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Studies of cultured populations can be invaluable to the conservation of imperiled species for which little is known and whose very low abundance in the wild can preclude studies with robust sample sizes. For example, in endangered delta smelt (Hypomesus transpacificus), conservation measures developed to mitigate population decline are hindered by a lack of knowledge regarding spawning behavior. However, the availability of a cultured population allows important questions about spawning to be addressed experimentally. We examined the effect of substrate type on spawning behavior and egg fertilization success in cultured H. transpacificus under laboratory conditions. We also examined the diel frequency of spawning and the sex and number of spawning participants. We found that the frequency of spawns and resulting egg fertilization success did not differ between sand, gravel, and control (acrylic) substrates. We also demonstrated that spawning was predominantly nocturnal, though rare diurnal spawns were observed. Of the five mature males and five ripe females available within a given trial, spawns included up to seven participants, but most frequently occurred between a single female and one or two males. Our results highlight the wide behavioral variation exhibited by H. transpacificus during spawning, particularly in terms of substrate use, diel timing, and participants. These findings provide the most detailed and experimentally robust data regarding H. transpacificus spawning behavior to date and thereby provide ongoing conservation efforts with much-needed information.

An improved 18-hour pre-culture procedure coupling with duplex polymerase chain reaction (PCR) method capable of detecting Salmonella in frozen chicken meat is herein reported. Two pairs of primers (TS5/TS11 and S18/S19) were used for the PCR method. Positive PCR results were obtained after the 18-hour preculture (6 h in lactose broth at 37℃ followed by 12 h in Rappaport-Vassiliadis broth at 42℃) for spiked raw and irradiated meats with or without freezing treatment. The applicability of this improved procedure was tested in 50 retail frozen and 50 non-frozen chicken drumstick samples, and the results were compared with those obtained with the BAM standard culture method. No significant difference was found (p≧0.05) between these two methods.

In December 2017, the National Academy of Neuropsychology convened an interorganizational Summit on Population Health Solutions for Assessing Cognitive Impairment in Geriatric Patients in Denver, Colorado. The Summit brought together representatives of a broad range of stakeholders invested in the care of older adults to focus on the topic of cognitive health and aging. Summit participants specifically examined questions of who should be screened for cognitive impairment and how they should be screened in medical settings. This is important in the context of an acute illness given that the presence of cognitive impairment can have significant implications for care and for the management of concomitant diseases as well as pose a major risk factor for dementia. Participants arrived at general principles to guide future screening approaches in medical populations and identified knowledge gaps to direct future research. Key learning points of the summit included: recognizing the importance of educating patients and healthcare providers about the value of assessing current and baseline cognition;emphasizing that any screening tool must be appropriately normalized and validated in the population in which it is used to obtain accurate information, including considerations of language, cultural factors, and education; andrecognizing the great potential, with appropriate caveats, of electronic health records to augment cognitive screening and tracking of changes in cognitive health over time. Summit Participants Deb Adler1, Christopher Alban, MD, MBA2, Mark Bondi, PhD3, Michelle Braun, PhD4, Xavier Cagigas, PhD5, Morgan Daven6, Robert L. Denney, PsyD7,8, Lisa Drozdick, PhD9, Norman L. Foster, MD10,11, Ula Hwang, MD12–15, Laurie Ivey, PsyD16, Grant Iverson, PhD7,17, Joel Kramer, PsyD18, Laura Lacritz, PhD7,19, Melinda Lantz, MD20, Lisa Latts, MD, MSPH, MBA21, Shari M. Ling, MD22, Ana Maria Lopez, MD23–26, Michael Malone, MD27,28, Lori Martin-Plank, PhD, MSN, MSPH, RN29, Katie Maslow, MSW30, Don Melady, MSc(Ed), MD31–33, Melissa Messer34, John Meyers, PsyD7, Charles E. McConnel, PhD19, Randi Most, PhD36, Margaret P. Norris, PhD37, William Perry, PhD7,85,39, Neil Pliskin, PhD40, David Shafer, MBA41, Nina Silverberg, PhD42, Tresa Roebuck-Spencer, PhD43,44, Colin M. Thomas, MD, MPH45, Laura Thornhill, JD46, Jean Tsai, MD, PhD10,47, Nirav Vakharia, MD48, Martin Waters, MSW49 Organizations Represented Alzheimer’s Association, Chicago, IL AMA/CPT Health Care Professionals Advisory Committee, Chicago, IL American Academy of Clinical Neuropsychology (AACN), Ann Arbor, MI American Academy of Neurology (AAN), Minneapolis, MN American Association of Geriatric Psychiatry (AAGP), McLean, VA American Association of Nurse Practitioners (AANP), Austin, TX American Board of Professional Neuropsychology (ABN), Sarasota, FL American College of Emergency Physicians (ACEP), Philadelphia, PA American College of Physicians (ACP), Philadelphia, PA American Geriatrics Society (AGS), New York, NY American Psychological Association (APA), Washington, DC Beacon Health Options, Boston, MA Canadian Association of Emergency Physicians, Ottawa, ON, Canada Collaborative Family Healthcare Association (CFHA), Rochester, New York Gerontological Society of America, Washington, DC Hispanic Neuropsychological Society (HNS), Los Angeles, CA IBM Watson Health, Denver, CO International Federation of Emergency Medicine, West Melbourne, Australia International Neuropsychological Society (INS), Salt Lake City, UT National Academy of Neuropsychology (NAN), Denver, CO Optum of UnitedHealth Group, Minneapolis, MN Pearson, New York City, New York Psychological Assessment Resources, Inc, Lutz, FL Society for Clinical Neuropsychology, Washington, DC U.S. Department of Veterans Affairs, Washington, DC *Please note that participation in the Summit does not constitute organizational endorsement of this report

