Differentiation of Lactobacillus Species by Molecular Typing

A total of 64 type, reference, clinical, health food, and stock isolates of microaerophilic Lactobacillus species were examined by restriction fragment length polymorphisms. Of particular interest were members of six of the eight species most commonly recovered from the vaginas of healthy premenopausal women, namely, Lactobacillus jensenii, L. casei, L. rhamnosus, L. acidophilus, L. plantarum, and L. fermentum. Six main groupings were identified on the basis of ribotyping. This technique was able to classify fresh isolates to the species level. In the case of the ribotype A grouping for L. rhamnosus, differences between strains were evident by chromosome typing (chromotyping). Many isolates did not possess plasmids. Six L. rhamnosus strains isolated from four different health food products appeared to be identical to L. rhamnosus ATCC 21052. The molecular typing system is useful for identifying and differentiating Lactobacillus isolates. Studies of strains of potential importance to the urogenital flora should include molecular characterization as a means of comparing genetic traits with those of strains whose characteristics associated with colonization and antagonism against pathogens have been defined.Lactobacilli colonizing human tissues have long been considered important for the maintenance of a healthy gastrointestinal tract (4) and urogenital tract (20, 21, 22, 25). The disruption of the lactobacillus flora has been associated with many urogenital infections, and as these afflict over 150 million women worldwide each year, this area of study is an important one. Indeed, many patients resort to taking health food products containing lactobacilli as a means of trying to maintain a healthy intestinal (and in some cases vaginal) flora.The typing of lactobacilli has generally been conducted by cell and colony morphology and biochemical tests. These techniques type bacteria based on their ability to ferment sugars and produce acids such as lactic acid and acetic acid (9). Unfortunately, these typing methods are not completely accurate, and strains which show intermediate characteristics are frequently encountered (9, 33).Many studies emphasize that the classification of lactobacilli is unsatisfactory and does not reflect the real phylogenetic relatedness of different strains and species (6, 19, 30). Several new genetic and chemotaxonomic approaches have been used during the last 14 years with an aim of improving the classification and identification of lactobacilli: for example, analysis of plasmid content (18), sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of whole-cell protein (19) and of total soluble cell protein (8), sequencing of rRNA (5, 6, 19), restriction endonuclease fingerprinting (14, 30), and DNA-DNA hybridization (6, 17, 19, 27). All of these approaches have improved the taxonomic knowledge of the generic and suprageneric relationships of lactobacilli. However, no analysis of urogenital or health food isolates of lactobacilli by molecular typing has been reported.Plasmid typing of Lactobacillus strains has been suggested as a taxonomic tool in a number of reports (27, 31), but there is evidence to suggest that it is not very effective (2, 12, 15, 28), since plasmids can be absent or unstable.Chromosome typing (restriction endonuclease fingerprinting of chromosomal DNA) (chromotyping) has been applied to the discrimination of strains of lactobacilli and has been found more specific and reproducible than plasmid content analysis (14). However, one of the disadvantages of chromotyping is that comparing electrophoretic patterns consisting of up to 100 bands is difficult. Hence, chromotyping alone may not be widely used for typing large numbers of strains.A sensitive but rapid method for species differentiation involves the use of ribotyping (1, 27), which combines Southern hybridization of chromosomal DNA fingerprints with the use of Escherichia coli rRNA probes, thereby discriminating between various species and individual strains of lactobacilli. The method involves the separation and identification of the rRNA genes present within the bacterial genome, which exist in various copy numbers (10, 13), making it possible to delineate species based on differences in the restriction fragment length polymorphisms of the rRNA genes. Of particular importance is the fact that specific regions of the rRNA genes have remained well conserved because of their functional importance, thus allowing the detection of a broad range of bacteria with 16S and 23S rRNA of E. coli as probes.The aim of this study was to develop a method to test the efficacy of ribotyping of Lactobacillus type and reference strains and to use this method to characterize a number of clinical, health food, and laboratory isolates.     

