neptune, a Krüppel-like transcription factor that participates in primitive erythropoiesis in Xenopus.

The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Krüppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates beta-globin expression during erythroid differentiation [2-6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptune's DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus.

Primitive erythropoiesis in the Xenopus embryo: the synergistic role of LMO-2, SCL and GATA-binding proteins.

Hematopoietic stem cells are derived from ventral mesoderm during vertebrate development. Gene targeting experiments in the mouse have demonstrated key roles for the basic helix-loop-helix transcription factor SCL and the GATA-binding protein GATA-1 in hematopoiesis. When overexpressed in Xenopus animal cap explants, SCL and GATA-1 are each capable of specifying mesoderm to become blood. Forced expression of either factor in whole embryos, however, does not lead to ectopic blood formation. This apparent paradox between animal cap assays and whole embryo phenotype has led to the hypothesis that additional factors are involved in specifying hematopoietic mesoderm. SCL and GATA-1 interact in a transcriptional complex with the LIM domain protein LMO-2. We have cloned the Xenopus homolog of LMO-2 and show that it is expressed in a similar pattern to SCL during development. LMO-2 can specify hematopoietic mesoderm in animal cap assays. SCL and LMO-2 act synergistically to expand the blood island when overexpressed in whole embryos. Furthermore, co-expression of GATA-1 with SCL and LMO-2 leads to embryos that are ventralized and have blood throughout the dorsal-ventral axis. The synergistic effect of SCL, LMO-2 and GATA-1, taken together with the findings that these factors can form a complex in vitro, suggests that this complex specifies mesoderm to become blood during embryogenesis.

Cooperative effects of growth factors involved in the induction of hematopoietic mesoderm.

Hematopoietic induction occurs on the ventral side of Xenopus gastrulae and is thought to be triggered by the growth factor bone morphogenetic protein 4 (BMP-4). To characterize this process, we developed a quantitative and sensitive assay for the induction of erythroid cells from totipotent ectoderm of the embryo. When high doses of BMP-4 were used in this explant assay, few erythroid cells were detected. In contrast, large numbers of differentiated erythroid cells were induced when ectoderm was treated with BMP-4 and the mesoderm inducers, activin, or fibroblast growth factor (FGF). Ectopic expression of GATA-1 also induced abundant erythroid cells in ectoderm treated with bFGF. This induction of erythroid cells by GATA-1 was blocked by coexpression with a dominant negative BMP-4 receptor, showing that GATA-1 requires the BMP signaling cascade to function. These results suggest that BMP-4 requires mesoderm induction to generate a program of gene expression, which regulates the specification of hematopoietic mesoderm by GATA factors.

Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins.

We have developed a technique, fast retrieval of gel shift activities (FROGS), that allows for the rapid isolation of proteins that interact with DNA. Using this technique, we have isolated two proteins that are structurally similar to Mix.1, a PAX class homeodomain protein with ventralizing activity in Xenopus. The Mix family of proteins are expressed during late blastula and gastrula stages of Xenopus development. During gastrulation, these genes are expressed at high levels in distinct, yet overlapping regions in mesoderm and endoderm. The members of the Mix family heterodimerize with each other and overexpression of each results in severe axial abnormalities. Mix.3 and Mix.4 can directly induce primitive ectoderm to become endoderm whereas Mix.1 cannot. Injection of Mix.3 or Mix.4 RNA in the whole embryo results in extensive ectopic endodermin mRNA expression. The expression of the Mix family homeoproteins is differentially regulated by activin, Vg1, BMP-4, and fibroblast growth factor, supporting a model in which the Mix homeoproteins are downstream effectors of growth factor signaling during endoderm and ventral mesoderm formation.

The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis.

