Passive avoidance learning in the earthworm (Lumbricus terrestris).

7 earthworms learned rapid approach to an earth goal at the end of a straight runway in 20 trials. Significant inhibition of approach occurred in 1 trial with aversive stimulation (saline goal). After 5 saline trials, readaptation to earth resulted in significant decreases in runway-approach time. A replication (69 Ss indicated that passive avoidance could be retained for 24 hr but not over 240 hr. Demonstration of 1-trial learning may be accounted for by the selection of salient stimuli that are particularly relevant in a cue-to-consequence relationship. (21 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Stretch-sensitive neural units in the body wall of the earthworm, Lumbricus terrestris L

In a previous study we have shown that an isometric handgrip is accompanied by a rapid decrease in vascular resistance in the resting (contralateral) forearm, lasting for about 1 min, which in all probability is neurogenic. 2. In the present study the distribution of the vasodilatation was investigated by analysing O2 saturation in a deep vein, draining mainly muscle tissue. Some possible neurogenic mechanisms for the vasodilatation were tested by repeating the handgrip after local beta- and alpha-adrenergic blockade of the resting forearm with propranolol and phentolamine, respectively. 3. Without blockades the forearm vascular resistance decreased and the deep vein O2 saturation increase to similar degrees on contralateral handgrip. Propranolol markedly reduced both the decrease in resistance and the increase in deep vein O2 saturation. Phentolamine did not alter the initial decrease in resistance, but with phentolamine resistance continued to decrease after 1 min. 4. It is concluded that the rapid decrease in vascular resistance in the resting forearm on contralateral handgrip takes place, to a great extent, in muscle. It is mediated by neurogenic beta-adrenergic mechanisms and if the contraction is prolonged it gradually changes to a vasoconstriction mediated by alpha-adrenergic mechanisms.     

A molecular model for the d chain of the giant haemoglobin from Lumbricus terrestris and its implications for subunit assembly

We have previously reported that rhodacyanine dyes, such as 1 and 2, exhibited a potent inhibitory effect on the growth of several tumor cells and that 4-oxothiazolidine (rhodanine) was an essential moiety for antitumor activity. On the basis of our foregoing work, two types of rhodacyanine dyes, which categorized into class I and II depending on the methine length, were synthesized and evaluated as a novel antitumor agent. Attention was particularly focused on the structure-activity study of two heteroaromatic rings. In class I, where the A rings were conjugated to rhodanine via two methine groups, compounds 1, 20, 23, and 24 were found to be efficacious in tumor-bearing nude mice model study, but they did not have the chemical properties (stability, solubility) suitable for clinical use. In contrast, in class II, where the A rings were directly conjugated to rhodanine, compounds 13 and 25, which possessed a benzothiazole moiety for the A ring, exhibited the favorable biological and chemical properties. Therefore, we decided to have a benzothiazole moiety as the A ring and introduce various heterocyclic groups for the B ring. As a result, the pyridinium ring was selected as the optimal moiety for the B ring (compound 13). Further, the variation of counteranion had a profound effect on solubility in water without influence on antitumor activity. Chloride anion was selected as the favorable anion with respect to synthetic method as well as solubility in water. Our study finally led us to the identification of compound 3 (MKT 077, 1-ethyl-2-[[3-ethyl-5-(methylbenzothiazolin-2-ylidene)-4-oxothi azolidin-2 -ylidene]methyl]pyridinium chloride) as the candidate for clinical trials and is currently subjected to further investigation as a potent antitumor agent in phase I clinical trial for the treatment of solid tumors.     

Identification of heavy metal induced changes in the expression patterns of the translationally controlled tumour protein (TCTP) in the earthworm Lumbricus rubellus1

This paper is concerned with the development of a software package for the automatic tuning of myoelectric prostheses. The package core consists of Fuzzy Logic Expert Systems (FLES) that embody skilled operator heuristics in the tuning of prosthesis control parameters. The prosthesis system is an artificial arm-hand system developed at the National Institute of Accidents at Work (INAIL) laboratories. The prosthesis is powered by an electric motor that is controlled by a microprocessor using myoelectric signals acquired from skin-surface electrodes placed on a muscle in the residual limb of the subject. The software package, Microprocessor Controlled Arm (MCA) Auto Tuning, is a tool for aiding both INAIL expert operators and unskilled persons in the controller parameter tuning procedure. Prosthesis control parameter setup and subsequent recurrent adjustments are fundamental for the correct working of the prosthesis, especially when we consider that myoelectric parameters may vary greatly with environmental modifications. The parameter adjustment requires the end-user to go to the manufacturer's laboratory for the control parameters setup because, generally, he/she does not have the necessary knowledge and instruments to do this at home. However, this procedure is not very practical and involves a waste of time for the technicians and uneasiness for the clients. The idea behind the MCA Auto Tuning package consists in translating technician expertise into an FLES knowledge database. The software interacts through a user-friendly graphic interface with an unskilled user, who is guided through a step-by-step procedure in the prosthesis parameter tuning that emulates the traditional expert-aided procedure. The adoption of this program on a large scale may yield considerable economic benefits and improve the service quality supplied to the users of prostheses. In fact, the time required to set the prosthesis parameters are remarkably reduced, as is the technician's working time. This is interpreted as minor costs for prostheses manufacturers and suppliers.     

Characterization of an RNA-binding domain in the bacteriophage phi 29 connector

The connector of bacteriophage phi 29 is known to promote the viral prohead assembly, to bind DNA, and to drive DNA packaging into preformed viral shells in an RNA-dependent process. In this report, the phi 29 connector protein, p10, is shown to bind RNA in a sequence-independent fashion, and to possess an RNA recognition motif comprised approximately the region between residues 21 and 94 of the p10 sequence. Substitution mutants in specific amino acids of the RNA-binding domain obtained by site-directed mutagenesis showed that amino acids Phe23, His57, Phe59, and Tyr61 are critical for RNA binding and, subsequently, for DNA packaging into proheads. Proteolytic modified forms of the phi 29 connector have allowed us to conclude that the DNA- and RNA-binding domains are separated within the p10 sequence. It is also shown that RNA is stably associated to DNA-filled proheads during the DNA-packaging process.     

Characterization of Trichomonas vaginalis virus proteins in the pathogenic protozoan T. vaginalis

PURPOSE: Children who have brain tumors are at risk for a variety of treatment-related sequelae, including neuropsychological and cognitive impairment, neurologic deficits, and neuroendocrinologic disturbances. We sought to determine the value of proton MR spectroscopy in assessing brain tissue remote from the tumor site to ascertain the effects of chemotherapy and radiation treatment in these patients. METHODS: Single-voxel proton MR spectra from 70 patients (111 spectra) and 11 healthy volunteers (11 spectra) were analyzed. NAA/Cr, NAA/Cho, and Cho/Cr ratios based on peak areas were obtained from nonneoplastic regions of the frontal lobe. The relationship between MR spectroscopic ratios and treatment was determined. RESULTS: NAA-containing ratios were decreased in patients as compared with control subjects. The presence of gadolinium-based contrast material did not cause significant changes in the ratios as compared with precontrast data. When chemotherapy was a component of a child's treatment protocol, we found a significant decline in NAA/Cr ratios. Patients who underwent both chemotherapy and radiation therapy showed a trend toward lower NAA-containing ratios if the chemotherapy was administered before the radiation therapy. Patients receiving whole-brain radiation had a trend toward lower NAA-containing ratios than did those who had only focal tumor treatment. CONCLUSION: In children with brain tumors, MR spectroscopy of brain tissue remote from the tumor reveals treatment-related biochemical changes.     

Characterization of serine and threonine phosphorylation sites in beta-elimination/ethanethiol addition-modified proteins by electrospray tandem mass spectrometry and database searching

A new method for the characterization of serine and threonine phosphorylation sites in proteins has been developed. After modification of a phosphoprotein by beta-elimination/ethanethiol addition and conversion of phosphoserine and phosphothreonine residues to S-ethylcysteinyl or beta-methyl-S-ethylcysteinyl residues, the modified protein was subjected to proteolytic digestion. Resulting digests were analyzed by a combination of microbore liquid chromatography, electrospray ionization tandem (MS/MS) ion trap mass spectrometry and database searching to identify original phosphorylated residues. The computer program utilized (SEQUEST) is capable of identifying peptides and modified residues from uninterpreted MS/MS spectra, and using this method, all of the five known phosphorylation sites in bovine beta-casein were identified. Application of the method to multiply phosphorylated human high molecular weight neurofilament protein (NF-H) resulted in the identification of 21 peptides and their modified residues and hence, the in vivo phosphorylation sites. These included 26 KSP and 1 KTP site, all of which occur in the KSP repeat C-terminal tail domain (residues 502-823). One site at residue 518 was previously uncharacterized. A novel non-KSP serine at residue 421 near the KLLEGEE region in a IPFSLPE motif was characterized as phosphorylated (or glycosylated). The 27 characterized phosphorylation sites occur at S/TP residues in the following motifs: KSPVKEE, KSPAEAK, KSPEKEE, KSPAEVK, KSPEKAK, KSPPEAK, KSPVKAE, and KTPAKEE. On the basis of kinase consensus sequences, all of these motifs, including the previously unreported KTPAKEE motif, can be phosphorylated by proline-directed kinases. Advantages of the new method vis-a-vis our previously reported method [Jaffe, H., Veeranna, Shetty, K. T., and Pant, H. C. (1998) Biochemistry 37, 3931-3940] include (i) production of diastereomers eluting at different retention times increased the chances of peptide identification, (ii) increased hydrophobicity and hence retention time of the modified peptides, (iii) facilitation of positive ion production, and (iv) increased susceptibility to tryptic digestion as a result of conversion of negatively charged phosphorylated residues to neutral S-ethylcysteine or beta-methyl-S-ethylcysteine residues.     

Characterization of the elastin binding domain in the cell-surface 25-kDa elastin-binding protein of staphylococcus aureus (EbpS)

Our previous studies have established that a cell-surface 25-kDa elastin-binding protein of Staphylococcus aureus (EbpS) mediates binding of this pathogen to the extracellular matrix protein elastin. Results from binding assays examining the activity of various EbpS fragments suggested that the elastin recognition domain is contained within the first 59 amino acids. In this report, we have used functional analyses with synthetic peptides and recombinant truncated forms of EbpS to localize the elastin binding domain to a 21-amino acid region contained within residues 14-34 of EbpS. Further evidence for the importance of this domain was obtained by demonstrating that the inhibitory activity of anti-EbpS antibodies on staphylococcal elastin binding was neutralized when these antibodies were pre-absorbed with a truncated recombinant EbpS construct containing residues 1-34. Overlapping synthetic peptides corresponding to EbpS residues 14-36 were then generated and tested for elastin binding activity to define further the elastin binding domain, and results from these studies showed that sequences spanning amino acids Gln14-Asp23, Asp17-Asp23, and Thr18-Glu34 inhibit binding of Staphylococcus aureus to elastin. Our analyses indicate that the hexameric sequence Thr18-Asn-Ser-His-Gln-Asp23 is the minimal sequence common to all active synthetic peptides, proteolytic fragments, and recombinant constructs of EbpS. Furthermore, substitution of Asp23 with Asn abrogated the blocking activity of the synthetic peptides, demonstrating the requirement for a charged amino acid at this location. The composite data indicate that staphylococcal elastin binding is mediated by a discrete domain defined by short peptide sequences in the amino-terminal extracellular region of EbpS.     

Localization of proteins HL29 and HL31 from Haloarcula marismortui within the 50 S ribosomal subunit by chemical crosslinking

Isolated 50 S ribosomal subunits from the halophilic archaebacterium Haloarcula marismortui were treated in situ with the homobifunctional and cleavable crosslinking reagent dithiobis(succinimidyl propionate) (12 A). Several crosslinked complexes were obtained. Among these were the protein pairs HmaL4-HL29 and HmaL18-HL31; HL29 and HL31 are ribosomal proteins without any equivalent in eubacterial ribosomes. The crosslinked protein pairs were isolated on a preparative scale by combining conventional ion-exchange chromatography and reverse phase high-pressure liquid chromatography. The monomeric proteins involved in crosslink formation were unambiguously identified by two-dimensional gel electrophoresis and N-terminal or internal protein sequencing. Due to the homology between HmaL4 and HmaL18 and their Escherichia coli counterparts, and the roughly known location of these proteins within the 50 S subunit, our results demonstrate that HL29 is probably located in the centre of the large subunit in the vicinity of the peptidyltransferase domain, whereas HL31 must be situated within the central protuberance close to the region of the 5 S RNA.     

Characterization of the leukotriene B4 receptor in porcine leukocytes. Separation and reconstitution with heterotrimeric GTP-binding proteins

Leukotriene B4 (LTB4) is a potent chemoattractant derived from arachidonic acid. When cDNAs for LTB4 receptor (BLT) were cloned it was found that they belong to a guanine nucleotide-binding regulatory protein (G-protein)-coupled receptor superfamily. However, purification of BLT from inflammatory cells and reconstitution with various types of G-proteins have not been successful. In the present study, BLT from porcine leukocytes was solubilized, separated from associated G-proteins by Ricinus communis agglutinin (RCA) 120 chromatography, and reconstituted with several endogenous and exogenous G-proteins, in combination with the fraction which contained endogenous phospholipids and Gbeta gamma. Kinetic studies of LTB4 were performed to determine the association with G-proteins. A partially purified BLT fraction (retained on an RCA120 column) free of G-proteins showed a lower affinity for LTB4 (Kd = 500 nm), but reconstitution of the BLT fraction with a G-protein-rich fraction (flow-through of an RCA column) increased the affinity for LTB4 10-fold (Kd = 50 nm). The partially purified BLT fraction was also reconstituted with exogenous G-proteins such as a heterotrimeric Gi2 purified from bovine brain or recombinant alpha subunits of Gi1, Gi2, Gi3, and Go expressed in Spodoptera frugiperda-9 cells. These increases in LTB4 bindings demonstrate that the BLT of porcine leukocytes can interact with pertussis toxin-sensitive G-proteins in vitro. The method is useful for the purification and reconstitution of other, as yet unisolated, G-protein-coupled receptors.     

Antirotaviral activity of milk proteins: lactoferrin prevents rotavirus infection in the enterocyte-like cell line HT-29

Different milk proteins were analyzed for their inhibitory effect on either rotavirus-mediated agglutination of human erythrocytes or rotavirus infection of the human enterocyte-like cell line HT-29. Proteins investigated were alpha-lactalbumin, beta-lactoglobulin, apo-lactoferrin, and Fe(3+)-lactoferrin, and their antiviral action was compared with the activity of mucin, a milk glycoprotein known to affect rotavirus infection. Results obtained demonstrated that beta-lactoglobulin, apo- and Fe(3+)-lactoferrin are able to inhibit the replication of rotavirus in a dose-dependent manner, apo-lactoferrin being the most active. It was shown that apo-lactoferrin hinders virus attachment to cell receptors since it is able to bind the viral particles and to prevent both rotavirus haemagglutination and viral binding to susceptible cells. Moreover, this protein markedly inhibited rotavirus antigen synthesis and yield in HT-29 cells when added during the viral adsorption step or when it was present in the first hours of infection, suggesting that this protein interferes with the early phases of rotavirus infection.     

Characterization of [3H]idazoxan binding proteins in solubilized membranes from rabbit and human liver

This study sought to determine the potential role of nitric oxide (NO) in N-methyl-D-aspartate (NMDA)-stimulated efflux of [14C] gamma-aminobutyric acid (GABA) and [3H]acetylcholine from striatal slices in vitro. In Mg2+-free buffer, NMDA-stimulated [14C]GABA and [3H]acetylcholine release were inhibited by the guanylate cyclase inhibitor, 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and, to a lesser extent, by the nitric oxide synthase inhibitor, nitroarginine (N-Arg). Since reversal of catecholamine transporters previously has been implicated in the mechanism underlying NO-induced catecholamine release, we used the GABA transport inhibitor, 1-(2-(((diphenylmethylene)imino)oxy)ethyl)-1,2,5,6-tetrahydro-3-py ridine-carboxylic acid hydrochloride (NNC-711), to address the role of GABA transport in NArg-sensitive NMDA-induced release. NNC-711 inhibited NMDA-stimulated [14C]GABA efflux by 50%, confirming our previous report that NMDA-stimulated GABA release is partially dependent on reversal of the transporter. The effect of N-Arg in the presence of NNC-711 was similar to its effect in the absence of the transport inhibitor, suggesting that reversal of the transporter is not involved in the NO component of NMDA-stimulated [14C]GABA release. These data suggest that glutamatergic transmission through striatal NMDA receptors is partially mediated through activation of the NO/guanylate cyclase pathway and that this mechanism may contribute to the tetrodotoxin sensitivity of NMDA-induced release of GABA and acetylcholine in the striatum.     

Localization and characterization of the 86- and 84-kDa heat shock proteins in Hepa 1c1c7 cells

The central region of the mouse MHC harbors a recombinational hot spot area. Most recombinations in this part of the complex take place between the Hsp70.1 gene and the G7 gene. This interval is of interest since structurally indistinguishable recombinant haplotypes do differ in functional behavior. Susceptibility to experimental allergic orchitis, which is controlled by the Orch-1 locus, is one example. We have analyzed the hot spot region at the molecular level in order to understand the molecular organization of this chromosomal segment. From a C57BL genomic library we constructed a cosmid contig bridging the interval between Hsp70.1 and G7. The Orch-1 gene maps to a 60-kb segment of DNA in which we found a new Hsp70 homologue, Hsp70.3. Thus, as in the human MHC, the central region of the mouse MHC harbors a cluster of three Hsp70 genes; Hsp70.1, Hsp70.3, and Hsc70t. Two other genes are located in this critical interval (G7b and G7a/Bat-6), and there might still be other undetected genes present in the region. Heat shock proteins play an important role in a large number of physiological processes and it is tempting to speculate that Hsc70t, which exhibits testis-specific expression, may be identical to Orch-1.     

Novel developmentally regulated phosphoinositide binding proteins from soybean whose expression bypasses the requirement for an essential phosphatidylinositol transfer protein in yeast

Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development. While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized. We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains. We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs. Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant. Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress. We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.     

Characterization of presynaptic proteins involved in synaptic vesicle exocytosis in the nervous system of Torpedo marmorata

Synaptobrevin, SNAP-25 and syntaxin (SNAP receptor proteins) are molecular components that play a key role in the exocytotic machinery of synaptic vesicles. Their presence, distribution and interactions are reported in central and peripheral nervous systems of the electric fish Torpedo marmorata. These three proteins form a protein complex in all the nervous system regions tested, including the electric lobe and the electric organ which is innervated by pure cholinergic nerve terminals. Immunoblot analysis revealed a double protein pattern of SNAP-25 in the anterior brain and cerebellum, although a single protein band corresponding to SNAP-25 was observed in the electromotor system. Moreover, SNAP-25 showed a differential distribution in the electromotor system. It was present along nerve fibres and terminals that innervated the electric organ but it was not detected in nerve terminals at the electric lobe. Immunoisolation experiments using anti-synaptobrevin antibodies showed a tissue-specific co-existence of SNAP-25 and syntaxin with synaptobrevin in the immunoisolated organelles. In conclusion, the molecular components of the exocytotic machinery are shown to be conserved in Torpedo, although some differences mainly on SNAP-25, suggest a potential diversity in the regulation of neurosecretion.     

Stability of determination and differentiation of the somatic sexual characters in Nereis pelagica L. (Annelida polychaeta)

Experiments of regeneration in situ or on grafts were carried out to test the stability of determination of the somatic sexual characters in Nereis pelagica. The sexual characters of the parapodial cirri (male and female swellings, male crenellations) are always expressed on stump or regenerate according to the genetic sex. Supernumary parapodia which developed at the site of junction of the heterologous faces of host and graft of opposite sexes, might have the characters of either sex; this result can be interpreted by supposing that a supernumary parapodium is derived from tissues of either host or graft, of the corresponding sex. Conversely, the pygidial papillae, which normally occur only on the heteronereid male, will appear on regenerating female pygidia, triggered to transform to the heteronereid condition naturally or by decerebration, with a frequency depending on the age of the regenerate, the length of the regenerate, and the genital condition of both the stump and the regenerate. It is concluded that the presence of the papillae is not a sex-specific character, but that their development is normally inhibited in the maturing female.     

Novel homologs of replication protein A in archaea: implications for the evolution of ssDNA-binding proteins

In Bacteria and Eukarya, ssDNA-binding proteins are central to most aspects of DNA metabolism. Until recently, however, no counterpart of an ssDNA-binding protein had been identified in the third domain of life, Archaea. Here, we report the discovery of a novel type of ssDNA-binding protein in the genomes of several archaeons. These proteins, in contrast to all known members of this protein family, possess four conserved DNA-binding sites within a single polypeptide or, in one case, two polypeptides. This peculiar structural organization allows us to propose a model for the evolution of this class of proteins.     

Characterization of the human SDHC gene encoding of the integral membrane proteins of succinate-quinone oxidoreductase in mitochondria

The neurological manifestations of multiple sclerosis (MS) have been considered to result from demyelination of axons with relative preservation of axonal integrity. This concept has been challenged recently by a landmark pathological study, published in the New England Journal of Medicine, which has demonstrated that axonal degeneration is also present. The authors of the study hypothesized that axonal degeneration is the pathological correlate of the irreversible neurological impairment in this disease. However, this hypothesis cannot be reconciled with the clinical results obtained with transcranial applications of AC pulsed electromagnetic fields (EMFs) of picotesla flux density which have shown rapid and sustained improvement of symptoms including normalization of evoked potential responses in patients with chronic progressive or secondary progressive MS without demyelinated areas first undergoing remyelination or transected axons undergoing regeneration. Biochemical studies have shown that MS patients are serotonergically depleted with the extent of cerebral depletion correlating with the degree of motor disability and a chronic progressive course. It is believed that progressive serotonergic neuronal atrophy with synaptic inactivation, not axonal degeneration, are the hallmarks of the disease and that administration of AC pulsed magnetic fields improves symptoms of MS partly through reactivation of serotonergic neurons and amplification of synaptic serotonergic transmission.