Cyclic di-GMP acts as a cell cycle oscillator to drive chromosome replication.

Fundamental to all living organisms is the capacity to coordinate cell division and cell differentiation to generate appropriate numbers of specialized cells. Whereas eukaryotes use cyclins and cyclin-dependent kinases to balance division with cell fate decisions, equivalent regulatory systems have not been described in bacteria. Moreover, the mechanisms used by bacteria to tune division in line with developmental programs are poorly understood. Here we show that Caulobacter crescentus, a bacterium with an asymmetric division cycle, uses oscillating levels of the second messenger cyclic diguanylate (c-di-GMP) to drive its cell cycle. We demonstrate that c-di-GMP directly binds to the essential cell cycle kinase CckA to inhibit kinase activity and stimulate phosphatase activity. An upshift of c-di-GMP during the G1-S transition switches CckA from the kinase to the phosphatase mode, thereby allowing replication initiation and cell cycle progression. Finally, we show that during division, c-di-GMP imposes spatial control on CckA to install the replication asymmetry of future daughter cells. These studies reveal c-di-GMP to be a cyclin-like molecule in bacteria that coordinates chromosome replication with cell morphogenesis in Caulobacter. The observation that c-di-GMP-mediated control is conserved in the plant pathogen Agrobacterium tumefaciens suggests a general mechanism through which this global regulator of bacterial virulence and persistence coordinates behaviour and cell proliferation.

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium.

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone-deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha-methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.

7-Dehydrocholesterol-dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome.

Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7(-/-) mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency.

Insertional activation of a promoterless thymidine kinase gene.

A plasmid carrying a promoterless herpes simplex virus thymidine kinase gene was transfected via calcium phosphate precipitation into LM (tk-) mouse fibroblast cells. The transfected gene was efficiently expressed, as the transfected cells grew perfectly well in selective hypoxanthine-aminopterin-thymidine medium, suggesting that the thymidine kinase-coding region became linked to a promoterlike element on integration into the recipient genome. To investigate the structure of the surrogate promoter, we first isolated the integrated gene from a genomic library. The nucleotide sequence of the DNA adjacent to the thymidine kinase-coding sequence was then determined. We found, first, that the integration of the transfected DNA apparently occurred by a blunt end ligation mechanism involving no obvious sequence similarities between integrated and recipient DNA and, second, that the 5'-flanking region included a TATA box, two CCAAT boxes, and a GC box element. However, the TATA box motif and the most proximal CCAAT box appeared to be sufficient for full promoter activity, as determined by the transfection efficiencies of appropriate plasmid constructs. Except for these canonical promoter elements, the surrogate promoter had no obvious similarities to known thymidine kinase gene promoters.

Localization of regulatory elements controlling human MYCN expression.

Expression of the cellular oncogene MYCN is restricted to few cell lineages and is highest during development both in mouse and in humans. In pursuit of elucidating the mechanisms underlying MYCN regulation we introduced a human MYCN clone (pNb-9) with approximately 2.5 kbp 5'- and 6 kbp 3'-flanking genomic sequences into different murine and human cell lines as well as into mice. In all cases we found a correlation between the expression of the exogenous and the endogenous MYCN. Among cell lines, only those expressing the endogenous gene also expressed the transfected gene. In the transgenic mice transcripts of the transgene were present in proportion to the transcripts of the endogenous MYCN gene with the highest level in the brain. Therefore, the genetic information necessary for regulated expression of MYCN appears to be contained in pNb-9. To localize the DNA-regions responsible for regulated expression, we generated MYCN/CAT hybrid genes with different portions of the putative MYCN promoter region linked to the reporter gene. Transient transfections into various murine and human cell lines identified three DNA regions apparently involved in the regulation of expression. One region about 200 bp upstream of the transcriptional start site is responsible for a basal level of MYCN expression. A second region located about 800 bp further upstream appears to be involved in cell type-specific gene activation. The third regulatory element is located at the 3' end of the first exon and/or in the first intron and may mediate tissue-specific down regulation of gene expression.

Syncope in hypertrophic cardiomyopathy: multivariate analysis of prognostic determinants.

