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PubMed2000

Human serine racemase: moleular cloning, genomic organization and functional analysis.

High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.

Amino Acid Sequence↗

PubMed2000

The use of transmigration and spermac stain to evaluate epididymal cat spermatozoa.

Epididymal spermatozoa of domestic cats were diluted with TEST medium and frozen. The parameters - estimated percentage of motile spermatozoa, concentration of spermatozoa, cell morphology and transmigration rate (TMR) - were evaluated before freezing and after thawing. Transmigration is a new method to measure the percentage of spermatozoa that consistently move forward, and has not been investigated with cat spermatozoa until now. Estimated percentage of motile spermatozoa averaged 65%, TMR was 76%, concentration of spermatozoa was 30,000 microl(-1) and the incidence of morphologically abnormal spermatozoa averaged 58% before freezing. After thawing, the estimated number of motile spermatozoa declined by 22%, but TMR remained at 76%. The TMR did not correlate with estimated motility but mostly was higher than the latter, which is postulated to be caused by the mobilizing effect of the countercurrent in the transmigration apparature. The estimated percentage of motile cells in the target chamber of the transmigration apparature was improved by using phosphate-buffered saline (PBS) as transmigration medium. Morphology was assessed both after fixation of spermatozoa in Hancock solution and after staining of smears with Spermac. Spermac did not stain all protoplasmic droplets but proved to be more suitable for the routine examination of acrosomal morphology after thawing.

Animals↗

PubMed1997

Metabolic aspects of HIV: associated wasting.

Despite recent progress in the treatment of human immunodeficiency virus (HIV) infection, wasting syndrome (WS) is now one of the major aspects of acquired immunodeficiency syndrome (AIDS). Malnutrition in HIV infected patients is characterised by a predominant loss of body cell mass (BCM), the amount of functional protoplasm in non-adipose tissue. This loss of BCM is correlated with a higher risk of AIDS events and a greater risk of mortality. If anorexia plays a major role in the development of the WS, some abnormalities in the metabolism drive the predominant loss of BCM. In the stable state, the resting energy expenditure (REE) is increased by about 10%. The REE is significantly correlated with the whole body protein turn over as measured by C13 leucine. This particular and only metabolic situation is associated with an increased insulin sensitivity and a high level in de novo hepatic lipogenesis. During periods of secondary infections, patients had a striking average weight loss, resulting from the combination of anorexia and dramatic elevated REE.

Basal Metabolism↗

PubMed1997

Developmental immunohistochemistry of growth inhibitory factor in normal brains and brains of patients with Down syndrome.

Growth inhibitory factor, a new metallothioneinlike protein, was investigated at postmortem examination in the brains of 18 patients with Down syndrome ranging in age from 18 weeks gestation to 50 years of age and in 20 age-matched normal controls by developmental immunohistochemistry. In the frontal cortex of both Down syndrome patients and controls, growth inhibitory factor immunoreactivity was localized in the cell bodies and processes of protoplasmic astrocytes from 18 weeks gestation, and these immunoreactive processes formed so dense a meshwork in the gray matter that they outlined neuronal perikarya as negative contours in the brain at age more than 16 years. The number of growth inhibitory factor-immunoreactive astrocytes exhibited a greater increase in layer 3 than in layer 2 in controls from 37 weeks gestation to 7 months of age, although there was no difference in the growth inhibitory factor-positive cell number between layers 2 and 3 in young Down syndrome patients. Therefore, growth inhibitory factor in astrocytes may be correlated with dendritic maturation of neurons. On the other hand, growth inhibitory factor-immunoreactive astrocytes in layer 2, where senile plaques are abundant, were smaller than those in layer 3 in adult Down syndrome patients from age 32 years. When senile plaques began to immunoreact with the amyloid precursor protein, the number of growth inhibitory factor-immunoreactive astrocytes decreased around senile plaques in elderly Down syndrome brains with the Alzheimer type of dementia. On the contrary, the number of glial fibrillary acidic protein-immunoreactive astrocytes around senile plaques increased. This loss of growth inhibitory factor around senile plaques may be correlated with neuronal loss or degeneration and lead to sprouting responses which may be involved in the formation of senile plaques.

Adolescent↗

PubMed2001

Signaling at the growth cone: the scientific progeny of Cajal meet in Madrid.

