Characterization of a purified beta-fructofuranosidase from Bifidobacterium infantis ATCC 15697.

AIMS: To characterize the beta-fructofuranosidase of Bifidobacterium infantis ATCC 15697 and to compare it with other bacterial beta-fructofuranosidases. METHODS AND RESULTS: The beta-fructofuranosidase of B. infantis ATCC 15697 was purified 46.8 times over the crude extract by anion exchange chromatography, ultrafiltration and gel filtration. The sequence of 15 amino acid residues of the NH2 terminal was determined. This enzyme was a monomeric protein (Mr 70 kDa) with beta-fructofuranosidase and invertase activities. The isoelectric point was pH 4.3, the optimum pH 6.0 and pKas (4.5 and 7.2) of two active groups were obtained. The activities were inhibited by Hg2+ and p-chloromercuribenzoic acid (pCMB). The optimal temperature was 37 degrees C and activities were unstable at 55 degrees C. beta-fructofuranosidase activity was more efficient than that of invertase with Vm/Km ratios of 0.65 and 0.025 x 10-3 l min(-1) mg(-1), respectively. The enzyme catalyses the hydrolysis of fructo-oligosaccharides, sucrose and inulin at relative velocities of 100, 10 and 6, respectively. CONCLUSIONS: The enzyme of B. infantis ATCC 15697 is an exo-inulinase which has beta-fructofuranosidase and invertase activities. This protein was different from the beta-fructofuranosidase of another strain of B. infantis (B. infantis JCM no. 7007). SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of bacterial beta-fructofuranosidases, especially from bifidobacteria, has been gained.

Lectin from Griffonia simplicifolia identifies an immature-appearing subpopulation of sensory hair cells in the avian utricle.

The sensory hair cells of the inner ear are coated with a variety of glycoproteins and glycolipids which can be identified by the binding of specific lectins. The present study examined the binding patterns of three lectins-Wheat Germ Agglutinin, Peanut Agglutinin, and lectin from Griffonia simplicifolia (Isoform B(4))-in the avian utricle. Each of the lectins exhibited a distinct pattern of hair cell labeling. Wheat Germ Agglutinin (WGA) appeared to label the ciliary bundles of all sensory hair cells. In contrast, the binding of Peanut Agglutinin (PNA) was mainly confined to the ciliary bundles of extrastriolar hair cells. Finally, lectin from Griffonia simplicifolia (GS-IB(4)) labeled a subpopulation of hair cells in all regions of the chick utricle. Those bundles were much smaller than the majority of ciliary bundles labeled by either WGA or PNA, and the density of GS-IB(4)-labeled bundles in the normal mature utricle was relatively low. Increased densities of GS-IB(4)-labeled hair cells were observed in the embryonic utricle and during the process of hair cell regeneration. The observations suggest that GS-IB(4) labels a glycoprotein that is expressed preferentially on the ciliary bundles of immature hair cells.

Ongoing cell death and immune influences on regeneration in the vestibular sensory organs.

Hair cells in the vestibular organs of birds have a relatively short life span. Mature hair cells appear to die spontaneously and are then quickly replaced by new hair cells that arise from the division of epithelial supporting cells. A similar regenerative mechanism also results in hair cell replacement after ototoxic damage. The cellular basis of hair cell turnover in the avian ear is not understood. We are investigating the signaling pathways that lead to hair cell death and the relationship between ongoing cell death and cell production. In addition, work from our lab and others has demonstrated that the avian inner ear contains a resident population of macrophages and that enhanced numbers of macrophages are recruited to sites of hair cells lesions. Those observations suggest that macrophages and their secretory products (cytokines) may be involved in hair cell regeneration. Consistent with that suggestion, we have found that treatment with the anti-inflammatory drug dexamethasone reduces regenerative cell proliferation in the avian ear, and that certain macrophage-secreted cytokines can influence the proliferation of vestibular supporting cells and the survival of statoacoustic neurons. Those results suggest a role for the immune system in the process of sensory regeneration in the inner ear.

