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Results for “proximity labeling”

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PubMed1977

The digestion and transmission of labelled immunoglobulin G by enterocytes of the proximal distal regions of the small intestine of young rats.

Density gradient centrifugation of samples prepared from proximal gut homogenates after intra-lumenal injection of 125I-labelled IgG, was used to prepare batches of IgG fragments according to sedimentation coefficients. 2. Ultrafiltration was employed to partition the radioactivity in the vascular compartments, viscera and carcasses of rats aged 14-15 days, 2 hr after the injection of standard doses of labelled IgG into the proximal and distal regions of the small intestine. 3. Radioactive samples prepared by these methods were re-introduced into young rats by intra-cardiac injection, and the rate at which they were removed from the vascular compartment was assessed. 4. Proximal enterocytes transmitted about 39% of the IgG which had been removed from the intestine in intact form. Most of this was retained in the vascular compartment; they degraded up to about 57% of the total removed into fragments less than 1000 mol. wt. and about 4% into intermediate sized fragments. 5. Distal enterocytes degraded almost 90% of the IgG processed into fragments less than 1000 mol. wt., about 8% as fragments greater than 100,000 mol. wt. 6. Fragments, of all sizes, were cleared rapidly from the circulation into the viscera and carcass. 7. The relevance of these results to protein transmission and digestion by the rat small intestine is discussed.

Animals↗

PubMed2023

Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.

The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

Mycobacterium tuberculosis↗

PubMed1979

Axonal transport in early experimental diabetes.

In the present study fast axonal transport was examined in streptozotocin rats with 4 weeks duration in diabetes. Tritiated leucine and 14C-labelled glucosamine were injected into the fifth lumbar ganglion and TCA-soluble as well as insoluble activity were measured in segments of the sciatic nerve at various time intervals. (1) Time from injection until start of fast axonal transport was prolonged in diabetic rats whereas anterograde transport velocity was unchanged. (2) Incorporation of labelled leucine was reduced by 40%, whereas labelled glucosamine incorporation was unchanged. (3) Alterations observed in accumulations of labelled glycoconjugates proximal and distal to a collection crush might represent a decreased amount of retrograde transported material. The changes found in protein and glycoconjugate synthesis and transport could be related to the early reduction in axon calibre and conduction velocity in peripheral nerve of streptozotocin-diabetic rat.

Animals↗

PubMed2026

Paradoxical non-catalytic kinase functions are driven by inhibitor-induced displacement of autoinhibitory domains.

ATP-competitive kinase inhibitors represent one of the largest classes of targeted anti-cancer drugs. While their primary mechanism is to block catalytic activity, they can also trigger paradoxical phenotypic effects that cannot be explained by catalytic inhibition alone. These observations point to a hidden layer of drug action that modulates non-catalytic kinase functions via changes in kinase conformation and protein-protein interactions (PPIs). Here, we developed a multimodal proteomics approach combining limited proteolysis coupled mass spectrometry on affinity-purified samples (AP-LiP-MS), AP-MS, and proximity labeling-MS to map inhibitor-induced conformation and PPI changes. We show that inhibitor binding causes structural rearrangements in the autoinhibitory domains (AIDs) of all tested kinases, consistent with a transition to an open, active-like kinase conformation. These structural shifts drive distinct kinase-protein interaction changes that control non-catalytic functions: sequestration of AMPK by inhibited CAMKK2 blocks phosphorylation by other kinases, CHEK1 inhibition causes dissociation from the mitochondrial protein CLPB and leads to mitochondrial fragmentation, and structural changes in inhibited PRKCA trigger rapid relocalization to cell junctions. Thus, we identify the ATP-binding site as a major organizing center of kinase conformation and interaction. Our work suggests that these on-target, off-mechanism effects are likely to occur in other kinases as well, and provides the analytical framework to systematically characterize a frequently overlooked phenomenon highly relevant for understanding drug side effects to guide the development of novel therapeutics.

Protein Kinase Inhibitors↗

PubMed1975

Kidney tubular cell proliferation in burned mice.