I had the good fortune to behold for the first time that fantastic ending of the growing axon. In my sections of the spinal cord of the three day chick embryo, this ending appeared as a concentration of protoplasm of conical form, endowed with amoeboid movements. It could be compared with a living battering ram, soft and flexible, which advances, pushing aside mechanically the obstacles which it finds in its path, until it reaches the region of its peripheral termination. This curious terminal club, I christened the growth cone.

Animals↗

PubMedDate not supplied

The Astrocentric Hypothesis: proposed role of astrocytes in consciousness and memory formation.

Consciousness is self-awareness. This process is closely associated with attention and working memory, a special form of short-term memory, which is vital when solving explicit task. Edelman has equated consciousness as the "remembered present" to highlight the importance of this form of memory (G.M. Edelman, Bright Air, Brilliant Fire, Basic Books, New York, 1992). The majority of other memories are recollections of past events that are encoded, stored, and brought back into consciousness if appropriate for solving new problems. Encoding prior experiences into memories is based on the salience of each event (A.R. Damasio, Descartes' Error, G.P. Putnam's Sons, New York, 1994; G.M. Edelman, Bright Air, Brilliant Fire, Basic Books, New York, 1992). It is proposed that protoplasmic astrocytes bind attended sensory information into consciousness and store encoded memories. This conclusion is supported by research conducted by gliobiologist over the past 15 years.

Animals↗

PubMed2001

Biochemical properties of porcine white adipose tissue mitochondria and relevance to fatty acid oxidation.

The capacity of white adipose tissue mitochondria to support a high beta-oxidative flux was investigated by comparison to liver mitochondria. Based on marker enzyme activities and electron microscopy, the relative purity of the isolated mitochondria was similar thus allowing a direct comparison on a protein basis. The results confirm the comparable capacity of adipose tissue and liver mitochondria for palmitoyl-carnitine oxidation. Relative to liver, both citrate synthase and alpha-ketoglutarate dehydrogenase were increased 7.87- and 10.38-fold, respectively. In contrast, adipose tissue NAD-isocitrate dehydrogenase was decreased (2.85-fold). Such modifications in the citric acid cycle are expected to severely restrict citrate oxidation in porcine adipose tissue. Except for cytochrome c oxidase, activities of the enzyme complexes comprising the electron transport chain were not significantly different. The decrease in adipose cytochrome c oxidase activity could partly be attributed to a decreased inner membrane as suggested by lipid and enzyme analysis. In addition, Western blotting indicated that adipose and liver mitochondria possess similar quantities of cytochrome c oxidase protein. Taken together these results indicate that not only is the white adipose tissue protoplasm relatively rich in mitochondria, but that these mitochondria contain comparable enzymatic machinery to support a relatively high beta-oxidative rate.

Adipose Tissue↗

PubMed2006

FISH and Calcofluor staining techniques to detect in situ filamentous fungal biofilms in water.

Filamentous fungi are a ubiquitous and diverse group of eukaryotic organisms and may contribute, along with bacteria, yeasts, protozoa and viruses, to the formation of biofilms in water distribution systems. However, fungal involvement in biofilms has not been demonstrated unambiguously. Furthermore, these fungi may be responsible for the production of tastes, odours and mycotoxins in drinking water making their early detection important. The detection of fme these problems a combination of two fluorescent techniques for direct detection was tested: (a) Fluorescence In Situ Hybridization (FISH) employing the universal rRNA probe EUK516, labelled with the red Cy3, followed by (b) staining with Calcofluor White MR2 fluorescent dye which stains fungal cell walls blue. Pure cultures of Penicillium brevicompactum were used to establish the methods followed by separate experiments with real water biofilm samples in PVC-C and cast iron coupons. FISH demonstrated eukaryotic microrganisms after approximately 5 h while the calcofluor method revealed chitinous filamentous structures in less than one hour. When the two methods were combined, additional resolution was obtained from the images of filamentous walls (blue) with intact protoplasm (red). In conclusion, FISH and Calcofluor staining provide rapid, direct and unambiguous information on the involvement of ff in biofilms which form in water.

Benzenesulfonates↗

PubMedDate not supplied

Arthroconidia in coccidioidoma: case report and literature review.