The leaves of Toona sinensis, a well-known traditional oriental medicine, have been prescribed for the treatment of enteritis and infection. Recently, aqueous extracts of Toona sinensis leaves (TSL-1) have demonstrated many biological effects both in vitro and in vivo. In the central nervous system, microglial activation and their proinflammatory responses are considered an important therapeutic strategy for neuroinflammatory disorders such as cerebral ischemia, Alzheimer's disease, and Parkinson's disease. The present study attempted to validate the effect of TSL-1 on microglia-mediated neuroinflammation stimulated by lipopolysaccharide (LPS). As inflammatory parameters, the production of nitric oxide (NO), inducible NO synthase, and tumor necrosis factor-α were evaluated. Our results demonstrate that TSL-1 suppresses LPS-induced NO production, tumor necrosis factor-α secretion, and inducible NO synthase protein expression in a concentration-dependent manner, without causing cytotoxicity. In addition, the inhibitory effects of TSL-1 in LPS-stimulated BV-2 microglia were extended to post-treatment suggesting the therapeutic potential of TSL-1. Therefore, this work provides the future evaluation of the role of TSL-1 in the treatment of neurodegenerative diseases by inhibition of inflammatory mediator production in activated microglia.

Cerebral vasospasm is the leading cause of mortality and morbidity in patients after aneurysmal subarachnoid hemorrhage (SAH). However, the mechanism and adequate treatment of vasospasm are still elusive. In the present study, we evaluate the effect and possible mechanism of progesterone on SAH-induced vasospasm in a two-hemorrhage rodent model of SAH. Progesterone (8 mg/kg) was subcutaneously injected in ovariectomized female Sprague-Dawley rats one hour after SAH induction. The degree of vasospasm was determined by averaging the cross-sectional areas of basilar artery 7 days after first SAH. Expressions of endothelial nitric oxide synthase (eNOS) and phosphorylated Akt (phospho-Akt) in basilar arteries were evaluated. Prior to perfusion fixation, there were no significant differences among the control and treated groups in physiological parameters recorded. Progesterone treatment significantly(P<0.01)attenuated SAH-induced vasospasm. The SAH-induced suppression of eNOS protein and phospho-Akt were relieved by progesterone treatment. This result further confirmed that progesterone is effective in preventing SAH-induced vasospasm. The beneficial effect of progesterone might be in part related to upregulation of expression of eNOS via Akt signaling pathway after SAH. Progesterone holds therapeutic promise in the treatment of cerebral vasospasm following SAH.

This article summarizes the results of 20 years (1991–2010) of investigation on food-borne disease outbreaks (FBDOs) collected by the Food and Drug Administration of the Department of Health in Taiwan. Among the 4284 FBDOs (82,342 cases) reported, the average annual number of 285 outbreaks during 2001–2010 was substantially greater than the average annual number of 143 outbreaks reported during 1991–2000. The average number of 15.5 cases per outbreak in 2001–2010 was lower than that in 1991–2000, which is 28.5 cases per outbreak. Small-scale FBDOs increased during 2001–2010. Good hygiene practices should be carried out at food service establishments and school kitchens that provide compound cooking food or meal boxes. The three most common bacterial etiology agents involved were Vibrio parahaemolyticus , Staphylococcus aureus, and Bacillus cereus . FBDOs resulting from B. cereus and Salmonella spp. exhibited an increasing trend during 2001–2010. Increased botulism FBDOs in 2001–2010 had been effectively controlled and reduced after governmental policy intervention. However, natural toxin-associated FBDOs are of concern as they can be fatal, especially the outbreaks associated with eating pufferfish by mistake.

