In remembrance of Eugene Ladislas Kanabrocki (18 April 1922 * 23 April 2009): a 'General' in the battle of penetrating the normal range and in revealing its relation to the cosmos.

Col. Eugene L. (Gene) Kanabrocki, PhD, commanding officer of the 361st Medical Laboratory of the U.S. Army Reserve, together with Col. Lawrence E. (Larry) Scheving, Professor at the University of Arkansas, initiated in May 1969 a linked cross-sectional (hybrid) study at Fort Sam Houston, TX, to examine the oscillatory (circadian) nature of many physiological variables in a group of 13 army men, 22-28 years of age, anticipating that such data would serve, as indeed they did, as time-specified reference values in future investigations of health and aging. In the initial study, 36 variables were examined around the clock in observations at 3-hour intervals. In subsequent 24-hour profiles, mapped in May of 1971 (mostly on new, young subjects, and not officially part of the Aging Project), 1979, 1988, 1993, 1998 and 2003, additional subjects and variables were included. The follow-up studies were conducted at the Hines VA Medical Center in Hines, Illinois. Of the original 13 subjects, four were measured in all 6 studies and another four in 5 of the 6 studies. Three of the eight became diabetic (Type II) and three had vascular circulatory problems. Presently, a bank of circadian data for 187 medically relevant variables of blood (plasma or serum), saliva, urine, vital signs and other variables on the same subjects covers a span of 34 years. Dr. Robert B. Sothern (RBS), of the University of Minnesota, USA, the major analyst of Gene's investigations, in addition to being an add-on subject as he was in three studies, set up the half-hourly monitoring of blood pressure (BP) and heart rate (HR) in the 2003 study that yielded the data suggesting that the standard deviations (SD) of systolic (S) and diastolic (D) BP and HR are infl uenced by a magnetic storm. Since the standard deviation rather than the amplitude of a vascular spectral component was affected, we may be dealing with a stochastic rather than frequency window-dependent resonance with a magnetic storm. Gene and RBS also found (p< 0.08) an about-decadal signature of solar activity in long-acting natriuretic peptide (LANP), vessel dilator (VSDL), insulin, LH, prolactin, T3 uptake and, most importantly, in melatonin (p=0.004), noted solely to constitute a stimulus for follow-up studies, even when resonance occurs in an anticipated Horrebow-Schwabe circadecadal window gauged by relative sunspot (Wolf) numbers and involves many endocrine variables, as anticipated on the basis of independent evidence in melatonin and cortisol. The wealth of circadian information collected in these studies by Gene constitutes a treasure trove of unique advances in the battle of the normal range, with solid contributions also by Prof. Germaine Cornélissen of the University of Minnesota, USA, and by Prof. Ramon C. Hermida of the University of Vigo, Spain.

Insulin and heregulin-beta1 upregulate guanylyl cyclase C expression in rat hepatocytes: reversal by phosphodiesterase-3 inhibition.

Guanylyl cyclase C (GC-C) is the receptor for the hormones guanylin and uroguanylin. Although primarily expressed in the rat intestine, GC-C is also expressed in the liver during neonatal or regenerative growth or during the acute phase response. Little is known about the hepatic regulation of GC-C expression. The influence of various hepatic growth or acute phase regulators on GC-C expression was evaluated by immunoblot analysis of protein from primary rat hepatocytes grown in a serum-free medium. Insulin and heregulin-beta1 strongly stimulated GC-C expression by 24 h of cell culture. Several different hormones and agents suppressed this action, including transforming growth factor beta (TGF-beta), as well as inhibitors of phosphatidylinositol 3-kinase (PI-3-kinase) and phosphodiesterase 3 (PDE-3, an insulin- and PI-3-kinase-dependent enzyme). The compartmental downregulation of cAMP levels by PDE-3 may be a critical step in the hormonal action that culminates in GC-C synthesis.

High salt intake increases uroguanylin expression in mouse kidney.