Kidney tubular cell proliferation after a 17%, sublethal, third-degree burn injury in mice was studied in vivo using tritiated thymidine. Changes in cell morphology were also followed. Total incorporation of 3H-TdR in renal cells was measured and autoradiographic analyses of the tubular cells in the renal cortex and in the stratum subcorticale were made 2 hours to 30 days after the injury. After the second day postburn there was a 3.5-fold stimulation in the total incorporation of 3H-TdR in DNA, and an 18-fold increase in the labelling index of tubular cells. The majority of the labelled cells were proximal tubular cells. After the third day postburn a moderate increase in the labelling index (7.5-fold) was observed; 40% of the labelled cells were distal tubular cells. No morphological signs of frank necrosis of the tubular cells could be observed in the burned mice within 3 days postburn, which indicates that the stimulated proliferation of the kidney tubular cells was induced by minor reversible tubular cell damage during the first few hours postburn. It is suggested that the tubular cells loose their normal growth-inhibiting factors through an altered cell wall which results in an abnormal stimulation of cell growth 2 days later. These findings support the idea that there are abnormalities in the regulation of cell growth in several organs after burn injury; in some tissues--as in the kidney--this results in a temporary insufficiency of organ function.

Animals↗

PubMed1977

An autoradiographic study of macromolecular syntheses in the epithelium of the ductus epididymidis in the mouse. II. Incorporation of L-fucose-1-3H.

Glycoprotein dynamism in the mouse epididymis was studied by means ofhistoautoradiography after injection of L-fucose-1-3H. The label was detected, at thirty minutes p.i., in the area occupied by Golgi apparatus in the epithelial cells. At 4 h p.i. the label was already present inthe lumen of ductus epididymidis. At this time interval, the luminal labelling was highest in the initial segment of the epididymis and decreased against the more distal segments considerably. At ten days p.i. very high labelling was detected in the luminal contents in the terminal segment of the ductus epididymidis and in ductus deferens, the labelling in the proximal segments of the epididymis being much lower. These observations suggested a wave of labelled glycoprotein in epididymal plasma passing through the epididymis after a fucose pulse. Higher labelling was detected in so-called "clrial was seen in epididymal and uterine spermatozoa, mostly in sperm tail region.

Animals↗

PubMed1979

Asymmetric labeling of amino lipids in liposomes.

Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H+ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin-K+ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents. When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching. These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surface, monitor vesicular fusion, and assess liposomal structure.

Amines↗

PubMed1978

Pyrene excimer fluorescence in rabbit skeletal alphaalphatropomyosin labeled with N-(1-pyrene)maleimide. A probe of sulfhydryl proximity and local chain separation.

Rabbit skeletal alphaalphatropomyosin was specificially labeled at cysteine 190 with the fluorescent reagent, N-(1-pyrene)maleimide. Spectroscopically different products were obtained by labeling at pH 6.0 (PyrI-alphaalphaTm) or pH 7.5 (PyrII-alphaalphaTm). PyrII-alphaalphaTm results from a secondary reaction between the N-(1-pyrene)succinimido moiety at cysteine 190 of PyrI-alphaalphaTm and a lysine group on the same chain, probably lysine 189. Pyrene excimer fluorescence was present in the native state but absent in the unfolded state of both products, thus verifying the proximity of the--SH groups and the chain register model for the structure of tropomyosin. Studies of the guanidinium chloride-dependent unfolding of PyrII-alphaalphaTm showed that loss of excimer fluorescence precedes unfolding, providing evidence for a region of preferential instability in the molecule near cysteine 190. This work suggests that N-(1-pyrene)maleimide could be used to probe both--SH proximity and local conformation in any protein if the presence of two or more proximal--SH groups is suspected.

Animals↗

PubMed1978

Two-dimensional gel electrophoresis of proteins in rapid axoplasmic transport.

Two-dimensional electrophoresis of proteins has allowed high resolution analysis of the protein species rapidly transported in the frog sciatic nerve. The 7th, 8th and 9th dorsal root ganglia were selectively labeled with [3H]leucine or [35S]methionine in one compartment of a lucite chamber. Transport of TCA-precipitable material was monitored in the spinal roots and sciatic nerve kept in another compartment. Fastest transport rates were 75-90 mm/day at 18 decrees C. Ligation of the nerve 30 mm distal to the 8th ganglion at the beginning of the experiment resulted in accumulation of label during a 24 h period. This material was subjected to two-dimensional electrophoresis (pI 5-8; mol.wt. 10(4)-10(5) daltons) in 3 mm nerve segments. Autoradiographs or fluorographs from segments proximal to the ligature yielded a pattern of about 140 spots. Of these, at least 60 were considered to be independent protein species. Neither actin nor tubulin were present among these rapidly-transported, labeled proteins. No pattern was observed from segments distal to the ligature. Blocking protein synthesis with 18 micrometer anisomycin reduced the accumulation of label proximal to the ligature by 98%. Direct labeling of nerve segments produced patterns significantly different from the pattern of transported proteins.

Animals↗

PubMed1976

Local reflex in microcirculation in human cutaneous tissue.