Twenty-nine consecutive patients with symptomatic hypertrophic cardiomyopathy and a mean age of 44.8 +/- 12.2 years (range 21 to 63) underwent complex invasive and noninvasive testing to identify a risk profile for syncope. Clinical, morphologic, electrophysiologic and hemodynamic variables at rest and at a symptom-limited pacing rate were analyzed for a significant association with syncope. Exact stepwise logistic regression analysis identified three variables as significant independent predictors of syncope in hypertrophic cardiomyopathy: 1) age less than 30 years (beta = 4.803; p = 0.0007); 2) left ventricular end-diastolic volume index less than 60 ml/m2 (beta = 3.302; p = 0.006); and 3) nonsustained ventricular tachycardia on 72 h ambulatory electrocardiographic monitoring (beta = 2.5909; p = 0.03). The combined occurrence of all three variables had a sensitivity and specificity of 100% in identifying eight patients with syncopal events. Thus, the risk for syncope in hypertrophic cardiomyopathy is high in young patients with the combination of low left ventricular filling volume and episodes of nonsustained ventricular tachycardia. This finding might also explain the mechanism of syncope in hypertrophic cardiomyopathy as low input-low output failure induced by a sudden increase in heart rate in the presence of a low filling volume.

Angiotensin-converting enzyme gene mutations, blood pressures, and cardiovascular homeostasis.

A common polymorphism of the angiotensin-converting enzyme (ACE) gene (ACE in humans, Ace in mice) is associated with differences in circulating ACE levels that may confer a differential risk for cardiovascular diseases. To study the effects of genetically determined changes in Ace gene function within a defined genetic and environmental background, we have studied mice having one, two, or three functional copies of the Ace gene at its normal chromosomal location. ACE activities in the serum increased progressively from 62% of normal in the one-copy animals to 144% of normal in the three-copy animals (P < 10(-15), n = 132). The blood pressures of the mice having from one to three copies of the Ace gene did not differ significantly, but the heart rates, heart weights, and renal tubulointerstitial volumes decreased significantly with increasing Ace gene copy number. The level of kidney renin mRNA in the one-copy mice was increased to 129 +/- 9% relative to that of the normal two-copy mice (100 +/- 4%, P = .01, n = 16). We conclude that significant homeostatic adaptations successfully normalize the blood pressures of mice that have quantitative changes in Ace gene function. Our results suggest only that quantitative changes in expression of the Ace gene will observably affect blood pressures when accompanied by additional environmental or genetic factors that together with Ace exceed the capacity of the homeostatic mechanisms.

Abnormal serotonergic development in a mouse model for the Smith-Lemli-Opitz syndrome: implications for autism.

The Smith-Lemli-Opitz syndrome (SLOS) is a malformation/mental retardation syndrome resulting from an inborn error in 3beta-hydroxysteroid Delta7-reductase (DHCR7), the terminal enzyme required for cholesterol biosynthesis. Using a targeting strategy designed to virtually eliminate Dhcr7 activity, we have created a SLOS mouse model that exhibits commissural deficiencies, hippocampal abnormalities, and hypermorphic development of serotonin (5-HT) neurons. The latter is of particular interest with respect to current evidence that serotonin plays a significant role in autism spectrum disorders and the recent clinical observation that 50% of SLOS patients present with autistic behavior. Immunohistochemical analyses have revealed a 306% increase in the area of 5-HT immunoreactivity (5-HT IR) in the hindbrains of mutant (Dhcr7-/-) mice as compared to age-matched wild type animals. Amount of 5-HT IR was measured as total area of IR per histological section. Additionally, a regional increase as high as 15-fold was observed for the most lateral sagittal hindbrain sections. In Dhcr7-/- mice, an expansion of 5-HT IR into the ventricular zone and floor plate region was observed. In addition, the rostral and caudal raphe groups exhibited a radial expansion in Dhcr7-/- mice, with 5-HT IR cells present in locations not seen in wild type mice. This increase in 5-HT IR appears to represent an increase in total number of 5-HT neurons and fibers. These observations may help explain the behavioral phenotype seen in SLOS, and provide clues for future therapeutic interventions that utilize pharmacological modulation of the serotonergic system.

Bursellopsis spaniopogon (Ciliophora: Prostomatida): II. Stomatogenesis as Revealed by Light Microscopy and Scanning Electron Microscopy and some Phylogenetic Implications Concerning Prostome Ciliates.

