VO2, VCO2, and RQ in a respiratory chamber: accurate estimation based on a new mathematical model using the Kalman-Bucy method.

A respiratory chamber is used for monitoring O(2) consumption (Vo(2)), CO(2) production (Vco(2)), and respiratory quotient (RQ) in humans, enabling long term (24-h) observation under free-living conditions. Computation of Vo(2) and Vco(2) is currently done by inversion of a mass balance equation, with no consideration of measurement errors and other uncertainties. To improve the accuracy of the results, a new mathematical model is suggested in the present study explicitly accounting for the presence of such uncertainties and error sources and enabling the use of optimal filtering methods. Experiments have been realized, injecting known gas quantities and estimating them using the proposed mathematical model and the Kalman-Bucy (KB) estimation method. The estimates obtained reproduce the known production rates much better than standard methods; in particular, the mean error when fitting the known production rates is 15.6 +/- 0.9 vs. 186 +/- 36 ml/min obtained using a conventional method. Experiments with 11 humans were carried out as well, where Vo(2) and Vco(2) were estimated. The variance of the estimation errors, produced by the KB method, appears relatively small and rapidly convergent. Spectral analysis is performed to assess the residual noise content in the estimates, revealing large improvement: 2.9 +/- 0.8 vs. 3,440 +/- 824 (ml/min)(2) and 1.8 +/- 0.5 vs. 2,057 +/- 532 (ml/min)(2), respectively, for Vo(2) and Vco(2) estimates. Consequently, the accuracy of the computed RQ is also highly improved (0.3 x 10(-4) vs. 800 x 10(-4)). The presented study demonstrates the validity of the proposed model and the improvement in the results when using a KB estimation method to resolve it.     

Galectin genes: Regulation of expression

In this review we have summarized the more recent studies on the expression of mammalian galectins. One interesting observation that can be made is that in most of microarrays and/or differential display analysis performed in recent years one or more galectins have been picked up. From a critical evaluation of the pertinent studies the main conclusion that can be drawn is that, although it is not yet clear whether the 14 galectins identified so far have functions in common, a striking common feature of all galectins is the strong modulation of their expression during development, differentiation stages and under different physiological or pathological conditions. This suggests that the expression of different galectins is finely tuned and possibly coordinated. In spite of these observations it is rather unexpected that very few studies have been performed on the molecular mechanisms governing the activity of galectin genes. Published in 2004.     

Fabrication and Characterization of Native and Oxidized Potato Starch Biodegradable Films

The need to replace conventional polymers due to environmental pollution caused by them has led to increased production of biodegradable polymers such as starch. Thus, the application possibilities of starch have increased. In this study, we produced and characterized biodegradable films derived from native and oxidized potato starch. The film-forming solution was prepared with different concentrations of extracted starch (native or oxidized) and a plasticizer (glycerol or sorbitol). Then, the mechanical, barrier, morphological, and structural properties of the films were characterized. The moisture content of the films varied from 15.35?±?1.31 to 21.78?±?0.49%. The elastic modulus of the films ranged from 219?±?14.97 to 2299?±?62.91 MPa. The film of oxidized starch plasticized with sorbitol in the lowest content was the most resistant and flexible; moreover, this film also presented lower water vapor permeability and low solubility in water. Fourier-transform infrared spectroscopic analysis of the biodegradable films indicated the presence of same functional groups as those of starch with bands in the same regions. The film thickness was lower for the films plasticized with glycerol whereas the color variation (Δ?) was lower for the ones plasticized with sorbitol. In case of both plasticizers, the increase in their content decreased the Δ? value. All the biodegradable films presented stability against water absorption owing to their low solubility in water. Morphological evaluation revealed the presence of partially gelatinized starch granules in the films. The roughness parameter (Rq) of the films varied from 3.39 to 10.9 nm, indicating that their surfaces are smooth. X-ray diffraction studies showed a B-type pattern for the starches, which is representative of tubers. Further, the films present higher relative crystallinity (RC) compared to the starches. The biodegradable starch films are uniform, transparent and with low solubility in water. The oxidation of starch and use of sorbitol as a plasticizer resulted in improved properties of the starch films, which is suitable for application.     

A novel double nucleotide substitution in the HMG box of the SRY gene associated with Swyer syndrome

We describe a novel double nucleotide substitution in the SRY gene of a 46,XY female with gonadal dysgenesis or Swyer syndrome. The SRY sequence was analysed by both the single-strand conformational polymorphism assay and direct DNA sequencing of products from the polymerase chain reaction. A double nucleotide substitution was identified at codon 18 of the conserved HMG box motif, causing an arginine to asparagine amino-acid substitution. The altered residue is situated in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. Since the mutation abolishes one HhaI recognition site, the results were confirmed by HhaI restriction mapping. No other mutations were found in the remaining regions of the gene. The corresponding DNA region from the patient’s brother was analysed and found to be normal. We conclude that the SRY mutation in the reported XY female occurred de novo and is associated with sex reversal. Received: 16 December 1996 / Accepted: 5 May 1997     

Diphosphatidylglycerol-induced changes in the organization of mitochondrial ATPase

1. On submitochondrial particles from bovine heart, diphosphatidylglycerol produced a selective solubilization of ATPase. The solubilized enzyme was purified further by ammonium sulfate fractionation and shown to have the same reconstitutive activity as coupling factor F₁ (Pullman, M.E., Penefsky, H. S., Datta, A. and Racker, E. (1960) J. Biol. Chem. 235, 3322–3329).

2. Diphosphatidylglycerol-treated submitochondrial particles retained large amounts of the phospholipid and showed a decreased ATPase activity. Once the excess of phospholipid was removed, soluble ATPase could be again reincorporated in an oligomycin-sensitive complex.

3. On Mg-ATP particles the solubilization of ATPase induced by diphosphatidylglycerol was preceded by a stimulation of oligomycin-sensitive ATPase which indicated a dissociation of F₁ from the ATPase inhibitor (Pullman, M. E. and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762–3769). Magnesium was required to obtain the oligomycin-sensitive stimulation whereas in the absence of magnesium the solubilization of ATPase was prevalent.

4. It is concluded that the decreased association of F₁ with the ATPase inhibitor produced by diphosphatidylglycerol, causes a labilization of ATPase-membrane interaction. Under particular conditions, e.g. a high amount of phospholipid and a low concentration of magnesium, this is followed by the detachment of ATPase.     

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.     

Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in intact human adenocarcinoma A549 cells.

Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.