11-Trans leukotriene C: A naturally occurring slow reacting substance

A slow reacting substance, produced by murine mastocytoma cells, has been shown to have the structure 5(S)-hydroxy-6(R)-$\text{S}$-glutathionyl-7,9,11-$\text{trans}\text{-14-}\text{cis}\text{-eicosatetraenoic acid (11-}\text{trans}$ leukotriene C, previously referred to as leukotriene C-2) by ultraviolet spectroscopy, amino acid analyses, lipoxygenase conversion and comparisions with a synthetic compound of known structure and stereochemistry.     

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.     

Immunohistochemical studies on the serotonergic innervation of the pia mater

Summary The direct innervation of the pial blood vessels by serotonin neurons has been demonstrated with a modified peroxidase-antiperoxidase technique in the mammalian central nervous system. The pia mater covering the ventrolateral surface of the medulla oblongata is innervated by numerous varicose serotonin fibers originating from the serotonin neurons of the lower brainstem. Scattered serotonin fibers were observed in the pia mater in every part of the brain and spinal cord.     

Conformational states of beta-lactamase: molten-globule states at acidic and alkaline pH with high salt

We present evidence that beta-lactamase is close to fully unfolded (i.e., random coil conformation) at low ionic strength at the extremes of pH and that the presence of salt causes a cooperative transition to a conformation with the properties of a molten globule, namely, a compact state with native-like secondary structure but disordered side chains (tertiary structure). The conformation of beta-lactamase I from Bacillus cereus was examined over the pH 1.5-12.5 region by circular dichroism (CD), tryptophan fluorescence, dynamic light scattering, and 1-anilino-8-naphthalenesulfonate (ANS) binding. Under conditions of low ionic strength (I = 0.05) beta-lactamase was unfolded below pH 2.5 and above pH 11.5, on the basis of the far-UV and near-UV CD and tryptophan fluorescence. However, at high ionic strength and low pH an intermediate conformation (state A) was observed, with a secondary structure content similar to that of the native protein but a largely disordered tertiary structure. The transition from the unfolded state (U) to state A induced by KCl was cooperative and had a midpoint at 0.12 M KCl (I = 0.17 M) at pH 1.6. A similar conformation (state B) was observed at high pH and high ionic strength. The transition from the alkaline U state to state B induced by KCl at pH 12.2 was cooperative and had a midpoint at 0.6 M KCl (I = 0.65 M). Light scattering measurements showed that state B was compact although somewhat expanded compared to the N state. The compactness of state A could not be determined due to its strong propensity to aggregate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin-3 is a novel neuropeptide: isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain

The molecular forms of endothelin (ET) related peptides were investigated in porcine brain by using high performance liquid chromatography coupled with three specific radioimmunoassays. ET-1 and its oxidized form were isolated and sequenced as in the case of porcine spinal cord. A very small amount of big ET-1 (1-39) and its C-terminal fragment (big ET-1 (22-39] were also detected. Furthermore, immunoreactive (ir)-ET-3 was isolated and sequenced; its partial primary structure was identical to that of human (rat) ET-3. The concentrations of ir-ET-1 and ir-ET-3 in porcine brain were 140 fmol/g tissue and 5 fmol/g tissue, respectively. These results indicate that besides ET-1, ET-3 is a novel neuropeptide in the central nervous system.     

Effects of 20 days horizontal bed rest on kinesthesia during knee flexion and two-point discrimination in skin of young subjects

Investigating the effect of 20 days bed rest (BR) on kinesthesia and two-point discrimination, 10 young volunteers participated in this study as subjects. Angle position sensation of right knee was measured in the prone position during flexion and extension monitored by a goniometer after two-point discrimination in skin was determined on the same lower leg. Flexed constant error was unchanged but directional constant error and absolute error were increased after BR (p<0.05). Two-point discrimination was unchanged after BR, which brought about a decline of the orientation of moving the joint indicated as over shooting of the angle during knee flexion, while it did not affect superficial sensation observed by two-point discrimination. Probably, an adjustment of deep sensation to the knee joint is lowered by the reduction in kinesthesia as well as the sensory disturbance during BR, which is independent on information from superficial sensation.     