Fermentations of fructo-oligosaccharides and their components by Bifidobacterium infantis ATCC 15697 on batch culture in semi-synthetic medium.

AIMS: To compare the physiological behaviour of Bifidobacterium infantis ATCC 15697 growing on synthetic oligofructose or its components. METHODS AND RESULTS: The studies were carried out in regulated or non-regulated batch cultures on semi-synthetic media. Differences between the carbohydrate utilization patterns with glucose, fructose, sucrose and fructo-oligosaccharides (FOS) were determined. Glucose was the preferred substrate for growth and biomass production, whereas fructose was the best for lactate and acetate production. With sucrose, biomass production reached the level obtained with glucose, whereas with FOS, more metabolites were produced, as with fructose. In a mixture of FOS, the shorter saccharides were used first and fructose was released in the medium. Fructofuranosidase, an enzyme necessary to hydrolyse FOS, was inducible by fructose. CONCLUSION: Glucose contained in FOS and sucrose might sustain growth and cell production, while fructose might enable the production of major metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the bifidogenic nature of oligofructose has been gained.

The supporting-cell antigen: a receptor-like protein tyrosine phosphatase expressed in the sensory epithelia of the avian inner ear.

After noise- or drug-induced hair-cell loss, the sensory epithelia of the avian inner ear can regenerate new hair cells. Few molecular markers are available for the supporting-cell precursors of the hair cells that regenerate, and little is known about the signaling mechanisms underlying this regenerative response. Hybridoma methodology was used to obtain a monoclonal antibody (mAb) that stains the apical surface of supporting cells in the sensory epithelia of the inner ear. The mAb recognizes the supporting-cell antigen (SCA), a protein that is also found on the apical surfaces of retinal Müller cells, renal tubule cells, and intestinal brush border cells. Expression screening and molecular cloning reveal that the SCA is a novel receptor-like protein tyrosine phosphatase (RPTP), sharing similarity with human density-enhanced phosphatase, an RPTP thought to have a role in the density-dependent arrest of cell growth. In response to hair-cell damage induced by noise in vivo or hair-cell loss caused by ototoxic drug treatment in vitro, some supporting cells show a dramatic decrease in SCA expression levels on their apical surface. This decrease occurs before supporting cells are known to first enter S-phase after trauma, indicating that it may be a primary rather than a secondary response to injury. These results indicate that the SCA is a signaling molecule that may influence the potential of nonsensory supporting cells to either proliferate or differentiate into hair cells.

Macrophage secretory products influence the survival of statoacoustic neurons.

Prior studies have shown that macrophages are recruited to sites of injury or infection in the sensory organs of the inner ear, but the effects of macrophages and their cytokine secretory products on the sensory structures of the ear are not known. In the present study, cultures of dissociated statoacoustic neurons were incubated with selected macrophage secretary products and the numbers of surviving neurons after 48 h in vitro were quantified. Results indicate that two macrophage secretary products, interleukin 1 and fibroblast growth factor 2, can enhance the survival of statoacoustic neurons, while another cytokine, tumour necrosis factor-alpha can diminish the survival of those neurons. Also, numerous macrophages were present in both cytokine-treated and control cultures. The findings suggest that macrophages may influence the survival of the sensory neurons of the inner ear.

Immune cytokines and dexamethasone influence sensory regeneration in the avian vestibular periphery.