Coccidioides immitis is a dimorphic fungus capable of causing a diverse spectrum of disease in humans. Although the diagnostic pathologic finding in tissue is a mature endosporulating spherule, hyphal structures can also be found in over 50% of pathologic specimens. This report presents a case of coccidioidomycosis in which there were no intact spherules, but characteristic barrel shaped arthrospores present in tissue and cultures positively identified the organism as C. immitis. This case was further complicated by presentation in a nonendemic area for the fungus. Coccidioides immitis is a soil fungus with two distinct phases: the saprophytic and the parasitic. This biphasic life cycle was first described by Ophöls,1 and more fully characterized by Baker and colleagues. The saprophytic stage occurs in the environment with the organism existing in a mycelial state. As the mold matures, barrel-shaped arthroconidia form and alternate with empty cells. The arthroconidia subsequently fracture from hyphae and are dispersed as an aerosol. The saprophytic cycle repeats itself upon infection of a new soil site. However, if the airborne arthroconidia are inhaled by a susceptible host, the parasitic phase is initiated. In the parasitic cycle, the arthrospore swells and becomes a rounded structure, known as a spherule. The protoplasm of the spherule divides to form large numbers of endospores. If the spherule ruptures, the endospores are released. Each endospore can develop into a new spherule, or, upon expulsion via contaminated secretions from the host, return to the environment where it transforms into its mycelial form. Although the characteristic histologic finding in coccidioidomycosis is the spherule with endospores, hyphal forms also can be found. The mycelial form of C. immitis cannot be definitively identified morphologically and requires confirmation by culture techniques or genetic probes. This report presents a pulmonary coccidioidoma in which arthroconidia were the predominant forms found in tissue.

Adult↗

PubMed1983

Moniezia expansa: the interproglottidal glands and their secretions.

Acetylcholinesterase (EC 3:1:1:7) and alkaline phosphatase (EC 3:1:3:1) were detected in secretions of Moniezia expansa maintained in vitro. Ultrastructural cytochemical studies demonstrated acetylcholinesterase activity on the surface of the microtriches at the base of the interproglittidal glands and in the gland lumen but not in the distal tegument or the gland cells. Alkaline phosphatase activity was demonstrated in the cytoplasm of the gland cells and especially in their protoplasmic connections with the distal tegument. Activity was also found in the distal tegument and the microtriches. It is suggested that the acetylcholinesterase secreted by M. expansa performs a metabolic role at the worm's surface.

Acetylcholinesterase↗

PubMed1975

Effect of gamma-irradiation on oocysts of Eimeria necatrix.

Effect of gamma radiation on oocysts of Eimeria necatrix was investigated. It was observed that oocysts exposed to 200 kR or above did not sporulate. Irratiation at 10-150 kR caused a progressive decrease in sporulation. Irradiation affected normal development of unsporulated oocysts as the zygote protoplasm divided into unequal masses or was shattered into granules. Increase in the intensity of irradiation of sporulated oocysts resulted in the progressive decrease in severity of the resultant infections in chicks and their effects - mortality, type of lesions developed, total oocyst production and immunity produced - were comparable with infections induced by decreasing the number of unirradiated oocysts. Infection produced by 1000 unirradiated oocysts was comparable with that resulting from 50 000 oocysts irradiated at 25 kR. Infection obtained with 20 000 unexposed oocysts approximated to that produced by 50 000 oocysts irradiated at 2-5 kR. It was concluded that irradiation abolished infectivity of the oocysts/sporozoites rather than bringing about attenuation of the parasite.

Animals↗

PubMed1991

Ultrastructural and electrophysiological changes associated with K(+)-evoked release of neurotransmitter at the synaptic terminals of skate photoreceptors.

