Optimization of the enzymatic hydrolysis of mussel meat.

UNLABELLED: Mussel meat was subjected to enzymatic hydrolysis using Protamex. The relationship of temperature (46 to 64 degrees C), enzyme : substrate ratio (0.48% to 5.52%), and pH (6.7 to 8.3) to the degree of hydrolysis were determined. The surface response methodology showed that the optimum conditions for enzymatic hydrolysis of mussel meat were pH 6.85, temperature 51 degrees C, and enzyme : substrate ratio of 4.5%. Under these conditions a degree of hydrolysis of 26.5% and protein recovery of 65% were obtained. The produced hydrolysate, under optimum condition, was characterized in terms of chemical composition, electrophoretic profile, and amino acid composition. PRACTICAL APPLICATION: The practical application of mussel meat hydrolysate is its use as flavoring in products such as soups, sauces, and special beverages. In addition, the product is partially digested and has great nutritional value due to its good amino acid profile and thus can be used as a food supplement in special diets.

Nutritive evaluation of a non-conventional leafy vegetable (Pereskia aculeata Miller).

Pereskia aculeata Miller is a native cactus that can be found in Brazil and is called 'ora-pro-nobis' (OPN). Many people from poor communities consume the dark green leaves of OPN as a vegetable. The objective of the present work was to evaluate the nutritional components in terms of proximate composition, minerals, vitamins, protein content and their in vitro protein digestibility. OPN leaves showed remarkable levels of total dietary fiber (39.1% dry basis), minerals (calcium, magnesium, manganese and zinc) and vitamins (vitamin A, vitamin C and folic acid). Among amino acids, tryptophan was the most abundant (20.5% of the total amino acids) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed small peptides, inferior to 6.5 kDa, and four major bands (61 kDa, 53 kDa, 33 kDa, and 15 kDa). The protein digestibility corrected amino acid score showed the lowest value of sulfur-amino acids (Met+Cys). OPN leaves could be considered a good source of minerals, vitamins and amino acids, and may serve as a potential functional ingredient.

Patterns of wave break during ventricular fibrillation in isolated swine right ventricle.

Several different patterns of wave break have been described by mapping of the tissue surface during fibrillation. However, it is not clear whether these surface patterns are caused by multiple distinct mechanisms or by a single mechanism. To determine the mechanism by which wave breaks are generated during ventricular fibrillation, we conducted optical mapping studies and single cell transmembrane potential recording in six isolated swine right ventricles (RV). Among 763 episodes of wave break (0.75 times x s(-1) x cm(-2)), optical maps showed three patterns: 80% due to a wave front encountering the refractory wave back of another wave, 11.5% due to wave fronts passing perpendicular to each other, and 8.5% due to a new (target) wave arising just beyond the refractory tail of a previous wave. Computer simulations of scroll waves in three-dimensional tissue showed that these surface patterns could be attributed to two fundamental mechanisms: head-tail interactions and filament break. We conclude that during sustained ventricular fibrillation in swine RV, surface patterns of wave break are produced by two fundamental mechanisms: head-tail interaction between waves and filament break.

Preventing ventricular fibrillation by flattening cardiac restitution.

Ventricular fibrillation is the leading cause of sudden cardiac death. In fibrillation, fragmented electrical waves meander erratically through the heart muscle, creating disordered and ineffective contraction. Theoretical and computer studies, as well as recent experimental evidence, have suggested that fibrillation is created and sustained by the property of restitution of the cardiac action potential duration (that is, its dependence on the previous diastolic interval). The restitution hypothesis states that steeply sloped restitution curves create unstable wave propagation that results in wave break, the event that is necessary for fibrillation. Here we present experimental evidence supporting this idea. In particular, we identify the action of the drug bretylium as a prototype for the future development of effective restitution-based antifibrillatory agents. We show that bretylium acts in accord with the restitution hypothesis: by flattening restitution curves, it prevents wave break and thus prevents fibrillation. It even converts existing fibrillation, either to a periodic state (ventricular tachycardia, which is much more easily controlled) or to quiescent healthy tissue.

Characterization of chromosomal aberrations in human gastric carcinoma cell lines using chromosome painting.

