TREM-1 modulation during early stages of dengue virus infection.

Uncontrolled and intricate production of inflammatory factors is the characteristic feature of dengue infection. The triggering receptor expressed in myeloid cells-1 (TREM-1), expressed on the surface of monocytes and neutrophils, is capable of enhancing and regulating the inflammatory response via the production of different mediators in bacterial and viral infections. Here, both the expression of TREM-1 on human monocytes and neutrophils from peripheral blood of dengue infected individuals, as well as the levels of the soluble form of TREM-1 (sTREM-1) in the sera of these patients were compared against healthy controls. A significant reduction of TREM-1 expression was observed in neutrophils during the first days of infection, followed by a gradual recovery throughout the course of infection. Also, sera from DENV-infected patients exhibited significantly higher sTREM-1 levels than healthy individuals. The difference was more pronounced during the first 5 days after the onset of symptoms. These findings highlight the dynamic process of TREM-1 expression during DENV infection. We hypothesized that increment of free sTREM-1 could be a compensatory mechanism aiming to counteract the inflammatory process elicited during DENV infection.

Fast NMDA receptor-mediated synaptic currents in neurons from mice lacking the epsilon2 (NR2B) subunit.

The N-methyl-D-aspartate (NMDA) receptor has been implicated in the formation of synaptic connections. To investigate the role of the epsilon2 (NR2B) NMDA receptor subunit, which is prominently expressed during early development, we used neurons from mice lacking this subunit. Although epsilon2(-/-) mice die soon after birth, we examined whether NMDA receptor targeting to the postsynaptic membrane was dependent on the epsilon2 subunit by rescuing hippocampal neurons from these mice and studying them in autaptic cultures. In voltage-clamp recordings, excitatory postsynaptic currents (EPSCs) from epsilon2(-/-) neurons expressed an NMDA receptor-mediated EPSC that was apparent as soon as synaptic activity developed. However, compared with wild-type neurons, NMDA receptor-mediated EPSC deactivation kinetics were much faster and were less sensitive to glycine, but were blocked by Mg(2+) or AP5. Whole cell currents from epsilon2(-/-) neurons were also more sensitive to block by low concentrations of Zn(2+) and much less sensitive to the epsilon2-specific antagonist ifenprodil than wild-type currents. The rapid NMDA receptor-mediated EPSC deactivation kinetics and the pharmacological profile from epsilon2(-/-) neurons are consistent with the expression of zeta1/epsilon1 diheteromeric receptors in excitatory hippocampal neurons from mice lacking the epsilon2 subunit. Thus epsilon1 can substitute for the epsilon2 subunit at synapses and epsilon2 is not required for targeting of NMDA receptors to the postsynaptic membrane.

The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro.

Activity-dependent synaptic rearrangements during CNS development require NMDA receptor activation. The control of NMDA receptor function by developmentally regulated subunit expression has been proposed as one mechanism for this receptor dependence. We examined the phenotype of synaptic and extrasynaptic NMDA receptors during the development of synaptic load using the NMDA receptor 2B (NR2B)-selective antagonist ifenprodil. In cultured rat hippocampal neurons when relatively few synapses had formed, the ifenprodil block of EPSCs was less than whole-cell currents, the latter of which included both synaptic and extrasynaptic receptors. At the same developmental stage, we found that extrasynaptic receptors outnumbered synaptic receptors by 3:1; thus whole-cell currents were dominated by the extrasynaptic population. We used the macroscopic kinetics of ifenprodil block to distinguish between the receptor populations. The ifenprodil kinetics of whole-cell currents from neurons before and during the development of synaptic load was comparable with that of whole-cell currents in HEK293 cells transfected with NR1 and NR2B cDNA, indicating that extrasynaptic receptors are largely NR1/NR2B heteromers. In contrast, synaptic receptors included both a highly ifenprodil-sensitive (NR1/NR2B) component as well as a second population with lower ifenprodil sensitivity; the reduced ifenprodil block of EPSCs was attributable to synaptic receptors with lower ifenprodil sensitivity rather than to the appearance of ifenprodil-insensitive (NR1/NR2A) receptors. Our data indicate that the synaptic NMDA receptor complement changes quickly after synapse formation. We suggest that synapses containing predominately NR1/NR2B heteromers represent "immature" sites, whereas mature sites express NMDA receptors with a distinct, presumably triheteromeric, subunit composition.