In stomatogenesis of Bursellopsis a new circumoral kinety as well as three brush rows have to be formed in the opisthe. The formation of these structures occurs in a stomatogenic field of six to twelve somatic kineties. These stomatogenic kineties can be separated into two groups: a left one of three to seven kineties, forming the brush primordia, and a right one of three to five kineties, forming the primordia of the circumoral kinety. Between both groups the presumptive oral opening develops. In the stomatogenic kineties forming the brush primordia, dikinetids develop by kinetosome proliferation. These dikinetids arrange side by side into three horizontal rows. By an excessive kinetosome proliferation in the somatic kineties lying left to these rows the whole organelle gets vertically orientated later on in stomatogenesis. In the right group of stomatogenic kineties short fragments, consisting of dikinetids, develop. These fragments undergo a clockwise circular migration around the presumptive oral opening. By joining head to tail they arrange into the circumoral kinety. The primordia of the brush and of the circumoral kinety originate in front of only a few somatic kineties on the ventral side of the cell ("merotelokinetal stomatogenesis"). The apical ("prostome") position of the oral apparatus is the result of the cell division and has to be considered as secondary. The term "prostome" is therefore not correct in a phylogenetic view. According to the "merotelokinetal" type of stomatogenesis a sister group relationship of the prorodontid and some colpodid ciliates is assumed.

Bursellopsis spaniopogon n. spec. (Ciliophora: Prostomatida): I. Infraciliature and ultrastructure of the somatic and oral cortex.

A description is given of a new species of the genus Bursellopsis Corliss, 1960. Its infraciliature is studied by means of Klein's dry silver technique and Fernández-Galiano's pyridinated silver-carbonate-method. The light microscopical results are supplemented by a comparative scanning and transmission electron microscope study, with special reference to the somatic cortex, the circum-oral ciliature, and the "dorsal brush". The kineties of the somatic cortex, consisting of monokinetids, cover only the anterior four fifths of the cell. Three fibrillar systems, a kinetodesmal fibre, a postciliary microtubular ribbon, a transverse microtubular ribbon and one parasomal sac are associated with each monokinetid. The circum-oral kinety consists of dikinetids bearing only stubs of cilia. The so-called "dorsal brush" consists of dikinetids, with only their anterior kinetosomes bearing "brush cilia", which are shorter than the somatic cilia. The biometric data of Bursellopsis spaniopogon are given and the ultrastructure of the new species is compared to light microscopical and ultrastructural data of Urotricha, Holopbrya and Prorodon. The systematic position of the genus Bursellopsis is discussed and an improved diagnosis of Bursellopsis is provided.

Nailfold capillaroscopy and laser Doppler fluxmetry for evaluation of Raynaud's phenomenon: how valid is the local cooling test?

BACKGROUND: Since there is a need for an objective parameter of microcirculation in follow-up of patients with Raynaud's syndrome we evaluated the reproducibility of the reaction to a cold exposure test with nailfold capillaroscopy and laser Doppler fluxmetry during a winter period. PATIENTS AND METHODS: 10 healthy subjects and 16 patients with primary Raynaud's syndrome were evaluated with nailfold capillaroscopy and laser Doppler fluxmetry of the finger tips during and after a standardized cold exposure stress test. The measurements were repeated in the controls within 2 weeks, in the patient population twice within 24 weeks. RESULTS: In the healthy controls the percentage of capillaries with flow stop was stable (r = 0.785) and the duration of flow stop was reproducible (r = 0.993). Both parameters were significantly lower in controls than in the patients. The flow stop durations in patients varied intra-individually to a great extent between the different weeks without any significant correlation. For all calculated laser Doppler perfusion and time parameters we did not find any significant differences between controls and patients nor any consistent correlations in the intra-individual comparison within the different weeks for both, controls and patients. CONCLUSION: Nailfold capillaroscopy during cold exposure is able to discriminate between healthy persons and patients with primary Raynaud's syndrome, but seems to be of minor value for follow-up evaluation of patients because of intraindividual variations. Laser Doppler fluxmetry is invalid for both purposes when using the applied cold exposure test.

Immunohistochemical and microarray analyses of a mouse model for the smith-lemli-opitz syndrome.

