Production of exopolysaccharides by Lactobacillus and Bifidobacterium strains of human origin, and metabolic activity of the producing bacteria in milk.

This work reports on the physicochemical characterization of 21 exopolysaccharides (EPS) produced by Lactobacillus and Bifidobacterium strains isolated from human intestinal microbiota, as well as the growth and metabolic activity of the EPS-producing strains in milk. The strains belong to the species Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus vaginalis, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. The molar mass distribution of EPS fractions showed 2 peaks of different sizes, which is a feature shared with some EPS from bacteria of food origin. In general, we detected an association between the EPS size distribution and the EPS-producing species, although because of the low numbers of human bacterial EPS tested, we could not conclusively establish a correlation. The main monosaccharide components of the EPS under study were glucose, galactose, and rhamnose, which are the same as those found in food polymers; however, the rhamnose and glucose ratios was generally higher than the galactose ratio in our human bacterial EPS. All EPS-producing strains were able to grow and acidify milk; most lactobacilli produced lactic acid as the main metabolite. The lactic acid-to-acetic acid ratio in bifidobacteria was 0.7, close to the theoretical ratio, indicating that the EPS-producing strains did not produce an excessive amount of acetic acid, which could adversely affect the sensory properties of fermented milks. With respect to their viscosity-intensifying ability, L. plantarum H2 and L. rhamnosus E41 and E43R were able to increase the viscosity of stirred, fermented milks to a similar extent as the EPS-producing Streptococcus thermophilus strain used as a positive control. Therefore, these human EPS-producing bacteria could be used as adjuncts in mixed cultures for the formulation of functional foods if probiotic characteristics could be demonstrated. This is the first article reporting the physicochemical characteristics of EPS isolated from human intestinal microbiota.

Human alpha fetoprotein enhances epidermal growth factor proliferative activity upon porcine granulosa cells in monolayer culture.

Alpha fetoprotein (AFP) is present at high concentrations in fetal fluids, certain neoplasias, and regenerating liver. Its physiological function remains largely unknown. Using a primary monolayer culture system, we investigated the proliferative activity of human (h) cord blood (CB) and highly purified AFP. hAFP, purified from hCB by Cibacron blue and immunoaffinity chromatography was homogeneous on SDS-PAGE and silver stain. Porcine granulosa cells from ovarian small follicles were cultured (25,000/cm2) for 2 days in medium (Ham's F-12:DMEM, 1:1) + 5% fetal calf serum (FCS) to facilitate attachment, followed by 6 days in medium containing: FCS, hCB or h amniotic fluid (1-20%)+/- EGF (10 ng/ml); or 0.25% plasma-derived serum (PDS) containing human low density lipoprotein (LDL, 25 ug/ml), +/- AFP (0.05-5 ug), and +/- EGF and IGF-I (10 ng/ml). In this system, single growth factors do not stimulate proliferation, a characteristic also exhibited by AFP. When combined with EGF, however, AFP dose-dependently increased proliferation to levels equal to that obtained with 10% FCS (2.3-fold increase vs PDS/LDL controls). When combined with EGF+IGF-I, AFP again dose-dependently increased proliferation to levels equal to that obtained with 10% FCS+EGF (6.7-fold increase vs controls). Purified human albumin used in place of AFP was not effective. TGF-a but not PDGF could replace the proliferative activity of EGF. These results suggest that AFP at physiological levels, although not itself mitogenic, can enhance the mitogenic activity of EGF and TGF-a and may function to modulate growth factor-mediated proliferation during development and neoplasia.

Inhibition of pituitary gonadotropin secretion by the gonadotropin-releasing hormone antagonist antide. II. Development of an in vitro bioassay for characterization of pharmacokinetics and pharmacodynamics of antide in circulation.