Using chromosome painting, a study of chromosomal abnormalities was performed in six gastric carcinoma cell lines (SNU-484, 601, 620, 638, 668, 719) from Korean patients. Each carcinoma cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. SNU-484, SNU-620, and SNU-668 had near-triploidy; SNU-601, SNU-638, and SNU-719 had near-diploidy. The origins of the marker chromosomes of these cell lines were identified by fluorescence in situ hybridization with constructed painting probes. In all of six cell lines, rearrangement of chromosome 17 resulting in partial deletion of 17p (and/or partial duplication of 17q) was found. The most frequent marker was a partial gain of chromosome 7 with the breakpoints on 7q22 and 7q31. The nonrandom rearrangements of chromosomes were also determined on 1q32, 5q11-q22, 8q, 14q22, 14q34, and 15q15; suggesting that they may be the candidate regions for the isolation of the genes related to gastric cancer.

Scroll wave dynamics in a three-dimensional cardiac tissue model: roles of restitution, thickness, and fiber rotation.

Scroll wave (vortex) breakup is hypothesized to underlie ventricular fibrillation, the leading cause of sudden cardiac death. We simulated scroll wave behaviors in a three-dimensional cardiac tissue model, using phase I of the Luo-Rudy (LR1) action potential model. The effects of action potential duration (APD) restitution, tissue thickness, filament twist, and fiber rotation were studied. We found that APD restitution is the major determinant of scroll wave behavior and that instabilities arising from APD restitution are the main determinants of scroll wave breakup in this cardiac model. We did not see a "thickness-induced instability" in the LR1 model, but a minimum thickness is required for scroll breakup in the presence of fiber rotation. The major effect of fiber rotation is to maintain twist in a scroll wave, promoting filament bending and thus scroll breakup. In addition, fiber rotation induces curvature in the scroll wave, which weakens conduction and further facilitates wave break.

Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti.

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024-1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27(Kip1) is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27(Kip1)-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27(Kip1)-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.

Determination of hair cell degeneration and hair cell death in neomycin treated cultures of the neonatal rat cochlea.

The spatial-temporal course of hair cell degeneration and hair cell death was examined in the mammalian cochlea following aminoglycoside treatment. Organotypic cultures were established from postnatal rats (P3) and treated with 1 mM neomycin sulfate for 12-48 h and analyzed using a live/dead assay under epifluorescence microscopy. Live hair cells were labeled with calcein, a probe whose fluorescence and cellular retention depends upon intracellular esterase activity and cell-membrane integrity, respectively. Hair cell death was determined by ethidium homodimer-1, a probe that can enter cells with compromised cell membranes only. Inside the cell it binds to DNA. Hair cell morphology was also examined using phalloidin labeling, scanning electron microscopy and semi-thin section analysis. Results showed that hair cell degeneration and hair cell death occurred in a time dependent gradient from base to apex. After 48 h of neomycin treatment, most apical hair cells survived while most basal hair cells died. Calcein labeling provides a sensitive functional assay for measuring hair cell survival.

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.

Extraction of extracellular L-asparaginase from Candida utilis.

L-Asparaginase was extracted from Candida utilis cells using various reducing agents, 2-mercaptoethanol, dithiothreitol, or cysteine. The extraction of the enzyme depended upon the kind and concentration of reducing agents, temperature, time of incubation, and pH of buffer used. The enzyme was typically extracted by incubating the cells at 50 degrees C for 4 h in extraction solution containing 20 mM 2-mercaptoethanol in 20 mM potassium phosphate buffer (pH 7.0). The enzyme can be extracted from either cell precipitate or cell culture broth. The yeast cells were viable after extraction of L-asparaginase.

Development of ventral cochlear nucleus projections to the superior olivary complex in gerbil.