OXYGEN MICROSENSOR STUDIES ON ZOOXANTHELLATE CLIONAID SPONGES FROM THE COSTA BRAVA,MEDITERRANEAN SEA1

Photosynthetic properties of two symbiotic demosponges were compared using Clark‐type oxygen microsensors. The putatively distinct sponge species, Cliona viridis (Schmidt, 1862) and Cliona nigricans (Schmidt, 1862) were discriminated by their mean megasclere lengths of 296 and 387 μm, respectively. Photosynthetic behavior was used to generate additional taxonomic information. Sponge–dinoflagellate symbioses were well adapted to low light due to the hosts' endolithic lifestyle. Both sponges reached light compensation and saturation at similar light levels with means close to 10 and 30 μmol photons·m^?2·s^?1, respectively. The gross photosynthetic activity was closely related to symbiont cell density in the sponge surface tissue. Mean symbiont densities, chl a content, and gross photosynthesis were about six times higher in C. viridis than in C. nigricans, with respective values of 3000 and 440 symbiont·mm^?2, 1.3 and 0.2 μg chl a·g^?1, and 5.4 and 1.0 μmol O₂·cm^?3·s^?1 gross photosynthesis. Net photosynthesis and respiration could not be calculated accurately from the oxygen gradients, because significant gas exchange occurs through the pumping activity. Thus, assumptions of diffusional oxygen exchange via the surface do not hold for sponges. Combined data of this study indicate that the metabolic activity of C. viridis depends on photosynthetic activity of its symbionts, whereas C. nigricans appears to have a higher pumping intensity and is more actively filter feeding. The difference in photosynthetic activities is not caused by different light adaptations but provides new evidence against the conspecifity of C. viridis and C. nigricans.     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus on different carbon sources

Poly--hydroxybutyrate was produced in shake cultures by Alcaligenes eutrophus H16 on fructose, xylose, and fumaric, itaconic, lactic and propionic acids in a three-stage process. The maximum polymer concentration of 6.9 g l^–1 (69% of cell dry matter) was obtained with 20g l^–1 of fructose with a volumetric productivity of about 0.22 g l^–1 h^–1 at 24h. Up to about 3 g l^–1 (about 50% of cell dry matter) of polymer was also produced on lactic and propionic acids as the sole carbon source during the production phase. In multivatiate optimization employing an orthogonal 2³-factorial central composite experimental design with fructose as the substrate in a single-stage process, the optimal initial fructose concentration decreased from 35 g l^–1 to 24 g l^–1 when the incubation time was increased from about 35 h to 96 h. The optimal shaking speed range was 90–113 rpm. Correspondence to: S. Linko     

Preliminary studies in numerical systematics of the Paramphistomoidea (Digenea) from domestic Artiodactyla of Northern India

The systematic relationships between nine taxa (species) of paramphistomes belonging to the families Cladorchiidae, Gastrodiscidae, Gastrothylacidae and Paramphistomidae from domestic buffaloes Bubalus bubalis and pigs Sus scrofa were examined using SEM revealed two types of papillae occurring commonly on the oral cone and less frequently around the gonopore and acetabulum. Non-pitted papillae occur on the oral cone in Paramphistomum epiclitum, Calicophoron calicophorum, Explanatum (=Gigantocotyle) explanatum, Orthocoelium scoliocoelium, Gastrothylax crumenifer and Olveria indica. These papillae are also evident around the gonopore in Calicophoron papillosum, O. scoliocoelium and O. indica, and the ventral pouch opening in G. crumenifer. Ciliated papillae occur on the oral cone in C. papillosum and Fischoederius elongatus. Morphological and immunological observations were employed to classify paramphistomes using phenetic and phylogenetic approaches to systematics. Classifications based on phenetic methods compared taxa according to overall similarity of their characteristics and produced largely discordant results. Classifications based on phylogenetic methods compared taxa according to the genealogical descent of their characteristics and produced largely concordant results. These are in agreement with the widely accepted scheme of classification proposed by Stiles & Goldberger (1910) in which Gastrothylacidae and Paramphistomidae occur as natural groups. Furthermore, the Gastrothylacidae appear to share common ancestry with a member of the Paramphistomidae and are consequently nested within the latter group.This study forms part of the requirements for a thesis submitted by the first author to Q.U.B. for the degree of Doctor of Philosophy.This study forms part of the requirements for a thesis submitted by the first author to Q.U.B. for the degree of Doctor of Philosophy.     

Complementation of the anti-TNP PFC response of N:NIH nude mice from nude dams by spleen cells of nude mice from heterozygous dams.

Type I and Type II nude (nu/nu) mice are borne and raised by nu/nu dams and +/nu dams, respectively, under SPF conditions. Splenocytes from Type I nude mice exhibit even smaller in vitro PFC responses to trinitrophenylated rabbit erythrocytes than splenocytes from Type II nude mice. Type II splenocytes complement the magnitude of the PFC response of Type I splenocytes. The helper cell-like activity contained in Type II splenocyte suspensions was increased by treating Type II nude donor mice with dibutyryl cyclic AMP, but was essentially unaffected by pretreatment of Type II donors with endotoxin (LPS). The helper-like activity of Type II splenocytes was suppressed by treatment with a rabbit antiserum (plus C′) raised against a mixture of neonatal mouse thymocytes (RAM-T). We interpret the data to indicate that Type II splenocytes contain a larger number of residual T cells and/or a more fully developed population of T precursor cells that initiate or that are capable of being induced to initiate a helper function.     

Management of colonic and rectal injuries.