The intestinal peptides, guanylin and uroguanylin, may have an important role in the endocrine control of renal function. Both peptides and their receptor, guanylyl cyclase C (GC-C), are also expressed within the kidney, suggesting that they may act locally in an autocrine/paracrine fashion. However, their physiological regulation within the kidney has not been studied. To begin to address this issue, we evaluated the distribution of uroguanylin and guanylin messenger RNA (mRNA) in the mouse nephron and the regulation of renal expression by changes in dietary salt/water intake. Expression was determined in 1) wild-type mice, 2) two strains of receptor-guanylyl cyclase-deficient mice (ANP-receptor-deficient, GC-A-/-, and GC-C-deficient mice); and 3) cultured renal epithelial (M-1) cells, by RT-PCR, Northern blotting and immunocytochemistry. Renal uroguanylin messenger RNA expression was higher than guanylin and had a different distribution pattern, with highest levels in the proximal tubules, whereas guanylin was mainly expressed in the collecting ducts. Uroguanylin expression was significantly lower in GC-C-/- mice than in GC-A-/- and wild-types, suggesting that absence of a receptor was able to down-regulate ligand expression. Salt-loading (1% NaCl in drinking water) increased uroguanylin-mRNA expression by >1.8-fold but had no effect on guanylin expression. Uroguanylin but not guanylin transcripts were detected in M-1 cells and increased in response to hypertonic media (+NaCl or mannitol). Our results indicate that high-salt intake increases uroguanylin but not guanylin expression in the mouse kidney. The synthesis of these peptides by tubular epithelium may contribute to the local control of renal function and its adaptation to dietary salt.

Biological clocks and the digestive system.

Circadian rhythms play a major role in regulating the digestive systems of many organisms. Cell proliferation, migration, differentiation, and even structure vary as a function of time of day in many different digestive organs (i.e., stomach, gut, liver, and pancreas) and cell types, resulting in regionally specific temporal variations in protein and gene expression. Feeding and light set the hands of the digestive clock(s). However, the clockwork has a genetic basis. During the last 10 years, new developments have emerged in our understanding of how cells keep time. Surprisingly, clock genes in mammals are expressed not only in specialized time keepers in the brain, but also in peripheral organs, suggesting that the ability to keep time may also belong to cells within the digestive system. This article reviews several classic examples of circadian variation in the digestive system, with an emphasis on rhythms in cell proliferation, function, and structure. It also briefly summarizes several new ideas about how cells in the brain and possibly the digestive system keep time.

Circadian regulation of uroguanylin and guanylin in the rat intestine.

Uroguanylin (UGN) and guanylin (GN) are the endogenous intestinal ligands for guanylyl cyclase C (GC-C). We examined the circadian expression of UGN, GN, and GC-C in the jejunum, ileum, and proximal colon of young adult rats by Northern blot analyses. These assays revealed that UGN is more abundant in the proximal small intestine, whereas GN and GC-C are more abundant in the proximal colon. mRNA levels showed significant circadian variation for UGN (3- to 18-fold peak/trough difference), GN (2.1- to 2.8-fold peak/trough difference), and GC-C (3- to 5-fold peak/trough difference). The maximal abundance occurred in the dark period for all three mRNAs, although peak UGN and GN expression occurred later in the dark period in the jejunum relative to the ileum and colon. Immunoblot analyses using monospecific polyclonal antibodies against UGN and GN prohormones confirmed the regional and circadian variation detected by Northern assays. Thus the expression of these genes is regulated not only by histological position but also by circadian time.

Regulation of intestinal tyrosine phosphorylation and programmed cell death by peroxovanadate.

Cell suspensions of ileal mucosa undergo a rapid and synchronized form of programmed cell death when cultured in a simple medium at 37 degrees C. Because tyrosine phosphorylation of proteins plays a crucial role in the signal transduction of many cellular processes, we examined its role in intestinal programmed cell death by use of immunoblot and immunohistochemical methods. We observed a 50-70% reduction in tyrosine phosphorylation during the initial 10 min of intestinal epithelial cell culture. We hypothesized that the inhibition of protein tyrosine phosphatases would increase protein tyrosine phosphorylation in these suspensions and decrease programmed cell death. A strong inhibitor of these phosphatases (peroxovanadate) but not a weaker one (sodium orthovanadate) abolished the DNA fragmentation/laddering normally seen in dying enterocytes. Peroxovanadate enhanced protein tyrosine phosphorylation of many intestinal proteins, dramatically increasing the dually phosphorylated and active form of mitogen-activated protein kinase. Immunohistochemistry revealed a particularly high level of increased tyrosine phosphorylation in the intestinal crypts in peroxovanadate-treated mucosa. Kinetic studies indicated that the pivotal time for protein tyrosine phosphatase inhibition occurred within 5 min of ex vivo culture, precisely when protein tyrosine phosphorylation declined. Our data suggest that tyrosine kinase inactivation or tyrosine phosphatase activation may initiate intestinal epithelial cell death.

