Telithromycin (HMR 3647) achieves high and sustained concentrations in tonsils of patients undergoing tonsillectomy.

Telithromycin, the first ketolide antimicrobial to be developed for clinical use, has potent activity against group A beta-haemolytic streptococci (GABHS), including macrolide-resistant strains. The penetration of telithromycin into tonsils was assessed in 22 adults undergoing tonsillectomy at 3, 12 or 24 h after the fourth dose of oral telithromycin 800 mg once daily. Telithromycin rapidly penetrated tonsillar tissues, achieving a mean concentration of 3.95 mg/kg at 3 h post dose, 3.4 times greater than the corresponding plasma concentration (1.22 mg/l. The mean tonsil:plasma concentration ratio increased to 13.1 at 24 h post dose, indicating slower elimination from tonsils than plasma. Tonsillar and plasma concentrations exceeded the MIC(50) for GABHS throughout the 24-h dosing period. These findings suggest that telithromycin may be an effective new alternative treatment for GABHS tonsillopharyngitis.

The allergenicity of soybean-based products is modified by food technologies.

BACKGROUND: Numerous products based on soybean are available and various food technologies are applied for their production. The allergenicity of natural soybean may be modified by these treatments. OBJECTIVES: To compare the allergenicity of native soybean proteins with those of soy milk and texturized protein products. To show additional allergens. METHODS: Three commercial products and two infant formulas were studied: Soybean flour, soy milk, texturized soy proteins, two infant formulas; the first containing total proteins and the second containing a soy protein hydrolysate. Sera from 9 patients allergic to soy protein were tested by immunoblotting (IB). IB inhibition was achieved by incubating sera with protein extract from soybean flour. RESULTS: The SDS-PAGE profile of soybean flour protein and soy milk showed a difference in the proportions of the various protein fractions, with a higher concentration of 37-kD protein in flour and 33-kD protein in milk. Infant formula 1 contained proteins with a molecular weight below 28 kD. The texturized extract contained high proportions of 31- to 34- and 38-kD proteins. Immunoblotting revealed a lack of allergenicity in infant formula. Sera recognizing the 38- and 50-kD proteins in texturized soy protein also recognized the 37- and 49-kD proteins in soybean flour and in soy milk, suggesting a protein glycation by texturization processes. The 30- to 34-kD band in texturized proteins was devoid of any allergenicity. This study seems to indicate that the 30-kD allergen (Gly m Bd 30) disappears during the production of texturized soy protein. CONCLUSION: All technologies applied to soybean-based products induce striking variation in the protein profile and allergenicity. Texturized protein could lack the major allergen Gly m Bd 30. Further studies or texturization might generate modified technologies in order to create hypoallergenic texturized proteins.

Action of boron at the molecular level: effects on transcription and translation in an acellular system.

It has been shown that boric acid has well-defined biological effects such as stimulation of wound healing in vivo, release of growth factors and cytokines, and increase of the extracellular matrice turnover. We examined its action at the molecular level, using cell-free systems of transcription (isolated placenta nuclei) and translation (wheat germ extract). We found that 10 mM boric acid greatly increased RNA synthesis, measured by absorbance at 260 nm (x 6.4) or by [3H]-UTP uptake (x 11). Full-length functional mRNA was produced because proteins of 14-80 kDa were translated. Among these proteins, factors involved in angiogenesis and, subsequently, in wound healing (VEGF and TGFbeta) were identified by slot blot, whereas growth factors such as FGF1 and TNFalpha were not detected. These results demonstrate that boron may contribute to biological cell activities at both the transcription and translation levels. However, the mechanism of action is still not known.

Free radical production and changes in superoxide dismutases associated with hypoxia/reoxygenation-induced apoptosis of embryonic rat forebrain neurons in culture.

Following hypoxia/reoxygenation (6h/96h), cultured neurons from the embryonic rat forebrain undergo delayed apoptosis. To evaluate the participation of oxidative stress and defense mechanisms, temporal evolution of intraneuronal free radical generation was monitored by flow cytometry using dihydrorhodamine 123, in parallel with the study of transcriptional, translational, and activity changes of the detoxifying enzymes Cu/Zn-SOD and Mn-SOD. Two distinct peaks of radical generation were depicted, at the time of reoxygenation (+ 27%) and 48 h later (+ 25%), respectively. Radical production was unaffected by caspase inhibitors YVAD-CHO or DEVD-CHO, which prevented neuronal damage, suggesting that caspase activation is not an upstream initiator of radicals in this model. Cell treatment by vitamin E (100 microM) displayed significant neuroprotection, whereas the superoxide generating system xanthine/xanthine oxidase induced apoptosis. Transcript and protein levels of both SODs were reduced 1 h after the onset of hypoxia, but activities were transiently stimulated. Reoxygenation was associated with an increased expression (139%), but a decreased activity (21%) of the inducible Mn-SOD, whereas Cu/Zn-SOD protein and activity were low and progressively increased until 48 h post-hypoxia, when the second rise in radicals occurred. In spite of a temporal regulation of SODs, which parallels radical formation, oxidative stress might account for neurotoxicity induced by hypoxia.