Prior studies have shown that macrophages are recruited to sites of hair cell lesions in the avian inner ear in vitro (Warchol, 1997) and in vivo (Bhave et al., 1998). Although the avian ear has a high capacity for sensory regeneration (Oberholtzer & Corwin, 1997; Stone et al., 1998), the role of macrophages in the regenerative process is uncertain. The present study examined the possible influence of macrophages and immune cytokines on regenerative proliferation in the avian utricle, one of the sensory endorgans of the vestibular system. Utricles from post-hatch chicks were placed in organ culture and hair cell lesions were created by incubation in neomycin. The cultures were then maintained for an additional 24-48 hours in vitro, and some cultures were treated with dexamethasone, which inhibits macrophage activation and cytokine production. Following fixation, resident macrophages were identified by immunoreactivity to CD68. Labeled macrophages were present in all specimens and increased numbers of macrophages were observed following neomycin treatment. Regenerative proliferation in dexamethasone-treated specimens was reduced by about 50%, relative to untreated controls. Additional experiments showed that two macrophage secretory products-TGF-alpha and TNF-alpha-enhanced the proliferation of utricular supporting cells. The results are consistent with a role for macrophages in hair cell regeneration.

Macrophage activity in organ cultures of the avian cochlea: demonstration of a resident population and recruitment to sites of hair cell lesions.

The factors that regulate the repair and regeneration of the sensory hair cells of the inner ear are not understood. Previous studies of hair cell injury in the lateral line sensory organs of amphibians and the cochleae of mammals have demonstrated that macrophages and other leukocytes are recruited to sites of hair cell lesions. The present study examined the distribution and activity of macrophages in organ cultures of the avian cochlea, a system whose regenerative abilities have been widely studied. Cochleae were removed from chicks and placed in organ culture, and precise hair cell lesions were created using a laser microbeam. Macrophages in the cultures were identified using histochemical, immunocytochemical, and morphologic criteria. It was found that (a) cultured cochleae contained a resident population of macrophages, and (b) increased numbers of macrophages were recruited to the sites of hair cell lesions. Furthermore, the latency of macrophage recruitment to lesions is consistent with a suggested role for macrophages in the initiation of hair cell regeneration.

Cell death, cell proliferation, and estimates of hair cell life spans in the vestibular organs of chicks.

We have examined the level of on-going cell death in the chick vestibular epithelia using the TUNEL method and compared this to the rate of on-going cell proliferation. Utricles contained 22.6 +/- 6.8 TUNEL-labeled cells (mean +/- s.e.m.) while saccules contained 15.1 +/- 4.0, with approximately 90% being labeled hair cells. In separate experiments, chicks were given a single injection of BrdU and killed 2 h later. Utricles contained 116.9 +/- 6.5 BrdU-labeled cells (mean +/- s.e.m.) and saccules contained 41.0 +/- 2.2. After 24 h in culture, utricles treated with 1 mM neomycin contained 115.5 +/- 38.9 TUNEL-labeled cells, an increase of 270% over controls. After 48 h, neomycin-treated saccules contained 40.9 +/- 7.8, an increase of 152% over controls. The majority of labeled cells were in the hair cell layer. Thus, neomycin exposure results in an apoptotic death of hair cells. The in vivo data measured here were used to estimate that the average life span of utricular hair cells in young chickens is approximately 20 days, in sharp contrast to the life spans assumed for hair cells in humans.

Regenerative proliferation in organ cultures of the avian cochlea: identification of the initial progenitors and determination of the latency of the proliferative response.

Sensory hair cells in the cochleae of birds are regenerated after the death of preexisting hair cells caused by acoustic over-stimulation or administration of ototoxic drugs. Regeneration involves renewed proliferation of cells in an epithelium that is otherwise mitotically quiescent. To determine the identity of the first cells that proliferate in response to the death of hair cells and to measure the latency of this proliferative response, we have studied hair-cell regeneration in organ culture. Cochleae from hatchling chicks were placed in culture, and hair cells were killed individually by a laser microbeam. The culture medium was then replaced with a medium that contained a labeled DNA precursor. The treated cochleae were incubated in the labeling media for different time periods before being fixed and processed for the visualization of proliferating cells. The first cells to initiate DNA replication in response to the death of hair cells were supporting cells within the cochlear sensory epithelium. All of the labeled supporting cells were located within 200 microns of the hair-cell lesions. These cells first entered S-phase approximately 16 hr after the death of hair cells. The results indicate that supporting cells are the precursors of regenerated hair cells and suggest that regenerative proliferation of supporting cells is triggered by signals that act locally within the damaged epithelium.

