Quantitative autoradiographic analysis of [I]-human CGRP binding sites in the dorsal horn of rat following chronic constriction injury or dorsal rhizotomy

Quantitative receptor autoradiography was used to examine the binding of [¹²⁵I]-human CGRP in the dorsal horn of the L4 spinal segment of rats with a chronic constriction injury (CCI) of the sciatic nerve or unilateral dorsal rhizotomies of spinal segments L1–L6. At the times selected for study, we found no change in the amount of CGRP binding in any areas examined following CCI. In contrast, our results showed a temporally related increase in the amount of CGRP binding in areas within laminae I–II and in lateral lamina V of the dorsal horn ipsilateral to the rhizotomies. These results indicate that CGRP binding sites are regulated, most likely, by changes in the release of CGRP. Further, our results suggest that the release of CGRP from primary afferent neurons is unchanged in animals with a CCI.     

Modulation of diphthamide synthesis by 5'-deoxy-5'-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5'-deoxy-5'-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Methionine adenosyltransferase activity in cultured cells and in human tissues

We have investigated methionine adenosyltransferase activity (MAT) in extracts of a variety of normal and malignant human tissues and cultured cell lines. MAT activity assayed from 17 different cultured cell lines varied to a great extent. Ramos (human, Burkitt's lymphoma) and EL4 (mouse, T cell lymphoma) cell showed MAT activity near 300 pmol/mg per min. Daudi (human, Burkitt's lymphoma) and almost all monolayer cells had MAT activity below 100 pmol/mg per min. Human peripheral blood lymphocytes had MAT activity of 36 pmol/mg permin. The MAT activity of the cell lines can be related to doubling time: cell lines with short doubling times have much higher MAT activity than other cell lines. A large variation in MAT activity in different human tissues was observed. In autopsy samples MAT activity was highest in the brain and in the colon. Malignant tissue samples gave much higher MAT activity than normal tissues. Lung cancer (carcinoma squamocellulare pulmonis) had MAT activity of 30.7 pmol/mg per min, while in normal lung it was 2.4 pmol/mg per min.     

Inactivation of liver S-adenosylhomocysteine hydrolase in vitro of rats treated with erythro-9-(2-hydroxynon-3-yl)adenine.

S-Adenosylhomocysteine hydrolase activity decreased in vitro time-dependently in liver homogenates obtained from rats treated in vivo with erythro-9-(2-hydroxynon-3-yl)adenine, a potent inhibitor of adenosine deaminase. The inhibitor in itself had no effect on the stability of the hydrolase. The inactivation of S-adenosylhomocysteine hydrolase was irreversible, proceeded fairly rapidly at a low temperature (0 degrees C) and showed first-order reaction kinetics. Adenosine was found to accumulate in these tissue homogenates during storage. Several lines of evidence suggest that adenosine caused the observed suicide-like inactivation post mortem. Pre-incubation of purified S-adenosylhomocysteine hydrolase at 0 degrees C with adenosine showed a half-maximal inactivation rate at 33 microM substrate concentration; the rate constant of inactivation was 0.01 min-1. Inactivation during tissue preparation and storage complicates the assay of S-adenosylhomocysteine hydrolase activity in samples that contain an inhibitor of adenosine deaminase. These results also suggest that the decrease of S-adenosylhomocysteine hydrolase activity reported to occur in several disturbances of purine metabolism should be re-examined to exclude the possibility of inactivation of the enzyme in vitro.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.     

Buried charged surface in proteins

BACKGROUND: The traditional picture of charged amino acids in globular proteins is that they are almost exclusively on the outside exposed to the solvent. Buried charges, when they do occur, are assumed to play an essential role in catalysis and ligand binding, or in stabilizing structure as, for instance, helix caps. RESULTS: By analyzing the amount and distribution of buried charged surface and charges in proteins over a broad range of protein sizes, we show that buried charge is much more common than is generally believed. We also show that the amount of buried charge rises with protein size in a manner which differs from other types of surfaces, especially aromatic and polar uncharged surfaces. In large proteins such as hemocyanin, 35% of all charges are greater than 75% buried. Furthermore, at all sizes few charged groups are fully exposed. As an experimental test, we show that replacement of the buried D178 of muconate lactonizing enzyme by N stabilizes the enzyme by 4.2 degrees C without any change in crystallographic structure. In addition, free energy calculations of stability support the experimental results. CONCLUSIONS: Nature may use charge burial to reduce protein stability; not all buried charges are fully stabilized by a prearranged protein environment. Consistent with this view, thermophilic proteins often have less buried charge. Modifying the amount of buried charge at carefully chosen sites may thus provide a general route for changing the thermophilicity or psychrophilicity of proteins.     