In managing a colonic or rectal injury the surgeon must decide whether it is acceptable to have feces passing over a suture line or anastomosis. If it is, resection and anastomosis or simple oversewing of the bowel can be done. If it is not, there are four choices: (a) closure of the wound, drainage and proximal diversion; (b) primary closure or resection and anastomosis of the wound with exteriorization; (c) formation of a double-barrelled colostomy; and (d) resection of the injured colon with formation of an end-colostomy and a mucosal fistula or a Hartmann procedure. The surgeon''s choice should be dictated by the severity of the injury, the degree of fecal contamination and the general condition of the patient.     

Prostaglandin synthesis in spleen cell cultures of mice injected with Corynebacterium parvum.

Spleen cells of C57BL/6 mice produced high amounts of PGE in vitro when tested 5 to 10 days after injection of heat-killed C. parvum organisms. Little or no PGE was produced by spleen cells from untreated mice or from mice injected with a strain of coryneform bacteria that does not stimulate the lymphoreticular system of mice. Significant release of PGE from spleen cells of C. parvum injected mice could be detected as early as 30 min after initiating the cultures and maximal levels were usually seen after 48 hr. Treatment by indomethacin completely abolished this PGE production. Removal of the adherent population from the spleen cell suspension resulted in markedly decreased levels of PGE, but PGE release of the remaining population was never completely abolished. These data suggest that the cells responsible for most of the PGE synthesis in this system were adherent cells, presumably macrophages. The levels of PGE produced in spleen cells of C. parvum-treated mice were further increased by in vitro addition of C. parvum. This effect could also be observed after addition of zymosan particles indicating that it was not an immunologically specific effect. The reported data suggest that prostaglandins may represent important mediator molecules of the described immunostimulatory and immunosuppressive effects of C. parvum.     

Reexamination of dopamine as the prolactin-release inhibiting factor (PIF): supplementary agent may be required for dopamine to function as the physiological PIF

A large number of studies have been performed concerning dopamine's inhibitory effect on prolactin release, but many of these studies have examined the effect of dopamine dissolved in a solution containing ascorbic acid. Ascorbic acid, routinely used to protect dopamine from oxidation, alone does not stimulate or inhibit prolactin release, but it can potentiate the inhibitory effect of dopamine in a static monolayer culture system by approximately 100 times. We have closely examined the inhibitory effect of dopamine on prolactin release in the absence of ascorbic acid using a perifusion system. Male rat adenohypophyses were dispersed with trypsin and cultured in a Petri dish to form cell clusters. Inhibition of prolactin release by dopamine (1 mumol/L) in the absence of ascorbic acid was sustained for only 63 min during the 2-h perifusion period. Following a 2-h period of incubation of dopamine in the same experimental solution, the dopamine concentration was reduced from 1 to 0.18 mumol/L, yet this "2-h-old dopamine" was still effective in inhibiting prolactin release (approximately 30 min). This result suggests that the lactotrophs may be desensitized by chronic exposure to a high concentration of dopamine in the absence of ascorbic acid. In contrast, when a low concentration of dopamine (3 nmol/L) containing ascorbic acid (0.1 mmol/L) was perifused, inhibition of prolactin release was sustained for the entire 2-h perifusion period. Although there may be a large number of explanations for dopamine's transient inhibitory effect on prolactin release, the present results suggest that dopamine may require supplementary agent(s) to effectively inhibit prolactin release and thus function as the prolactin release inhibitory factor (PIF).(ABSTRACT TRUNCATED AT 250 WORDS)     

The crystal and molecular structure of 1,6-anhydro-β-d-mannopyranose

The crystal structure of 1,6-anhydro-β-d-mannopyranose, C₆H₁₀O₅, is orthorhombic, P2₁2₁2₁, with a = 10.971(2), b = 13.935(3), c = 9.012(1) Å, V = 1377.76 »³ (MoKα, λ = 0.7107 Å), Z = 8, D_x = 1.563 M.gm⁻³, D_m = 1.565 M.gm⁻³. the structure was solved by MULTAN and refined to R(F) = 0.043 for 2355 reflections. The two symmetry-independent molecules in the unit cell have similar conformations, except for the orientation of one of the three hydroxyl groups. The conformation of the pyranose rings is ¹C₄ distorted towards E_o, and that of the anhydro rings is E. There are significant differences between the two molecules in two of the four C---O bond-lengths. These occur where there are important differences in the hydrogen-bonding environment of the oxygen atoms. The molecules are hydrogen-bonded by three linear and three bifurcated O---H···O interactions which form four-membered loops linked into infinite chains. Empirical force-field calculations with MMI-CARB reproduced the geometry of the molecules within the variations observed experimentally between the two molecules, except for a C---O bond in one of the molecules. The effect of excluding the anomeric effect from the theoretical calculations was not significant. Calculations for an intramolecularly hydrogen-bonded molecule were also carried out as a model for the molecules in a non-polar solvent.