Background Apoptosis is implicated in vasospasm and the long-term sequelae of subarachnoid hemorrhage (SAH). This study tested the hypothesis that attenuation of SAH-induced apoptosis after 17β-estradiol (E2) treatment is associated with an increase in phosphorylation of Akt via estrogen receptor-α (ER-α) in rats. Materials and methods We examined the expression of phospho-Akt, ERα and ERβ, and apoptosis in cerebral cortex, hippocampus, and dentate gyrus in a two-hemorrhage SAH model in rats. We subcutaneously implanted other rats with a silicone rubber tube containing E2; they received daily injections of nonselective estrogen receptor antagonist (ICI 182,780), selective ERα-selective antagonist (methyl-piperidino-pyrazole), or ERβ-selective antagonist (R,R-tetrahydrochrysene) after the first hemorrhage. Results At 7 d after the first SAH, protein levels of phospho-Akt and ERα were significantly decreased and caspase-3 was significantly increased in the dentate gyrus. The cell death assay revealed that DNA fragmentation was significantly increased in the dentate gyrus. Those actions were reversed by E2 and blocked by ICI 182,780 and methyl-piperidino-pyrazole, but not R,R-tetrahydrochrysene. However, there were no significant changes in the expression of the protein levels of phospho-Akt, ERα, ERβ, and caspase-3, and DNA fragmentation after SAH. Conclusions The present study shows that a beneficial effect of E2 in attenuating SAH-induced apoptosis is associated with activation of the expression of phospho-Akt and ERα, and alteration in caspase-3 protein expression via an ERα-dependent mechanism in the dentate gyrus. These data support further the investigation of E2 in the treatment of SAH in humans.

Rapid eye movement (REM) sleep behaviour disorder (RBD) is characterised by complex motor enactment of dreams and is a potential prodromal marker of Parkinson9s disease (PD). Of note, patients with PD observed during RBD episodes exhibit improved motor function, relative to baseline states during wake periods. Here, we review recent epidemiological and mechanistic findings supporting the prodromal value of RBD for PD, incorporating clinical and electrophysiological studies. Explanations for the improved motor function during RBD episodes are evaluated in light of recent publications. In addition, we present preliminary findings describing changes in the activity of the basal ganglia across the sleep–wake cycle that contribute to our understanding of RBD.

Hypoxic-ischemic brain damage often results from a combination of cardiogenic and respiratory failure. Whether or not hypoxia in the absence of ischemia is injurious to the brain has been a topic of research. An example of hypoxia without ischemia is found in obstructive sleep apnea (OSA), which causes recurrent nocturnal oxygen desaturations. Furthermore, it is a pervasive problem in the general population, particularly in people with common disorders such as obesity or diabetes. Mounting evidence in the past decade indicates that cerebrovascular disease, specifically stroke, and neurobehavioral consequences, including excessive daytime sleepiness and cognitive deficits, are prevalent in people with OSA, at great costs to the individual well-being, public health, and the economy. Investigation of the two disease associations poses similar and unique challenges. Predictors of these sequelae need to be better defined. The apnea-hypopnea index, the most common measure of OSA, has proven to be variably related to stroke and cognitive impairment. The role of individual markers, whether they are comorbidities or differences in inherent cognitive reserve, also is incompletely understood. This review discusses the burgeoning literature on the neurological and neurobehavioral sequelae of OSA and highlights the future avenues of research in the field.

Object Cerebral vasospasm remains a major complication in patients who have suffered a subarachnoid hemorrhage (SAH). Previous studies have shown that 17β-estradiol (E2) attenuates experimental SAH–induced cerebral vasospasm. Moreover, E2 has been shown to reduce neuronal apoptosis and secondary injury following cerebral ischemia. Adenosine A1 receptor (AR-A1) expression is increased following ischemia and may represent an endogenous neuroprotective effect. This study was designed to evaluate the efficacy of E2 in preventing cerebral vasospasm and reducing secondary injury, as evidenced by DNA fragmentation and AR-A1 expression, following SAH. Methods A double-hemorrhage model of SAH in rats was used, and the degree of vasospasm was determined by averaging the cross-sectional areas of the basilar artery 7 days after the first SAH. A cell death assay was used to detect apoptosis. Changes in the protein expression of AR-A1 in the cerebral cortex, hippocampus, and dentate gyrus were compared with levels in normal controls and E2-treated groups (subcutaneous E2, 0.3 mg/ml). Results The administration of E2 prevented vasospasm (p < 0.05). Seven days after the first SAH, DNA fragmentation and protein levels of AR-A1 were significantly increased in the dentate gyrus. The E2 treatment decreased DNA fragmentation and prevented the increase in AR-A1 expression in the dentate gyrus. There were no significant changes in DNA fragmentation and the expression of AR-A1 after SAH in the cerebral cortex and hippocampus in the animals in the control and E2-treated groups. Conclusions The E2 was effective in attenuating SAH-induced cerebral vasospasm, decreasing apoptosis in the dentate gyrus, and reducing the expression of AR-A1 in the dentate gyrus after SAH. Interestingly, E2 appears to effectively prevent cerebral vasospasm subsequent to SAH as well as attenuate secondary injury by reducing both apoptosis and a compensatory increase in AR-A1 expression in the dentate gyrus.