Visual pigments in a living fossil,the Australian lungfish <Emphasis Type="Italic">Neoceratodus forsteri</Emphasis>

Background  

One of the greatest challenges facing the early land vertebrates was the need to effectively interpret a terrestrial environment. Interpretation was based on ocular adaptations evolved for an aquatic environment millions of years earlier. The Australian lungfish Neoceratodus forsteri is thought to be the closest living relative to the first terrestrial vertebrate, and yet nothing is known about the visual pigments present in lungfish or the early tetrapods.     

NMR and crystal structures of the Pyrococcus horikoshii RadA intein guide a strategy for engineering a highly efficient and promiscuous intein

In protein splicing, an intervening protein sequence (intein) in the host protein excises itself out and ligates two split host protein sequences (exteins) to produce a mature host protein. Inteins require the involvement for the splicing of the first residue of the extein that follows the intein (which is Cys, Ser, or Thr). Other extein residues near the splicing junctions could modulate splicing efficiency even when they are not directly involved in catalysis. Mutual interdependence between this molecular parasite (intein) and its host protein (exteins) is not beneficial for intein spread but could be advantageous for intein survival during evolution. Elucidating extein-intein dependency has increasingly become important since inteins are recognized as useful biotechnological tools for protein ligation. We determined the structures of one of inteins with high splicing efficiency, the RadA intein from Pyrococcus horikoshii (PhoRadA). The solution NMR structure and the crystal structures elucidated the structural basis for its high efficiency and directed our efforts of engineering that led to rational design of a functional minimized RadA intein. The crystal structure of the minimized RadA intein also revealed the precise interactions between N-extein and the intein. We systematically analyzed the effects at the -1 position of N-extein and were able to significantly improve the splicing efficiency of a less robust splicing variant by eliminating the unfavorable extein-intein interactions observed in the structure. This work provides an example of how unveiling structure-function relationships of inteins offer a promising way of improving their properties as better tools for protein engineering.     

Acute toxicity effects of ibuprofen on behaviour and haematological parameters of African catfish Clarias gariepinus (Burchell, 1822)

Indiscriminate discharge of pharmaceutical waste into the aquatic ecosystem may pose serious health challenges to aquatic biota. The effect of acute exposure to ibuprofen was evaluated using changes in behaviour and haematological parameters under static bio-assay method in Clarias gariepinus. Test specimens were exposed to acute concentrations of ibuprofen (0.28, 0.33, 0.38, 0.43 and 0.48 mg l^?1) for 24, 48, 72 and 96 h durations respectively. Behavioural and phenotypic changes were observed in surviving fish. There were significant (p < 0.05) concentration and duration-dependent increases in erythrocyte (RBC), haemoglobin (Hb), pack cell volume (PCV) and leukocytes (WBC) in treated fish compared to the control. Insignificant decreases (p > 0.05) in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were observed in treated fish compared to the control. Ibuprofen elicited dose and duration- dependent decrease in neutrophil counts with the decreases being significant (p < 0.05) in the higher doses of 0.43 and 0.48 mg l^?1. Ibuprofen did not elicit any significant changes in monocytes, basophils and eosinophils. Changes observed in this study showed that ibuprofen negatively affected the health of the fish and we recommend that discharge of ibuprofen into the aquatic environment should be monitored and controlled.     

Dynorphin increases in the dorsal spinal cord in rats with a painful peripheral neuropathy

It is known that painful tissue injury evokes an increase in dynorphin in spinal neurons. It is not known, however, whether dynorphinergic systems respond similarly to the pain that accompanies peripheral neuropathy. Radioimmunoassays and immunocytochemistry were used to evaluate changes in dynorphin A(1-8) in the spinal cord of rats with a painful peripheral neuropathy. The neuropathy is the result of a constriction injury that is created by tying loose ligatures around the common sciatic nerve. Signs of abnormal pain sensations, hyperalgesia, allodynia (pain after normally innocuous stimuli), and spontaneous pain (or dysesthesia), are first detected 2-5 days after injury, reach peak severity in about 10 days, and persist for 2-3 months (Bennett, G. J.; Xie, Y.-K. Pain 33:87-107; 1988). Dynorphin increased by 5 days in cells in laminae I-II and V-VII in the lumbar spinal cord ipsilateral to the injury. This increase, maximal at 10 days (262%), was still present 20 days after the injury but was now seen only in neurons in the deep laminae (V-VII). Thus, the spinal dynorphinergic system appears to respond to neuropathic pain. Furthermore, our results suggest that dynorphinergic cells in the superficial and deep laminae may have different roles in nociception.