Pigment-dispersing factor sets the night state of the medulla bilateral neurons in the optic lobe of the cricket,Gryllus bimaculatus

Pigment-dispersing factor (PDF) is an octadeca-neuropeptide widely distributed in the insect brain and suggested to be involved in the insect circadian systems. We have examined its effects on the neuronal activity of the brain efferents in the optic stalk including medulla bilateral neurons (MBNs) in the cricket, Gryllus bimaculatus. The MBNs are visually responding interneurons connecting the bilateral medulla, which show a clear day/night change in their light responsiveness that is greater during the night. Microinjection of PDF into the optic lobe induced a significant increase in the spontaneous activity of the brain efferents and the photo-responsiveness of the MBNs during the day, while little change was induced during the night. The enhancing effects began to occur about 20 min after the injection and another 10 min was necessary to reach the maximal level. The effects of PDF were dose-dependent. When 22 nl of anti-Gryllus-PDF (1:200) IgG was injected into the medulla, the photo-responsiveness of the MBNs was suppressed in both the day and the night with greater magnitude during the night. No significant suppression was induced by injection of the same amount of IgG from normal rabbit serum. These results suggest that in the cricket optic lobe, PDF is released during the night and enhances MBNs' photo-responsiveness to set their night state.     

Effects of 5,7-DHT Injection into the Optic Lobe on the Circadian Locomotor Rhythm in the Cricket, Gryllus bimaculatus

The effect of direct 5,7-dihydroxytryptamine (5,7-DHT) injection into the medulla region of the optic lobe on the locomotor activity was investigated in the adult male cricket, Gryllus bimaculatus. After a 6 hr phase advance of a light-dark cycle, the 5,7-DHT injected animals needed significantly longer time for resynchronization to the new cycle (6.55 +/- 0.62 days) than the control, Ringer's solution injected animals (3.17 +/- 0.15 days; P < 0.001, t-test). Light induced a bout of activity (i.e., masking effect) when light-dark cycle was phase advanced by 6 hr and the duration of the masking effect was significantly longer in 5,7-DHT injected animals. An initial bout of the nocturnal activity was significantly greater in the 5,7-DHT injected animal. Under constant darkness, the freerunning periods of both groups were not significantly different. Under constant light, a significantly higher percentage of 5,7-DHT injected animals showed arrhythmicity compared with the control group. An analysis carried by high-pressure liquid chromatography with electro-chemical detection (HPLC-ECD) revealed that the serotonin content in the optic lobe was significantly reduced to less than 50% in the 5,7-DHT injected animals, even one month after the injection. These results suggest that serotonin plays important roles in the regulation of circadian locomotor rhythms of the cricket mainly by regulating the sensitivity of the photoreceptive system.     

Light and temperature cooperate to regulate the circadian locomotor rhythm of wild type and period mutants of Drosophila melanogaster

In the wild type (Canton-S) and period mutant flies of Drosophila melanogaster, we examined the effects of light and temperature on the circadian locomotor rhythm. Under light dark cycles, the wild type and per(S) flies were diurnal at 25 degrees C. However, at 30 degrees C, the daytime activity commonly decreased to form a rather nocturnal pattern, and ultradian rhythms of a 2 approximately 4h period were observed more frequently than at 25 degrees C. The change in activity pattern was more clearly observed in per(0) flies, suggesting that these temperature dependent changes in activity pattern are mainly attributable to the system other than the circadian clock. In a 12h 30 degrees C:12h 25 degrees C temperature cycle (HTLT12:12), per(0) flies were active during the thermophase in constant darkness (DD) but during the cryophase in constant light (LL). The results of experiments with per(0);eya flies suggest that the compound eye is the main source of the photic information for this reversal. Wild type and per(0) flies were synchronized to HTLT12:12 both under LL and DD, while per(S) and per(L) flies were synchronized only in LL. This suggests that the circadian clock is entrainable to the temperature cycle, but the entrainability is reduced in the per(S) and per(L) flies to this particular thermoperiod length, and that temperature cycle forces the clock to move in LL, where the rhythm is believed to be stopped at constant temperature.     

