Levels Of Endogenous Activity Research Articles

A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling.

Hundreds of human proteins are modified by reversible palmitoylation of cysteine residues (S-palmitoylation), but the regulation of depalmitoylation is poorly understood. Here, we develop “depalmitoylation probes” (DPPs), small molecule fluorophores to monitor the endogenous activity levels of “erasers” of S-palmitoylation, acyl-protein thioesterases (APTs). Live-cell analysis with DPPs reveals rapid growth factor-mediated inhibition of the depalmitoylation activity of APTs, exposing a novel regulatory mechanism of dynamic lipid signaling.

History of the long-wavelength topography of Venus

A comparison of the global geological and topographic maps of Venus allows analysis of the topographic configuration of the major stratigraphic units forming the planet’s surface. The main positive relief features on Venus are manifested as prominences of tectonized units, which usually pertain to the initial episodes in the observable portion of its geological history (the beginning of the Guineverian period). The main morphostructural unit in this category is tessera, the main massifs of which constitute a special class of plateau-like highlands up to several kilometers high and tens to hundreds or thousands of kilometers wide. The younger shield and regional plains are confined to the regional slopes of the highlands formed by ancient tectonized units and are concentrated in regional lowlands. This indicates that regional lowlands (hundreds to thousands of kilometers across) as well as the highlands of ancient tectonized units had been formed in the early stages of the geological history of Venus. The estimates for the absolute model age of regional plains, which fill the lowlands, indicate that the formation of this material unit marks the first third of the observable geological history. Thus, the long-wavelength topographic pattern of Venus had formed, in general, in the early stages of the geological history, during the first third of it. In the final stages, which cover the next two thirds of the observable geological history of Venus (the Atlian period), the formation of long-wavelength topographic features is confined to dome-shaped highs, which make a minor contribution (about 15%) to the overall long-wavelength topographic pattern of Venus. This is evidence of a sharp fall in the level of endogenous activity after the emplacement of regional plains, with the style of the activity having changed from intensive crustal dislocations to extensive yet isolated uplifts of the thick thermal lithosphere.

Plasma active matrix metalloproteinase 9 and indices of diastolic function in patients with preserved systolic function

BackgroundThis study aimed to investigate whether the endogenous active levels of MMP-9 or tissue inhibitor of metalloproteinases-1 (TIMP-1) were related to indices of diastolic dysfunction (DD) in the setting of contemporary treatment of coronary artery disease (CAD). Methods and resultsWe prospectively studied 116 patients with CAD and preserved left ventricular LV systolic function (ejection fraction≥45%). All patients were free of heart failure symptoms at recruitment and underwent percutaneous intervention (PCI) of culprit lesions. Demographic and angiographic characteristics were collected. Plasma samples were analysed for the active form of MMP-9 and TIMP-1 using enzyme-linked immunosorbent assay-based isoform sensitive assays. Conventional and tissue Doppler-echocardiographic assessment of diastolic filling was undertaken with measurements of maximal early (E) and late (A) transmitral velocities in diastole, E/A ratio, E-wave deceleration time, isovolumic relaxation time, peak systolic (S), diastolic (D) and atrial reversal velocities of pulmonary venous flow, S/D fraction, time difference between A and duration of atrial reversal flow, early diastolic peak velocities of the lateral mitral annulus (E′) and E/E′. Active MMP-9 level was higher in patients with more severe phases of DD (normal [n=22]: median 0.57ng/ml; mild [n=19] 0.83ng/ml; mild-moderate [n=41] 0.64ng/ml; moderate or severe [n=34] 1.63ng/ml; p<0.0001 for trend). Three month post-PCI elevated levels of active MMP-9 had an adjusted odds ratio of 11.2 (2.3–56.0, p<0.004) for association with moderate or severe DD. ConclusionElevated active MMP-9 level is associated with more severe DD in patients with CAD and preserved systolic function, which may indicate abnormal extracellular matrix metabolism in myocardial ischaemia.

