Crystallization and Preliminary X-Ray Analysis of Neuropsin, a Serine Protease Expressed in the Limbic System of Mouse Brain

Neuropsin (M_r25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that crystal form I belongs to triclinic space groupP1 with unit cell dimensionsa= 97.16 Å,b= 97.12 Å,c= 46.75 Å and α = 99.17°, β = 99.77°, γ = 117.35°. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space groupP1 but has unit cell dimensions ofa= 38.40 Å,b= 55.16 Å,c= 65.37 Å and α = 95.38°, β = 89.98°, γ = 110.46° with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis. Intensity data to 3.1 Å resolution for form I and to 2.2 Å resolution for form II have been collected.     

Changes in predominant energy substrate after hepatectomy

The changes in the energy substrate utilized by the remnant liver were studied in relation to the changes in the cellular energy status of 25 and 70% hepatectomized rabbits. In 25% hepatectomized rabbits, the energy charge $\text{(}\text{(}\text{ATP}\text{+0.5}\text{ADP}\text{)}\text{(}\text{ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}\text{)}$ level of the remnant liver remained unchanged, the energy substrate of which was predominantly glucose, rather than fatty acid. In contrast, in 70% hepatectomized rabbits, the energy production by the mitochondria was mainly dependent upon fatty acid oxidation at the early period after hepatectomy when the energy charge level decreased remarkably, and then upon glucose oxidation, concomitant with the restoration of the energy charge. It is suggested that the changes in the energy substrate utilized are closely related to those in the energy charge level and the mitochondrial phosphorylative activity of the remnant liver following hepatectomy.     

Isolated Bovine Spinal Motoneurons Have Specific Ganglioside Antigens Recognized by Sera from Patients with Motor Neuron Disease and Motor Neuropathy

The gangliosides GM1 and GD1b have recently been reported to be potential target antigens in human motor neuron disease (MND) or motor neuropathy. The mechanism for selective motoneuron and motor nerve impairment by the antibodies directed against these gangliosides, however, is not fully understood. We recently investigated the ganglioside composition of isolated bovine spinal motoneurons and found that the ganglioside pattern of the isolated motoneurons was extremely complex. GM1, GD1a, GD1b, and GT1b, which are major ganglioside components of CNS tissues, were only minor species in motoneurons. Among the various ganglioside species in motoneurons, several were immunoreactive to sera from patients with MND and motor neuropathy. One of these gangliosides was purified from bovine spinal cord and characterized as N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)] by compositional analysis, fast atom bombardment mass spectra, and the use of specific antibodies. Among seven sera with anti-GM1 antibody activities, five sera reacted with GM1(NeuGc) and two did not. Two other gangliosides, which were recognized by another patient's serum, appeared to be specific for motoneurons. We conclude that motoneurons contained, in addition to the known ganglioside antigens GM1 and GD1b, other specific ganglioside antigens that could be recognized by sera from patients with MND and motor neuropathy.     

Characteristics of surface mechanomyogram are dependent on development of fusion of motor units in humans.

The purpose of this study was to test whether surface mechanomyogram (MMG) recorded on the skin reflects the contractile properties of individual motor units in humans. Eight motor units in the medial gastrocnemius muscle were identified, and trains of stimulation at 5, 10, 15, and 20 Hz were delivered to each isolated motor unit. There was a significant positive correlation between the duration of MMG and twitch duration. MMG amplitude decreased with increasing stimulation frequency. Reductions in MMG amplitude were in parallel with the reductions in force fluctuations, and the rate of change in both was positively correlated across the motor units. Rate of change in MMG amplitude against force was negatively correlated to half relaxation time and twitch duration. Similar negative correlations were found between force fluctuations and contractile properties. These results provide evidence supporting a direct relation between MMG and contractile properties of individual motor units within the gastrocnemius muscle, indicating that surface MMG is dependent on the contractile properties of the activated motor units in humans.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

Changes in contractile properties and neuromuscular propagation evaluated by simultaneous mechanomyogram and electromyogram during experimentally induced hypothermia.

The present study examined, whether or not mechanomyogram (MMG) amplitude and frequency component could reflect the contractile properties of the triceps surae muscles, composed of relatively slow soleus (SOL) and fast medial gastrocnemius (MG), during experimentally induced hypothermia condition. In eight male subjects, lying in prone position, supramaximal single twitch and repetitive electrical stimulations at 10 Hz were applied at the intramuscular temperatures of control (34 degrees C), 15, 20, and 25 degrees C, respectively. The hypothermia induced substantial reduction in muscle contractile properties, e.g. prolonged twitch contraction and half relaxation times, resulted in a highly significant reduction in the fluctuation of force signal during the repetitive stimulations. These changes were almost mirrored by the similar and significant reductions in the MMG amplitude in both SOL and MG. Power spectrum analysis revealed that peak frequency components of MMG and fluctuation of force were almost matched with the applied stimulation frequencies, independent of the temperature condition. These results strongly suggest that MMG analysis could be employed to study muscle contractile properties varying across different physiological conditions.     

TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.