In vivo transduction of central neurons using recombinant Sindbis virus: Golgi-like labeling of dendrites and axons with membrane-targeted fluorescent proteins.

A new recombinant virus which labeled the infected neurons in a Golgi stain-like fashion was developed. The virus was based on a replication-defective Sindbis virus and was designed to express green fluorescent protein with a palmitoylation signal (palGFP). When the virus was injected into the ventrobasal thalamic nuclei, many neurons were visualized with the fluorescence of palGFP in the injection site. The labeling was enhanced by immunocytochemical staining with an antibody to green fluorescent protein to show the entire configuration of the dendrites. Thalamocortical axons of the infected neurons were also intensely immunostained in the somatosensory cortex. In contrast to palGFP, when DsRed with the same palmitoylation signal (palDsRed) was introduced into neurons with the Sindbis virus, palDsRed neither visualized the infected neurons in a Golgi stain-like manner nor stained projecting axons in the cerebral cortex. The palDsRed appeared to be aggregated or accumulated in some organelles in the infected neurons. Anterograde labeling with palGFP Sindbis virus was very intense, not only in thalamocortical neurons but also in callosal, striatonigral, and nigrostriatal neurons. Occasionally there were retrogradely labeled neurons that showed Golgi stain-like images. These results indicate that palGFP Sindbis virus can be used as an excellent anterograde tracer in the central nervous system.     

Increased uncoupling protein-2 and -3 gene expressions in skeletal muscle of STZ-induced diabetic rats

Streptozotocin (STZ)-induced diabetic animals are vulnerable to cold stress. Uncoupling proteins (UCPs) play an important role in regulating thermogenesis. We investigated the gene expressions of UCPs in brown adipose tissue (BAT), white adipose tissue (WAT), liver and gastrocnemius muscle of STZ-diabetic rats using Northern blot. UCP-1, -2 and -3 mRNA expressions in BAT were all remarkably lower in STZ-diabetic rats than those in control rats. Both UCP-2 and -3 gene expressions in gastrocnemius muscle were substantially elevated in STZ-diabetic rats and insulin treatment restored UCP gene expressions to normal levels. These results suggest that in STZ-diabetic rats, the overexpression of UCP-2 and UCP-3 in skeletal muscle provides a defense against hypothermogenesis caused by decreased UCPs in BAT.     

Mycobacteriocins produced by rapidly growing mycobacteria are Tween-hydrolyzing esterases.

Smegmatocin, a protein produced by Mycobacterium smegmatis ATCC 14468, was found to have an esterase activity, hydrolyzing Tween 80, polyoxyethylene sorbitan monooleate, added to the assay medium for various "bacteriocins" from mycobacteria. Because M. diernhoferi ATCC 19340 (indicator strain for smegmatocin) is highly susceptible to oleic acid and smegmatocin requires Tween 80 for manifestation of its anti-M. diernhoferi activity, it is likely that smegmatocin-mediated antimicrobial action is caused by oleic acid generated by hydrolysis of Tween 80 by the inherent esterase action of smegmatocin. Other mycobacteriocins from rapidly growing mycobacteria also have inherent esterase activity against Tween 80 and require Tween 80 for expression of antimycobacterial action. Smegmatocin was found to hydrolyze various polyoxyethylene (sorbitan) fatty acyl esters but not sorbitan monooleate and glyceryl esters.     

An alpha-L-arabinofuranosidase from Trichoderma reesei containing a noncatalytic xylan-binding domain.