High-performance liquid chromatographic analysis of muscular interstitial arginine and norepinephrine kinetics. A microdialysis study in rats.

Complex interactions between the L-arginine/nitric oxide synthase (NOS) pathway and the sympathetic nervous system have been reported. Methods capable of measuring L-arginine and norepinephrine (NE) have mainly been reported for plasma. We report the use of the microdialysis technique combined with high-performance liquid chromatography (HPLC) for measurement of both L-arginine and NE within the same tissue microdialysis sample. The microdialysis probe consisted of linear flexible probes (membrane length: 10 mm, outside diameter: 290 microm, molecular weight cut-off 50 kDa). The method used for L-arginine measurement was HPLC with fluorescence detection, giving a within-run and a between-day coefficient of variation of 2.9 and 12.8%, respectively. The detection limit was 0.5 pM/20 microl injected for L-/D-arginine. The method used for NE measurement was HPLC with electrochemical detection. The coefficients of variation were 4% for within-assay precision and 7.5% for between-assay precision. The detection limit for NE was 1 fmol/20 microl injected. The microdialysis technique coupled with HPLC system was validated in vivo to measure muscular interstitial concentrations of both arginine and NE under baseline conditions and after intravenous infusion of 500 mg/kg of L-arginine or D-arginine. In conclusion, the microdialysis technique coupled to HPLC allows the simultaneous measurements of both L-arginine and NE within the same tissue microenvironment and will enable the study of the complex interactions between the L-arginine/NO pathway and sympathetic nervous system within the interstitial space of different organs.

[Mucopolysaccharidosis type I in Morocco: clinical features and genetic profile]. / Mucopolysaccharidose de type I au Maroc: manifestations cliniques et profil génétique.

BACKGROUND: Mucopolysaccharidoses (MPS) are inherited metabolic disorders due to lysosomal enzyme deficiencies, leading to glycosaminoglycan accumulation in lysosomes of different tissues. The aim of this study was to characterize MPS types, particularly MPS I, which are difficult to differentiate by clinical features. PATIENTS AND METHODS: Over a period of three years (June 1996-May 1999), 16 Moroccan patients (3-20 years old) with MPS were investigated. Twelve of them came from the Souss region. In subjects with suspected clinical MPS I or II, the diagnosis was confirmed by biochemical investigations, which included the quantification of total glycosaminoglycans (GAGs) released in urine, their identification, and the assay of alpha-L-iduronidase activity in leucocytes. A molecular analysis was performed in parallel, to provide the genetic proof of the diagnosis. RESULTS: These 16 patients belonged to 12 families, nine of which were consanguineous (75%). Twelve patients had Hurler syndrome and three had Hurler/Scheie's syndrome; no case of Scheie's syndrome was observed. Short stature, coarse face, organomegaly, hernia, cardiac disease, mental delay and dysostosis were observed in variable degrees. We report three cases without corneal clouding. Increased total urinary GAGs, identified as dermatan sulfate and heparan sulfate by thin-layer chromatography and total deficiency of alpha-L-iduronidase activity, were noted in studied subjects. At the molecular level the P533R mutation was detected in 24 among 26 alleles studied. CONCLUSION: It is now possible to perform the screening of MPS I and II in Morocco by analysis of clinical, radiologic observations and biological investigation. The predominance of P533R mutation could permit the screening of healthy heterozygotes and genetic counselling for families of Moroccan descent.

Screening of new antioxidant molecules using flow cytometry.

We present a flow cytometry technique to evaluate the antioxidative properties of molecules on living cells, using a stable murine-murine hybridoma (Mark 3) cell line routinely cultured. Using this technique, intracellular superoxide anions and peroxides were evaluated with dihydrorhodamine (DHR-123) and dichlorofluorescein diacetate (DCFH-DA), respectively. When cells were first incubated for 10 min with either H(2)O(2) or the xanthine (X)/xanthine oxidase (XO) system, this flow cytometric technique was capable of evaluating the oxidative stress on cells. Twenty-one new analogues of ellipticine were synthesized and tested for their antioxidative properties compared to vitamin E and Ebselen used as references. A good statistical reflection of the antioxidative activities of these molecules was achieved by analyzing 35 000 cells in each experiment. Among them, the selenated molecule 18 was found to be 10 times more active than Ebselen but 10 000 times less active than vitamin E. Moreover, eight compounds showed glutathione peroxidase-like activities.

