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A potential error in modifications of the [14C]2-deoxyglucose technique.

Although the technique developed by Sokoloff et al. employing [14C]2-deoxyglucose as tracer, provides measurements of local cerebral glucose utilization, various modifications of the original technique are presently in widespread use. We have examined the validity of a crucial simplifying assumption which is an integral feature of many of these modifications; namely, that all the radioisotope in the CNS at the moment of sacrifice is present as 2-deoxyglucose-6-phosphate. By employing the kinetic model developed by Sokoloff et al. and arterial plasma [14C]2-deoxyglucose and glucose histories, the proportion of total radioactivity present in the CNS as unphosphorylated 2-deoxyglucose was calculated and found to vary from 5 to 50% of the total amount of isotope present in the CNS under different experimental conditions. It can be shown to be dependent upon (a) the rate of cerebral glucose utilization, (b) the time which elapses from administration of the radioisotope until the sacrifice, (c) the route by which the isotope is administered, and (d) arterial plasma glucose levels. Thus, modifications of the original 2-deoxyglucose technique have introduced a potential source of error which is not a feature of the approach of Sokoloff and his associates.

Animals↗

Modifications in the responsiveness of rat ventrobasal thalamic neurons at different stages of carrageenin-produced inflammation.

The present study was aimed at analyzing the responsiveness of the ventrobasal (VB) thalamic neurons in rats presenting with a hyperalgic carrageenin-produced inflammation. The following were studied: the responses of the same VB neuron, before and 15-145 min after the plantar injection of carrageenin in a part of its receptive field (RF) (acute phase); the responses of VB neurons located in the thalamus contralateral to the hyperalgesic inflamed paw, 24-96 h after the injection (subacute phase); and the effect of a local anesthetic injected in the inflamed paw, and that of an intravenous injection of Aspirin, on neuronal response modifications. Responses of VB neurons initially activated by light tactile stimuli (group 1; n = 4) and by moderate joint stimulation (group 3; n = 4) were not modified in the early period following the carrageenin injection. By contrast, in the first few minutes following the injection. VB neurons exclusively driven by noxious mechanical and thermal stimuli (group 2; n = 23), exhibited a clear enhancement of their responses, which persisted during the observation period. These modifications were also observed for responses obtained from part of the RF remote from the injection site; moreover there was an extension of the RF to areas distant from the injured paw. The local injection of an anesthetic (Xylocaine) in this paw, suppressed the modifications of responses of group 2 neurons, elicited not only from the injected paw, but also from the remote parts of the RF. At this time Aspirin was almost inefficient (even at the dose of 100 mg/kg) on responses of these group 2 neurons. In the subacute phase responses of 72 somatosensory neurons were analyzed. Twenty-five of 72 responded to rapid repetitive light tactile stimulation applied on a small contralateral RF (group 1); their responses were similar to those encountered in a normal situation. Thirty-three of 72 neurons responded to intense mechanical stimuli such as pinches (group 2). For half of them the response characteristics were similar to those described in the normal rat; for the other half responses appeared 'faded': short duration; absence of after-discharge; poor reproducibility. Fourteen of 72 neurons responded to moderate stimulation of the joints, deep tissues and/or surrounding cutaneous areas of the inflamed paw (group 3). Their RF was mostly unilateral, i.e. contralateral to the recording site; the responses were sustained during the stimulation but rarely exhibited after-discharge.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

Drastic changes of ventromedial medulla neuronal properties induced by barbiturate anesthesia. II. Modifications of the single-unit activity produced by Brevital, a short-acting barbiturate in the awake, freely moving rat.

In the preceding study, we have found that pentobarbital, a powerful barbiturate substance, strongly modified the ventromedial medulla (VMM) physiology in relation to nociception: indeed, in the same rats, during time-separated similar VMM penetrations, we have recorded, under pentobarbital, 'new' neuronal groups as compared to the awake state, such as the units exclusively driven (excited or inhibited) by cutaneous innocuous or noxious stimulations and the multimodal multireceptive neurons inhibited by non-noxious and noxious stimuli. Still under pentobarbital, we have also recorded the same units found as the rats were awake, i.e., the multimodal multireceptive neurons exclusively excited by various innocuous and noxious stimuli. However, the spontaneous and nociceptive activities of these units were strongly modified as compared to awake animals. Using Brevital (a short-acting barbiturate substance) administration, we have, in the present study, tried to understand the mechanisms underlying these drastic modifications. In particular, one of the questions was whether or not the 'new' neuronal classes recorded under anesthesia resulted from a modification of the physiological properties of the unique VMM neuronal group potentially involved in nociception in awake animals: the multimodal multireceptive units. By following the VMM neuronal activities either before and after or after Brevital administration until recovery from anesthesia, we have determined that the units exclusively driven by innocuous stimulation might result from a modification of the multimodal multireceptive neurons. Alternatively, the multireceptive units inhibited by peripheral stimulations are possibly totally different neurons, silent when the animals are awake.

Anesthesia↗

Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation.

Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.

Carbohydrates↗

Oat lipids-induced covalent DNA modifications (I-compounds) in female Sprague-Dawley rats, as determined by 32P-postlabeling.

Previous studies have shown that the presence of oats in the diet contributes to formation of I-compounds (age-dependent covalent DNA modifications detected by 32P-postlabeling assay) in female Sprague-Dawley rat liver DNA. The current study explored the possible ingredients in oats responsible for the observed effects on DNA. Feeding AIN-76A diet containing 5% oat lipids (obtained by methanol extraction and dissolved in trioctanoin) in place of corn oil for 2 months successfully induced the formation of 3 oats-specific (spots 2-4) and 4 natural ingredient diet-specific I-compounds (spots 6-9) in liver DNA. Barley, an oatlike cereal, induced 3 of these spots at very low intensities but not the 3 oats-specific I-spots. Oral administration of oat lipids to weanling rats of both sexes for 7 days elicited trace amounts of the oats-specific spots and spot 9 in liver DNA. However, when oat lipids were given at 6 or 9 weeks of age, the oats-specific spots were detected at high levels in female but not in male rats. These oats-related DNA modifications were also present in 6-week-old female rats which had received oat lipids p.o. for 2 or 3 days or i.p. for 4 days. Rats given trioctanoin or extracts from natural ingredient Wayne diet (lacking oats) did not show any of these spots. On the other hand, rats treated with extracts from an oats-containing Teklad diet displayed a trace amount of one of these I-compounds. Oat lipids did not induce any extra spots in rat kidney DNA. Feeding of AIN diet supplemented with oats to female Syrian hamsters did not elicit any renal or hepatic DNA alterations, as detected by 32P-postlabeling. Rats fed oat lipids-supplemented AIN diet or Purina diet showed the highest levels of I-compounds overall in liver among all dietary groups and these two groups also had significantly higher hepatic DNA synthesis rates. Oat lipids enhanced kidney DNA synthesis also. The total hepatic or renal cytochrome P-450 contents were not significantly affected by different diets. These results demonstrate a novel link between a natural dietary ingredient and covalent DNA modifications and shed light on the origins of certain I-compounds.

Animals↗

Oxidative modification of Lp(a) causes changes in the structure and biological properties of apo(a).

We previously showed that oxidative modification converted Lp(a) particles to a form readily recognized by macrophage scavenger receptor(s). This was mediated in part by apo(a) changes indicated by a stronger negative charge protein fragmentation and increased immunoreactivity to monoclonal antibody K07, which also cross-reacted with plasminogen (Pg). The present study demonstrated that the expression of K07 and K09 epitopes of apo(a) increased markedly during time-dependent oxidative modification of Lp(a) by copper ions. Incubation of oxidized Lp(a) with the monocytoid U937 cell line showed that these particles competed more effectively with 125I-type 2 Pg binding to specific cell surface receptors than native Lp(a). This study suggests that oxidative Lp(a) modification causes significant changes in apo(a) conformation, resulting in the enhanced interaction of these particles with macrophage scavenger receptors and Pg binding sites.

Apolipoproteins↗

Serum high density lipoprotein cholesterol determination: a simple modification.

A simple modification to the method of Burnstein et al. (Burnstein, M., Scholnick, H.R. and Morfin, R. (1970) J. Lip. Res. 11, 583) [1] for the measurement of serum high-density lipoprotein (HDL) cholesterol concentrations is reported. The modification obviates the need for a refrigerated centrifuge and makes the method suitable for routine use in almost any laboratory. Good correlation was obtained for values in 80 healthy adults using the method as originally described by Burnstein et al. [1] and then with the modification. The presented method was linear and precise and the study showed that it was not necessary to fast before giving a blood sample for the assay. It was found that HDL cholesterols were higher in women than in men, but did not change significantly with age in either group.

Adult↗

A study on post-synthetic modifications in alfa-alfa enolase (EC 4.2.1.11) brought about by a human serum protein.

Human alfa-alfa enolase is modified in the blood circulation by a serum protein for which the name 'modifying protein' is proposed. The protein occurs in every human serum tested and appears to be the same protein that is responsible for the post-synthetic modification in the M subunit of creatine kinase. Three alfa-alfa enolase forms, the original one plus two modified forms can be found in serum both in vivo and after in vitro incubation. The original alfa-alfa enolase is modified completely within a few hours in vitro in a pH-controlled human serum matrix at 37 degrees C. As the modification also takes place in vivo it is theoretically possible to acquire information about the activity of a disease process by doing one single determination of the amount of circulating alfa-alfa 3 enolase. A mechanism is proposed for the modification, where at first one of the two alfa chains is modified resulting in the alfa-alfa 2 form. Ultimately the second alfa chain is also modified. The alfa-alfa 1 form seems to be the completely modified alfa-alfa form. The three enolase forms differ in their isoelectric points but have similar Michaelis-Menten constants.