L-Sorbose, an excellent cellulase and xylanase inducer from Trichoderma reesei PC-3-7, also induced alpha-L-arabinofuranosidase (alpha-AF) activity. An alpha-AF induced by L-sorbose was purified to homogeneity, and its molecular mass was revealed to be 35 kDa (AF35), which was not consistent with that of the previously reported alpha-AF. Another species, with a molecular mass of 53 kDa (AF53), which is identical to that of the reported alpha-AF, was obtained by a different purification procedure. Acid treatment of the ammonium sulfate-precipitated fraction at pH 3.0 in the purification steps or pepsin treatment of the purified AF53 reduced the molecular mass to 35 kDa. Both purified enzymes have the same enzymological properties, such as pH and temperature effects on activity and kinetic parameters for p-nitrophenyl-alpha-L-arabinofuranoside (pNPA). Moreover, the N-terminal amino acid sequences of these enzymes were identical with that of the reported alpha-AF. Therefore, it is obvious that AF35 results from the proteolytic cleavage of the C-terminal region of AF53. Although AF35 and AF53 showed the same catalytic constant with pNPA, the former showed drastically reduced specific activity against oat spelt xylan compared to the latter. Furthermore, AF53 was bound to xylan rather than to crystalline cellulose (Avicel), but AF35 could not be bound to any of the glycans. These results suggest that AF53 is a modular glycanase, which consists of an N-terminal catalytic domain and a C-terminal noncatalytic xylan-binding domain.     

Ultrastructure of orexin-1 receptor immunoreactivities in the spinal cord dorsal horn

The ultrastructural properties of orexin 1-receptor-like immunoreactive (OX1R-LI) neurons in the dorsal horn of the rat spinal cord were examined using light and electron microscopy techniques. At the light microscopy level, the most heavily immunostained OX1R-LI neurons were found in the ventral horn of the spinal cord, while some immunostained profiles, including nerve fibers and small neurons, were also found in the dorsal horn. At the electron microscopy level, OX1R-LI perikarya were identified containing numerous dense-cored vesicles which were more heavily immunostained than any other organelles. Similar vesicles were also found within the axon terminals of the OX1R-LI neurons. The perikarya and dendrites of some of the OX1R-LI neurons could be seen receiving synapses from immunonegative axon terminals. These synapses were found mostly asymmetric in shape. Occasionally, some OX1R-LI axon terminals were found making synapses on dendrites that were OX1R-LI in some cases and immunonegative in others. The synapses made by OX1R-LI axon terminals were found both asymmetric and symmetric in appearance. The results provide solid morphological evidence that OX1R is transported in the dense-cored vesicles from the perikarya to axon terminals and that OX1R-LI neurons in the dorsal horn of the spinal cord have complex synaptic relationships both with other OX1R-LI neurons as well as other neuron types.     

Role of ATP-sensitive K+ channels in electrophysiological alterations during myocardial ischemia: a study using Kir6.2-null mice

The role of cardiac ATP-sensitive K(+) (K(ATP)) channels in ischemia-induced electrophysiological alterations has not been thoroughly established. Using mice with homozygous knockout (KO) of Kir6.2 (a pore-forming subunit of cardiac K(ATP) channel) gene, we investigated the potential contribution of K(ATP) channels to electrophysiological alterations and extracellular K(+) accumulation during myocardial ischemia. Coronary-perfused mouse left ventricular muscles were stimulated at 5 Hz and subjected to no-flow ischemia. Transmembrane potential and extracellular K(+) concentration ([K(+)](o)) were measured by using conventional and K(+)-selective microelectrodes, respectively. In wild-type (WT) hearts, action potential duration (APD) at 90% repolarization (APD(90)) was significantly decreased by 70.1 +/- 5.2% after 10 min of ischemia (n = 6, P < 0.05). Such ischemia-induced shortening of APD(90) did not occur in Kir6.2-deficient (Kir6.2 KO) hearts. Resting membrane potential in WT and Kir6.2 KO hearts similarly decreased by 16.8 +/- 5.6 (n = 7, P < 0.05) and 15.0 +/- 1.7 (n = 6, P < 0.05) mV, respectively. The [K(+)](o) in WT hearts increased within the first 5 min of ischemia by 6.9 +/- 2.5 mM (n = 6, P < 0.05) and then reached a plateau. However, the extracellular K(+) accumulation similarly occurred in Kir6.2 KO hearts and the degree of [K(+)](o) increase was comparable to that in WT hearts (by 7.0 +/- 1.7 mM, n = 6, P < 0.05). In Kir6.2 KO hearts, time-dependent slowing of conduction was more pronounced compared with WT hearts. In conclusion, the present study using Kir6.2 KO hearts provides evidence that the activation of K(ATP) channels contributes to the shortening of APD, whereas it is not the primary cause of extracellular K(+) accumulation during early myocardial ischemia.