function were shown to occur at CPH concentrations that did not affect cell growth or viability. The results show that the actions of CPH do not appear to be dependent upon the existence of operational glucose signalling mechanisms for insulin synthesis and secretion.

Repression of somatostatin gene transcription mediated by two promoter silencer elements.

We report that expression of the somatostatin gene in pancreatic islets and in non-islet cells is negatively regulated by two proximal silencer elements, PS1 and PS2. Transient transfection assays showed that PS1 decreases somatostatin gene promoter activity stimulated by an upstream enhancer in the islet D-cell line RIN-1027-B2, but not in the islet B-cell line RIN-1046-38, whereas PS2 inhibits gene transcription both B- and D-cell lines. In BHK fibroblasts, both PS1 and PS2 independently inhibit somatostatin gene in non-islet cells. DNA-binding studies revealed that both PS1 and PS2 bind similar nuclear protein complexes in islet and non-islet cells (120 and 130 kDa). PS1 also binds a 100-kDa protein present in islet B- and D-cell lines. In addition, both PS1 and PS2 bind three D-cell-specific proteins (40, 43 and 45 kDa). These observations support a direct involvement of both positive and negative transcriptional control mechanisms in the regulation of the islet cell-specific expression of the somatostatin gene.

In vitro antioxidant effects of estrogens with a hindered 3-OH function on the copper-induced oxidation of low density lipoprotein.

Estrogens with some bulky alkyl substituents in both the 2- and 4-positions have been synthesized and evaluated for the ability to inhibit the in vitro oxidation of low density lipoprotein as determined by the thiobarbituric reactive substances method. The present compounds with bulky groups in either the 2- or the 4-position (but not both the 2- and 4-) were especially effective as antioxidants, having IC50 values lower than either estradiol or probucol; however, they do not bind to the estrogen receptor with any great affinities (RBA < 0.1 versus estradiol). This separation of antioxidant efficacy from estrogenicity may allow these compounds to serve as useful probes for ascertaining the relative importance of these effects in the cardioprotective role played by estrogens.

Marizomib, a proteasome inhibitor for all seasons: preclinical profile and a framework for clinical trials.

The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique ß-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, chemotherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside.