Antennal movements induced by odour and central projection of the antennal neurones in the honey-bee

Movements of the antennae induced by odour were investigated. Odour presented to the antenna of one side induced both antennae to move to that side. The EMGs recorded from the flexor muscles of both scapes showed that the latency of the movement of the ipsilateral flagellum when induced by odour was about 71 msec shorter than that of the contralateral flagellum. Recording electrical activities from the antennal nerve showed that there are more than 14 neurones in the antenno-motor externus.The distribution of the antennal nerve in the brain was investigated histologically by the injection of fluorescent dye. Antennal sensory neurones terminated at the glomeruli in the antennal lobe, in the dorsal lobe, in the protocerebrum, and in the commissural part of the suboesophageal ganglion. Injection of the fluorescent dye into the antennal nerve after degeneration of the antennal sensory neurones showed that the antennal motoneurones run in the ventral side of the antennal and dorsal lobes, and terminate in the marginal region of the ipsilateral oesophageal connective.The difference in latency of odour-induced flagellar movements is discussed in relation to the histological results and the unitary responses in the brain reported previously.     

Kinetic studies of mold α-galactosidase on PNPG hydrolysis

The kinetic properties of α-galactosidase of Mortierella vinacea were investigated in detail using PNPG (p-nitrophenyl-α-D -galactopyranoside) as a substrate. Consequently, the enzyme was markedly inhibited not only by the substrate, but also by the galactose hydrolized. The initial rate of reaction at sufficiently high substrate concentrations, however, did not fall to zero and did approach a finite value. Galactose behaved as a mixed inhibitor and was neither totally competitive nor totally noncompetitive. A rate equation was obtained from a generalized equation derived from a kinetic model which took both the inhibitions into consideration. The constants used in the equation were appropriately estimated. The calculated rate agreed fairly well with the observed initial rate. Moreover, the PNPG hydrolysis progressing in a batch system was found to be approximately representable by simple first order kinetics in which the rate constant was dependent on the initial substrate concentration.     

Pre-existing glomerular immune complexes induce polymorphonuclear cell recruitment through an Fc receptor-dependent respiratory burst: potential role in the perpetuation of immune nephritis

In immune complex (IC) diseases, FcR are essential molecules facilitating polymorphonuclear cell (PMN) recruitment and effector functions at the IC site. Although FcR-dependent initial tethering and FcR/integrin-dependent PMN accumulation were postulated, their underlying mechanisms remain unclear. We here addressed potential mechanisms involved in PMN recruitment in acute IC glomerulonephritis (nephrotoxic nephritis). Since some renal cells may be recruited from bone marrow (BM) lineages, reconstitution studies with BM chimeras and PMN transfer between wild-type (WT) and FcR-deficient mice (gamma(-/-)) were performed. Severe glomerular damage was induced in WT and W gamma chimeras (BM from WT to irradiated gamma(-/-)), while it was absent in gamma(-/-) and gamma W chimeras (gamma(-/-) BM to WT). Moreover, WT PMN transfer, but not gamma(-/-) PMN, reconstituted the disease in gamma(-/-), indicating that FcR on resident cells is not a prerequisite for PMN recruitment in this disease. Surprisingly, transferred WT PMN were recruited coincidentally with NF-kappa B activation and TNF-alpha overexpression even in glomeruli with preformed IC (nephrotoxic Ab administered 3 days previously), suggesting that PMN can initially be recruited via its own FcR without previous chemoattractant release. Furthermore, H(2)O(2) inhibition by catalase attenuated the acute WT PMN recruitment and the induction of NF-kappa B and TNF-alpha much more than integrin (CD18) blockade, indicating a role for the respiratory burst before integrin-dependent accumulation. In coculture experiments with IC-stimulated PMN and glomeruli, PMN caused acute glomerular TNF-alpha expression predominantly via FcR-mediated H(2)O(2) production. In conclusion, glomerular IC, even preformed, can cause PMN recruitment and injury through PMN FcR-mediated respiratory burst during initial PMN tethering to IC.     