In vertebrates, hematopoietic and vascular progenitors develop from ventral mesoderm. The first primitive wave of hematopoiesis yields embryonic red blood cells, whereas progenitor cells of subsequent definitive waves form all hematopoietic cell lineages. In this report we examine the development of hematopoietic and vasculogenic cells in normal zebrafish and characterize defects in cloche and spadetail mutant embryos. The zebrafish homologs of lmo2, c-myb, fli1, flk1, and flt4 have been cloned and characterized in this study. Expression of these genes identifies embryonic regions that contain hematopoietic and vascular progenitor cells. The expression of c-myb also identifies definitive hematopoietic cells in the ventral wall of the dorsal aorta. Analysis of b316 mutant embryos that carry a deletion of the c-myb gene demonstrates that c-myb is not required for primitive erythropoiesis in zebrafish even though it is expressed in these cells. Both cloche and spadetail mutant embryos have defects in primitive hematopoiesis and definitive hematopoiesis. The cloche mutants also have significant decreases in vascular gene expression, whereas spadetail mutants expressed normal levels of these genes. These studies demonstrate that the molecular mechanisms that regulate hematopoiesis and vasculogenesis have been conserved throughout vertebrate evolution and the clo and spt genes are key regulators of these programs.

Transcriptional regulation of blood formation during Xenopus development.

Hematopoiesis is the generation, proliferation and differentiation of hematopoietic stem cells (HSCs) into the erythroid, myeloid and lymphoid lineages. The HSC is a ventral mesodermal derivative that arises due to inductive and patterning processes triggered by bone morphogenetic protein 4 (BMP-4). Recent studies in Xenopus laevis have identified families of transcription factors that participate in hematopoietic development. Transcription factor families, such as the SMADs, Mixs and Vents pattern mesoderm to a ventral fate which ultimately results in the expression of hematopoietic-specific transcription factors such as GATA-1 and SCL. This transcriptional regulatory network enforces the correct temporal and spatial expression of the blood program.

Surgical creation of portacaval shunts during temporary intestinal arterial and venous occlusion in dogs.

OBJECTIVE: To surgically create complete portacaval shunts in dogs during temporary arrest of intestinal arterial and portal venous blood flow. DESIGN: Complete portacaval anastomoses were surgically created, and liver function was evaluated for 14 to 18 weeks after surgery. ANIMALS: 32 adult mixed-breed dogs of either sex. PROCEDURE: Administration of deferoxamine and temporary intestinal arterial occlusion were used to minimize the intestinal cellular damage resulting from the complete, temporary arrest of portal venous blood flow during creation of the portacaval anastomosis. Side-to-side, appositional anastomoses ( > 2 cm diameter) were formed between the portal vein and caudal vena cava. Dogs were observed daily for signs of hepatic encephalopathy, and food intake was recorded. Body weight was recorded weekly. Preprandial plasma ammonia, serum urea nitrogen, and glucose concentrations and sulfobromophthalein retention were measured monthly. The dogs were euthanatized, and necropsy was performed 14 to 18 weeks after surgery. RESULTS: 30 of 32 dogs recovered without complications. Complete portosystemic shunting was documented by increased plasma ammonia concentration, decreased serum urea nitrogen and glucose concentrations, prolonged sulfobromophthalein retention (P < 0.01), and inspection at necropsy. CONCLUSION: This method of providing temporary, complete arrest of portal venous blood flow was helpful in allowing accurate, appositional portacaval anastomoses to be created that remained patent for 14 to 18 weeks. CLINICAL RELEVANCE: This method of providing temporary, complete arrest of portal venous blood flow may prove useful in clinical surgery when temporary arrest of portal blood flow is desired.

Microcytosis and iron status in dogs with surgically induced portosystemic shunts.