Bathing the skate retina in a Ringer solution containing a high concentration (100 mM) of potassium ions depolarized the visual cells, depleted the receptor terminals of synaptic vesicles, and suppressed completely the b-wave of the ERG and the intracellularly recorded response of horizontal cells (the S-potential). The depletion of synaptic vesicles was accompanied by a large increase in the extent of the plasma membrane resulting in distortion of the normal terminal profile, i.e. distension of the basal surface and elaborate infolding of protoplasmic extensions. Morphometric analysis showed that despite the changes in vesicle content and terminal structure, the combined linear extent of the vesicular and plasma membranes was unchanged from control (superfusion with normal Ringer solution); the increase in plasma membrane was equivalent to the observed loss of vesicular membrane. When returned to a normal Ringer solution, the terminals rapidly began to reform, and in about 10 min they were morphologically indistinguishable from receptor terminals seen in control preparations. After 30 min in the normal Ringer solution, the amount of membrane associated with the vesicles and the plasma membrane had reverted to control values, and once again the total membrane estimated morphometrically remained essentially the same. Thus, there is an efficient mechanism at the photoreceptor terminal for the recycling of vesicle membrane following exocytosis. The K(+)-induced depletion of synaptic vesicles was paralleled by a precipitous loss of responsivity in both the b-wave of the ERG and the S-potential of the horizontal cells. However, after 30-min exposure to the high K+ and a return to normal Ringer solution, the recovery of electrophysiological activity followed a much slower time course from that associated with the structural changes; 60 min or longer were required for the potentials to exhibit maximum response amplitudes. It appears that the rate-limiting step in restoring normal synaptic function following massive depletion of vesicular stores is transmitter resynthesis and vesicle loading rather than vesicle recycling.

Animals↗

PubMed2003

Nuclei, micronuclei and appendages in tri- and tetraradiate conidia of Cornutispora and four other coelomycete genera.

The distribution and behaviour of nuclei in conidia of 11 coelomycete species with tri- and tetraradiate conidia and belonging to five genera has been investigated: Cornutispora (C. ciliata, C. intermedia, C. lichenicola, C. limaciformis, and C. pittii), Eriosporella (E. calami), Furcaspora (F. abieticola, F. pinicola), Suttoniella (S. eriobotryae, S. gaubae), and Tetranacrium (T. gramineum). They have been studied by the HCl-Giemsa technique using dried, preserved material including holotypes and isotypes with ages ranging from 3 to 116 yr. Conidia of Cornutispora species showed different ploidy levels, and C. limaciformis showed a very high (> 90%) frequency of stable and viable micronuclei with an unusual type of ploidy level, occurring naturally. Frequency of ploidy levels in nuclei within conidia of Cornutispora species appeared to be associated with changes in gross conidial morphology. This is the first report of micronuclei in coelomycetes. The types of appendages on arms or parts of conidia have been studied using various stains including erythrosin in ammonia and a modified Leifson's flagella staining technique. In Furcaspora species the apical and basal conidial appendages are cellular maintaining protoplasmic continuity with the arms on which they are sited. The results have been compared with those of Crucellisporium species which have tetraradiate conidia. The new species, Cornutispora intermedia, C. pittii and Furcaspora abieticola spp. nov. are described, and illustrated, and a key to all known Cornutispora species is provided.

Cell Nucleus↗

PubMed1999

Topographic Imaging of Chromium-Coated Frozen-Hydrated Cell and Macromolecular Complexes by In-Lens Field Emission Scanning Electron Microscopy.

: An in-lens Schottky field emission scanning electron microscope (SEM) combined with a transmission electron microscope (TEM)-type cold-stage and a chromium (Cr) sputter-coating system was developed to rapidly prepare and cryo-image biological specimens to attain accurate nanometer-level structural information. High-resolution topographic images at high primary magnification (>/=200,000x) were digitally recorded with very short dwell times and without beam damage. Plunge freezing in ethane, followed by fracturing, Cr coating, and in-lens cryo-high-resolution scanning electron microscope (HRSEM) imaging directly revealed macromolecular features of yeast cells, platelets, and cell-free elastin analogues. The "vitreous" nature of bulk water in its solid state appeared featureless in cryo-HRSEM images, suggesting that if ice crystals were present they would be </=2-3 nm (the approximate instrument resolution on cryo-specimens). Compared to technically difficult and indirect freeze-fracture TEM replicas, cryo-HRSEM samples are fully hydrated, unfixed, noncryoprotected specimens immersed in featureless ice. The time necessary to cryo-immobilize the specimen and record the image is <3 hr. The hexagonal arrays of intramembrane particles on the protoplasmic face of yeast cells and differences in surface morphology between thrombin-stimulated and quiescent platelets were assessed. A clear interface line between collapsed elastin fibril lacework and vitreous lakes was commonly observed. These experiments demonstrate the feasibility of this technique to rapidly evaluate macromolecular features in cryofixed cells and cell-free systems.