Human prorenin has "gate and handle" regions for its non-proteolytic activation

We investigated the mechanism for non-proteolytic activation of human prorenin using five kinds of antibodies. Each of the antigens, L1PPTDTTTFKRI11P, T7PFKRIFLKRMP17P, I11PFLKRMPSIRESLKER26P, M16PPSIRESLKER26P, and G27PVDMARLGPEWSQPM41P, was designed from the tertiary structure of predicted prorenin. These antibodies were labeled anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, for their binding specificities. Inactive recombinant human prorenin (0.1 nM) bound to various concentrations of anti-01/06, anti-11/26, and anti-27/41 antibodies at 4 degrees C with equilibrium dissociation constants of 138, 41, and 22 nM, respectively. However, intact prorenin (0.1 nM) did not show significant binding to 200 nM anti-07/10 and anti-16/26 antibodies for 20 h. Ninety percent of prorenin (0.1 nM) was found to be non-proteolytically activated by incubation with anti-11/26 antibodies (200 nM) at 4 degrees C for 20 h. Prorenin was not active even under complex with either anti-01/06 or anti-27/41 antibodies. Prorenin was also reversibly activated at pH 3.3 and 4 degrees C for 25 h. The acid-activated prorenin bound to anti-07/10 and anti-16/26 antibodies as well as to anti-01/06, anti-11/15, and anti-27/41 antibodies at neutral pH and 4 degrees C in 2 h. Their dissociation constants were 13, 40, 8.6, 3.6, and 14 nM, respectively. The acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 degrees C in 50 h. Anti-07/10 and anti-11/26 antibodies inhibited such re-inactivation at 25 degrees C by more than 90% and 50%, respectively, whereas other kinds of antibodies did not prevent the re-inactivation at 25 degrees C. These results indicate that prorenin has "gate" (T7PFKR10P) and "handle" (I11PFLKR15P) regions critical for its non-proteolytic activation.     

Impaired pressure sensation in mice lacking TRPV4

The sensation of pressure, mechanosensation, in vertebrates remains poorly understood on the molecular level. The ion channel TRPV4 is in the TRP family and is a candidate for a mechanosensitive calcium-permeable channel. It is located in dorsal root ganglia. In the present study, we show that disrupting the Trpv4 gene in mice markedly reduced the sensitivity of the tail to pressure and acidic nociception. The threshold to noxious stimuli and the conduction velocity of myelinated nerve responding to stimuli were also impaired. Activation of unmyelinated nerve was undetected. However, the mouse still retained olfaction, taste sensation, and heat avoidance. The TRPV4 channel expressed in vitro in Chinese hamster ovary cells was opened by low pH, citrate, and inflation but not by heat or capsaicin. These data identify the TRPV4 channel as essential for the normal detection of pressure and as a receptor of the high-threshold mechanosensory complex.     

Involvement of syntaxin 7 in human gastric epithelial cell vacuolation induced by the Helicobacter pylori-produced cytotoxin VacA

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.     

IcsB,secreted via the type III secretion system,is chaperoned by IpgA and required at the post-invasion stage of Shigella pathogenicity

Shigella deliver a subset of effector proteins such as IpaA, IpaB and IpaC via the type III secretion system (TTSS) into host cells during the infection of colonic epithelial cells. Many bacterial effectors including some from Shigella require specific chaperones for protection from degradation and targeting to the TTSS. In this study, we have investigated the role of the icsB gene located upstream of the ipaBCDA operon in Shigella infection because the role of IcsB as a virulence factor remains unknown. Here, we found that the IcsB protein is secreted via the TTSS of Shigella in vitro and in vivo. We show that IpgA protein encoded by ipgA, the gene immediately downstream of icsB, serves as the chaperone required for the stabilization and secretion of IcsB. We have shown that IcsB binds to IpgA in bacterial cytosol and the binding site is in the middle of the IcsB protein. Intriguingly, although its significance in Shigella pathogenicity is as yet unclear, the icsB gene can be read-through into the ipgA gene to create a translational fusion protein. Furthermore, the contribution of IcsB to the pathogenicity of Shigella was demonstrated by plaque-forming assay and the Sereny test. The ability of the icsB mutant to form plaques was greatly reduced compared with that of the wild type in MDCK cell monolayers. Furthermore, when guinea pig eyes were infected with a non-polar icsB mutant, the bacteria failed to provoke keratoconjunctivitis. These results suggest that IcsB is secreted via the TTSS, chaperoned by IpgA, and required at the post-invasion stage of Shigella pathogenicity     

Globin and linker sequences of the giant extracellular hemoglobin from the leech Macrobdella decora

A detailed electrospray ionization mass spectrometric study of the 3.5-MDa hexagonal bilayer hemoglobin (HBL Hb) from the pond leech Macrobdella decora has shown it to consist of at least six 17-kDa globin chains, of which two are monomeric and the remaining four occur as disulfide-bonded heterodimers, and three 24-kDa nonglobin linker chains (Weber et al., J. Mol. Biol. 251: 703–720, 1995). The cDNA sequences of the five major constituent chains, globin chains IIA, IIB, B, and C and linker chain L1, are reported here. The globins and linkers share 30%–50% and 20%–30% identity, respectively, with other annelid sequences. Furthermore, IIB and C align with strain A of annelid sequences, whereas IIA and B align with the strain B sequences. Although chains B and C are monomeric, chains IIA and IIB form the main disulfide-bonded dimer. They also have some unusual features: the distal His (E7) is replaced by Phe in IIA, and the highly conserved CD1Phe is replaced by Leu in IIB. In spite of these unusual features, the functional properties of Macrobdella Hb are comparable to those of other HBL Hbs. A phylogenetic analysis of the globin sequences from Macrobdella, the polychaete Tylorrhynchus, the oligochaete Lumbricus, and the vestimentiferan Lamellibrachia, indicates that the two strains originated by gene duplication followed by additional duplication of each of the two strains. The mutation rate of the linkers appeared to be faster than that of the globin chains. The phylogenetic trees constructed using the Maximum Likelihood, Neighbor-Joining and Fitch methods showed the Macrobdella globin sequences to be closest to Lumbricus, in agreement with a view of annelid evolution in which the divergence of the polychaetes occurred before the divergence of the leeches from oligochaetes.     

The Hsp90 Inhibitor Geldanamycin Abrogates Colocalization of eIF4E and eIF4E-Transporter into Stress Granules and Association of eIF4E with eIF4G

The eukaryotic translation initiation factor eIF4E plays a critical role in the control of translation initiation through binding to the mRNA 5′ cap structure. eIF4E is also a component of processing bodies and stress granules, which are two types of cytoplasmic RNA granule in which translationally inactivated mRNAs accumulate. We found that treatment with the Hsp90 inhibitor geldanamycin leads to a substantial reduction in the number of HeLa cells that contain processing bodies. In contrast, stress granules are not disrupted but seem to be only partially affected by the inhibition of Hsp90. However, it is striking that eIF4E as well as its binding partner eIF4E transporter (4E-T), which mediates the import of eIF4E into the nucleus, are obviously lost from stress granules. Furthermore, the amount of eIF4G that is associated with the cap via eIF4E is reduced by geldanamycin treatment. Thus, the chaperone activity of Hsp90 probably contributes to the correct localization of eIF4E and 4E-T to stress granules and also to the interaction between eIF4E and eIF4G, both of which may be needed for eIF4E to acquire the physiological functionality that underlies the mechanism of translation initiation.     

Alcadein Cleavages by Amyloid ��-Precursor Protein (APP) ��- and ��-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related ��-Secretase Dysfunction

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc_α, Alc_β, and Alc_γ. The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP α-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent γ-secretase complex, thereby generating “APP p3-like” and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc_α, p3-Alc_β, and p3-Alc_γ, whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor β-amyloid species Aβ42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc_α, p3-Alc_β, and p3-Alc_γ were not equivalent, suggesting that one type of γ-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect γ-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.