Dietary restriction regulates brain acetylcholinesterase in female mice as a function of age

In the present study, the normal endogenous activity level of acetylcholinesterase (AChE) was investigated in cerebral hemispheres and cerebellum of female mice as a function of age. The effects of 24-h fasting and refeeding, and dietary restriction (DR) on AChE activity and its protein expression patterns were also investigated in young (1-month) and old (18-month) mice. Our results show that the activity (U/mg protein) and level of AChE protein in the cerebral hemispheres of young mice is decreased significantly on 24-h fasting which reverses back on refeeding. On the other hand, DR produces an accumulative effect; thereby it decreases the activity of this enzyme in the cerebral hemispheres of both the young and old mice and the degree of reduction is of different magnitude, dictated in an age- and brain region-specific manner. Our findings suggest that DR regulates the activity of this enzyme which may be useful in related neurodegenerative disease conditions.

Plasma active matrix metalloproteinase 9 associated to diastolic dysfunction in patients with coronary artery disease

Circulating levels of total matrix metalloproteinases (MMP) have been associated with diastolic dysfunction and heart failure [1,2]. This study aimed to investigate the relationship of the endogenous active levels of MMP-1,-2,-3 and -9 or tissue inhibitor of metalloproteinases-1 (TIMP-1) and diastolic dysfunction (DD) in the setting of coronary artery disease (CAD).

Antithetical effects of corticosterone and dibutyryl cAMP on adenosine deaminase in the gastrointestinal tract of chicken during postnatal development

Adenosine deaminase (ADA; EC 3.5.4.4) is a purine catabolic enzyme causing hydrolytic deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine. In the present study, the normal endogenous activity level of ADA was investigated in different regions of the gastrointestinal tract (GIT) during postnatal development of chicken. The effects of corticosterone and dibutyryl cAMP (Bt(2)-cAMP) were studied at two selected postnatal ages. The results indicated a significantly high level of ADA at day 1 in all the regions of GIT, which then declined (-34% in esophagus, -35% in crop, and -48% in small intestine) at day 10 and remained fairly constant till day 90. While in the proventiculus, the activity of ADA decreased (-30%) at day 30 and showed further decline (-52%) at day 90 as compared to day 1. Corticosterone was seen to significantly decrease (-23 to 79%) the activity level, depending on the regions of GIT studied except proventriculus. The magnitude of decline was more pronounced at day 60 compared to day 10. Bt(2)-cAMP, on the other hand, caused a significant increase (+21 to 67%) in the activity level of ADA again depending on the regions of GIT studied except crop. Western blot analyses also depicted that the decrease and/or increase, respectively, of ADA activity by corticosterone and Bt(2)-cAMP was at the ADA protein level. In conclusion, the study suggests that the ADA activity level is highest at day 1 in all the regions of chicken GIT and could be reduced or enhanced by corticosterone and dibutyryl cAMP, respectively, in an age-specific manner.

Cyclic Cushing's syndrome: an overview

Cyclic Cushing's syndrome (CS) is a disorder in which glucocorticoid levels are alternately normal and high, the latter occurring in episodes that can last from a few days to several months. It is more common in children than in adults. Cyclic CS may be either of the two different forms of CS (ACTH-dependent or -independent CS). Clinically, it may present with one or many symptoms, depending on the duration of disease activity and the timing of the fluctuations. A serotoninergic influence, cyclic changes in central dopaminergic tone, spontaneous episodic hemorrhage in the tumor, and the action of inflammatory cytokines with antitumor properties are some of the mechanisms suggested to explain the physiopathology of this phenomenon but the exact mechanism remains to be clarified. The cyclic pattern of hypercortisolism can delay the final diagnosis of CS and make it difficult to interpret the results of dynamic tests. Patients may have paradoxical responses to dexamethasone that can reflect increasing or decreasing levels of endogenous activity. Hormone assessments have to be repeated periodically when a diagnosis of CS is suspected. The cyclic pattern can also interfere with medical treatment because patients may show unexpected clinical and biochemical signs of hypocortisolism when cortisol secretion cyclically returns to normal, so an accurate follow-up is mandatory in these patients.