Assay of a seric human hexapeptide (HWESAS) using a monoclonal antibody and ELISA.

Human serum contains low-molecular-weight growth factors potentiating some in vitro biological effects of IGF-I and IGF-II and recently two peptides were mainly identified: HWESAS and WGHE. In order to determine seric HWESAS concentration, a specific monoclonal antibody against HWESAS was prepared. Its specificity was studied by inhibition tests: this antibody cross-reacts with Y-HWESAS, Cys-HWESAS. It does not react with HWESAS when its COOH is blocked, or with HWE, WGHE and tryptophan or with C3f (SSKITHRIHWESASLLR) which is a fragment of human complement containing HWESAS motif. Its affinity was measured by non competitive enzyme immunoassay (3.89+/-2.44.10(8) M(-1)). Then, this antibody was used in enzyme-linked immunosorbent assay (ELISA) and the preliminary assays were performed to detect HWESAS in serum. In contrast to healthy subjects, patients with chronic renal failure exhibited undetectable concentration of hexapeptide while after successful renal transplantation values increased to reach levels found in healthy subjects and varying according to post-operative evolution. These data are a strong hint that the kidney plays an important role in the production of this hexapeptide and underly the clinical interest of HWESAS detection in renal pathology.

Bovine beta-lactoglobulin receptors on transformed mammalian cells (hybridomas MARK-3): characterization by flow cytometry.

Flow cytometry was used to demonstrate the presence of beta-lactoglobulin (betaLG) receptors on living murine hybridoma MARK-3 cells using a fluorescein isothiocyanate-betaLG conjugate (FITC-betaLG: molar ratio of 5:1). A site occupation curve was produced using a shift in the mean channel fluorescence at various concentrations of FITC-betaLG. The binding of labelled ligand was concentration dependent and was inhibited by unlabelled betaLG. The on-rate constant was 3.2x10(2) M(-1) min(-1) and the off-rate constant was 0.002 min(-1). Scatchard plot analysis gave a dissociation constant (K(d)) of 44+/-21x10(-7) and 39+/-24x10(-5) M (n=3). Flow cytometry indicated that at least 15% of the FITC-betaLG were internalized for 5 min and that internalization was temperature- and time-dependent. The internalization was confirmed by 3-D fluorescence microscopy (CELLScan system).

Evolution of lactate/pyruvate and arterial ketone body ratios in the early course of catecholamine-treated septic shock.

OBJECTIVES: To measure arterial lactate/pyruvate (L/P) and arterial ketone body ratios as reflection of cytoplasmic and mitochondrial redox state at different stages of catecholamine-treated septic shock and compare them with normal and pathologic values obtained in patients in shock who have decreased oxygen transport (cardiogenic shock), and to assess the relationship between the time course of lactate, L/P ratio, and mortality in septic shock. DESIGN: Prospective, observational human study. SETTING: A university intensive care unit. PATIENTS: Sixty consecutive adult patients who developed septic shock and lactic acidosis requiring the administration of vasopressors. Twenty patients in the intensive care unit without shock, sepsis, and hypoxia and with normal lactate values and 10 patients with cardiogenic shock were also studied. MEASUREMENTS: Hemodynamic measurements, arterial and mixed venous blood gases, arterial lactate and pyruvate concentrations, and arterial ketone body ratio were measured within 4 hrs after the introduction of catecholamine and 24 hrs later. MAIN RESULTS: Fifteen patients (25%) died within the first 24 hrs of septic shock, and these early fatalities had a higher blood lactate (12.2+/-3 versus 4.6+/-1.3 mmol/L; p<.01) concentration and a higher L/P ratio (37+/-4 versus 20+/-1; p<.01) than those who died later. No difference was found for arterial ketone body ratio (0.41+/-0.1 versus 0.50+/-0.06). Forty-five patients survived >24 hrs including 25 survivors and 20 nonsurvivors. Although there was no difference between survivors and nonsurvivors in initial lactate concentration (4.1+/-0.4 and 4.6+/-0.3, respectively), L/P ratio (19+/-1 and 20+/-1, respectively), and arterial ketone body ratio (0.5+/-0.06 and 0.52+/-0.07, respectively), blood lactate and L/P ratio significantly decreased during the first 24 hrs in the survivors (2.8+/-0.4 and 14+/-1, respectively; p<.05). and were stable in the nonsurvivors (4+/-0.3 and 22+/-1, respectively) Although returning to normal values after 24 hrs in survivors and nonsurvivors, arterial ketone body ratio was higher in survivors (1.72+/-0.17 versus 1.09+/-0.15; p<.05). Lactate and L/P ratio were closely correlated (r2 = .8, p<.0001). In the cardiogenic shock group, lactate concentration was 4+/-1 mmol/L, L/P ratio was 40+/-6, and arterial ketone body ratio was 0.2+/-0.05. The mortality rate was 60%. CONCLUSIONS: The main result of the present study is that hemodynamically unstable patients with sepsis needing catecholamine therapy had a lactic acidosis with an elevated L/P ratio and a decreased arterial ketone body ratio, suggesting a decrease in cytoplasmic and mitochondrial redox state. The duration of lactic acidosis is associated with the development of multiple organ failure and death.