Studies in sepsis suggest that mitochondria mediate multiple organ dysfunction, including cardiac failure; however, the underlying molecular mechanisms remain elusive. This study examined changes in mitochondrial membrane integrity, antioxidant activities, and oxidative stress in the heart after infectious challenge (intratracheal Streptococcus pneumoniae, 4 x 10(6) colony-forming units). Inflammation responses also were examined.Cardiac tissues were harvested from Sprague-Dawley rats 4, 8, 12, and 24 h after bacterial challenge (or intratracheal vehicle for sham-treated animals) and homogenized, followed by preparation of subcellular fractions (mitochondrial, cytosol, and nuclei) or whole-tissue lysate. We examined mitochondrial outer membrane damage and cytochrome C translocation to evaluate mitochondrial integrity, mitochondrial lipid and protein oxidation to assess oxidative stress, and mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities to estimate antioxidant defense. In addition, we measured nuclear factor-kappa B (NF-kappaB) activation in myocardium and cytokine production to investigate inflammatory responses to septic challenge.Oxidation of mitochondrial protein and lipid was evident 4 h through 24 h after bacterial challenge. Mitochondrial outer membrane damage and cytochrome C release were accompanied by down-regulation of mitochondrial SOD and GPx activity. After bacterial challenge, systemic and myocardial cytokine production increased progressively, and NF-kappaB was activated gradually.Sepsis impaired cardiac mitochondria by damaging membrane integrity, increasing oxidative stress, and altering defenses against reactive oxygen species. These alterations occur earlier than or simultaneously with inflammatory responses in myocardium after infectious challenge, suggesting that mitochondria play a role in modulating inflammation in sepsis.

A depot drug delivery system, fibrin encapsulated liposome-in-chitosan matrix (FLCM), has been developed to deliver a water-soluble drug which is configured by a porous chitosan matrix containing a bovine fibrin network encapsulated different surface properties of liposomes. Quinacrine (QR), a water-soluble, low-molecular weight fluorescent marker, is used as a model drug to evaluate the delivery characteristics of the system. The SEM photographs show that the fibrin network adheres to the surfaces and pores of the chitosan matrix of a FLCM system. The QR release periods of the FLCM are sustained for about four times longer than those of QR encapsulated into the liposomes. However, the QR release periods and profiles of the FLCM are influenced by the surface properties of liposomes. The release of QR from FLCM is sustained for 9 days for neutral liposomes and only 5 days for PEG modified liposomes (PEG-liposome). After crosslinking the fibrin network of the FLCM with 0.5% of glutaldehyde, the release of QR is further sustained for 17 days with good linear profiles (e.g., 13 days) and with 50% of reduced burst release compared with those of without crosslinking, indicating that the stability of the fibrin network plays an important role on QR release of the system. More interestingly, the release periods and profiles of QR of the FLCM system are highly similar to those of Tirofiban, low-molecular weight of a water-soluble clinical cardiovascular drug, although the study has been done by human platelet poor plasma instead of bovine fibrinogen as a source of fibrin network. It suggests that the QR is a suitable model for investigating the drug delivery behaviors for water-soluble, low-molecular weight drugs of the FLCM. In conclusion, with QR as a model drug, FLCM with crosslinked fibrin network can effectively sustain the release of QR for 17 days but the release profiles are influenced by the surface properties of encapsulated liposomes. This study suggests that FLCM may have the potential as a depot drug delivery system for water-soluble drugs.

Using isogenic recombinant murine coronaviruses expressing wild-type murine hepatitis virus strain 4 (MHV-4) or MHV-A59 spike glycoproteins or chimeric MHV-4/MHV-A59 spike glycoproteins, we have demonstrated the biological functionality of the N-terminus of the spike, encompassing the receptor binding domain (RBD). We have used two assays, one an in vitro liposome binding assay and the other a tissue culture replication assay. The liposome binding assay shows that interaction of the receptor with spikes on virions at 37°C causes a conformational change that makes the virions hydrophobic so that they bind to liposomes (B. D. Zelus, J. H. Schickli, D. M. Blau, S. R. Weiss, and K. V. Holmes, J. Virol. 77: 830-840, 2003). Recombinant viruses with spikes containing the RBD of either MHV-A59 or MHV-4 readily associated with liposomes at 37°C in the presence of soluble mCEACAM1 a , except for S 4 R, which expresses the entire wild-type MHV-4 spike and associated only inefficiently with liposomes following incubation with soluble mCEACAM1 a . In contrast, soluble mCEACAM1 b allowed viruses with the MHV-A59 RBD to associate with liposomes more efficiently than did viruses with the MHV-4 RBD. In the second assay, which requires virus entry and replication, all recombinant viruses replicated efficiently in BHK cells expressing mCEACAM1 a . In BHK cells expressing mCEACAM1 b , only viruses expressing chimeric spikes with the MHV-A59 RBD could replicate, while replication of viruses expressing chimeric spikes with the MHV-4 RBD was undetectable. Despite having the MHV-4 RBD, S 4 R replicated in BHK cells expressing mCEACAM1 b ; this is most probably due to spread via CEACAM1 receptor-independent cell-to-cell fusion, an activity displayed only by S 4 R among the recombinant viruses studied here. These data suggest that the RBD domain and the rest of the spike must coevolve to optimize function in viral entry and spread.