Previous data from this laboratory revealed a rapid and unexpectedly long inhibition of pituitary gonadotropin secretion in ovariectomized monkeys after a single high dose injection of the GnRH antagonist antide. This extended action of antide may correlate with an extended presence of antide in the peripheral circulation. We have reported on use of a RRA for antide in serum; however, during such a prolonged presence in the body, the possibility of catabolic loss of biological activity remained to be analyzed. In the present study, we have developed an in vitro pituitary cell bioassay for antide to investigate the pharmacokinetics and possible mechanism(s) contributory to its long action. Dispersed anterior pituitary cells from adult female rats were plated (48 h; 5 x 10(5) cells/well), washed, and incubated with 0.024-6 ng antide for 24 h. Media were removed, and cells were washed twice and then incubated with GnRH (1 x 10(-8) M) plus antide standards or serum samples for 4 h. Before antide injection into long term ovariectomized monkeys, peripheral GnRH antagonist levels were undetectable. One day after a single injection (3.0 mg/kg, sc, in 50% propylene glycol-water), the level of antide was 31 +/- 13 ng/ml (n = 3). Thereafter, antide levels declined slowly and were still detectable (greater than 1.4 ng/ml) in two of three monkeys 31 days after injection. After iv administration (3.0 mg/kg; n = 2), peripheral antide levels followed a similar pharmacokinetic profile and declined slowly. Detectable antide concentrations were still present 36 days after single iv injection in both monkeys. The circulating half-lives of antide were 1.7 and 14.5 days for the first and second phases, respectively. Peripheral LH levels were suppressed to the limits of detectability within 1 day and slowly recovered to pretreatment levels within 30 +/- 5 days after sc or iv antide treatment. The ratio of bioactive antide to antide levels measured by RRA was similar throughout the study (chi = 1.24 +/- 0.09; range, 0.40-2.22), although there was a trend toward an increased B/R ratio at the end of the study. In summary, we have developed an in vitro bioassay using cultured rat pituitary cells to measure biologically active antide concentrations in peripheral circulation after sc and iv treatments. The prolonged action of antide on pituitary gonadotropin secretion in vivo is apparently due to the continued presence of biologically active antide in circulation after a single injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolonged duration of gonadotropin inhibition by a third generation GnRH antagonist.

The dose-response effects of a single administration of Nal-Lys-GnRHant (antagonist) on serum LH and FSH concentrations were compared to the effects of Nal-Glu-GnRHant in monkeys. Twenty ovariectomized monkeys were divided into four sc treatment groups: a) 1.0 mg/kg Nal-Glu-GnRHant; or Nal-Lys-GnRHant at b) 0.3; c) 1.0; d) 3.0 mg/kg. Each monkey received vehicle (propylene glycol/water, 1:1) on day 0, followed by an antagonist preparation on day 11. Serum LH and FSH were measured by RIA; serum LH was also measured by in vitro bioassay. The short-term effects were similar among the four treatment groups. Typically, serum LH declined (p less than 0.05) within 4 to 8 h, achieving maximal reduction by 24 h. Serum FSH levels declined more slowly, but were significantly reduced by 24 h (p less than 0.05). Recovery during the study interval to pretreatment control values occurred in only two groups: a) Nal-Glu-GnRHant (1.0 mg/kg) by day 4 post-treatment and b) Nal-Lys-GnRHant (0.3 mg/kg) by day 2 post-treatment. Monkeys receiving 1.0 or 3.0 mg/kg Nal-Lys-GnRHant had a prolonged inhibition of serum LH and FSH levels. In all animals, serum FSH and LH returned to control levels within 2 months. The duration of gonadotropin inhibition was also prolonged when the Nal-Lys-GnRHant was administered iv. In contrast, Nal-Glu-GnRHant reduced serum LH and FSH for 3 days or less in all monkeys. The serum bioassayable LH levels paralleled those of immunoassayable LH. The prolonged inhibition of gonadotropin secretion following Nal-Lys-GnRHant distinguishes its action from those of previous GnRH antagonists and make this compound of great interest for clinical investigations.

Extended presence of antide (Nal-Lys GnRH antagonist) in circulation: prolonged duration of gonadotropin inhibition may derive from antide binding to serum proteins.

Previous data from this laboratory revealed a rapid (-12h) and unexpectedly long (-30 days) inhibition of pituitary gonadotropin secretion after a single injection of Antide (Nal-Lys GnRH antagonist) in ovariectomized (OVX) monkeys. Although the apparent mechanism of action of Antide is competitive occupancy of GnRH receptors, the etiology of the prolonged action is unknown. Here, we report development of a radioreceptor assay to measure circulating Antide levels to determine the mechanism(s) of its long duration of action. Five long-term OVX monkeys were injected with Antide (3.0 mg/kg). Blood samples were collected daily for 30 days, and thereafter on alternate days until day 60. Following sc or iv Antide injection, peripheral luteinizing hormone (LH) levels declined from 281 +/- 19 ng/ml to 29 +/- 3 ng/ml within one day (P less than 0.05). LH levels slowly recovered to pretreatment levels within 35 +/- 7 days. Peripheral Antide levels were 16,531 +/- 4,432 ng/ml within 15 minutes following iv injection, and 52 +/- 21 ng/ml at 1 day after sc Antide injection. Interestingly, thereafter clearance of Antide-from the peripheral circulation was very slow, with an apparent t1/2 (second phase) of 6.5 days following iv administration. Detectable Antide levels were present in the peripheral circulation for more than one month in all five monkeys. In a second experiment, incubation of 125I-Tyro Antide with OVX monkey serum resulted in binding of the labelled peptide to serum proteins and reduction of 125I-Tyro Antide binding to pituitary receptors. Following gel permeation chromatography, greater than 70% of the radioactivity was associated with a 66 kDa protein(s). In conclusion, the prolonged duration of gonadotropin inhibition by Antide seems to derive from the long circulatory half-life of this molecule. In turn, this extended action of Antide may be manifest, at least in part, by binding to serum protein(s) that serves as a built-in peripheral depot release mechanism.