Experimental milk formulae with reduced protein content and desialylated milk proteins: influence on the faecal flora and the growth of term newborn infants.

We have assessed the growth, tolerance and the faecal flora composition in healthy infants on different feeding regimens. Four groups of infants were fed exclusively on mother's milk, a standard formula and two experimental formulae. The first experimental formula consisted of a milk with a reduced protein content (1.2 g/100 ml), the second in a formula with the same protein content and with milk proteins desialylated by mild acid hydrolysis. The aim of the study was to test whether lowering the protein content and/or modifying the proteins by desialylation would favour the development of a bifidus flora. A bifidus flora was detected in 60% of breastfed infants at 1 month of life. All formulae employed during the study failed to induce a prevalence of colonization with bifidobacteria at 1 month of age. The two experimental milk formulae were well tolerated, but the infant growth rate was slightly lower as compared to the breastfed infants and the infants fed the standard formula. The presence in milk formulae of pre-digested and desialylated proteins can offer some advantages in term of digestibility and mimic a physiological intestinal mechanism of the infant.

[3-13C] gamma-linolenic acid: a new probe for 13C nuclear magnetic resonance studies of arachidonic acid synthesis in the suckling rat.

Our objective was to develop a suitable probe to study metabolism of polyunsaturated fatty acids by 13C nuclear magnetic resonance (NMR) in the suckling rat pup. [3-13C] gamma-Linolenic acid was chemically synthesized, and a 20 mg (Experiment 1) or 5 mg (Experiment 2) dose was injected into the stomachs of 6-10-day-old suckling rat pups that were then killed over a 192 h (8 d) time course. 13C NMR showed that 13C in gamma-linolenate peaked in liver total lipids by 12-h post-dosing and that [5-13C]-arachidonic acid peaked in both brain and liver total lipids 48-96 h post-dosing. 13C enrichment in brain gamma-linolenic acid was not detected by NMR, but gas chromatography-combustion-isotope ratio mass spectrometry showed that its mass enrichment in brain phospholipids at 48-96 h post-dosing was 1-2% of that in brain arachidonic acid. 13C was present in liver and brain cholesterol and in perchloric acid-extractable water-soluble metabolites in the brain, liver and carcass. We conclude that low but measurable amounts of exogenous gamma-linolenic acid do access the suckling rat brain in vivo. The slow time course of [5-13C] arachidonic acid appearance in the brain suggests most of it was probably transported there after synthesis elsewhere, probably in the liver. Some carbon from gamma-linolenic acid is also incorporated into lipid products other than n-6 long-chain polyunsaturated fatty acids.

Detection of four human milk groups with respect to Lewis blood group dependent oligosaccharides.

Neutral oligosaccharides in human milk samples from approximately 50 women were analysed applying a recently developed high-pH anion-exchange chromatographic method. Three different oligosaccharide patterns could be detected in accordance with milk groups that had been already described. These oligosaccharide groups correspond to the Lewis blood types Le(a-b+), Le(a+b-) and Le(a-b-). In addition to these oligosaccharide patterns, a new carbohydrate pattern was detected in a milk sample from a Le(a-b-) individual. Here, only nonfucosylated oligosaccharides and compounds bearing alpha1,3 linked fucosyl residues were found, whereas structures with alpha1,2 and alpha1,4 fucosyl linkages were missing. This finding led to the hypothesis that there are four different oligosaccharide milk groups that fit well to the genetic basis of the Lewis blood group system.

Influence of two infant formulas and human milk on the development of the faecal flora in newborn infants.