The cleavage and fusion properties of recombinant murine hepatitis viruses (MHV) were examined to assess the role of the cleavage signal in determining the extent of S protein cleavage, and the correlation between cleavage and induction of cell-to-cell fusion. Targeted recombination was used to introduce amino acid substitutions into the cleavage signal of the fusion glycoprotein (spike or S protein) of MHV strain A59. The recombinants were then used to address the question of the importance of S protein cleavage and viral-mediated cell-to-cell fusion on pathogenicity. Our data indicate that cleavage of spike is not solely determined by the amino acid sequence at the cleavage site, but may also depend on sequences removed from the cleavage site. In addition, efficient cell-to-cell fusion is not necessary for virulence.

Purpose To report the ophthalmic findings and response to treatment in a patient with glutamic-acid decarboxylase antibodies. Design Case report. Methods A 55-year-old woman developed progressive, painful, low back muscle spasms, vertical diplopia, downbeating nystagmus, and asymmetric appendicular ataxia. Results Downbeating nystagmus was present in primary gaze with an alternating skew deviation in lateral gaze. Serum and cerebrospinal fluid GAD antibodies were detected. Treatment with diazepam led to resolution of spasticity, whereas repeated courses of intravenous immunoglobulin improved cerebellar function, including appendicular ataxia and downbeating nystagmus. Conclusions Patients with GAD antibodies may have elements of both Stiff-person syndrome (muscle rigidity and spasms) and prominent cerebellar dysfunction. Treatment with diazepam rapidly improved Stiff-person symptoms, whereas IVIg was partially effective at the early stage of cerebellar dysfunction.

Skin rash and depigmentation are common in patients with meningitis and meningoencephalitis. Skin changes must always be evaluated in conjunction with the clinical symptoms, signs, brain imaging, and laboratory abnormalities, particularly the features of the CSF pleocytosis. The purpose of this montage is to help the clinician identify a specific etiologic agent as early as possible.

We describe a patient with zoster paresis and an MRI that revealed extensive spinal cord lesions from the upper cervical to the lower thoracic spinal cord. Importantly, the patient reported considerable spontaneous improvement in strength 2-3 weeks after zoster. This report reveals a previously undescribed remarkable preponderance of MRI lesions far beyond the site of zoster rash and focal lower motor neuron weakness.

The cause of multiple sclerosis (MS) is unknown. Despite indications from epidemiological and identical-twin studies that MS is infectious, no virus or other infectious agent has been tightly linked to disease. The isolation of Chlamydia pneumoniae from the cerebrospinal fluid (CSF) of MS patients and the detection of both Chlamydia-specific DNA and antibody in MS CSF have been reported. Other analyses of brain and CSF have shown no significant difference in C. pneumoniae-specific DNA or antibody between MS and control subjects. Recent work has revealed intrathecal production of C. pneumoniae-specific IgG in only 24% of MS patients compared with 5% of control patients. More importantly, the major CSF oligoclonal bands from MS patients did not react to C. pneumoniae.

The C-terminal domain of the fibrinogen gamma chain (gammaC) has been shown to bind to the integrins alphaIIbbeta3, alphaMbeta2 and alphaVbeta3. It has also been reported that a peptide derived from the alphaMbeta2-binding site of gammaC can suppress an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Here we have truncated gammaC at position 399 to remove the prothrombotic alphaIIbbeta3-binding site. We show that this truncated version of gammaC, termed gammaC399tr, can bind to activated T cells. In addition, T cells incubated with gammaC399tr secreted less IFN-gamma when stimulated with antigen and APC; however, cytokine secretion was unaltered when T cells were stimulated non-specifically with a mixture of anti-CD3 and anti-CD28 antibodies. Thus, only antigen-dependent T cell activation is inhibited by gammaC399tr. When administered intraperitoneally, gammaC399tr potently inhibited actively induced EAE and reversed ongoing disease. We hypothesize that the ability of gammaC399tr to inhibit autoreactive immune responses is a result of its ability to bind integrins. This activity was not solely dependent on the alphaMbeta2 integrin-binding site. When polyalanine was substituted for the alphaMbeta2-binding site, the resulting gammaC390polyA was still able to inhibit EAE. To our knowledge, this is the first demonstration that T cells can bind to fibrin (ogen), an important extracellular matrix protein that is deposited at sites of inflammation. Our results also identify gammaC399tr as a novel therapeutic molecule.