Plant peptide deformylase: a novel selectable marker and herbicide target based on essential cotranslational chloroplast protein processing

Transgenic tobacco plants expressing three different forms of Arabidopsis plant peptide deformylase (AtDEF1.1, AtDEF1.2 and AtDEF2; EC 3.5.1.88) were evaluated for resistance to actinonin, a naturally occurring peptide deformylase inhibitor. Over-expression of either AtDEF1.2 or AtDEF2 resulted in resistance to actinonin, but over-expression of AtDEF1.1 did not. Immunological analyses demonstrated that AtDEF1.2 and AtDEF2 enzymes were present in both stromal and thylakoid fractions in chloroplasts, but AtDEF1.1 was localized to mitochondria. The highest enzyme activity was associated with stromal AtDEF2, which was approximately 180-fold greater than the level of endogenous activity in the host plant. Resistance to actinonin cosegregated with kanamycin resistance in Atdef1.2-D and Atdef2-D transgenic plants. Here, we demonstrate that the combination of plant peptide deformylase and peptide deformylase inhibitors may represent a native gene selectable marker system for chloroplast and nuclear transformation vectors, and also suggest plant peptide deformylase as a potential broad-spectrum herbicide target.

Early effects of embryonic movement: 'a shot out of the dark'.

It has long been appreciated that studying the embryonic chick in ovo provides a variety of advantages, including the potential to control the embryo's environment and its movement independently of maternal influences. This allowed early workers to identify movement as a pivotal factor in the development of the locomotor apparatus. With an increasing focus on the earliest detectable movements, we have exploited this system by developing novel models and schemes to examine the influence of defined periods of movement during musculoskeletal development. Utilizing drugs with known neuromuscular actions to provoke hyperactivity (4-aminopyridine, AP) and either rigid (decamethonium bromide, DMB) or flaccid (pancuronium bromide, PB) paralysis, we have examined the role of movement in joint, osteochondral and muscle development. Our initial studies focusing on the joint showed that AP-induced hyperactivity had little, if any, effect on the timing or scope of joint cavity elaboration, suggesting that endogenous activity levels provide sufficient stimulus, and additional mobilization is without effect. By contrast, imposition of either rigid or flaccid paralysis prior to cavity formation completely blocked this process and, with time, produced fusion of cartilaginous elements and formation of continuous single cartilaginous rods across locations where joints would ordinarily form. The effect of these distinct forms of paralysis differed, however, when treatment was initiated after formation of an overt cavity; rigid, but not flaccid, paralysis partly conserved precavitated joints. This observation suggests that 'static' loading derived from 'spastic' rigidity can act to preserve joint cavities. Another facet of these studies was the observation that DMB-induced rigid paralysis produces a uniform and specific pattern of limb deformity whereas PB generated a diverse range of fixed positional deformities. Both also reduced limb growth, with different developmental periods preferentially modifying specific osteochondral components. Changes in cartilage and bone growth induced by 3-day periods of flaccid immobilization, imposed at distinct developmental phases, provides support for a diminution in cartilage elaboration at an early phase and for a relatively delayed influence of movement on osteogenesis, invoking critical periods during which the developing skeleton becomes receptive to the impact of movement. Immobilization also exerts differential impact along the proximo-distal axis of the limb. Finally, our preliminary results support the possibility that embryonic hyperactivity influences the potential for postnatal muscle growth.

Measuring gelatinase activity in colorectal cancer

Aims: Gelatinases (MMP-2, -9) are the most extensively studied MMPs in cancer. The aim of the study was to determine the levels of active and latent forms of the gelatinases in paired colorectal tumour and normal tissue (n =77) and correlate these with pathological stage. Methods: Gelatinase levels were compared following the techniques of gelatin zymography (active and latent) and the novel gelatinase activity assays (total and endogenous/active). Results: Both latent and active MMP-2 and MMP-9 lysis bands (zymography) and both total (active and latent) MMP-9 and endogenous (active) MMP-9 and MMP-2 levels (activity assays) were greater in tumour than normal colorectal tissue. Total MMP-2 and MMP-9 levels as determined by activity assays correlated with both the Dukes staging (e.g. total MMP-9 in tumours: adenoma, 1.0 (0.3–3.6); Dukes A, 9.6 (2.4–35.4); Dukes B, 14.7 (1.5–103.9); Dukes C, 22.3 (2.2–57.9) and Dukes D, 37.4 (2.1–47.0) ng/mg protein) and with lymphatic invasion (e.g. total MMP-9; in tumours which had undergone lymphatic invasion, 22.7 (2.1–57.9) and those with no lymphatic invasion, 14.0 (1.5–103.9) ng/mg protein). Conclusions: Both gelatinases were upregulated in tumour tissue, however total and not endogenous active levels correlated with the pathological stage of the tumour. Therefore gelatinases may only be activated when required for tumour invasion and metastasis.