Dying enterocytes downregulate signaling pathways converging on Ras: rescue by protease inhibition.

Organ and cell cultures of the small intestine serve as excellent in vitro models for programmed cell death (PCD). Cells cultured in serum-free, minimal medium rapidly died, as evidenced by histological changes, internucleosomal DNA cleavage, and TdT-mediated dUTP nick end labeling. Cell death was pervasive, although nonepithelial cells within the fibrovascular villus core were spared. PCD did not require a functional p53 gene. Serine and cysteine protease inhibitors, but not FCS, suppressed it. Relative to structural and functional proteins, dying enterocytes rapidly downregulated Ras-convergent proteins, including epidermal growth factor receptor, Erb-B2, and the son of sevenless guanine nucleotide exchangers. Reductions in the steady-state levels of both protein and mRNA were observed. These reductions were prevented by a combination of death-defying serine and caspase inhibitors, indicating a requirement for the initiation of death. Thus, during catastrophic PCD, intestinal epithelial cells delete cell surface signaling pathways responsible for Ras activation.

Circadian regulation of CREB transcription factor in mouse esophagus.

Very little is known about the circadian regulation of cell entry into the S and M phases of the cell cycle. Yet, in the mouse esophagus, a seven- to ninefold increase in DNA synthesis coincides with nocturnal feeding. The phosphorylation of the cAMP response element binding protein (CREB), a transcriptional factor, may regulate hypothalamic circadian rhythms in the brain. Here, we investigate the circadian regulation of CREB and Ser-133-phospho-CREB (PCREB) in the mouse esophagus by immunocytochemical and biochemical methods. We found that, during the dark phase, coincident with the onset of feeding and increased DNA synthesis, esophageal CREB and PCREB expression decreased. Although CREB-like immunoreactivity (CREB-lir) was expressed in many different cell types, it was concentrated in the mucosa, particularly in the replicating basal cell layer. The injection of epidermal growth factor, at a dosage known to maximally stimulate esophageal DNA synthesis in a 4- to 8-h period, rapidly decreased PCREB levels within 10 min of injection. We speculate that PCREB-lir may be involved in the circadian regulation of cell cycle events in the intact mouse esophagus.

Differential processing of guanylyl cyclase C along villus-crypt axis of rat small intestine.

Many strains of enterotoxigenic Escherichia coli produce a heat-stable peptide enterotoxin (STa) that binds to the intestinal receptor guanylyl cyclase C (GC-C). STa receptors are structurally heterogeneous, but the molecular events causing this heterogeneity remain obscure. We examined the influence of cell position along the villus-crypt axis on STa receptor heterogeneity by fractionating EDTA-dissociated cells that detached in a villus-to-crypt direction. STa affinity labeling experiments revealed that the initially released villus "tip" fraction had four major STa binding proteins (STBPs), with relative molecular weight (M(r)) of 150,000, 135,000, 125,000, and 95,000, that did not react with a GC-C carboxy-terminal antibody. Yet succeeding villus cell fractions had major immunoreactive STBPs with M(r) of 275,000 and 250,000. Limited proteolysis of these larger GC-C isoforms produced 1) smaller STBPs that had M(r) similar to those in the initial villus fraction, 2) a 65,000 M(r) protein GC-C isoform that did not bind STa, and 3) elevated basal and STa-induced cyclase activity. Our data show that STBP structural heterogeneity in the intact intestine arises largely from multisite proteolytic processing of GC-C.

Structure, glycosylation, and localization of rat intestinal guanylyl cyclase C: modulation by fasting.