Object Apoptosis is implicated in vasospasm and long-term sequelae of subarachnoid hemorrhage (SAH). The authors observed that 17β-estradiol (E2) can attenuate cerebral vasospasm, lower endothelin-1 production, and preserve normal endothelial nitric oxide synthase expression by reduction of inducible NO synthase expression in experimental SAH. The authors investigated the potential antiapoptotic effects of E2 in an experimental rat model of SAH. Methods The authors examined the antiapoptotic effects of E2 in a double-hemorrhage SAH model in male Sprague-Dawley rats. The rats underwent subcutaneous implantation of a Silastic tube containing corn oil either with or without E2, and some E2-treated animals also received ICI 182,780 (a nonselective estrogen receptor [ER] antagonist) for 7 days after SAH. The degree of vasospasm was determined by averaging the cross-sectional areas of the basilar artery 7 days after SAH. The expression of apoptotic indicators, including TNF-α, caspase 3, Bcl-2, Bax, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL), and cell death assays were used for detection of apoptosis. Results Treatment with E2 significantly attenuated SAH-induced vasospasm. Seven days after the induction of SAH, positive TUNEL-staining was seen, and DNA fragmentation was increased in the dentate gyrus. Increased TNF-α and cleaved caspase-3 protein expression and decreased Bcl-2 protein expression in the dentate gyrus were also observed. These changes were reversed with E2-treatment but not in the presence of ICI 182,780. However, the expression of Bax did not change after SAH either with or without E2 treatment. Conclusions The authors found that E2 appears to confer an antiapoptotic effect that reduces secondary brain injury after SAH via estrogen receptor–dependent mechanisms. This finding provides support for possible future applications of E2 treatment for the reduction of secondary injury after SAH in patients.

Cerebral vasospasm remains a major complication in patients who have suffered a subarachnoid hemorrhage (SAH). Previous studies have shown that 17beta-estradiol (E2) attenuates experimental SAH-induced cerebral vasospasm. Moreover, E2 has been shown to reduce neuronal apoptosis and secondary injury following cerebral ischemia. Adenosine A1 receptor (AR-A1) expression is increased following ischemia and may represent an endogenous neuroprotective effect. This study was designed to evaluate the efficacy of E2 in preventing cerebral vasospasm and reducing secondary injury, as evidenced by DNA fragmentation and AR-A1 expression, following SAH.A double-hemorrhage model of SAH in rats was used, and the degree of vasospasm was determined by averaging the cross-sectional areas of the basilar artery 7 days after the first SAH. A cell death assay was used to detect apoptosis. Changes in the protein expression of AR-A1 in the cerebral cortex, hippocampus, and dentate gyrus were compared with levels in normal controls and E2-treated groups (subcutaneous E2, 0.3 mg/ml).The administration of E2 prevented vasospasm (p0.05). Seven days after the first SAH, DNA fragmentation and protein levels of AR-A1 were significantly increased in the dentate gyrus. The E2 treatment decreased DNA fragmentation and prevented the increase in AR-A1 expression in the dentate gyrus. There were no significant changes in DNA fragmentation and the expression of AR-A1 after SAH in the cerebral cortex and hippocampus in the animals in the control and E2-treated groups.The E2 was effective in attenuating SAH-induced cerebral vasospasm, decreasing apoptosis in the dentate gyrus, and reducing the expression of AR-A1 in the dentate gyrus after SAH. Interestingly, E2 appears to effectively prevent cerebral vasospasm subsequent to SAH as well as attenuate secondary injury by reducing both apoptosis and a compensatory increase in AR-A1 expression in the dentate gyrus.

Background Our previous study showed that 17β-estradiol (E2) and an adenosine A2A receptor (AR-A2A) agonist could attenuate subarachnoid hemorrhage (SAH)-induced cerebral vasospasm via preventing the augmentation of iNOS expression and preserving the normal eNOS expression. This study tests the hypothesis that E2 attenuates SAH-induced vasospasm and apoptosis by activating adenosine AR-A2A and extracellular signal-regulated kinase 1 and 2 (ERK1/2), and by altering antiapoptotic and proapoptotic protein expression (Bcl-2 and Bax, respectively). Materials and Methods The two-hemorrhage SAH model in rat was used. Animals were treated with E2 with or without a nonselective estrogen receptor (ER) antagonist (ICI182,780). The cross sectional areas of the basilar artery and terminal dUTP nick-end labeling (TUNEL) were used to determine the degree of vasospasm and apoptosis, respectively. The expressions of Bcl-2, Bax, AR-A2A, and ERK1/2 in the cerebral cortex, hippocampus, and dentate gyrus were investigated. Results E2 significantly attenuated vasospasm. Seven days after the first SAH, TUNEL scores were significantly increased, and protein levels of AR-A2A, ERK1/2, and Bcl-2 were significantly decreased in the dentate gyrus only but not in the cortex and hippocampus. These changes were reversed by E2 while ICI182,780 abrogated the antiapoptotic and anti-spastic effects of E2. The expression of Bax did not change in the dentate gyrus after SAH with or without treatment. Conclusions The down-regulated AR-A2A and ERK may play a role in vasospasm and apoptosis after SAH. The beneficial effect of E2 in the attenuating SAH-induced vasospasm and apoptosis may be due to an increased expression of AR-A2A and ERK via ER-dependent mechanisms. These data may support further investigation of E2 in the treatment of SAH in humans.