Constitutive expression of the steroid sulfatase gene supports the growth of MCF-7 human breast cancer cells in vitro and in vivo.

Many human breast tumors are driven by high intratumor concentrations of 17beta-estradiol that appear to be locally synthesized. The role of aromatase is well established, but the possible contribution of the steroid sulfatase (STS), which liberates estrogens from their biologically inactive sulfates, has been inadequately assessed and remains unclear. To evaluate the role of STS further, we transduced estrogen-dependent MCF-7 human breast cancer cells with a retroviral vector directing the constitutive expression of the human STS gene. Gene integration was confirmed by Southern hybridization, production of the appropriately sized messenger RNA by Northern hybridization, and expression of functional protein by metabolism of [(3)H]estrone sulfate to [(3)H]estrone. Maximum velocity estimates of estrone formation are 64.2 pmol estrone/mg protein.h in STS-transduced cells (STS Clone 20), levels comparable to those seen in some human breast tumors. Lower levels of endogenous activity are seen in MCF-7 cells (13.0 pmol estrone/mg protein.h) and in cells transduced with vector lacking the STS gene (Vector 3 cells; 12.0 pmol estrone/mg protein.h). 17beta-Estradiol sulfate induces expression of the progesterone receptor messenger RNA only in STS Clone 20 cells, whereas estrone sulfate produces the greatest stimulation of anchorage-independent growth in these cells. STS Clone 20 cells retain responsiveness to antiestrogens, which block the ability of estrogen sulfate to increase the proportion of cells in both the S and G(2)/M phases of the cell cycle. Consistent with these in vitro observations, only STS Clone 20 cells exhibit a significant increase in the proportion of proliferating tumors in nude ovariectomized mice supplemented with 17beta-estradiol sulfate. The primary activity in vivo appears to be from intratumor STS, rather than hepatic STS. Surprisingly, 17beta-estradiol sulfate appears more effective than 17beta-estradiol when both are administered at comparable concentrations. This effect, which is seen only in STS Clone 20 cells, may reflect differences in the cellular pharmacology of exogenous estrogens compared with those released by the activity of intracellular STS. These studies directly demonstrate that intratumor STS activity can support estrogen-dependent tumorigenicity in an experimental model and may contribute to the promotion of human breast tumors.

A further justification for the negligence rule

We use a principal-agent framework to reexamine the implications of the negligence and strict liability rules when the tortfeasor is an agency. We assume a unilateral care situation and consider both the cases of moral hazard and of adverse selection. In both instances the negligence rule is shown to Pareto dominate the strict liability rule when the activity level is exogenously given. We find a simple condition that guarantees that the result extends to an endogenous activity level. We also examine the case where this condition is not satisfied.

Diet and carbohydrate digestion in the yellow-spotted longicorn beetle Psacothea hilaris

Larval and adult Psacothea hilaris feed on mulberry wood and leaves, respectively. High levels of endogenous activity against the major dietary carbohydrates, cellulose, hemicellulose, starch and soluble sugars were secreted in the gut of larvae and adults. Activity against pectin was also high and multiple polygalacturonase (EC 3.2.1.15) components were secreted in the gut of larvae. One glycanase component, β-EG1, which was primarily an endo- β-1,4-glucanase (EC 3.2.1.4) and another, β-EG2, which was mostly an endo- β-1,4-xylanase (EC 3.2.1.8), were also secreted, while at least four additional components hydrolysed laminarin, lichenin and crystalline cellulose. The β-glycosidase component β-GD1 was associated with most of the β-mannosidase (EC 3.2.1.25) and β-xylosidase (EC 3.2.1.37) activity secreted in the gut of larvae, while another, β-GD2, was a β-glucosidase (EC 3.2.1.21), the activity of which was directed against cellobiose and other β-linked disaccharides, and a β-fucosidase (EC 3.2.1.38). A β-galactosidase (EC 3.2.1.23), which did not hydrolyse lactose, was also secreted, as were distinct β- N-acetylhexosaminidase (EC 3.2.1.52), trehalase (EC 3.2.1.28), α- L-arabinosidase (EC 3.2.1.55), α-galactosidase (EC 3.2.1.22) and a minimum of four α-glucosidase (EC 3.2.1.20) components, one of which was also likely to be associated with a peak of α-mannosidase (EC 3.2.1.24) activity. The α-glucosidase components varied in their specificity for α-linked disaccharides, but none was active against sucrose, which was hydrolysed by a β-fructofuranosidase (EC 3.2.1.26) component. Overall average levels of activity in larvae were twice those of adults, but the secretion of individual carbohydrases in both was not regulated in response to the relative abundance of particular carbohydrate components in their respective diets.

