Selective Deletion of PTEN in Dopamine Neurons Leads to Trophic Effects and Adaptation of Striatal Medium Spiny Projecting Neurons

The widespread distribution of the tumor suppressor PTEN in the nervous system suggests a role in a broad range of brain functions. PTEN negatively regulates the signaling pathways initiated by protein kinase B (Akt) thereby regulating signals for growth, proliferation and cell survival. Pten deletion in the mouse brain has revealed its role in controlling cell size and number. In this study, we used Cre-loxP technology to specifically inactivate Pten in dopamine (DA) neurons (Pten KO mice). The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinson''s disease, and other neurodegenerative disorders.     

Differential expression and cellular distribution of γ‐tubulin and βIII‐tubulin in medulloblastomas and human medulloblastoma cell lines

The breakdown of the blood–brain barrier (BBB) has been considered to be a key step in the disease process of a number of neurological disorders such as cerebral ischemia and Alzheimer's disease. Many in vitro BBB models derived from animal tissues have been established to elucidate the mechanism of BBB insufficiency. However, only a few human immortalized in vitro BBB models have been reported. In the present study, a temperature‐sensitive SV40‐T antigen was introduced to immortalize cells using a retrovirus to obtain a better human in vitro BBB model which sustains physiological properties. This endothelial cell (EC) line, termed TY08, showed a spindle‐shaped morphology. The cells expressed all key tight junctional proteins, such as occludin, claudin‐5, zonula occludens (ZO)‐1 and ZO‐2 at their cell‐to‐cell boundaries, and had low permeability to inulin across its monolayer. The cells also expressed various influx and efflux transporters and exhibited the functional expression of p‐glycoprotein. Furthermore, the TY08 cells grew and proliferated well under the permissive temperature and stopped growing under the non‐permissive temperature to serve as physiological ECs forming the BBB. Thus, conditionally immortalized TY08 cells retaining the in vivo BBB functions should facilitate analyses for determining the pathophysiology of various neurological diseases. J. Cell. Physiol. 225: 519–528, 2010. © 2010 Wiley‐Liss, Inc.     

Osteoarthritis and nutrition. From nutraceuticals to functional foods: a systematic review of the scientific evidence

The scientific and medical community remains skeptical regarding the efficacy of nutrition for osteoarthritis despite their broad acceptation by patients. In this context, this paper systematically reviews human clinical trials evaluating the effects of nutritional compounds on osteoarthritis. We searched the Medline, Embase, and Biosis databases from their inception to September 2005 using the terms random, double-blind method, trial, study, placebo, and osteoarthritis. We selected all peer-reviewed articles reporting the results of randomised human clinical trials (RCTs) in osteoarthritis that investigated the effects of oral interventions based on natural molecules. Studies on glucosamine and chondroitin sulfate were excluded. The quality of the RCTs was assessed with an osteoarthritic-specific standardised set of 12 criteria and a validated instrument. A best-evidence synthesis was used to categorise the scientific evidence behind each nutritional compound as good, moderate, or limited. A summary of the most relevant in vitro and animal studies is used to shed light on the potential mechanisms of action. Inclusion criteria were met by 53 RCTs out of the 2,026 identified studies. Good evidence was found for avocado soybean unsaponifiables. Moderate evidence was found for methylsulfonylmethane and SKI306X, a cocktail of plant extracts. Limited evidence was found for the Chinese plant extract Duhuo Jisheng Wan, cetyl myristoleate, lipids from green-lipped mussels, and plant extracts from Harpagophytum procumbens. Overall, scientific evidence exists for some specific nutritional interventions to provide symptom relief to osteoarthritic patients. It remains to be investigated whether nutritional compounds can have structure-modifying effects.     

Maternal melatonin influences rates of somatic and reproductive organs postnatal development of male rat offspring

Female rat dams, housed in 12L:12D photoperiod, were pinealectomized or injected daily 1(1/2) h before onset of darkness with 250 mg melatonin/100 g BW., during pregnancy; control and pinealectomized dams received a placebo. Somatic, reproductive organs and gonadotropins levels luteinizing hormone (LH) and follicle stimulating hormone (FSH) of male offspring were examined at the following phases of their sexual development: neonate, infantile, juvenile or prepubertal and pubertal periods. Pinealectomy of the mother produced an altered developmental pattern in the offspring (PIN-X offspring). During the infantile period when pups are lacking maternal melatonin and their own melatonin rhythm is not yet established, a delayed growth of body and testis weights was observed. After the second week of life, from 15 to 25 days of age, coinciding with the initiation of the melatonin rhythm, a speed-up growth of body and testes was observed, followed by a delayed growth from 25 to 30 days, in the juvenile period; this also coinciding with reduced LH levels observed at 30 days of age. Indeed, in PIN-X offspring significantly greater growth rate was observed during the pubertal period than in control offspring, which could be due to the increase in LH secretion up to normal values observed in the PIN-X offspring. Seminal vesicles of the PIN-X offspring also showed delayed growth, which was overcome at the pubertal period. Melatonin (MEL) treatment during pregnancy produced minor alterations in postnatal development of the reproductive tract. Only increased pituitary gland weight was observed at 15 and decreased at 25 days of age. At 25 days of age, MEL offspring reached the highest LH values, and at 30 days of age, PIN-X offspring still show low values. Which suggests that other factors than the endocrine activity of the gland are affecting the somatic growth of the pituitary gland. Seminal vesicles weight was delayed at 25 days of age in the MEL offspring. These results indicate that maternal melatonin is necessary for a normal somatic growth and postnatal development of reproductive organs of the offspring.     

Cytologic features of solitary fibrous tumor of the parotid gland. A case report

BACKGROUND: Solitary fibrous tumor (SFT) is a rare neoplasm that most commonly involves the pleura but is increasingly recognized at other locations, including lung, liver, thyroid and parotid glands, with only a few reports describing its cytologic features. CASE: The fine needle aspiration biopsy (FNAB) features of an SFT located in the left parotid gland of a 34-year-old woman were compared to the histologic and immunohistochemical aspects of the lesion. CONCLUSION: This case confirms that SFT has distinct cytomorphologic features and that FNAB can be helpful in its diagnosis.     

The Wzz (Cld) Protein in Escherichia coli: Amino Acid Sequence Variation Determines O-Antigen Chain Length Specificity

The O antigen is a polymer with a repeated unit. The chain length in most Escherichia coli strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values. wzz (cld/rol) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned wzz genes from E. coli strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric wzz genes and site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E. coli O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in wzz. It is concluded that the O-antigen chain length heterogeneity in E. coli strains is the result of amino acid sequence variation of the Wzz protein.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide- lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol- P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.      

High-throughput film-densitometry: an efficient approach to generate large data sets

A film-handling machine (robot) has been built which can, in conjunction with a commercially available film densitometer, exchange and digitize over 300 electron micrographs per day. Implementation of robotic film handling effectively eliminates the delay and tedium associated with digitizing images when data are initially recorded on photographic film. The modulation transfer function (MTF) of the commercially available densitometer is significantly worse than that of a high-end, scientific microdensitometer. Nevertheless, its signal-to-noise ratio (S/N) is quite excellent, allowing substantial restoration of the output to "near-to-perfect" performance. Due to the large area of the standard electron microscope film that can be digitized by the commercial densitometer (up to 10,000 x 13,680 pixels with an appropriately coded holder), automated film digitization offers a fast and inexpensive alternative to high-end CCD cameras as a means of acquiring large amounts of image data in electron microscopy.