The Smith-Lemli-Opitz syndrome is a mental retardation/malformation syndrome with behavioral components of autism. It is caused by a deficiency in 3beta-hydroxysteroid-Delta7-reductase (DHCR7), the enzyme required for the terminal enzymatic step of cholesterol biosynthesis. The availability of Smith-Lemli-Opitz syndrome mouse models has made it possible to investigate the genesis of the malformations associated with this syndrome. Dhcr7 gene modification (Dhcr7-/-) results in neonatal lethality and multiple organ system malformations. Pathology includes cleft palate, pulmonary hypoplasia, cyanosis, impaired cortical response to glutamate, and hypermorphic development of hindbrain serotonergic neurons. For the current study, hindbrain regions microdissected from gestational day 14 Dhcr7-/-, Dhcr7+/- and Dhcr7+/+ fetuses were processed for expression profiling analyses using Affymetrix oligonucleotide arrays and filtered using statistical significance (S-score) of change in gene expression. Of the 12,000 genes analyzed, 91 were upregulated and 98 were downregulated in the Dhcr7-/- hindbrains when compared to wild-type animals. Fewer affected genes, representing a reduced affect on these pathways, were identified in heterozygous animals. Hierarchical clustering identified altered expression of genes associated with cholesterol homeostasis, cell cycle control and apoptosis, neurodifferentiation and embryogenesis, transcription and translation, cellular transport, neurodegeneration, and neuronal cytoskeleton. Of particular interest, Dhcr7 gene modification elicited dynamic changes in genes involved in axonal guidance. In support of the microarray findings, immunohistochemical analyses of the netrin/deleted in colorectal cancer axon guidance pathway illustrated midline commissural deficiencies and hippocampal pathfinding errors in Dhcr7-/- mice. The results of these studies aid in providing insight into the genesis of human cholesterol-related birth defects and neurodevelopmental disorders and highlight specific areas for future investigation.

Performance on a nonsense syllable test for normal and hearing-impaired subjects.

The Nonsense Syllable Test (NST) developed by Edgerton and Danhauer in 1979 was administered to 22 normal-hearing and 26 hearing-impaired Ss. Tape recordings from Auditec of St. Louis were presented at 5 SLs from 25 to 65 db re: SRT. Responses were transcribed phonetically using both auditory and visual cues and were scored by the phoneme method. Interjudge scorer reliability was high (94%). Performance vs level functions were plotted and analyzed for differences both within and among groups. The NST differentiated between the normals and the hearing-impaired Ss. Percent-correct discrimination scores for the hearing-impaired Ss were about 10 to 15 points poorer than for the normals. The results were favorably compared to the earlier version of the NST and the potential clinical use of this taped version was discussed.

Pyrimidine nucleoside analogues.X. 5-Substituted 1-(1,3-dihydroxypropyl-2) uracils.

A convenient method is suggested for synthesis of uracil-1-malonic acid diethyl ester by alkylating 2,4-bis(trimethylsilyl) uracil with bromo-malonic acid diethyl ester. This compound has been shown to hydrolyze with NaOH yielding either uracil or uracil-1-acetic acid, depending on reaction conditions. Similarly, thymine-1-malinic acid diethyl ester and 5-fluorouracil-1,3-dimalonic acid tetraethyl ester were obtained. 1-(1,3-Dihydroxypropyl)uracil has been obtained by reducing uracil-1-malonic acid diethyl ester with NaBH4.

Aortic wall damage in mice unable to synthesize ascorbic acid.

By inactivating the gene for L-gulono-gamma-lactone oxidase, a key enzyme in ascorbic acid synthesis, we have generated mice that, like humans, depend on dietary vitamin C. Regular chow, containing about 110 mg/kg of vitamin C, is unable to support the growth of the mutant mice, which require L-ascorbic acid supplemented in their drinking water (330 mg/liter). Upon withdrawal of supplementation, plasma and tissue ascorbic acid levels decreased to 10-15% of normal within 2 weeks, and after 5 weeks the mutants became anemic, began to lose weight, and die. Plasma total antioxidative capacities were approximately 37% normal in homozygotes after feeding the unsupplemented diet for 3-5 weeks. As plasma ascorbic acid decreased, small, but significant, increases in total cholesterol and decreases in high density lipoprotein cholesterol were observed. The most striking effects of the marginal dietary vitamin C were alterations in the wall of aorta, evidenced by the disruption of elastic laminae, smooth muscle cell proliferation, and focal endothelial desquamation of the luminal surface. Thus, marginal vitamin C deficiency affects the vascular integrity of mice unable to synthesize ascorbic acid, with potentially profound effects on the pathogenesis of vascular diseases. Breeding the vitamin C-dependent mice with mice carrying defined genetic mutations will provide numerous opportunities for systematic studies of the role of antioxidants in health and disease.