Effect of novel CAAX peptidomimetic farnesyltransferase inhibitor on angiogenesis in vitro and in vivo.

Ras oncogenes can contribute to tumour development by stimulating vascular endothelial growth factor (VEGF)-dependent angiogenesis. The effect of Ras on angiogenesis may be affected by farnesyltransferase inhibitors (FTI) since farnesylation of Ras is required for its biological activity. In this paper we evaluated the effect of A-170634, a novel and potent CAAX FTI on angiogenesis. Human umbilical vein endothelial cell (HUVEC) tube formation and VEGF secretion were used to assess the effect of A-170634 on angiogenesis in vitro. In vivo, nude mice were injected with the K-ras mutant colon carcinoma cell line HCT116 and treated subcutaneously with A-170634 using osmotic minipump infusion for 10 days. The effect of A-170634 on corneal angiogenesis in vivo was assessed using pellets containing hydron, VEGF, A-170634 or vehicle. In vitro, A-170634 selectively inhibited farnesyltransferase activity over the closely related geranylgeranyltransferase I, inhibited Ras processing, blocked anchorage-dependent and -independent growth of HCT116 K-ras mutated cells, decreased HUVEC capillary structure formation, decreased VEGF secretion from tumour cells and HUVEC growth stimulating activity in a dose-dependent manner. In vivo, tumour growth was decreased by 30% and vascularisation in and around the tumours was reduced by 41% following drug-treatment with no apparent toxicity to the animals. VEGF-induced corneal neovascularisation was reduced by 80% following A-170634 treatment for 7 days. The data presented here demonstrated that A-170634 was a potent and selective peptidomimetic CAAX FTI with anti-angiogenic properties. These results implied that A-170634 may affect tumour growth in vivo by one or more antitumour pathways.

Differences in cell wall polysaccharides of several species of Eupenicillium.

A beta-(1-5)-galactofuran was isolated and characterized from fraction F1S (alkali- and water-soluble) of the cell wall of most of the species of Eupenicillium. In E. cryptum, E. euglaucum and E. nepalense the galactan contained galactofuranose with different linkages in addition to beta-(1-5). Fraction F1I (alkali-soluble, water-insoluble) was an alpha-glucan in certain species while in others it was a beta-glucan. Xylose was detected in some species in F1I or in F3 (alkali-soluble at 70 degrees C). The most abundant fraction (F4), resistant to the alkali treatment, was a beta-glucan-chitin complex. Excepting this component, the beta-(1-5)-galactofuran was the polysaccharide which appeared more frequently in the cell wall of species of Eupenicillium and it may have chemotaxonomic relevance.

Chemical and structural similarities in wall polysaccharides of some Penicillium, Eupenicillium and Aspergillus species.

Various fractions were extracted from cell-wall material of Eupenicillium crustaceum, Penicillium brevi-compactum, P. decumbens, Aspergillus flavipes and A. ochraceus. The most characteristic fractions, which may have chemotaxonomic relevance, were F1I, an alpha-(1-3) glucan (alkalisoluble, water-insoluble), which amounted to 16.2-32.5% of the cell-wall material, and F1S (alkali and water-soluble) which represented 2.5-6.2% of the cell-wall material and was identified as a beta-(1-5) galactan. 13C-NMR spectra of the F1S fractions showed the same pattern for all the fungal species, characteristic of beta-(1-5) linked galactofuranose.

Domiciliary biting frequency and blood ingestion of the Chagas's disease vector Rhodnius prolixus Ståhl (Hemiptera: Reduviidae), in Venezuela.