Guanylyl cyclase C (GC-C), an intestinal receptor guanylyl cyclase, binds diarrhea-producing bacterial ligands such as the Escherichia coli heat stable enterotoxin. We examined the regulatory influence of feeding and fasting on the expression, structure, and biochemical properties of GC-C. When solubilized at 4 degrees C under nonreducing conditions, GC-C from both fed and fasted rats migrated on 7% sodium dodecyl sulfate-polyacrylamide electrophoretic gels as two extremely large aggregates that barely penetrated the stacking and resolving gels. Chemical reduction of disulfide linkages disaggregated GC-C in fed but not fasted rat samples, causing it to migrate as smaller forms (approximately 220 and 240 kDa). Although GC-C aggregates from fasted rats resisted this disaggregating effect of chemical reduction, they rapidly acquired it within 90 min of refeeding. When solubilized at denaturing temperatures (95 degrees C) under reducing conditions, GC-C aggregates largely disassembled into four smaller proteins (relative molecular weight approximately 140,000, 131,000, 85,000, and 65,000). However, the 131-kDa glycoprotein was disproportionately increased in fasted rat membranes. This unit and the 220-kDa unit were sensitive to endoglycosidase H. Subcellular fractionation and immunohistochemical studies revealed a major redistribution of GC-C from surface to intracellular enterocyte sites during fasting.

Guanylyl cyclase C is up-regulated by nonparenchymal cells and hepatocytes in regenerating rat liver.

Guanylyl cyclase C (GC-C) is the receptor for the heat-stable enterotoxin produced by bacteria as well as for the newly discovered mammalian hormones guanylin and uroguanylin. Ligand activation of GC-C causes it to produce cyclic GMP inside target cells. Although once thought to be restricted to the intestine, GC-C mRNA has recently been detected in other tissues. We now examine the expression, localization, and activation of this glycoprotein after partial hepatectomy in rats. By immunoblot analysis, GC-C protein appeared as early as 4 h after partial hepatectomy, reached its maximal expression (a 30-fold increase) between 24 and 48 h, and returned to low baseline levels at 96 h. During the regenerative period, we detected two GC-C isoforms that differed in their size, temporal expression, and carbohydrase sensitivities. We showed that 131- and 140-kDa GC-C isoforms represented immature and mature GC-C glycoforms on the basis of endoglycosidase H and PNGase sensitivities. Cell separation experiments revealed that the nonparenchymal cell fractions of regenerating liver contained four times as much GC-C as purified hepatocytes. Immunohistochemistry confirmed these findings. The exuberant expression of GC-C by nonparenchymal cells and, to a lesser extent, hepatocytes suggests a role for cyclic GMP in liver regeneration.

Decreased transcription of the sodium-phosphate transporter gene in the hypophosphatemic mouse.

Recently, it has been hypothesized that the proximal tubular Na(+)-Pi transporter may play a role in murine X-linked hypophosphatemic vitamin D-resistant rickets. In the present investigation, Western blot analysis of renal brush-border membrane proteins, utilizing polyclonal antisera raised against the mouse Na(+)-Pi transporter, revealed a predominant band at 87 kDa in normal and hypophosphatemic (Hyp) mice. The intensity of this band was reduced in the Hyp mouse by 4.5-fold (Hyp/normal = 0.22 +/- 0.04, n = 3, P < 0.05). Additionally, immunohistochemical analysis of kidney cortex in both mice localized the protein to the apical membrane of the proximal tubules. Relative transcription rates of the Na(+)-Pi transporter gene in the normal and Hyp mouse were then investigated. Nuclear run-on assays showed a 51 +/- 0.02% decreased rate of transcription of the Na(+)-Pi transporter gene in the Hyp mice (n = 3). Thus abnormal transcriptional control of this gene in the Hyp mouse likely plays a role in X-linked hypophosphatemia.

Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors.

The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxysuccinimide ester. The derivatized toxin (BST) has an apparent Kd of 11.7 +/- 10 nM for rat brush border receptors. We used BST in an affinity panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST-binding proteins. BST was then used with streptavidin-gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors are concentrated in villus enterocytes, while guanylin appears to be produced at the base of the crypts.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronobiology as it relates to toxicology, pharmacology, and chemotherapy.

Examples of circadian (daily) rhythms in man are presented and discussed. This is followed by presentation of experimental data indicating how the temporal organization affects the response of the host to potentially toxic agents, namely amphetamine and sodium pentobarbital. Data also are presented indicating that one unequivocally can improve therapeutic efficacy using the L1210 mouse leukemia model. This was accomplished by taking into consideration the circadian host toxicity response to anticancer agents, namely cytosine arabinoside, adriamycin, and cyclophosphamide. The subject monitored was the "cure rate" subsequent to treatment of different tumor loads.