Blood Proteins↗

Marked deciliation and insufficient secretory modification of endometrial surface during treatment with a new progestogen-estrogen combination.

Endometrial modifications induced by SHB 209 AE, a new progestogen-estrogen combination, was investigated using scanning and transmission electron microscopy, in order to demonstrate specific variations of the endometrial surface and if these contribute to the contraceptive effects of the preparation. Endometrial biopsies were performed in 27 volunteers who had taken SHB 209 AE during the preceding 3 months. Endometrial biopsies of 15 normomenstruating volunteers were used as controls. Each fragment was subdivided for scanning and transmission electron microscopy examination. The ciliated cells were always significantly fewer in the various phases of the treatment cycle than in the control endometria although, as in the controls, their number and ratio with nonciliated cells was greater mid-way through the cycle. Ciliogenesis was always notably delayed compared to the normal endometria during the different treatment cycle phases. Secretory modifications appeared prematurely but remained incomplete through the treatment cycle. These modifications caused total and characteristic alterations of the mucosal architecture and cellular maturation that comprised the implantation phase.

Adolescent↗

Effect of chemical modifications on freeze denaturation of lactate dehydrogenase.

Lactate dehydrogenase (LDH) was chemically ethyl-acetimidated (EA-), dimethyladipimidated (DMA-), carbamylated, acetylated, acetoacetylated, or succinylated in order to alter the ionic charges on the epsilon-amino group of lysine residues. Acetylation, acetoacetylation, and succinylation, which change the positive charge at the lysine side chains to a negative one, inactivated the enzymic activity, but the rest of the modifications exerted no such inactivating effects. The active modified enzymes were subjected to freeze denaturation study, using the enzymic activity as an indication of the degree of the denaturation. The active enzymes were diluted with deionized water and stored in a freezer (-23 degrees C) for 1-3 days. Enzymic activity was assayed immediately after thawing. All the modified enzymes retained their activity even after the 3-day frozen storage, while the control or native enzyme lost its activity within 1 day of storage. Furthermore, the modified LDHs freeze-stored in 0.2 M monosodium glutamate (MSG) or 0.2 M lysine-hydrochloride (Lys-HCl) retained their activity. The cryoprotective effects exerted by the modifications and by 0.2 M MSG seemed to be synergistic, whereas those exerted by the modifications and by 0.2 M Lys-HCl did not. The mechanisms of cryoprotection and freeze denaturation are discussed in relationship with the cryoprotective effect exerted by already known cryoprotectants, such as sucrose or dimethyl sulfoxide.

Animals↗

Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum.

Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.

Acetylglucosaminidase↗

Defects in protein modification precede developmental defects in l(3)c21RRW630, a temperature-sensitive Drosophila developmental mutant.

The temperature-sensitive Drosophila developmental mutation, l(3)c21RRW630 (abbreviated RW630) disturbs oogenesis and has a maternal effect on embryogenesis. At restrictive temperature, RW630 alters post-translational modification of three abundant proteins. To examine the causal relationship between these biochemical defects and the developmental defects in RW630, a series of temperature-shift experiments was performed. It was found that defects in protein modification could be detected in RW630 ovaries after RW630 females had been exposed to restrictive temperature for 1 day. RW630 females treated in this fashion produce embryos which contain a low level of unmodified proteins. Nevertheless, these embryos hatch at a normal rate. Since these ovaries and these embryos are developmentally normal, but do show defects in protein modification, it is unlikely that the RW630 developmental defects cause the biochemical defects in RW630. It is more likely that accumulation of unmodified proteins after extended exposure to restrictive temperature produces the developmental defects in RW630.

Animals↗

Egg-induced modifications of the zona pellucida of mouse eggs: effects of microinjected inositol 1,4,5-trisphosphate.

Mouse eggs microinjected with physiological concentrations of inositol 1,4,5-trisphosphate (IP3) do not emit the second polar body, form a pronucleus, or display a fertilization-associated set of changes in the pattern of protein synthesis. IP3-injected eggs, however, display a conversion of the zona pellucida glycoprotein ZP2 to ZP2f. The effect is concentration-dependent with an EC50 (effective concentration, 50%) of 5-10 nM and also occurs in the presence of reduced levels of extracellular calcium. The egg-induced zona pellucida modification is not elicited by several other inositol phosphates that are not able to release calcium from intracellular stores in other systems. Analysis of individual eggs microinjected with IP3 reveals a strong correlation between a reduced binding of sperm to the zona pellucida and the ZP2 to ZP2f conversion. In addition, solubilized zonae pellucidae isolated from IP3-injected eggs possess reduced levels of acrosome reaction-inducing activity. These egg-induced modifications of the zona pellucida--reduced sperm receptor and acrosome reaction-inducing activities and the ZP2 to ZP2f conversion--elicited by microinjected-IP3 are similar to those that occur following fertilization. Results of these experiments suggest that IP3 generated in response to fertilization may play a role in the egg-induced modifications of the zona pellucida that result in the polyspermy block.