This study investigated whether the surface quantities of the additives and protein delivery characteristics for protein-loaded PLGA 85 (85:15), PLGA75 (75:25) and PLGA 50 (50:50) microspheres with hydrophobic additives, phosphatidylcholine (PC) as a model, are affected by the different ratios of lactide and glycolide in PLGA polymers. By applying phospholipids analysis, surface PC quantities of the microspheres are 2.1 +/- 1.2, 1.2 +/- 0.3 and 1.0 +/- 0.7 Pg (n = 3) per mg of PLGA 85, PLGA 75 and PLGA 50 microspheres with PC addition, respectively, that are affected by the ratio of lactide and glycolide in PLGA. The addition of PC causes the decreased encapsulation efficiency (E.E.) of albumin of the microspheres and the decreased percentages of E.E. are negative correspondent to ratio of lactide and glycolide in PLGA (e.g. -13.5, -20.2 and -24.5% for PLGA 85, PLGA 75 and PLGA 50, respectively) compared with those without addition. The cumulative releases of albumin were influenced by the ratio of lactide and glycolide in PLGA for both PC addition and non-addition microspheres. Moreover, the additions of PC reduce the initial burst and cumulative releases of albumin of the microspheres compared with those of non-added ones. Further investigation suggests that resistant water uptake of PC added microspheres may play a role on affecting those reduced protein release behaviours. In conclusion, the different ratios of lactide and glycolide in PLGA affect the surface quantities of PC, E.E. and cumulative release profiles of albumin for the PLGA microspheres with PC addition. The results of the study may help the better practical applications for protein delivery of the microspheres with hydrophobic additives.

A depot drug delivery system, fibrin encapsulated liposome-in-chitosan matrix (FLCM), has been developed to deliver a water-soluble drug which is configured by a porous chitosan matrix containing a bovine fibrin network encapsulated different surface properties of liposomes. Quinacrine (QR), a water-soluble, low-molecular weight fluorescent marker, is used as a model drug to evaluate the delivery characteristics of the system. The SEM photographs show that the fibrin network adheres to the surfaces and pores of the chitosan matrix of a FLCM system. The QR release periods of the FLCM are sustained for about four times longer than those of QR encapsulated into the liposomes. However, the QR release periods and profiles of the FLCM are influenced by the surface properties of liposomes. The release of QR from FLCM is sustained for 9 days for neutral liposomes and only 5 days for PEG modified liposomes (PEG-liposome). After crosslinking the fibrin network of the FLCM with 0.5% of glutaldehyde, the release of QR is further sustained for 17 days with good linear profiles (e.g., 13 days) and with 50% of reduced burst release compared with those of without crosslinking, indicating that the stability of the fibrin network plays an important role on QR release of the system. More interestingly, the release periods and profiles of QR of the FLCM system are highly similar to those of Tirofiban, low-molecular weight of a water-soluble clinical cardiovascular drug, although the study has been done by human platelet poor plasma instead of bovine fibrinogen as a source of fibrin network. It suggests that the QR is a suitable model for investigating the drug delivery behaviors for water-soluble, low-molecular weight drugs of the FLCM. In conclusion, with QR as a model drug, FLCM with crosslinked fibrin network can effectively sustain the release of QR for 17 days but the release profiles are influenced by the surface properties of encapsulated liposomes. This study suggests that FLCM may have the potential as a depot drug delivery system for water-soluble drugs.

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.

Early in infection, the mouse hepatitis virus (MHV) envelope glycoprotein spike (S) binds a cell surface receptor glycoprotein and is postulated to undergo a conformational change to reveal a hydrophobic domain, promoting the fusion of the viral envelope and the cell membrane. The actual steps of MHV entry and their effects on pathogenesis are largely unknown. By binding truncated S1 peptides to receptor glycoproteins blotted on membranes, the putative receptor binding domain (RBD) was mapped to the N-terminal 330 amino acids of S (Kubo et al., 1994). In this study, we have generated two groups of chimeric recombinant viruses in an isogenic background in order to study the role of the RBD in pathogenesis.

We have identified a cDNA encoding an isoform of the 116-kDa subunit of the bovine vacuolar proton translocating ATPase. The predicted protein sequence of the new isoform, designated a2, consists of 854 amino acids with a calculated molecular mass of 98,010 Da; it has approximately 50% identity to the original isoform (a1) we described (Peng, S.-B., Crider, B. P., Xie, X.-S., and Stone, D.K. (1994) J. Biol. Chem. 269, 17262-17266). Sequence comparison indicates that the a2 isoform is the bovine homologue of a 116-kDa polypeptide identified in mouse as an immune regulatory factor (Lee, C.-K., Ghoshal, K., and Beaman, K.D. (1990) Mol. Immunol. 27, 1137-1144). The bovine a1 and a2 isoforms share strikingly similar structures with hydrophilic amino-terminal halves that are composed of more than 30% charged residues and hydrophobic carboxyl-terminal halves that contain 6-8 transmembrane regions. Northern blot analysis demonstrates that isoform a2 is highly expressed in lung, kidney, and spleen. To determine the possible role of the a2 isoform in vacuolar proton pump function, we purified from bovine lung a vacuolar pump proton channel (VO) containing isoform a2. This VO conducts bafilomycin-sensitive proton flow after reconstitution and acid activation, and supports proton pumping activity after assembly with the catalytic sector (V1) of vacuolar-type proton translocating ATPase (V-ATPase) and sub-58-kDa doublet, a 50-57-kDa polypeptide heterodimer required for V-ATPase function. These data indicate that the a2 isoform of the 116-kDa polypeptide functions as part of the proton channel of V-ATPases.