Microcytosis is common in dogs with congenital portosystemic shunts (PSS) and acquired liver disease. The objective of this study was to determine if microcytosis could be induced in normal dogs by surgical creation of PSS, and to characterize the changes in hematology and iron status. Hematocrit, mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration decreased linearly from 45.5%, 69.1 fL, 22.8 g/dL and 33.1% to 39.5%, 55.9 fL, 17.8 g/dL and 31.9%, respectively, 18 weeks after creation of PSS. The erythrocyte count did not change, but red cell distribution widths indicated a shift to a heterogenous population with decreased volume. Mean cell volume and mean cell hemoglobin decreased rapidly after induction of PSS and were significantly (P < .05) different from presurgery values within 2 weeks. Serum iron and copper concentrations and total iron binding capacity were decreased in dogs with PSS. Liver iron concentration doubled after creation of PSS, with the majority of stainable iron located in Kupffer cells. The changes in erythrocyte indices and measures of iron status in dogs with surgically induced PSS were similar to those in dogs with congenital PSS. Microcystosis developed rapidly in dogs after induction of PSS. These results indicate that iron deficiency was not the cause of microcytosis in these dogs.

Apparent dietary protein requirement of dogs with portosystemic shunt.

Current medical management of dogs with portosystemic shunt (PSS) includes dietary protein restriction. After establishment of baseline values, 32 dogs underwent portosystemic anastomosis to induce PSS. They were assigned to 1 of 4 dietary treatments, and given 11 or 24% crude protein (CP); 20% of the protein was derived from branched chain or aromatic amino acids. The apparent digestibility of CP and of total digestible energy were not affected by PSS. The apparent digestibility of fat decreased from 92% to 85% in dogs with PSS (P < 0.01). Across all diets, the apparent dietary protein requirement (ADPR) was 2.07 g of CP/kg of body weight/d in clinically normal dogs and 2.11 g of CP/kg/d after PSS. Dietary amino acid composition had no effect on ADPR. The ADPR for dogs fed the 11% protein diets was 1.69 g of CP/kg/d in clinically normal dogs and 1.62 g of CP/kg/d after PSS, whereas the ADPR in dogs fed the 24% protein diets was 3.94 g of CP/kg/d before PSS and 3.31 g of CP/kg/d after PSS. Serum total protein, urea nitrogen, and albumin concentrations were lower in dogs with PSS fed the 11% protein diets, compared with those fed the 24% protein diets. We conclude that there is no difference in ADPR in dogs with PSS; however, the low protein intake of 1.62 g of CP/kg/d appeared inadequate to maintain normal protein stores. Dietary protein that provides at least 2.1 g of CP/kg/d is recommended for dogs with PSS.

Infertility in a ewe as a result of ovotestis.

A ewe with apparently normal estrous cycles was unable to conceive as a result of a single ovotestis. The ovotestis was diagnosed on the basis of results from laparoscopic examination, human chorionic gonadotropin stimulation, and histologic examination of the gonad. The report reinforces the necessity of considering all causes of infertility in affected sheep, and not simply infective causes.

Comparison of procedures for assessing adequacy of dog foods.

Dog foods with similar claims for nutritional adequacy were tested by chemical analysis and the American Association of Feed Control Officials' growth trial. All foods tested were similar chemically, however, dogs given one regionally marketed food had lower growth rate and food efficiency as well as suboptimal PCV and hemoglobin values during the growth trial. Pups fed this diet also had clinical signs typical of zinc and copper deficiencies. We concluded that American Association of Feed Control Officials' approved feeding tests provide valid assessment of pet food quality, and procedures involving only chemical analysis or calculated values may not.

A simplified technique for chronic cerebrospinal fluid collection in the awake dog.

A modified design for a guide tube to facilitate collection of cerebrospinal fluid from the lateral ventricle of awake dogs was developed. The modifications allowed for rapid, inexpensive and secure implantation. The tested guide tubes remained in place and patent for 20 weeks.

Influence of endotoxin-induced fever on the pharmacokinetics of gentamicin in ewes.