Age‐ and Tissue‐specific regulation of chicken inorganic pyrophosphatase

The activities and hormone regulatory patterns of inorganic pyrophosphatase (PPiase) of male Rhode Island Red (RIR) chickens were studied at two different postnatal ages (5- and 90-day). The endogenous activity level was found to be significantly higher in the immature (5-day) groups for all the tissues (liver, kidney and brain) studied as compared to that of mature (90-day). Hydrocortisone (HC) administration significantly inhibited the PPiase activity in the immature chicken liver and did not affect the enzyme in kidney and brain at either age. In contrast, insulin increased significantly the activity of PPiase in the liver of immature chicken. Kidney PPiase, however, was unaffected to insulin treatment at immature age, while it showed increased activity in mature group. On the other hand, brain PPiase activity was significantly increased at both the ages studied. These findings indicate an age- and tissue-specific regulation of PPiase activity by hydrocortisone and insulin in chicken.

Inhibition of SKF 89976–A of the y–aminobutyric acid release from primary neuronal chick cultures

Neuronal cultures were made from the 8-d-old embryonic chick telencephalon. The primary culture model was further improved, the medium composition was modified, and the cells grown for 10 d, which allowed the development of relatively differentiated neurones. A superfusion protocol was developed and applied to study the release of [3H]-gamma-aminobutyric acid ([3H]GABA). High endogenous activity levels of glutamate decarboxylase (GAD) and of a Ca-dependent potassium stimulated [3H]GABA release were used as criteria for GABAergic differentiation. The influence of the non-substrate inhibitor of GABA transport, SKF 89976-A, on the GABA release, was studied using the primary neuronal culture. The release was found to be inhibited by SKF 89976-A at higher concentrations (= 400 microM).

Defective differentiation of natural killer cells in SJL mice. Role of the thymus.

As previously shown, three distinct phenotypes exist in murine natural killer (NK) cell activity when it is evaluated by the endogenous levels of activity and the susceptibility to augmentation by interferon (IFN) and IFN inducers. The "low" phenotype has low levels of activity which can be poorly augmented by IFN, as in mice of SJL strain. The "inducible" phenotype exhibits low endogenous levels but can vigorously respond to IFN-mediated augmentation, as in A.SW strain. The "high" NK phenotype shows high levels of endogenous activity which can be augmented to still higher levels by IFN, as in B10.S mice. Since SJL mice with congenital absence of the thymus (nude) were of the inducible type, the effect of neonatal thymectomy was examined in the present study. Neonatal thymectomy was found to convert the low phenotype of SJL mice to the inducible, mimicking the effect of nu/nu genotype. Thymectomy as late as 25 days after birth was effective, but retransplantation of a syngeneic newborn or adult thymus, or thymocytes, failed to reverse the effect of thymectomy. The poor responsiveness of NK activity to IFN in SJL, therefore, is extrinsic to the NK cell lineage and is attributable to suppression or maturational block of NK cell differentiation by the thymus during the first few weeks of neonatal life. A series of experiments with bone marrow chimeras showed that the SJL recipients did not allow the expression of inducible or high phenotype by bone marrow progenitors from allogeneic donors with either phenotype. Therefore, the SJL recipients provide an environment which suppresses not only the development of IFN-sensitive NK cell precursors, but also the levels of endogenous NK cell activity. SJL bone marrow cells gave rise to NK activity of inducible phenotype in B10.S recipients, confirming the crucial role of the environment in which NK cell differentiation takes place.