The establishment of the faecal flora of 39 full-term infants fed exclusively on breast milk (n = 20) or with two different modern adapted cow's milk formulas (n = 19) was studied during the first 3 months of life. One formula investigated was based on 100% bovine casein as the protein source whereas the other formula contained bovine milk proteins with a whey/casein ratio of 60:40. A faecal flora rich in bifidobacteria was found in all study groups; the growth of putrefactive bacteria (especially Bacteroides spp.), however, was limited. In formula-fed infants, significantly higher bacterial counts of enterococci and clostridia were detected compared to breast milk-fed infants. Similarities and differences due to the feeding regimen were particularly reflected in the pattern of the anaerobic bacterial species. Bifidobacterium bifidum, B. infantis and B. breve constituted the majority of the bifidobacterial flora independent of the type of milk feeding. Other bifidobacterial species such as B. longum, B. adolescentis, B. parabifidum and B. pseudo-catenulatum were detected in high numbers and at low frequencies in breastfed infants. The latter three were observed in infants fed the whey/casein formula as well. It seems that infants fed a casein formula develop a faecal flora more like that of breastfed infants concerning Lactobacillus spp. (especially L. fermentum and L. brevis).

Transneuronal WGA-HRP: can it demonstrate development of ocular dominance patches and effects of monocular deprivation?

We have attempted to use intraocular injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to label ocular dominance patches in developing layer 4 of cat visual cortex. The cortices of animals killed at 49 days or later showed normal ocular dominance patches similar to those seen in [3H]proline material. Animals killed at 42 days showed some patches, but also showed unsegregated regions. Animals killed younger were difficult to stain and did not have patches. We also examined the ability of the WGA-HRP technique to demonstrate the effects of monocular deprivation (MD). MD for the first 3 months of life produced expansion of the afferents from the nondeprived eye and retraction of the patches from the deprived eye. One week of MD at about 5 weeks of age produced an expansion of the patches innervated by the nondeprived eye, but did not obviously affect the patches innervated by the deprived eye. We conclude that WGA-HRP is useful for examining the effects of long-term MD on ocular dominance patches but not for following the development of segregation. Its advantages over the [3H]proline technique are that it does not require a delay of many weeks before the sections can be examined and is much less expensive.

The complex formed between Tet repressor and tetracycline-Mg2+ reveals mechanism of antibiotic resistance.

In recent years Gram-negative bacteria have developed several resistance mechanisms against the broad-spectrum antibiotic tetracycline (Tc). The most abundant mechanism involves a membrane-associated protein (TetA) that exports the antibiotic out of the bacterial cell before it can attach to the ribosomes and inhibit polypeptide elongation. The expression of the TetA protein is regulated by the Tet repressor (TetR). It occurs as a homodimer and binds with two alpha-helix-turn-alpha-helix motifs (HTH) to two tandemly orientated DNA operators, thereby blocking the expression of the associated genes, one encoding for TetA and the other for TetR. If Tc in complex with a divalent cation binds to TetR, a conformational change occurs and the induced TetR is then unable to bind to DNA. TetR of class D, TEtRD, was cocrystallized with tetracycline (7HTc) and Mg2+ in space group I4(1)22 and studied by X-ray diffraction. One TetRD monomer occupies the crystal asymmetric unit, and the dimer is formed by a crystallographic 2-fold rotation. The crystal structure was determined by multiple isomorphous replacement at 2.5 A resolution, and on this basis the structure of the nearly isomorphous complex with 7-chlorotetracycline, TetRD/(Mg 7CITc)+, has been refined to an R-factor of 18.3% using all reflections to 2.1 A resolution. TetRD folds into ten alpha-helices with connecting turns and loops. The N-terminal three alpha-helices of the repressor form the DNA-binding domain, including the HTH with an inverse orientation compared with HTH in other DNA-binding proteins. The distance of 39 A between the two recognition helices explains the inability of the induced TetR to bind to B-form DNA. The core of the protein is formed by helices alpha 5 to alpha 10. It is responsible for dimerization and contains, for each monomer, a binding pocket that accommodates Tc in the presence of a divalent cation. The structure of the TetRD/(Mg 7CITc)+ complex reveals the octahedral coordination of Mg2+ by Tc (chelating O-11, and O-12), His100 N epsilon and by three water molecules; in addition there is an extended network of hydrogen bonding and van der Waals interactions formed between 7CITc and TetR. The detailed view of the Tc-binding pocket and the interactions between the antibiotic and the repressor offers the first solid basis for rational tetracycline design, with the aim of circumventing resistance.