Treatment of primary Raynaud's syndrome with traditional Chinese acupuncture.

OBJECTIVE: Evaluation of the effects of a standardized acupuncture treatment in primary Raynaud's syndrome. DESIGN: A controlled randomized prospective study. SETTING: A winter period of 23 weeks, angiological clinic of Hannover Medical School. SUBJECTS: Thirty-three patients with primary Raynaud's syndrome (16 control, 17 treatment). INTERVENTIONS: The patients of the treatment group were given seven acupuncture treatments during the weeks 10 and 11 of the observation period. MAIN OUTCOME MEASURES: All patients kept a diary throughout the entire observation period noting daily frequency, duration and severity of their vasospastic attacks. A local cooling test combined with nailfold capillaroscopy was performed for all patients at baseline (week 1) and in weeks 12 and 23, recording flowstop reactions of the nailfold capillaries. RESULTS: The treated patients showed a significant decrease in the frequency of attacks from 1.4 day-1 to 0.6 day-1, P < 0.01 (control 1.6 to 1.2, P = 0.08). The overall reduction of attacks was 63% (control 27%, P = 0.03). The mean duration of the capillary flowstop reaction decreased from 71 to 24 s (week 1 vs. week 12, P = 0.001) and 38 s (week 1 vs. week 23, P = 0.02) respectively. In the control group the changes were not significant. CONCLUSIONS: These findings suggest that traditional Chinese acupuncture is a reasonable alternative in treating patients with primary Raynaud's syndrome.

[Risk of syncope in hypertrophic cardiomyopathy: a multivariate analysis of prognostic variables]. / Synkopenrisiko bei hypertropher Kardiomyopathie: multivariate Analyse prognostischer Variablen.

Twenty-nine consecutive patients with symptomatic hypertrophic cardiomyopathy and a mean age of 44.8 +/- 12.2 years (range 21 to 63 years) underwent complex invasive and noninvasive testing in an attempt to identify a risk profile for syncope. Clinical, morphologic and electrophysiologic as well as functional parameters at rest and at symptom-limited pacing rate were analyzed for significant association with syncope. Exact stepwise logistic regression analysis identified three variables as significant independent predictors of syncope in hypertrophic cardiomyopathy: 1) age less than 30 years (beta = 4.803; p = 0.0007); 2) left ventricular end-diastolic volume index less than 60 ml/m2 (beta = 3.302; p = 0.006) and 3) non-sustained ventricular tachycardia on 72 h ambulatory ECG monitoring (beta = 2.5909; p = 0.03). The combined occurrence of all three variables had a 100% sensitivity and specificity to identify eight patients with syncopal events. Thus, the risk for syncope in hypertrophic cardiomyopathy is high in young patients with the combination of low left ventricular filling volume and episodes of non-sustained ventricular tachycardia. This finding might also explain the mechanism of syncope in hypertrophic cardiomyopathy as low-input-low-output failure induced by a sudden increase in heart rate in presence of a low filling volume.

[Non-steroidal anti-inflammatory agents increase intestinal permeability]. / Nicht-steroidale Antiphlogistika erhöhen die Darmpermeabilität.

51Cr-EDTA activity was measured in urine and blood of patients receiving non-steroidal anti-inflammatory (NSA) drug treatment and of healthy subjects and other patients (controls), after oral intake of 51Cr-EDTA, for the purpose of deciding whether NSA treatment increases urinary excretion of oral 51Cr-EDTA as an expression of increased intestinal permeability. 51Cr-EDTA activity in urine and blood of patients with rheumatoid arthritis (13) being treated with NSA was significantly higher (similar to results in 13 patients with Crohn's disease) than that of a control group (14) of patients with rheumatoid arthritis without such treatment (9) and patients without rheumatic disease (5). Both in patients with rheumatoid arthritis receiving NSA drugs and patients with Crohn's disease there was a highly significant correlation between urinary and blood activity. There was no effect of NSA drugs on renal function. The results indicate that NSA drugs increase interenterocytic permeability to an extent comparable to permeability abnormalities in Crohn's disease.