Growth factors as potential drugs for the sensory epithelia of the ear.

The highly ordered structures of the hearing and balance organs of vertebrate ears go through a coordinated sequence of cellular and morphogenetic events. It is to be expected that protein growth factors and other extracellular signals will regulate many events during embryonic development of the ear, including the induction of the ear, the specific induction of sensory epithelia, the proliferation of the cells that form the sensory epithelia, the differentiation of the sensory and supporting cells, and the attraction and maintenance of innervation. After embryonic development, growth factors will support cell survival and innervation of new sensory cells. In damaged sensory epithelia, supplementation of the normal growth factors in these tissues has the potential to influence cellular responses to trauma, to reduce cell death and to promote the replacement of dead cells through renewed proliferation and differentiation, so as to improve hearing and balance health via preventive and restorative treatments. Assessment of the influences of specific growth factors on the sensory epithelia of vertebrate ears is at an early stage: this paper provides a brief account of what we know from studies of normal and experimentally manipulated epithelia, discusses the current questions and suggests directions for future studies.

Supporting cells in isolated sensory epithelia of avian utricles proliferate in serum-free culture.

Sheets of sensory epithelia were isolated from the utricles of chicks and cultured in serum-free media and in media that contained serum. The proliferation of epithelial supporting cells was assayed using the mitotic tracer bromodeoxyuridine. Similar levels of supporting cell proliferation were observed in epithelia maintained in serum-free and serum-containing media. The results suggest that the vestibular epithelia of birds contain whatever mitogens are necessary for the continued proliferation of epithelial supporting cells.

Supporting cells in avian vestibular organs proliferate in serum-free culture.

Explants of saccules and utricles taken from hatchling chicks were cultured in medium that contained fetal bovine serum and in serum-free medium. The mitotic tracers [3H]thymidine and bromo-deoxyuridine were added to the media to label proliferating cells. High numbers of labeled supporting cells were found in cultures that were maintained in both serum-containing and serum-free media. After seven days in culture, some of the labeled cells had begun to differentiate as hair cells. The results suggest that any mitogenic factors necessary for supporting cell proliferation and the factors required for the initial stages of hair cell differentiation are produced by cells contained within explants of the vestibular sensory epithelia.

The developing organ of Corti contains retinoic acid and forms supernumerary hair cells in response to exogenous retinoic acid in culture.

The mammalian organ of Corti has one of the most highly ordered patterns of cells in any vertebrate sensory epithelium. A single row of inner hair cells and three or four rows of outer hair cells extend along its length. The factors that regulate the formation of this strict pattern are unknown. In order to determine whether retinoic acid plays a role during the development of the organ of Corti, exogenous retinoic acid was added to embryonic mouse cochleae in vitro. Exogenous retinoic acid significantly increased the number of cells that developed as hair cells and resulted in large regions of supernumerary hair cells and supporting cells containing two rows of inner hair cells and up to 11 rows of outer hair cells. The effects of retinoic acid were dependent on concentration and on the timing of its addition. Western blot analysis indicated that cellular retinoic acid binding protein (CRABP) was present in the sensory epithelium of the embryonic cochlea. The amount of CRABP apparently increased between embryonic day 14 and postnatal day 1, but CRABP was not detectable in sensory epithelia from adults. A retinoic acid reporter cell line was used to demonstrate that retinoic acid was also present in the developing organ of Corti between embryonic day 14 and postnatal day 1, and was also present in adult cochleae at least in the vicinity of the modiolus. These results suggest that retinoic acid is involved in the normal development of the organ of Corti and that the effect of retinoic acid may be to induce a population of prosensory cells to become competent to differentiate as hair cells and supporting cells.

Regenerative proliferation in inner ear sensory epithelia from adult guinea pigs and humans.