The pharmacokinetics of gentamicin (3 mg/kg of body weight) were evaluated in 6 adult ewes before and after fever was induced with Escherichia coli endotoxin (1 micrograms/kg). In the ewes with normal rectal temperature, significant (P less than 0.05) increases in rectal temperature occurred before gentamicin injection and during the first 2 hours. Other mild clinical signs of fever also were present. In the same ewes with endotoxin-induced fever, statistically (P less than 0.05) increased gentamicin concentrations occurred at 15 and 40 minutes and at 6 hours after injection of gentamicin. Changes were not observed in the apparent volume of distribution calculated by the area method, the volume of distribution at steady state, the overall biological half-life, or body clearance. Significant (P less than 0.05) reductions occurred in the zero time intercept for distribution, the distribution rate constant, the concentration in plasma at time of injection, the volume of the peripheral compartment, and the first order transfer rate constants; only the volume of the central compartment was increased. Total amounts of gentamicin were increased in the central compartment and decreased in the peripheral or tissue compartment.

The effect of diet on fecal moisture, osmolarity of fecal extracts, products of bacterial fermentation and loss of minerals in feces of weaned pigs.

Litters of pigs were allotted to one of three dietary treatments. Treatment 1 (T1) consisted of a corn-soybean meal starter diet. Pigs fed treatment 2 (T2) received a steamed, rolled oat groats-casein diet and pigs in treatment 3 (T3) remained with the sow. Four pigs/treatment were used to investigate the difference in performance and the cause of post-weaning diarrhea associated with early weaning of pigs at 4 wk of age to a starter diet. Fecal moisture, osmolarity, acetic acid, lactic acid and glucose contents were all good indicators of dietary differences because of treatment X age interactions. These variables increased faster in fecal extracts from pigs fed the corn-soybean meal diet. Lactic acid, volatile fatty acid (VFA) levels, glucose and pH values were indicative of a more active bacterial fermentation in pigs receiving T1 than in those receiving T2 or pigs remaining with the sow (T3). Excess minerals appear to contribute significantly to the osmolarity of fecal material. Of the anions, lactate was the main contributor to the osmolarity of feces of T1 pigs, followed by P, VFA, of which acetic acid contributed 70%, and Cl. The main cations were K, Na and Ca. In T2, P was the main anion, followed by lactate, VFA and Cl, while the main cations were Na, K and Ca. Minerals seemed to be the major osmotic particles in fecal extracts of pigs remaining with the sow. Phosphorus was the major anionic contributor to osmolarity, followed by VFA, Cl and lactic acid. Potassium was the major cation, followed by Na and Ca.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum erythromycin concentration in sheep fed two diets.

Serum erythromycin concentrations following a single IV injection (12.5 mg/kg) were compared in 6 sheep during feeding of a grass hay diet and a concentrate diet. The concentrate diet resulted in a mean decrease of at least 1 pH unit in forestomach fluid, but did not significantly alter either the serum drug concentrations or half-life. Variable antibacterial activity was present in forestomach fluid as early as 1 or 2 hours after injection. This study indicates that ion-trapping in forestomach fluid as a result of normal dietary changes does not require therapeutic dosage adjustment.

Hepatic metabolite concentration in lactic acidotic sheep.

The concentration of hepatic metabolites associated with energy-producing pathways and hepatic oxygen consumption were measured in clinically normal and lactic acidotic sheep. Significantly (P less than 0.005) lower pyruvate concentrations in livers of acidotic sheep produced significantly larger L-lactate to pyruvate concentration ratios. Glycogen concentrations were significantly (P less than 0.005) lower in liver from acidotic sheep. Oxygen consumption by liver from acidotic sheep was depressed when compared with oxygen consumption by normal sheep liver. Seemingly, limited available carbohydrate substrate and not tissue oxygenation may be limiting aerobic glycolysis in liver of lactic acidotic sheep.

Effects of promethazine on hemorrhagic shock in the dog.

The effects of pre- and post-treatment with promethazine, an antihistamine, in hemorrhage-shocked dogs were investigated. Survival to 6 hours was enhanced in the dogs given (IM administration) the H1-receptor antagonist at 1 hour before hemorrhage was induced when compared with survivals of saline-treated control dogs (3 of 5 dogs vs 1 of 5 dogs). Furthermore, 4 of 5 dogs given (IV administration) promethazine at 30 minutes after the initial hemorrhage survived. Promethazine treatment by either route increased mean arterial pressure among surviving dogs throughout the 6-hour experimental period.