Blood flow in cutaneous tissue measured by the local 133Xenon washout technique decreased about 35 per cent during venous stasis of 40 mmHg in three normal subjects. The response was unaffected by block of the nerve three cm proximally to the labeled area. When the tissue was infiltrated with lidocaine or with phentolamine, blood flow remained constant, indicating that the decrease in blood flow is due to an arteriolar vasoconstrictor response to increase in venous transmural pressure. Local venous stasis elicited a vasoconstrictor response in an adjoining area not affected by the stasis. The response was blocked by lidocaine applied to the side of stasis. In 2 chronically sympathectomized patients, the vasoconstrictor response was abolished in the denervated limbs but present on the non-operated side (1 patient with unilateral sympathectomy). The results indicate that the vasoconstrictor response to an increase in venous transmural pressure is due to a local nervous mechanism involving sympathetic adrenergic fibres, most likely a sympathetic axon reflex.

Blood Pressure↗

PubMed1977

Formation and turnover of plasma membrane glycoproteins in kidney tubules of young rats and adult mice, as shown by radioautography after an injection of 3H-fucose.

The formation and turnover of the glycoproteins of the plasma membrane have been investigated by quantitative radioautography in the kidney tubules of young rats and adult mice killed at various time intervals after an intravenous injection of 3H-fucose. In young (40 g) rats killed five to ten minutes after the injection, radioautographs of distal tubule cells show that the Golgi apparatus contained about 85% of the cell label. By 30 hours, only 8% of the label remained in this organnele, whereas 67% was in the plasma membrane, indicating that most of the label had migrated from Golgi apparatus to this membrane. Similarly, in proximal tubule cells, about 82% of the label was initially in the Golgi apparatus, but less than 2% remained at 30 hours, at which time 78% was in the plasma membrane. In the latter cells, the apical tubules and vacuoles became heavily labeled before the apical microvilli did and, therefore, may be involved in the transit of label from the Golgi apparatus to the microvillous membrane. The results are interpreted to mean that, in kidney tubule cells, the Golgi apparatus is the site of a continuous incorporation of fucose into glycoproteins and that these migrate to the plasma membrane. In fully formed cells, such a conclusion would imply a continuous turnover of plasma membrane glycoproteins. However, in the rapidly growing kidney of young rats many new cells are added daily, the growth of which might involve net addition as well as turnover of glycoproteins. Accordingly, the experiment has been repeated in adult mice, in which the cells are assumed to be fully formed. Furthermore, since turnover implies eventual decrease of incorporated label, some of the animals have been killed at longer intervals, up to 27 days after injection. In these adult mice, as in young rats, prompt Golgi uptake and subsequent migration of label to the plasma membrane were observed in distal and proximal tubules cells. With time the label content of the plasma membrane decreased gradually, and by 27 days had virtually disappeared. From grain counts, it is concluded that the mean half-life of glycoproteins in the apical membrane of distal tubule cells is about two days, whereas in both the apical and basal membranes of proximal tubule cells, it is slightly over three days.

Age Factors↗

PubMed1976

Studies on the control of myelinogenesis. II. Evidence for neuronal regulation of myelin production.

Tritiated thymidine has been used as a nuclear marker to trace the origin of Schwann cells, sited in the distal stump of a severed unmyelinated nerve, which are able to elaborate myelin around axons regenerating from an anastomosed proximal stump of a severed myelinated nerve. Two types of cross-anastomosis experiments were performed in young, adult rats: (1) the proximal stump of a myelinated sternohyoid nerve was labeled (5 mCi/kg body weight) selectively over a 4-day period of predetermined maximal thymidine uptake and two days later, after flushing the animal repeatedly with cold thymidine, the unmyelinated cervical sympathetic trunk was transected and its unlabeled distal stump linked to the proximal stump of the labeled sternohyoid nerve; (2) the distal stump of an unmyelinated cervical sympathetic trunk was labeled selectively over a 5-day period of predetermined maximal uptake and two days later, after flushing with cold thymidine, the myelinated sternohyoid nerve was severed and its unlabeled proximal stump linked to the labeled distal stump of the cervical sympathetic trunk. The fate of the labeled cells in each type of anastomosis was determined 3 weeks later by autoradiography and liquid scintillation spectrometry. In the first type, a small amount of label had migrated from proximal stumps but labeled Schwann cells were not found in successfully anastomosed distal stumps. In the second type, labeled Schwann cells were seen in the cervical sympathetic trunk in association with myelinated and non-myelinated axons regenerating from the sternohyoid nerve. These data suggest that the presence or absence of myelin formation by a Schwann cell is controlled by some property of the axon with which it is associated. