Molecular cloning and sequence analysis of full-length cDNAs encoding new group of Cyn d 1 isoallergens

BACKGROUND: Cyn d 1, the major allergen of Bermuda grass pollen, contains some acidic/basic isoforms. The N-terminal amino acid sequences of some acidic Cyn d 1 isoforms were found to be different from those of Cyn d 1 cDNA clones identified previously. METHODS: A predicted 17-meric oligonucleotide probe was designed to fish the unidentified isoallergen cDNAs out of BGP cDNA library. The reactive clones were isolated and verified by sequencing. Two of them were expressed in the yeast Pichia pastoris to obtain recombinant Cyn d 1 proteins. RESULTS: All four cDNA clones encode the full-length Cyn d 1 with mature proteins of 244 amino acid residues. A 97-99% identity was found among the deduced amino acids of these four clones while an 86% identity was elicited between the four clones and the ones previously identified. The predicted isoelectric focusing (pI) values of the newly identified Cyn d 1s are acidic while pIs of the previously identified Cyn d 1s are basic. The two recombinant acidic Cyn d 1 proteins possess the epitopes recognized by mouse and rabbit polyclonal anti-Cyn d 1 antibodies, and have human IgE-binding capacity as revealed by immunodot assay. CONCLUSIONS: The present study identified full-length cDNAs encoding new isoallergens of Cyn d 1, and separated Cyn d 1 gene into an acidic group and a basic group.     

Fluoxetine and 8-OH-DPAT in the lateral septum enhances and impairs retention of an inhibitory avoidance response in rats.

The present study investigated the role of lateral septal serotonin (5HT) in memory consolidation and the subtype of 5HT receptors involved in this process. Rats with cannulae implanted bilaterally into the lateral septum were trained in an inhibitory avoidance task. Immediately after training, the septal serotonergic function was manipulated by pharmacological agents selectively blocking 5HT reuptake (fluoxetine and zimelidine), antagonizing 5HT2 receptors (ketanserin and ritanserin), or activating 5HT1A receptors, respectively. Results indicated that direct fluoxetine infusions into the lateral septum at a dose of 6 micrograms/0.5 microliter and zimelidine at a dose of 5 micrograms/0.5 microliter both markedly enhanced memory. Intralateral septal injections of ketanserin (0.3 microgram/0.5 microliter and 0.5 microgram/0.5 microliter) and ritanserin (0.3 microgram/0.5 microliter and 0.6 microgram/0.5 microliter) did not have a significant effect by themselves on memory, and neither did they attenuate the memory-facilitating effect of fluoxetine in the same area. Intralateral septal infusions of 8-hydroxy-2-(di-n-propylamino)tetralin at 5 micrograms/0.5 microliter significantly impaired memory retention. These findings altogether support the notion that the lateral septal nuclei of rats are involved in the memory processes of inhibitory avoidance learning. Furthermore, postsynaptic 5HT receptor activation (not the 5HT2 receptor subtype) probably exerts a facilitatory effect while presynaptic 5HT1A receptor activation exerts an impairing effect on the memory consolidation process, probably due to autoreceptor inhibition of 5HT release.     

E(f)-current contributes to whole-cell calcium current in low calcium in frog sympathetic neurons

Because Ca(2+) plays diverse roles in intracellular signaling in neurons, several types of calcium channels are employed to control Ca(2+) influx in these cells. Our experiments focus on resolving the paradox of why whole-cell current has not been observed under typical recording conditions for one type of calcium channel that is highly expressed in frog sympathetic neurons. These channels, referred to as E(f)-channels, are present in the membrane at a density greater than the channels that carry approximately 90% of whole-cell current in low Ba(2+); but, E(f)-current has not been detected in low Ba(2+). Using Ca(2+) instead of Ba(2+) as the charge carrier, we recorded a possible E-type current in frog sympathetic neurons. The current was resistant to specific blockers of N-, L-, and P/Q-type calcium channels but was more sensitive to Ni(2+) block than was N- or L-current. Current amplitude in Ca(2+) is slightly greater than that in Ba(2+). In 3 mM Ca(2+), the current contributed approximately 12% of total current at peak voltage and increased at voltages more hyperpolarized to the peak, reaching approximately 40% at -30 mV, where whole-cell current starts to activate. The presence of E(f)-current in 3 mM Ca(2+) suggests a potential role for E(f)-channels in regulating calcium influx into sympathetic neurons.     