Supporting cells in the vestibular sensory epithelia from the ears of mature guinea pigs and adult humans proliferate in vitro after treatments with aminoglycoside antibiotics that cause sensory hair cells to die. After 4 weeks in culture, the epithelia contained new cells with some characteristics of immature hair cells. These findings are in contrast to expectations based on previous studies, which had suggested that hair cell loss is irreversible in mammals. The loss of hair cells is responsible for hearing and balance deficits that affect millions of people.

Hair cell development.

Neurobiologists have been challenged by the desire to understand how the highly specialized ultrastructure of the sensory hair cells of the ear develops, how patterns of phenotypically distinct hair cells are formed and regenerate, and how their specific neural connections are formed. Recent research has addressed some of these challenges at the level of cell and molecular biology, focusing on cell proliferation in hair cell epithelia, the mechanisms that control hair cell differentiation, and the developmental interdependencies between hair cells and neurons. The initial identification of some of the homeobox genes and growth factors that are involved in hair cell development has occurred during the past year.

Hair cell regeneration: the identities of progenitor cells, potential triggers and instructive cues.

Hair cells are produced and accumulate in the ears of fish and amphibians as they grow during postembryonic life; hair cell regeneration occurs in lateral line organs in those groups and in the cochlea in birds. Continuous time-lapse microscopy has directly demonstrated that supporting cells divide to give rise to hair cells during regeneration in lateral line neuromasts. Supporting cells also appear to give rise to hair cells during regeneration in the avian ear, but additional cell types have been proposed as hair cell progenitors. Alternative interpretations of current evidence are discussed in relation to the possibility that supporting cells may be the common progenitor in all cases of hair cell regeneration. The regenerative proliferation of hair cells in birds occurs in populations of cells that are mitotically quiescent in undamaged ears. Evidence suggests that the extrusion of damaged hair cells and the breaking of intercellular junctional adhesions may be a trigger for regenerative proliferation. The potential triggering influence of phagocytes is also discussed. The differentiation of replacement cells during regeneration in the cochlea may be regulated by surface interactions between cells. A model that could account for the reconstitution of the mosaic pattern of hair cells and supporting cells is proposed.

Neural coding in the chick cochlear nucleus.

Physiological recordings were made from single units in the two divisions of the chick cochlear nucleus-nucleus angularis (NA) and nucleus magnocellularis (NM). Sound evoked responses were obtained in an effort to quantify functional differences between the two nuclei. In particular, it was of interest to determine if nucleus angularis and magnocellularis code for separate features of sound stimuli, such as temporal and intensity information. The principal findings are: 1. Spontaneous activity patterns in the two nuclei are very different. Neurons in nucleus angularis tend to have low spontaneous discharge rates while magnocellular units have high levels of spontaneous firing. 2. Frequency tuning curves recorded in both nuclei are similar in form, although the best thresholds of NA units are about 10 dB more sensitive than their NM counterparts across the entire frequency range. A wide spread of neural thresholds is evident in both NA and NM. 3. Large driven increases in discharge rate are seen in both NA and NM. Rate intensity functions from NM units are all monotonic, while a substantial percentage (22%) of NA units respond to increased sound level in a nonmonotonic fashion. 4. Most NA units with characteristic frequencies (CF) above 1000 Hz respond to sound stimuli at CF as 'choppers', while units with CF's below 1000 Hz are 'primary-like'. Several 'onset' units are also seen in NA. In contrast, all NM units show 'primary-like' response. 5. Units in both nuclei with CF's below 1000 Hz show strong neural phase-locking to stimuli at their CF. Above 1000 Hz, few NA units are phase-locked, while phase-locking in NM extends to 2000 Hz. 6. These results are discussed with reference to the hypothesis that NM initiates a neural pathway which codes temporal information while NA is involved primarily with intensity coding, similar in principle to the segregation of function seen in the cochlear nucleus of the barn owl (Sullivan and Konishi 1984).