Historical comments on chronobiology with emphasis on cell proliferation, the concept of free running, cancer chemotherapy and experimental design.

A brief historical summary is presented regarding the emergence, over the past several decades, of chronobiology as the newest of the integrating discipline of biology. The emphasis is on the circadian system which normally is synchronized to the 24 h environmental light-dark cycle. In the absence of a suitable synchronizer, the system free runs on its own endogenous genetically determined frequency, which usually only approximates 24 h. Since the metabolic system changes rhythmically in time it follows that an organism such as man is biochemically and physiologically a different entity at different circadian stages; therefore it reacts differently to an identical stimulus given at different times. Different stimuli such as anticancer agents are examples considered clearly timed treatment has been shown to significantly improve therapeutic efficacy, data will be presented using the L1210 mouse leukemic model. Moreover data is presented showing that to ignore such rhythmic fluctuation when designing experiments that such can bring about experimental error and false interpretation. The common "same time of day" sampling does not take care of the rhythmic problem!

Postinsertional processing of sucrase-alpha-dextrinase precursor to authentic subunits: multiple step cleavage by trypsin.

Sucrase-alpha-dextrinase, a hybrid digestive carbohydrase of the intestinal brush border, is initially synthesized and transported to the surface membrane as a single-chain glycoprotein, P, which is then cleaved to alpha- and beta-subunits, presumably by one or more pancreatic proteases. However, efforts to convert P under controlled conditions to authentic alpha and beta have been unsuccessful. Sucrase-dextrinase immunoprecipitates from rats intraintestinally labeled with [3H]leucine or [35S]methionine without presence of biliary-pancreatic secretions revealed only the 230-kDa P precursor. Restoration of intestinal flow converted the brush border P to the alpha- (140 kDa) and beta- (125 kDa) subunits. Biliary plus pancreatic secretions facilitated this postinsertional cleavage, but bile alone played no role in conversion. When isolated brush borders, prelabeled in vivo, were exposed to a mixture of pancreatic proteases at physiological concentrations, P was converted to authentic alpha and beta, but only trypsin was responsible for the conversion. Kinetic analysis in prelabeled isolated brush-border vesicles revealed the appearance of several intermediate species (205-145 kDa) produced either by endogenous membrane proteases or by trypsin itself. Reconstituted duodenal luminal contents yielded a fragmentation pattern identical to that produced by trypsin alone. Trypsin was necessary and sufficient for processing of the intermediate precursors to the final authentic alpha- and beta-subunits. Based on the alpha- to beta radioactivity ratio and the known amino acid composition of the subunits, differential cleavage occurred with relatively greater production of the beta-subunit (alpha-to-beta molar ratio = 0.77). The conversion of P to the alpha- and beta-units, rather than occurring in a single step after membrane insertion, is differentially catalyzed by trypsin trimming to unequal amounts of the subunits involving a complex series of cleavage steps.

Structural and functional correlates of sucrase-alpha-dextrinase in intact brush border membranes.

The structure and catalytic function of rat intestinal sucrase-alpha-dextrinase (sucrase-isomaltase) were characterized in intact brush border membranes by differential denaturation in 1% SDS at 4, 37, 45, 55, and 100 degrees C, analysis by acrylamide electrophoresis, and subsequent renaturation by transfer to nitrocellulose and in situ analyses of immunoactivity and catalytic activity (immunoblotting and catalytic blotting). Both the sucrase and alpha-dextrinase activities were associated with two mature oligomers, with sucrase predominantly in a 250-260-kDa unit and dextrinase in a 330-350-kDa unit. While sucrase activity declined progressively in response to increasing temperature to 45 degrees C due to loss of active sites, alpha-dextrinase activity increased reciprocally (Vmax +176%). Three principal monomeric products of postinsertional processing comprise the oligomers: alpha, 140 kDa, which carries the sucrase active site; beta, 125 kDa, harboring the dextrinase active site; and gamma, 110 kDa, produced by removal of 185 amino acid residues from the N-terminus of the alpha. Rather than being a simple hybrid dimer, membrane-associated sucrase-alpha-dextrinase appears to consist of two major oligomeric forms having complex structural associations that dramatically affect the availability of the active catalytic sites at the brush border membrane surface.