Detection and deletion of motion artifacts in electrogastrogram using feature analysis and neural networks

Electrogastrogram is a surface measurement of gastric myoelectrical activity, and electrogastrography has been an attractive method for physiological and pathophysiological studies of the stomach due to its nonivasive nature. Motion artifacts, however, ruin the electrogastrogram (EGG), and make the analysis very difficult and sometimes even impossible. They must be eliminated from EGG signals before analysis. Up to now, this can only be done by visual inspection, which is not only time-consuming but also subjective. In this study, a method using feature analysis and neural networks has been developed to realize automatic detection and elimination of the motion artifacts in EGG recordings by computer. Experiments were conducted to investigate the characteristics of different motion artifacts. Useful features were extracted, and different combinations of the features used as the input of the neural network were compared to obtain the optimal performance for the detection of motion artifacts using the artificial neural network.     

Changes in HIV-1 incidence in heroin users in Guangxi Province, China

Guangxi Province, China recently experienced an outbreak of HIV-1 infection among heroin users. We studied HIV-1 incidence rates and associated risk factors for HIV-1 infection among heroin users residing in Pingxiang City. A total of 318 heroin users were followed from February 1998 through January 1999 (median follow-up: 8.1 months). Of these, 130 were prospectively followed from January through September 1999 (median follow-up: 8.3 months). HIV-1 and hepatitis C virus (HCV) incidence rates for each period were calculated. A generalized estimating equation approach was implemented to identify independent risk factors associated with HIV-1 infection across both periods. Among 318 study participants, 97.2% were men. The median age was 22 years. Approximately 60% reported sharing needles. HIV-1 prevalence at baseline was 15.4%. During the first follow-up period, HIV-1 incidence was 2.38 per 100 person years (py), and HCV incidence was 26.8 per 100 py. During the second follow-up period, HIV-1 incidence was 6.86 per 100 py, and HCV incidence was 28.9 per 100 py. After controlling for age and other factors, HCV seropositivity, history of sexually transmitted diseases, and sharing needles were independently associated with HIV-1 infection. These data suggest that HIV-1 incidence was rising over time in Pingxiang City, Guangxi Province. The high incidence of HCV heightens the importance of enhanced prevention programs to reduce injection and needle sharing among heroin users.     

Molecular and biochemical mechanisms ofPasteurella haemolyticaleukotoxin-induced cell death

Pasteurella haemolyticaleukotoxin (LKT) is a member of the RTX family of pore-forming toxins that kill bovine immune cells. Several studies have suggested that RTX toxins kill target cells by the induction of apoptosis. In the present study, BL3 bovine leukaemia cells were exposed to LKT and assessed by molecular and flow cytometric techniques that measure different aspects of apoptotic cell death. The intoxicated cells demonstrated morphological, light scatter and Hoechst 33258 staining characteristics consistent with cells undergoing apoptosis. The cells also exhibited internucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, both indicators of apoptosis. LKT-treated cells bound annexin-V-FITC indicating that phosphatidylserine groups were translocated from the inner to the outer leaflet of the cell membrane. The effect of LKT on cells was dose dependent and inhibitable by incubation with anti-LKT monoclonal antibody. Finally, an early step for induction of apoptosis appears to be the binding of LKT to a β2 integrin since pre-incubating cells with anti-β2 integrin antibodies inhibited LKT-induced apoptosis. This study provides new insights into understanding the pathogenesis of bovine pasteurellosis and could lead to the development of both preventative and therapeutic strategies for disease management.     

Lack of FasL expression in cultured human retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from tissue sections, were positive for FasL, indicating a discrepancy between RPE cells in vitro and in vivo with regard to this molecule.