The development of MK-801, kainate, AMPA, and muscimol binding sites in cat visual cortex.

Previous work using homogenate binding has shown that the development of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imin e maleate (MK-801) binding in cat visual cortex increases from 21 days to 42 days, the height of the plastic period, and decreases in adulthood. We have studied the generality of this finding by examining the development of NMDA binding sites in several brain regions and by examining the development of other binding sites in the visual cortex. After confirming the original finding, we extended it by showing that the sensitivity of MK-801 binding sites to glutamate and glycine decreases when the cat becomes an adult. We then examined the regional specificity of MK-801 binding. Retinal binding did not change significantly with age. Binding in both visual cortex and hippocampus increased significantly from 7 days to 42 days regardless of whether binding was measured per milligram wet weight or per milligram protein. The decline from 42 days to adulthood was less dramatic in the hippocampus than in the visual cortex and was statistically significant only when binding was measured per milligram protein. Saturation analyses also showed a difference in the two structures. Bmax in the visual cortex, but not in the hippocampus, decreased from 42 days to adulthood. To determine whether these developmental changes were specific to MK-801 binding sites, we compared the age-dependent binding of MK-801, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and muscimol. Like MK-801, kainate binding increased from 7 days to 42 days and decreased from 42 days to adulthood. AMPA and muscimol binding showed a similar increase in binding from 7 days to 42 days but did not decrease significantly from 42 days to adulthood. Displacement experiments suggest that AMPA and kainate bind to separate sites. The 42-day peak in NMDA and kainate binding suggests that their associated receptors may have a role in determining the plastic period of visual cortex.

Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance.

The most frequently occurring resistance of Gram-negative bacteria against tetracyclines is triggered by drug recognition of the Tet repressor. This causes dissociation of the repressor-operator DNA complex and enables expression of the resistance protein TetA, which is responsible for active efflux of tetracycline. The 2.5 angstrom resolution crystal structure of the homodimeric Tet repressor complexed with tetracycline-magnesium reveals detailed drug recognition. The orientation of the operator-binding helix-turn-helix motifs of the repressor is inverted in comparison with other DNA binding proteins. The repressor-drug complex is unable to interact with DNA because the separation of the DNA binding motifs is 5 angstroms wider than usually observed.

Variations of neutral oligosaccharides and lactose in human milk during the feeding.

There exist only few data concerning the variation of oligosaccharides in human milk. In this study the variations of neutral oligosaccharides and of lactose in human milk during the feeding were determined from five women at day 8 and at day 57 post partum. The milk of the investigated feedings was divided in four parts of equal volumes during sampling; the concentrations of neutral oligosaccharide fractions were determined by gel permeation chromatography on Fractogel TSK HW 40 (S) columns. No significant differences in the concentrations of the neutral oligosaccharide groups monofucosyllactoses, difucosyllactose, lacto-N-tetraoses, monofucosyllacto-N-tetraoses and difucosyllacto-N-tetraoses and of lactose were found in the four milk parts. The results of this study favor the use of so-called mid-feed samples, a simple and convenient sampling method for analytical studies of human milk. Mid-feed samples are representative of the whole feeding as concerned for neutral oligosaccharides.

Computer simulations and experimental studies of gel mobility patterns for weak and strong non-cooperative protein binding to two targets on the same DNA: application to binding of tet repressor variants to multiple and single tet operator sites.