Demolition of a rural house in the State of Cojedes, Venezuela, provided a collection of 7.934 Rhodnius prolixus of which a random sample of 1,415 was weighed within 48 hours. The field weights, coupled with laboratory information of weight loss (in %) with time, average blood ingestion and meal size sufficient to promote moulting, were used to estimate biting rate under domiciliary conditions. The results show that in this particularly highly infested house, the R. prolixus population bites, on the average, at a rate of 58 times/person/day, draining blood at a rate of about 100 cm3/person/month; this meant a total of 1.2 litres/month from the 11 people inhabiting the house. It was found that the more advanced R. prolixus is in its development, the more aggressive it is in securing its meal: 15, 25, 30, 59 and 77% of fed insects of instar 1 through 5, respectively, were able to achieve moulting with only one meal. Applying the estimated biting rate to R. prolixus collections of other 13 demolished houses, with more typical insect population densities, an average biting rate of 9 bites/person/day was obtained; this value was, however, extremely variable, ranging from 0.2 bites/person/day (once every five days) to 33 bites/person/day.

Human mammary tumor cell proliferation: primary role of platelet-derived growth factor and possible synergism with human alpha-fetoprotein.

Human mammary medullary carcinoma cells (passages 16 to 21) were cultured for 2 days to allow for attachment, followed by 6 days of culture in either fetal calf serum, human cord blood, human amniotic fluid, or growth factors in the presence or absence of purified human alpha-fetoprotein (AFP). When growth factors were tested alone, only platelet-derived growth factor produced a significant increase in cell proliferation. Although up to 40% amniotic fluid had no effect on cell proliferation, human cord blood was two-fold more potent than fetal calf serum at similar concentrations. The addition of 10 ng/ml of platelet-derived growth factor increased the proliferative activity of human cord blood 1.5- to 2.5-fold. Ablation of endogenous AFP by affinity chromatography reduced the proliferative activity of cord blood by 75%. Similarly, the mitogenic activity of cord blood plus platelet-derived growth factor was reduced by 56% when AFP was removed. Purified AFP dose-dependently enhanced the proliferative activity of platelet-derived growth factor. This synergistic effect was specific for platelet-derived growth factor. We conclude that platelet-derived growth factor is a major growth factor controlling the proliferation of these tumor cells and that AFP may enhance growth factor proliferative activity and human mammary tumor growth.

Thermolytical techniques to characterize fungal polysaccharides and bacterial lipopolysaccharides.

The present work constitutes an entirely novel contribution in the scope of microbiology and especially in taxonomy, introducing thermolysis curves as a rapid method of characterization of fungal polysaccharides and bacterial lipopolysaccharides. The thermal analysis techniques applied were thermogravimetry and derivative thermogravimetry (TG-DTG), differential thermal analysis (DTA), and differential scanning calorimetry (DSC). Each thermogram of a sample is represented by one or a few temperatures and, in DSC, by complementary enthalpy data. The temperatures of the thermograms from structurally unknown polysaccharides are compared with those used as references, and thus, information on their composition, linkage types, and anomeric configuration can be deduced. The situation is more complicated for bacterial lipopolysaccharides, but in whatever mode, a structural estimation is always possible. In the course of the development and validation of the thermal method, structural findings on relative stabilities of linkage types (valuable in carbohydrate research) have been recognized and are therefore also described in this work.

Probing studies on multiple dose effects of antide (Nal-Lys) GnRH antagonist in ovariectomized monkeys.

This study was designed to extend evaluation of the long-acting effects of a "third generation" Antide (Nal-Lys) GnRH antagonist on gonadotropin secretion in ovariectomized (OVX) monkeys, with special attention to recrudescence of pituitary gonadotropin secretion after multiple dose treatments, as well as pituitary secretory responsiveness to GnRH. The duration of FSH/LH inhibition by Antide was dose-dependent, as well as being much longer than for Nal-Glu GnRHant; however, full recrudescence of gonadotropin secretion, albeit gradual, did occur. The acute LH secretory response to serial iv boluses of GnRH, in the face of GnRHant-induced suppression of gonadotropin secretion, was transiently accelerated and biologically active. Thereafter, the state of FSH/LH inhibition was resumed chronically. Thus, treatment with Antide produced profound long-term inhibition of tonic gonadotropin levels, yet hyper-responsiveness to exogenous GnRH administration was maintained throughout.

Chemical structure and conformational features of cell-wall polysaccharides isolated from Aphanoascus mephitalus and related species.