Initiation of Protein Synthesis in Vitro by a Clostridial System: I. SPECIFICITY IN THE TRANSLATION OF NATURAL MESSENGER RIBONUCLEIC ACIDS

Abstract The homologous ribosomal systems from Escherichia coli and Clostridium pasteurianum were compared for their ability to translate f2 RNA, formaldehyde-treated f2 RNA, T4 early messenger RNA, E. coli messenger RNA, and C. pasteurianum messenger RNA in protein synthesis assays in vitro. The E. coli ribosomal system translated all five of these messengers, while the C. pasteurianum ribosomal system translated only the C. pasteurianum messenger RNA. The two ribosomal systems also had different characteristic magnesium profiles and exhibited different levels of endogenous activity in the protein synthesis assays. The messenger RNA responsible for the C. pasteurianum endogenous activity was shown to occur in the salt-washed ribosomes and not in the initiation factor fraction. The formaldehyde-treated f2 RNA and the C. pasteurianum messenger RNA exhibited different characteristics from the other types of messenger RNAs in their behavior with the E. coli ribosomal system in the protein synthesis assays. The two homologous ribosomal systems were also tested for their ability to bind [14C]formyl-Met-tRNA in response to different types of mRNA. The E. coli system bound fMet-tRNA in response to synthetic poly(A, G, U), f2 RNA, T4 early mRNA, and C. pasteurianum mRNA. The C. pasteurianum ribosomal system bound fMet-tRNA in response to poly(A, G, U) and C. pasteurianum mRNA but not f2 RNA or T4 early mRNA.

Particulate glucan synthetase activity: Generation and inactivation after treatments with indoleacetic acid and cycloheximide

Growing regions from epicotyls of Pisum sativum L. var Alaska contain a particulate enzyme which transfers glucose from guanosine diphosphate glucose to alkali-soluble and -insoluble products (glucan synthetase activity). When the epicotyl is decapitated to remove the source of natural hormone, the tissue below ceases growth and loses synthetase activity as well as the capacity to continue forming cellulose in vivo. If indoleacetic acid (IAA) is added to the cut apex, massive amounts of cellulose are deposited in the next few days. Particulate glucan synthetase activity is either maintained or greatly increased depending on whether endogenous activity levels are relatively high or low at the time of hormone addition. These effects appear to be due in part to IAA-dependent generation of a protein essential for synthetase activity since they are severely inhibited by concentrations of cycloheximide which are effective at preventing protein synthesis. Nevertheless, the addition of cycloheximide alone to the epicotyl reduces the rate of disappearance of synthetase activity, i.e., a protective effect. Also, a soluble thermolabile component is present in the aging epicotyl which promotes loss of synthetase activity when added to the particulate enzyme in vitro. Accordingly, turnover of pea glucan synthetase activity may be controlled in part by an inactivating protein which is itself subject to turnover.

Effect of isoniazid on mycobacterial polyphenylalanine synthesis

1. 1. A cell-free system from Mycobacterium smegmatis ATCC-607 capable of polypeptide synthesis is described. Although the system exhibited a low level of endogenous activity, it showed a marked stimulation in phenylalanine incorporation mediated by polyuridylic acid (100 μg) at 15 mM magnesium. 2. 2. In such a system, addition of isoniazid was found to alter the translation rate of polyuridylic acid stimulation. 3. 3. Evidence is presented showing that variation in the synthesis of polyphenylalanine in the presence of isoniazid is due to an effect on the rate of charging of the aminoacyl-tRNA.

Protein synthesis in erythroid cells III. Monoribosome and polyribosome function in the cell-free system

The capacity of reticulocyte polyribosomes of various size and of 80-S ribosomes to synthesize protein were examined before and after dialysis of ribosomes against buffer solutions free of Mg 2+. Following dialysis, polyribosomes are partly dissociated with a shift in the maximum endogenous capacity to form protein to polyribosomes of smaller size. Evidence is presented that 80-S ribosomes are heterogeneous with respect to (a) endogenous capacity to synthesize protein, and (b) response to poly-U. 80-S ribosomes were recovered from reticulocytes with little or no endogenous activity and little activity after addition of poly-U. Dialysis yielded 80-S ribosomes with higher levels of endogenous activity and 80-S ribosomes more responsive to poly-U. Dialyzed 80-S ribosomes were competent to synthesize protein with labeled peptides and a molecular weight corresponding to that of globin, without detectable polyribosome formation. In contrast, poly-U formed polyribosomes with 80-S ribosomes.