A series of computer simulations of gel patterns assuming non-cooperative binding of a protein to two targets on the same DNA fragment was performed and applied to interprete gel mobility shift experiments of Tet repressor-tet operator binding. While a high binding affinity leads to the expected distribution of free DNA, DNA bound by one repressor dimer and DNA bound by two repressor dimers, a lower affinity or an increased electrophoresis time results in the loss of the band corresponding to the singly occupied complex. The doubly occupied complex remains stable under these conditions. This phenomenon is typical for protein binding to DNA fragments with two identical sites. It results from statistical disproportionation of the singly occupied complex in the gel. The lack of the singly occupied complex is commonly taken to indicate cooperative binding, however, our analysis shows clearly, that cooperativity is not needed to interprete these results. Tet repressor proteins and small DNA fragments with two tet operator sites have been prepared from four classes of tetracycline resistance determinants. The results of gel mobility shift analyses of various complexes of these compounds confirm the predictions. Furthermore, calculated gel patterns assuming different gel mobilities of the two singly occupied complexes show discrete bands only if the electrophoresis time is shorter than the inverse of the microscopic dissociation rate constant. Simulations assuming increasing dissociation rates predict that the two bands first merge into one, which then disappears. This behavior was verified by gel mobility analyses of Tet repressor-tet operator titrations at increased salt concentrations as well as by direct footprinting of the complexes in the gel. It is concluded that comparison of the intensities of the single and the double occupation bands allow a rough estimation of the dissociation rate constant. On this basis the sixteen possible Tet repressor-tet operator combinations can be ordered with decreasing binding affinities by a simple gel shift experiment. The implications of these results for gel mobility analyses of other protein-DNA complexes are discussed.

Tet repressor binding induced curvature of tet operator DNA.

Tet repressor dimer binds to two tet operator sites spaced by 30 bp in the Tn10 encoded tet regulatory DNA. The effect of repressor binding on the gel mobility of circular permutated DNA fragments containing either one or both operator sequences is reported. The EcoRI induced bending of DNA is used to compare the results with other protein binding induced structural perturbations of DNA. Tet repressor bends a DNA fragment with a single tet operator to an angle of 42 degrees +/- 7 degrees. The apparent bend angle of DNA fragments containing the tandem tet operator arrangement occupied by two Tet repressor dimers turns out to be 52 degrees +/- 9 degrees. These results are interpreted with respect to the end to end distances of the bent DNA fragments. They indicate that either the intervening tet regulatory DNA between the operators or the bound operator sequences themselves contain additional perturbations from the canonical B-DNA structure. This finding is discussed in the light of previously obtained results from CD, neutron scattering, and electrooptical studies.

The quaternary structure of Tet repressors bound to the Tn10-encoded tet gene control region determined by neutron solution scattering.

The spatial arrangement of Tet repressor dimer, both free and in complex with an 80 bp DNA fragment spanning the wild-type Tn10-encoded tet transcriptional control sequence containing a tandem repeat of two operators, has been determined by neutron small-angle scattering. The active, free Tet repressor dimer is an elongated and flat molecule with a maximum dimension of 11 +/- 1.5 mm which can be approximated by an ellipsoid with the half-axes 6 nm, 2.5 nm and 1 nm. The overall conformation undergoes no detectable change when the repressor dimer is bound to a DNA fragment containing a single tet operator. The normal distance between the centre of gravity of the protein and the DNA axis is 3.0 +/- 0.1 nm, indicating that the repressor dimer is mainly located on one side of the DNA. When bound to the wild type tet control DNA, the two repressor dimers have a centre-to-centre distance of 11.0 +/- 0.5 nm. Their minimal distance is 5 +/- 2 nm. Protein-protein contacts via loop formation of the DNA by repressor binding is excluded. The repressors are well separated and have no direct contact. A model is proposed where the two repressor dimers are located on opposite sides of the DNA and the DNA is not strongly bent in the complex.

Identification and nucleotide sequence of the class E tet regulatory elements and operator and inducer binding of the encoded purified Tet repressor.