Journal Article↗

PubMed1989

Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy.

We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particles that tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of approximately 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of approximately 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P beta' phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. The membrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger than that of the ring itself.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2004

Exploring molecular and mechanical gradients in structural bioscaffolds.

Most organisms consist of a functionally adaptive assemblage of hard and soft tissues. Despite the obvious advantages of reinforcing soft protoplasm with a hard scaffold, such composites can lead to tremendous mechanical stresses where the two meet. Although little is known about how nature relieves these stresses, it is generally agreed that fundamental insights about molecular adaptation at hard/soft interfaces could profoundly influence how we think about biomaterials. Based on two noncellular tissues, mussel byssus and polychaete jaws, recent studies suggest that one natural strategy to minimize interfacial stresses between adjoining stiff and soft tissue appears to be the creation of a "fuzzy" boundary, which avoids abrupt changes in mechanical properties. Instead there is a gradual mechanical change that accompanies the transcendence from stiff to soft and vice versa. In byssal threads, the biochemical medium for achieving such a gradual mechanical change involves the elegant use of collagen-based self-assembling block copolymers. There are three distinct diblock copolymer types in which one block is always collagenous, whereas the other can be either elastin-like (soft), amorphous polyglycine (intermediate), or silk-like (stiff). Gradients of these are made by an incrementally titrated expression of the three proteins in secretory cells the titration phenotype of which is linked to their location. Thus, reflecting exactly the composition of each thread, the distal cells secrete primarily the silk- and polyglycine-collagen diblocks, whereas the proximal cells secrete the elastin- and polyglycine-collagen diblocks. Those cells in between exhibit gradations of collagens with silk or elastin blocks. Spontaneous self-assembly appears to be by pH triggered metal binding by histidine (HIS)-rich sequences at both the amino and carboxy termini of the diblocks. In the polychaete jaws, HIS-rich sequences are expanded into a major block domain. Histidine predominates at over 20 mol % near the distal tip and diminishes to about 5 mol % near the proximal base. The abundance of histidine is directly correlated to transition metal content (Zn or Cu) as well as hardness determined by nanoindentation. EXAFS analyses of the jaws indicate that transition metals such as Zn are directly bound to histidine ligands and may serve as cross-linkers.

Animals↗

PubMed2000

T.H. Huxley's criticism of German cell theory: an epigenetic and physiological interpretation of cell structure.

In 1853, the young Thomas Henry Huxley published a long review of German cell theory in which he roundly criticized the basic tenets of the Schleiden-Schwann model of the cell. Although historians of cytology have dismissed Huxley's criticism as based on an erroneous interpretation of cell physiology, the review is better understood as a contribution to embryology. "The Cell-theory" presents Huxley's "epigenetic" interpretation of histological organization emerging from changes in the protoplasm to replace the "preformationist" cell theory of Schleiden and Schwann (as modified by Albert von Kolliker), which posited the nucleus as the seat of organic vitality. Huxley's views influenced a number of British biologists, who continued to oppose German cell theory well into the twentieth century. Yet Huxley was pivotal in introducing the new German program of "scientific zoology" to Britain in the early 1850s, championing its empiricist methodology as a means to enact broad disciplinary and institutional reforms in British natural history.

Biology↗

PubMed2000

D-amino acids as putative neurotransmitters: focus on D-serine.

Of the twenty amino acids in the mammalian body, only serine and aspartate occur in D-configuration as well as L-configuration in significant amount. D-serine is selectively concentrated in the brain, localized to protoplasmic astrocytes that ensheath synapses and distributed similarly to N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. D-serine has been found to function as an endogenous ligand for the "glycine" site of the NMDA receptor. Evidences for this include the greater potency of D-serine to activate this site than glycine, and D-amino acid oxidase, which degrades D-serine as well as other neutral D-amino acids, markedly attenuates NMDA neurotransmission. D-serine is also formed by serine racemase, a recently cloned enzyme that converts L-serine to D-serine. Thus, in many ways D-serine fulfills criteria for defining its functionality as a neurotransmitter and challenges the dogma relating to neurotransmission, for it is the "unnatural" isomeric form of an amino acid derived from glia rather than neurons.

Amino Acids↗