Development and validation of a methodology for intracellular pH measurements of hybridoma cells under bioreactor culture conditions.

The intracellular pH (pH(i)) is an important factor in the regulation of different cellular processes. It might therefore be used as a marker of the physiological state of cells cultivated in a bioreactor environment. We developed and validated therefore a methodology that permits a reproducible and reliable pH(i) measurement under such bioreactor culture conditions, contrary to earlier reported measurements, carried out on cells resuspended in buffers under nongrowth conditions. The hybridoma cells were sampled from the culture, stained with the pH-sensitive dye BCECF-AM (BCECF = 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein), and analyzed by flow cytometry. Such a measurement is perfectible to changes of the cells between the moment of sampling and of final analysis on the flow cytometer. All intermittent steps were for this reason studied in detail, either to determine the optimal conditions to be used or to characterize their influence on the final pH(i) value measured. Additional experiments were carried out, showing the representativeness of the measured pH(i) value for the pH(i) the cells possess really in the culture at the moment of sampling.

Mucopolysaccharidosis type I: characterization of a common mutation that causes Hurler syndrome in Moroccan subjects.

A group of 13 Moroccan patients with MPS I and their families, including three siblings and twin siblings, was screened for mutations of the alpha-L-iduronidase gene using fluorescence-assisted mismatch analysis (FAMA) and cycle sequencing of PCR products. The P533R mutation, which is rare in Europeans, was identified in 92% of mutant alleles (24/26). This is the highest frequency of this mutation detected in patients with Hurler syndrome. None of the patients carried the W402X or Q70X alleles, the most common MPS I mutations in Europeans. These results suggest that the P533R mutation constitutes the genetic lesion which results in MPS I in people of Moroccan descent and provides yet more evidence for the uneven geographical distribution of mutations in MPS I.

Proteic composition of corpora amylacea in the bovine mammary gland.

Histochemical studies of mammary gland sections obtained from healthy lactating Prim' Holstein cows contained numerous corpora amylacea, mainly located in active alveoli. Observations by electron microscopy revealed a cauliflower shape, with a fibrillar or multilayered organization. Mineral studies confirmed the presence of high calcium levels (12.3% of dry matter) and phosphorus (7.4%) in the corpora amylacea composition. These bodies stained positive to Von Kossa silver nitrate and to Periodic acid-Schiff. However, depending on the gland of origin, corpora amylacea stained positive or negative to Congo red. Histochemical studies seemed, therefore, insufficient to determine the presence or absence of amyloid. The amount of total protein varied by approximately 25%. Immunoblotting and analysis of the amino acid sequence of a peptidic fragment obtained from corpora amylacea gave clear evidence of the occurrence of caseins, beta-lactoglobulin and alpha-lactalbumin. However, the comparison between the amino acid composition of corpora amylacea and those of the main milk proteins indicated the presence of other proteins. Electrophoretic analysis also gave evidence of the presence of several other proteins, i.e. glycoproteins. Therefore, it is probable that corpora amylacea composition is much more complex than previously reported.

Neomycin: microbiological assay or liquid chromatography?