The regulatory region and repressor (tetRE) gene from the class E tetracycline resistance determinant previously isolated from Enterobacteriaceae have been identified and completely sequenced. The regulatory region is located between the resistance gene and the tetR gene which have opposite polarity. The tetR gene encodes a protein consisting of 211 amino acids with a calculated molecular weight of 23.6 kDa. Cloning of the tetR gene under transcriptional control of the lambda PL promoter leads to overexpression of a polypeptide with an apparent molecular weight of 26 kDa. The purified protein binds sequence specifically to DNA fragments containing putative tet operators. This property is lost in the presence of tetracycline. The relationship of the tetRE sequence to four known tetracycline resistance determinants is discussed.

Structure of the Tet repressor and Tet repressor-operator complexes in solution from electrooptical measurements and hydrodynamic simulations.

The Tet repressor protein and tet operator DNA fragments and their complexes have been analyzed by electrooptical procedures. The protein shows a positive linear dichroism at 280 nm, a negative linear dichroism at 248 nm, and a strong permanent dipole moment of 3.5 X 10(-27) C m, which is independent of the salt concentration within experimental accuracy. Its rotation time constant of 40 ns indicates an elongated structure, which is consistent with a prolate ellipsoid of 100 A for the long axis and 40 A for the short axis. The time constant can also be fitted by a cylinder of length 78 A and diameter 37 A, which is consistent with nuclease protection data reported on repressor-operator complexes, if the cylinder axis is aligned parallel to the DNA axis. Addition of tetracycline induces changes of the limit dichroism but very little change of the rotation time constant. The rotation time constants observed for the operator DNA fragments show some deviations from the values expected from their contour length; however, these deviations remain relatively small. Formation of repressor-operator complexes leads to some increase of the DNA rotation time constants. Simulations by bead models demonstrate that these time constants can be explained without any major change of the hydrodynamic dimension of the components. The data for the complexes are fitted by bead models with smooth bending of the DNA corresponding to a radius of curvature of 500 A, but at the given accuracy, we cannot rule out that the DNA in the complex remains straight or is bent to a smaller radius of approximately 400 A. Thus, binding of the Tet repressor, which is a helix-turn-helix protein as judged from its sequence, to its operator seems to induce minor bending but does not induce strong bending of the DNA double helix.

Dynamics of repressor-operator recognition: the Tn10-encoded tetracycline resistance control.

Binding of the Tet repressor to nonspecific and specific DNA leads to quenching of the Tet fluorescence by approximately 22% and approximately 35%, respectively. This effect is used for a direct, quantitative characterization of the binding equilibria and dynamics involved in the recognition of the operator by its repressor. From the dependence of the nonspecific binding constant on the ion concentration, it is concluded that nonspecific binding is almost completely driven by the entropy change resulting from the release of three to four Na+ ions from the double helix upon protein binding. Formation of the specific complex is driven by a higher entropy term resulting from the release of seven to eight Na+ ions and in addition by a free energy term of -33 kJ/mol from nonelectrostatic interactions, which are attributed to the specific contacts. The dynamics of the repressor-operator recognition are resolved by stopped-flow measurements at various salt concentrations and for different DNA chain lengths into two separate steps. The first step follows a second-order mechanism and results in an intermediate complex associated with formation of about three to four electrostatic contacts between protein and DNA; apparently, this complex is equivalent to the nonspecific complex. The existence of an intermediate is also indicated by experiments in mixed Na+-Mg2+ buffers, which can be described with high accuracy by competition of Mg2+ and protein. The intermediate complex is formed at a rate of 3 X 10(8) M-1 s-1 and is converted in the second reaction step to the specific complex with a rate constant of 6 X 10(4) s-1, which is almost independent of the salt concentration. Our interpretation and the parameters obtained from our model are confirmed by competition of nonspecific DNA with operator DNA for repressor binding. The observed maximal rate constant of 3 X 10(8) M-1 s-1 is very close to theoretical predictions for the association without a sliding mechanism. The very small dependence of the observed rate constants on the chain length shows that the Tet repressor is not able to slide over any substantial distance even at low salt concentrations. The question of a potential contribution from sliding under our experimental conditions is critically discussed. The absence of sliding in the case of the Tet repressor under physiological conditions is compared with the high sliding efficiency of the lac repressor and is discussed with respect to possible molecular mechanisms of sliding in relation to biological function.