Acrosome↗

Chemical modifications of pokeweed antiviral protein: effects upon ribosome inactivation, antiviral activity and cytotoxicity.

Pokeweed antiviral protein (PAP) is a protein known to inactivate eukaryotic ribosomes by an unknown enzymatic action and inhibit the production of mammalian viruses in tissue culture. This protein was subjected to a variety of chemical modifications to determine their effects upon ribosomal inactivation, antiviral action, and cytotoxicity. It was found that modifications of a number of different amino acid residues had similar effects upon all 3 activities. Also the inactivation of PAP with diethylpyrocarbonate was not due to its reaction with a histidine residue but to a modification of an unidentified amino acid residue.

Animals↗

Sulfhydryl group modification of photoreceptor G-protein prevents its light-induced binding to rhodopsin.

The effect of sulfhydryl modification on the light-induced interaction between rhodopsin and the peripheral GTP-binding protein of the photoreceptor membrane (G-protein) has been investigated by time-resolved near-infrared light-scattering and polyacrylamide gel electrophoresis. It has been found that the modification of rhodopsin with the alkylating agent N-ethylmaleimide (NEM) does not affect its light-induced interaction with the G-protein. Modification of G-protein with NEM or other sulfhydryl agents prevents any light-induced binding to rhodopsin. Dark-association of G to the membrane as well as the light-induced complex with rhodopsin (once formed) is insensitive to NEM.

Alkylation↗

Affinity modification of E1-form of Na+, K+-ATPase revealed Asp-710 in the catalytic site.

An alkylating ATP analogue, gamma-[4-(N-2-chlorethyl-N-methylamino)]benzylamide ATP (C1RATP), covalently binds to the catalytic alpha-subunit of Na+, K+-ATPase yielding a product resistant to hydrolysis by the enzyme and inhibiting the ATP-hydrolysing activity. The Na+-form of the membrane-bound Na+, K+-ATPase modified with C1RATP was hydrolysed by pepsin under conditions providing maximum stability of the modification product (4 degrees C, pH 1.5). The modified peptide was isolated by HPLC and its amino acid sequence was found to involve residues 706-713 of the alpha-subunit polypeptide chain. This fragment located near the gamma-phosphate of ATP is a component of the active site. It is highly homologous with corresponding regions of the catalytic subunits of all the known E1-E2 ATPases. In the Na+-(or E1-)enzyme form Asp-710 is the target of modification. Evidently E1- and E2-enzymes have different targets in C1RATP modification, i.e. the polypeptide chain regions near the ATP gamma-phosphate in the enzyme active site differ somewhat in their conformations.

Adenosine Triphosphatases↗

Tyrosine sulfation is not the last modification of entactin before its secretion from 3T3-L1 adipocytes.

Tyrosine sulfation of entactin was studied by labeling of 3T3-L1 adipocytes with [35S]methionine or H2 35SO4 in the presence or absence of tunicamycin or monensin. Four precursors (EN1-4) at different steps of modification were detected in addition to mature entactin. Under normal conditions, EN2 and mature entactin were intracellular species, and the latter was sulfated and secreted. Inhibition of co-translational transfer of N-linked oligosaccharides by tunicamycin produced EN1 and EN3 as intracellular species, and EN3 was sulfated and secreted. Interruption of protein transport from medial to trans (distal) Golgi cisternae by monensin, and consequent blockage of terminal glycosylation caused intracellular accumulation of EN4. EN4 was sulfated and of different size compared to mature entactin. These facts suggested that tyrosine sulfation of entactin occurs in medial Golgi cisternae and is not the last modification before its secretion. Our results appeared inconsistent with recent observations by Baeuerle and Huttner [(1987) J. Cell Biol. 105, 2655-2664] that tyrosine sulfation of IgM occurred within the trans (distal) Golgi cisternae as the last modification before its exit from the Golgi complex.

Adipose Tissue↗

Bioluminescence of the Ca(2+)-binding photoprotein, aequorin, after histidine modification.

Modification studies of the 5 histidine residues in aequorin employing site-directed mutagenesis and diethyl pyrocarbonate suggested that His169 may be the site of binding of molecular oxygen in aequorin. The modification of this residue led to complete loss of activity, whereas modification of the remaining 4 histidine residues yielded mutant aequorins with varying bioluminescence activities.

Aequorin↗