The spinal antinociceptive effect of FR140423 is mediated through kyotorphin receptors

We investigated the antinociceptive effect of a novel anti-inflammatory and analgesic drug, 3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)phenyl]pyraz ole (FR140423), in the tail-pinch test in mice, and evaluated the mechanism of action of FR140423 using L-leucyl-L-arginine (Leu-Arg), a kyotorphin (endogenous Met-enkephalin releaser) receptor antagonist, L-NG-nitroarginine methylester (L-NAME), an inhibitor of nitric oxide (NO) synthase, and methylene blue (MB), an inhibitor of activation of guanylate cyclase. Oral administration of FR140423, at doses of 5-80 mg/kg, produced a dose-dependent antinociceptive effect with an ED50 value of 18 mg/kg. This antinociception was reversed by intrathecal (i.t.) (10 microg/mouse), but not by intracerebroventricular (i.c.v.) (100 microg/mouse), injection of Leu-Arg. Moreover, the antinociceptive effect of i.t. injection of FR140423 with an ED50 value of 3.7 microg/mouse was completely antagonized by co-administered Leu-Arg 10 microg/mouse. However, L-NAME (2000 mg/kg s.c.) and MB (200 mg/kg s.c.) did not antagonize the antinociception of FR140423. These findings suggest that FR140423 plays a role in nociceptive modulation in the spinal cord, being antinociceptive via the kyotorphin-Met-enkephalin pathway but not via the peripheral NO-cyclic GMP pathway.     

Heme oxygenase-2 is neuroprotective in cerebral ischemia

Heme oxygenase (HO) is believed to be a potent antioxidant enzyme in the nervous system; it degrades heme from heme-containing proteins, giving rise to carbon monoxide, iron, and biliverdin, which is rapidly reduced to bilirubin. The first identified isoform of the enzyme, HO1, is an inducible heat-shock protein expressed in high levels in peripheral organs and barely detectable under normal conditions in the brain, whereas HO2 is constitutive and most highly concentrated in the brain. Interestingly, although HO2 is constitutively expressed, its activity can be modulated by phosphorylation. We demonstrated that bilirubin, formed from HO2, is neuroprotectant, as neurotoxicity is augmented in neuronal cultures from mice with targeted deletion of HO2 (HO2(-/-)) and reversed by low concentrations of bilirubin. We now show that neural damage following middle cerebral artery occlusion (MCAO) and reperfusion, a model of focal ischemia of vascular stroke, is substantially worsened in HO2(-/-) animals. By contrast, stroke damage is not significantly altered in HO1(-/-) mice, despite their greater debility. Neural damage following intracranial injections of N-methyl-d-aspartate (NMDA) is also accentuated in HO2(-/-) animals. These findings establish HO2 as an endogenous neuroprotective system in the brain whose pharmacologic manipulation may have therapeutic relevance.     

An extremely acidic amino-terminal presequence of the precursor for the human mitochondrial hinge protein

Mitochondrial hinge protein is a subunit of ubiquinol-cytochrome-c reductase in the respiratory chain and 'hinges' cytochrome c with cytochrome c1. The protein is encoded in the nuclear genome, synthesized in the cytosol and then imported into the mitochondria. The cDNA of the human hinge protein has been cloned and its nucleotide sequence was determined. The deduced primary structure of the amino-terminal presequence consists of 13 amino acid residues, of which 4 amino acids are acidic and only one is basic. Since the presequences of most other precursors are rich in basic amino acids, this sequence is unique for targeting mitochondria. Expression of the gene was repressed in the presence of a phorbol ester in human promyelocyticleukemia cells (HL-60), and this repression was greater than that of the ADP/ATP translocator. These findings suggest that the hinge protein, the expression of which is well regulated, is imported into mitochondria via a specific pathway.