The postnatal development of the projection from the ventral cochlear nucleus to the principal nuclei in the superior olivary complex in gerbil (Meriones unguiculatus) was studied in an age-graded series of pups ranging from 0 to 18 days old. Small crystals of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were inserted into the ventral cochlear nucleus of aldehyde-fixed brains, and the labeled projections were examined with epifluorescence microscopy. Selected sections were photooxidized in a solution of diaminobenzidine and subsequently processed for electron microscopy to examine the development of labeled synapses in the target nuclei. Horseradish peroxidase was injected into the ventral cochlear nucleus of adult gerbils to assess the form and persistence of projections observed in the neonatal animals. In addition, electrophysiological responses to acoustic stimuli of single units in the adult auditory brainstem were analyzed to confirm the functionality of the novel projection from the ventral cochlear nucleus to the contralateral lateral superior olive. By the day of birth (P0), developing axons from the ventral cochlear nucleus have already established highly ordered pathways to the three primary nuclei of the superior olivary complex: the ipsilateral lateral superior olive, the contralateral medial nucleus of the trapezoid body, and at the lateral and medial dendrites of the ipsilateral and contralateral medial superior olive, respectively. Developing axons from the ventral cochlear nucleus that innervated the contralateral medial nucleus of the trapezoid body lacked the terminal morphology characteristic of the calyx of Held, but began to adopt a more characteristic form on P5. The mature calyx appeared around P14-16. Exuberant developmental projections to topographically inappropriate areas of the superior olivary complex were not observed at the postnatal ages studied. In addition to the projections of the ventral cochlear nucleus to the superior olivary complex described in other species, we observed the development and maintenance of a major direct projection from the ventral cochlear nucleus to the contralateral lateral superior olive. On P0, ventral cochlear nucleus axons decussate in the dorsal trapezoid body, form a plexus at the dorsal edge of the contralateral medial superior olive, and enter the ventrolateral limb of the contralateral lateral superior olive. Over the next 2 weeks, fascicles of fibers form on the hilar and ventral aspects of the ventrolateral limb. Fibers arising from these fascicles form converging, but nonoverlapping, arborizations within the ventrolateral limb at right angles to the curvature of the nucleus. The medial region was devoid of labeled axons.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of ectopic projections from the ventral cochlear nucleus to the superior olivary complex induced by neonatal ablation of the contralateral cochlea.

The ability of an animal to localize a sound in space requires the precise innervation of the superior olivary complex by the ventral cochlear nuclei on each side of the lower brainstem. This precise pattern of innervation could require an immutable recognition of appropriate targets by afferent processes arising from these nuclei. This possibility was investigated by destroying one cochlea of gerbil pups (Meriones unguiculatus) on the second postnatal day and assessing the projections from the ventral cochlear nucleus (VCN) on the unablated side to the superior olivary complex during the subsequent 2 weeks and after the animals had reached maturity. A crystal of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was inserted into VCN on the unablated side in animals ranging in age from 3 to 14 days. To assess the permanence of any altered pattern of innervation, horseradish peroxidase was injected into VCN on the unablated side in adult, neonatally ablated animals. Finally, electrophysiological responses to acoustic stimuli delivered to the ear on the unablated side were recorded in the superior olivary complex of adult animals to assess whether altered innervation patterns were functional. Normative data were derived from our accompanying study of the development of VCN projections to the superior olivary complex in normal gerbils (Kil et al., this issue). Whereas VCN normally projects to the lateral aspect of the ipsilateral medial superior olive and to the medial aspect of the contralateral medial superior olive in control animals, in experimental animals VCN on the unablated side projects to both sides of these nuclei. Whereas in the gerbil, VCN normally projects only to the hilar area and to the ventrolateral limb of the contralateral lateral superior olive, in experimental animals VCN on the unablated side projects throughout this nucleus. This induced projection is specific in that the efferents to each limb of the contralateral nucleus are linked to the normal projection to the homotopic region of the ipsilateral nucleus. Whereas VCN innervates the contralateral medial nucleus of the trapezoid body in control animals, in experimental animals VCN on the unablated side provides calyces of Held in the ipsilateral nucleus as well. The induced projections to these three major subnuclei of the superior olivary complex first appear within 24 hours of the cochlear ablation and continue to develop over at least the subsequent 11 days. Thus, prior to the day when the cochlea becomes functional, VCN has established specific ectopic projections to loci normally innervated by VCN on the ablated side.(ABSTRACT TRUNCATED AT 400 WORDS)

Magnetic resonance imaging of perilymphatic fistula.

Perilymphatic fistula (PLF) is considered to be a most challenging otologic issue. There are no currently agreed upon objective tests for the diagnosis of PLF. In an effort to improve diagnostic accuracy in patients with suspected PLF, a novel diagnostic test involving magnetic resonance (MR) contrast imaging was designed. An experimental PLF was created in the cochlear round window membrane of healthy adult cats. Since cochlear perilymph is thought to be an ultrafiltrate of cerebral spinal fluid, gadodiamide (gadolinium-diethylenetriamine penta-acetic acid [DTPA] bismethylamide), a nonionic paramagnetic contrast agent, was injected intrathecally in an attempt to enhance imaging of the created fistula. Post-contrast images of the fistualized cochlea demonstrate a significant increase in the signal intensity of the cochlear perilymph with pooling of enhanced perilymph observed in the ipsilateral mastoid bulla. Magnetic resonance contrast imaging may prove to be a valuable technique in human studies involving perilymphatic fistula.