Alkaline phosphatase and peptidase activities in Caco-2 cells: Differential response to triiodothyronine

Summary Caco-2 cell human colon adenocarcinoma cell line was used to study the hormonal regulation of small intestinal epithelial cell differentiation. We had previously shown that insulin-transferrin-selenium and triiodothyronine (5 × 10⁻⁸ M)-supplemented medium can best replace serum after 2 days of culture for both the maintenance and differentiation of Caco-2 cells. The present study demonstrates that precoating petri dishes with complete serum allows the growth and differentiation of Caco-2 cells seeded directly in serum-free medium. On the other hand, precoating with dialyzed serum inhibits alkaline phosphatase and dipeptidyl-dipeptidase IV activities by more than 50%. The results obtained with complete serum-precoated culture plates indicate that there is no synergy between insulin and triiodothyronine because cells maintained in transferrin-selenium and triiodothyronine-supplemented medium, with or without insulin, express comparable enzyme activities. Moreover, large increases in alkaline phosphatase and dipeptidyl-dipeptidase IV activities were observed when triiodothyronine was added to the culture medium by the time confluency was reached. In contrast, γ-glutamyltransferase was lowered to a greater extent when triiodothyronine was present from the beginning of culture. These findings show that triiodothyronine preferentially stimulates alkaline phosphatase and dipeptidyl-dipeptidase IV activities during the differentiation period whereas it selectively inhibits γ-glutamyltransferase during the proliferation phase. Triiodothyronine acts in a dose-dependent manner.     

Bacterial genome mapping by two-dimensional pulsed-field gel electrophoresis (2D-PFGE)

Macrorestriction mapping is often the first step toward a thorough physical and genetic characterization of a bacterial genome. The problem of deducing the order of partially or completely digested macrorestriction fragments to yield a physical genome map may readily be solved by applying twodimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. These powerful methods are quick and technically easy to perform; specifically, they are independent of DNA probes and should therefore be applicable to any bacterial species irrespective of its prior genetic characterization. In this article, detailed step-by-step protocols are given to set up, run, and evaluate 2D pulsed-field gels. Two basic methods are described: partial/complete 2D gels of one restriction enzyme and complete/complete 2D gels of two different restriction enzymes. Other topics include preparation of bacterial genomic DNA, screening for suitable rare-cutting restriction enzymes and determination of optimal running conditions. Accompanied by many notes, these protocols are meant to offer the novice a sound and rapid access to these important methods.     

4-Hydroxy-2-Nonenal Levels Increase in the Plasma of Patients with Adult Respiratory Distress Syndrome as Linoleic Acid Appears to Fall

Gas chromatography-mass spectrometry has been applied to the analysis of plasma linoleic acid and one of its oxidation products, 4-hydroxy-2-nonenal (HNE), in adult patients with the acute respiratory distress syndrome (ARDS). Peak areas of total ion chromatograms showed there to be negative correlations between loss of linoleic acid and formation of HNE (measured by selective ion monitoring) in 7 out 10 patients studied. When HNE was quantitated by selective ion monitoring, with reference to a pure standard of HNE and an internal standard of nonanoic acid, ARDS patients showed significantly increased levels of HNE (0.412 ± 0.023 nmol/ml) compared with normal healthy controls (0.205 ± 0.018 nmol/ml).     

Aluminum alters calcium transport in plasma membrane and endoplasmic reticulum from rat brain

Calcium is actively transported into intracellular organelles and out of the cytoplasm by Ca²⁺/Mg²⁺-ATPases located in the endoplasmic reticulum and plasma membranes. We studied the effects of aluminum on calcium transport in the adult rat brain. We examined ⁴⁵Ca-uptake in microsomes and Ca²⁺-ATPase activity in microsomes and synaptosomes isolated from the frontal cortex and cerebellum of adult male Long-Evans rats. ATP-dependent⁴⁵Ca-uptake was similar in microsomes from both brain regions. The addition of 50-800 μM AICI₃ resulted in a concentration-dependent inhibition of ⁴⁵Ca-uptake. Mg²⁺-dependent Ca²⁺-ATPase activity was significantly lower in synaptosomes compared to microsomes in both frontal cortex and cerebellum. In contrast to the uptake studies, AICI³ stimulated Mg²⁺-dependent Ca²⁺-ATPase activity in both microsomes and synaptosomes from both brain regions. To determine the relationship between aluminum and Mg²⁺, we measured ATPase activity in the presence of increasing concentrations of Mg²⁺ or AICI³. Maximal ATPase activity was obtained between 3 and 6 mM Mg²⁺. When we substituted AICI³ for Mg²⁺, ATPase activity was also stimulated in a concentration-dependent manner, but to a greater extent than with Mg²⁺. One interpretation of these data is that aluminum acts at multiple sites to displace both Mg²⁺ and Ca²⁺, increasing the activity of the Ca²⁺-ATPase, but disrupting transport of calcium.     