In a multicentre study involving six laboratories, a microbiological assay was performed on three neomycin samples containing respectively, 0.12, 2.1 and 11% (m/m) of neomycin C, as well on a pure neomycin C sample. The potency was determined according to the European Pharmacopoeia method but using a neomycin B base standard. The relative standard deviations between laboratories (RSD) on the potencies varied from 4.8 to 50%, depending on the sample examined. The RSD increased with the neomycin C content of the samples and the highest RSD values were observed for the pure neomycin C sample. The activity of neomycin C relative to neomycin B was found to be 62% by diffusion (RSD:41%) and 56% by turbidimetry (RSD: 50%). This confirmed that the presence of neomycin C in a neomycin sample influences the reproducibility of the microbiological assay. T estimate the influence of this effect on official standard, their composition was verified by liquid chromatography. The neomycin C base content of the standards varied between 0.4 and 5.8% (m/m). Based on the results obtained and on formerly published reports discussing problems encountered with microbiological assay of neomycin, it is proposed to introduce liquid chromatography in official monographs to replace microbiological assay.

Purification from human plasma of a hexapeptide that potentiates the sulfation and mitogenic activities of insulin-like growth factors.

The human plasma contains small peptide molecules known as low molecular weight growth factors synergistically increasing certain biological actions of insulin-like growth factors. In the present work we isolated and characterized a hexapeptide with HWESAS as structure. This purified peptide was absolutely necessary for the sulfation activity of insulin-like growth factor-I on chick embryo pelvic cartilages and improved the mitogenic activity of both insulin-like growth factors. The effects of this hexapeptide were confirmed by using the homologous synthetic peptide, that exhibited similar biological effects. Other synthetic peptides with structure derived from hexapeptide were shown to be active: the pentapeptide HWESA appeared more potent than the tripeptide HWE, which is about 170 to 200 times less active than the hexapeptide. The sequence of hexapeptide HWESAS is identified in only one human protein that is C3f, a fragment of C3 complement.

Plasma membrane receptor for beta-lactoglobulin and retinol-binding protein in murine hybridomas.

The aim of the present work was to study the binding of [125I]-BLGA (beta-lactoglobulin variant A) to the plasma membrane fraction of hybrid cells. This binding increased as a function of time with on-rate and off-rate constant at 4.47 +/- 0.18 x 10(6) M-1 min-1 and 0.17 +/- 0.07 min-1, respectively (n = 3). The saturation study showed a single binding site type corresponding to a Kd at 8.26 +/- 2.98 nM and 14.02 +/- 2.61 x 10(12) sites per mg of the plasma membrane protein (n = 3). Competitive of binding BLGA was observed with BLGA, complexed with retinol and also with RBP (retinol-binding protein). Gel filtration of [125I]-BLGA incubated with Triton X-100 solubilized membrane showed the formation of a ligand-receptor complex. Cross-linking of the tracer to plasma membrane showed a complex with a M(r) at 69 kDa, suggesting a receptor M(r) of 51 kDa, as seen by autoradiography of SDS-PAGE.

Boron modulates extracellular matrix and TNF alpha synthesis in human fibroblasts.

Boric acid was not mitogenic for human fibroblasts and it did not change cell viability until 0.5% (w/v). Boric acid treatment affected the metabolism of human dermal fibroblasts in culture, decreasing the synthesis of extracellular matrix macromolecules such as proteoglycans, collagen, and total proteins. It also increased the release of these molecules into the culture medium. The principal proteins secreted into the medium after boric acid treatment had molecular masses of 90, 70, 58, 49, and 43 kDa and faint bands were detected by electrophoresis between 14 and 30 kDa. hsp 70 and TNF alpha were detected among the secreted proteins by immunoblotting, and the amount of TNF alpha released was quantified by radioimmunoassay. Total mRNA levels were higher after boric acid treatment and peaked after 6 h of treatment. TNF alpha mRNA was undetectable in unstimulated fibroblasts and two TNF alpha mRNA bands were detected after stimulation: immature mRNA (4.8 kb) and mature TNF alpha mRNA (1.9 kb). Thus, the effects of boric acid observed in wound repair in vivo may be due to TNF alpha synthesis and secretion.

Applications of infrared spectroscopy to medical biology.

This short review presents the great power of Fourier transform infrared (FT-IR) spectroscopy in the field of medicinal biology. New spectrometers as well as new accessories for sampling have significantly contributed to extend the application fields of the infrared techniques. During the past few years, it has been pointed out that FT-IR spectroscopy can be used to identify bacteria, to characterize neoplasic changes from biopsies, to recognize the various forms of arthritis by analysis of synovial fluid. This paper intends also to illustrate two clinical chemistry applications: the determination of the fecal lipids by FT-IR spectroscopy with an attenuated total reflectance accessory (ATR) and the measurement of the carbone monoxide in blood.