The structures of cell-wall mannans isolated from Aphanoascus mephitalus, A. fulvescens, A. verrucosus, and A. reticulisporus have been investigated by chemical analyses and 1D and 2D 1H and 13C NMR techniques. It was found that all of them consists of a relatively simple comb-like structure of the disaccharide repeating block [-->6)-[alpha-Manp-(1-->2)]-alpha-Manp-(1-->]. The conformations around the alpha-(1-->2) and alpha-(1-->6) linkages in these kinds of polymers were also studied by using molecular mechanics and dynamics calculations, together with NOE data. The results are similar to those found within the oligosaccharide chains of glycoproteins, with a well-defined conformation for the alpha-(1-->2) linkage and a certain restriction around the alpha-(1-->6) bonding imposed by the 2-substitution.

Structural studies of fungal cell-wall polysaccharides from two strains of Talaromyces flavus.

The water-soluble cell-wall polysaccharides isolated from strains CBS 352.72 and 310.38 of Talaromyces flavus have been investigated by chemical analyses and NMR studies. Two different skeletons coexist, having the structures: [formula:see text]. The small differences between the polysaccharides isolated from both strains are probably due to slight diminution of branching in strain 352.72, as compared with strain 310.38.

Structural investigation of two cell-wall polysaccharides of Penicillium expansum strains.

The structure of two cell-wall polysaccharides isolated from three different strains of Penicillium expansum, the type species of the genus, have been established by 1D and 2D NMR spectroscopy, and also by methylation analyses. The water-soluble polysaccharide F1S-B consisted of a linear tetrasaccharide repeating unit with the following structure: [-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-(1-->5)-beta-D-Gal f-(1-->5)-beta-D- Galf-(1-->]n The alkali-soluble polysaccharide F1I is a (1-->3)-alpha-D-glucan.

Structure of a cell wall polysaccharide isolated from Hypocrea gelatinosa.

The structure of a polysaccharide isolated from the cell wall of Hypocrea gelatinosa has been investigated by means of chemical analyses and 1D and 2D NMR spectroscopy. The polysacharide has an irregular structure, idealized as follows: [carbohydrate structure in text].

Structure of complex cell wall polysaccharides isolated from Trichoderma and Hypocrea species.

The structure of fungal polysaccharides isolated from the cell wall of Trichoderma reesei, T. koningii, and Hypocrea psychrophila, have been investigated by means of chemical analyses and 1D and 2D NMR spectroscopy. The polysaccharides have an irregular structure, idealized as follows: [formula: see text] The proportions of the different side chains vary from a species to another, being n above some three times larger in H. psychrophila than in T. reesei or T. koningii.

Structural elucidation of acidic fungal polysaccharides isolated from the cell-wall of genera Cylindrocladium and Calonectria.

The structure of acidic fungal polysaccharides, isolated from the cell wall of Cylindrocladium and Calonectria species, has been investigated by chemical analysis, methylation and reductive cleavage analyses, and 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharides have an idealized repeating unit: [formula: see text] linked to a small mannan core (< 5%), with n = 3 for Cylindrocladium penicilloides and C. quinqueseptatum and n = 5 for Calonectria theae, C. crotalariae, and C. colhounii.

Autoerotic asphyxial death initially misinterpreted as suicide and a review of the literature.

Death during autoerotic activities is of special concern to law enforcement officials, medical examiners, the family of the decedent, and society in general. As in the probing of any violent demise, accurate identification, management, and preservation of all physical evidence; complete photographic documentation of the scene and the body; reconstruction of the scene; and interviews with the family and acquaintances (psychological autopsy) are mandatory for proper study, evaluation, and interpretation of the case. Because of a lack of knowledge on the part of many people, including professionals, about these activities and the fact that many autoerotic fatalities share common characteristics with suicide and homicide, these cases are often misinterpreted. The authors present a case of autoerotic accidental asphyxial death which was initially misinterpreted as suicide.

[The surgical treatment of critical aortic stenosis in a child by the Ross-Konno technic]. / Tratamiento quirúrgico de la estenosis aórtica crítica en el niño mediante la técnica de Ross-Konno.

A twenty-nine-day old male infant suffering from critical aortic stenosis underwent aortic valvotomy by cardiopulmonary bypass. At three years of age the aortic stenosis recurred and the child underwent a balloon aortic valvuloplasty, but developed severe aortic insufficiency after the procedure. The critical condition of the patient made aortic valve replacement mandatory. The surgical technique consisted of aortoventriculoplasty with infundibular and valve pulmonary autograft for substituting the aortic root (Ross-Konno technique). As for as we know this is the first report on the Ross-Konno procedure in Spanish journals.