Plant communities on anthropogenically disturbed sites on the Chukotka Peninsula,Russia

Abstract. The vegetation of anthropogenically disturbed sites on the Chukotka Peninsula was investigated in 1986 and 1990. 102 sites had been bulldozed free of vegetation and soil between 2 and 20 yr ago and revegetated. Anthropogenic vegetation differs considerably from natural tundra. The total plant cover decreases, lichens are absent, moss composition changes, and graminoids predominate. A floristic classification was developed in order to describe the diversity of anthropogenic communities formed on these sites. Four associations were distinguished, all belonging to the alliance Poion glaucomalacanthae. An additional alliance characterized by Alopecurus alpinus is suggested for colder sites, but it has not yet been described. There is a correspondence of associations to substrate types. The association Phippsietum algidae was found only on loam substrate. The Artemisietum glomeratae and Saxifragetum punctatae are connected with rubble or gravel. The graminoid association Arctagrostietum arundinaceae is generally widespread in disturbed habitats and occurs on a variety of substrate types.     

Effects of hypothalamic microinjection of PGE2 on body temperature and sympathetic nervous activities in the rabbit

The febrile response and sympathetic nervous response to hypothalamic microinjections of prostaglandin E₂ (PGE₂) were investigated in anesthetized rabbits. Microninjection of PGE₂ (500–1000 ng) caused an increase in rectal temperature of more than 0.3°C in 13 of 50 loci in the preoptic and anterior hypothalamic area (PO/AH). At 8 of these 13 loci, PGE₂ elicited response patterns in the sympathetic nervous system, such as an increase in cutaneous sympathetic nervous activity and decrease in renal sympathetic nervous activity. This pattern of sympathetic nervous responses was induced with a simultaneous increase in rectal temperature of more than 0.5°C. The 8 loci were distributed in the preoptic area, especially in the vicinity of the supraoptic nucleus. Electrolytic lesions of this region were made bilaterally, and intracerebroventricular injection of PGE₂ (8 µg/kg) was found to inhibit fever and sympathetic activity. The results demonstrate that the action of PGE₂ is responsible for the response patterns of sympathetic twigs during fever. The preoptic area, especially in the vicinity of the supraoptic nucleus, is most sensitive to PGE₂ for the patternized response of sympathetic neurons and fever.     

The fecundity patterns of S and L type rotifers of Brachionus plicatilis

Fecundity patterns of S and L type rotifers Brachionus plicatilis, which were previously found to have distinct growth ability, were analyzed using data of age-specific fecundity. The data were obtained by individual cultures of S and L type strains at 17, 24 and 34 °C. The pattern was analyzed by using normal probability functions. When the age was transformed into logarithmic value, the S and L types had an identical pattern of fecundity at every temperature. This fact indicates that the difference of the growth response to the temperature between S and L type strains stemmed only from the differences in net reproduction rate, not from the pattern.     

Effects of D2O on permeation and gating in the Ca2+-activated potassium channel fromChara

We studied the effects of H₂O/D₂O substitution on the permeation and gating of the large conductance Ca²⁺-activated K⁺ channels inChara gymnophylla droplet membrane using the patchclamp technique. The selectivity sequence of the channel was: K⁺>Rb⁺≫Li⁺, Na⁺, Cs⁺ and Cl⁻. The conductance of this channel in symmetric 100mm KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D₂O as compared to H₂O. The blockade of channel conductance by cytosolic Ca²⁺ weakened in D₂O as a result of a decrease in zero voltage Ca²⁺ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D₂O primarily due to the change in Ca²⁺ binding to the channel during the activation step. The Hill coefficient for Ca²⁺ binding was 3 in D₂O and around 1 in H₂O. The values of the Ca²⁺ binding constant in the open channel conformation were 0.6 and 6 μm in H₂O and D₂O, respectively, while the binding in the closed conformation was much less affected by D₂O. The H₂O/D₂O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1mm internal Ca²⁺.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

The evolution of long interspersed repeated DNA (L1, LINE 1) as revealed by the analysis of an ancient rodent L1 DNA family

Summary All modern mammals contain a distinctive, highly repeated (⩾50,000 members) family of long interspersed repeated DNA called the L1 (LINE 1) family. While the modern L1 families were derived from a common ancestor that predated the mammalian radiation ∼80 million years ago, most of the members of these families were generated within the last 5 million years. However, recently we demonstrated that modern murine (Old World rats and mice) genomes share an older long interspersed repeated DNA family that we called Lx. Here we report our analysis of the DNA sequence of Lx family members and the relationship of this family to the modern L1 families in mouse and rat. The extent of DNA sequence divergence between Lx members indicates that the Lx amplification occurred about 12 million years ago, around the time of the murine radiation. Parsimony analysis revealed that Lx elements were ancestral to both the modern rat and mouse L1 families. However, we found that few if any of the evolutionary intermediates between the Lx and the modern L1 families were extensively amplified. Because the modern L1 families have evolved under selective pressure, the evolutionary intermediates must have been capable of replication. Therefore, replicationcompetent L1 elements can reside in genomes without undergoing extensive amplification. We discuss the bearing of our findings on the evolution of L1 DNA elements and the mammalian genome.