The clathrin-coated vesicle H+-ATPase is composed of a peripheral catalytic sector (VC) and an integral membrane proton channel (VB), both of which are multiple subunit complexes. This study was conducted to determine if subunit F, previously identified in vacuolar proton pumps of tobacco hornworm and yeast, was present in mammalian pumps. Using a polymerase chain reaction-based strategy, we have isolated and sequenced cDNA clones from bovine and rat brain cDNA libraries. A full-length clone from rat brain encodes a 119-amino acid polypeptide with a predicted molecular mass of 13, 370 Da and with approximately 72 and 49% identity to subunit F of tobacco hornworm and yeast, respectively. Southern and Northern blot analyses indicate that the protein is encoded by a single gene. An anti-peptide antibody, directed against deduced protein sequence, was affinity-purified and shown to react with a 14-kDa polypeptide that is present in a highly purified pump prepared from clathrin-coated vesicles and also isolated VC. When stripped clathrin-coated vacuolars and purified chromaffin granule membranes were treated with KI in the presence of ATP, the 14-kDa subunit was released from both membranes, further indicating that it is part of the peripheral catalytic sector. In addition, direct sequencing of this 14-kDa component of the coated vacuolar proton pump confirmed its identity as a subunit F homologue.

The registration of shapes in two images appears as an indispensable step in many applications such as analysis of dynamic scenes involving shape matching. In this paper, we compute the corresponding shape specific points, which are invariant in rotation, scale, and translation, between two misaligned shapes by morphological operations. We find that erosion is enough to determine the specific points between two nontrivial binary shapes to be registered.

We have recently reported the reconstitution of the clathrin-coated vesicle proton-translocating complex with liposomes prepared from ethanol-extracted crude bovine brain lipids. Reconstitution of proton pumping by the isolated proton ATPase has now been achieved with liposomes prepared from pure lipids. Optimal proton pumping, as assessed by ATP-generated acridine orange quenching and 32Pi-ATP exchange, is achieved with liposomes prepared from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol at a weight ratio of 40:26.5:7.5:26, and a lipid-to-protein weight ratio of 200:1. Under such conditions, the extent of decrease of ATP-generated acridine orange quenching is 150-fold greater than that of native clathrin-coated vesicles. Cholesterol is required to stabilize the proteoliposome. Phosphatidylserine, which is most effective in activating ATP-hydrolytic activity of the solubilized enzyme, is not obligatory for reconstitution of proton pumping.

Toona sinensis (TS) leaves are used as a vegetable and in traditional Chinese medicine. However, in vivo experiments regarding the anti-inflammatory function of TS leaves have not previously been conducted. The aim of this study was to investigate the potential role of TS leaf extract (TSL) in the prevention of sepsis-induced lung injury in vivo and on macrophage activation in vitro. The results showed that oral gavage pretreatment with TSL in rats for 30 days improved the survival of cecal ligation and puncture-induced sepsis, potentially by attenuating sepsis-induced histological lung damage rather than inflammatory cell infiltration. Furthermore, we demonstrated that pretreatment with TSL attenuated the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase, thereby inhibiting nitric oxide production and release in murine macrophage-like RAW 264.7 cells. Interestingly, TSL did not affect the LPS-induced release of other cytokines (e.g., tumor necrosis factor α and interleukin 1β) but increased LPS-induced heme-oxygenase-1 expression. In conclusion, the study provides preliminary data for TSL on cecal ligation and puncture-induced sepsis. The beneficial impact of TSL needs extensive study to get solid evidence.

Human endothelial cells in culture synthesize prostaglandins and release these products into the culture medium. The major products of arachidonic acid metabolism were identified by high pressure liquid chromatography or thin layer chromatography, and release of prostaglandins was measured by radioimmunoassays. Addition of histamine or bradykinin enhanced release of prostaglandins in both arterial and venous endothelial cells. Other vasoactive compunds including angiotensin II, vasopressin, substance P, epinephrine, norepinephrine, or isoproterenol were ineffective. Release of prostaglandins by histamine was concentration-related, and involved H 1 receptors, as determined by addition of histamine antagonists. Incubation of endothelial cells with C-arachidonic acid resulted in a time-dependent uptake into cell lipids, where most of the radioactivity was incorporated into phosphatidyl choline and neutral lipids. Endothelian cells released 14 C_arachidonic acid as well as 14 C-prostaglandins in response to either histamine or bradykinin. The enhanced release of 14 C-prostaglandins was inhibited by either indomethacin or mepacrine, but 14 C-arachidonic acid release was inhibited only by mepacrine. We conclude that the vasoactive compounds, histamine and bradykinin, stimulate formation of prostaglandins in endothelial cells by the release of arachidonic acid from phospholipids of the cell membrane.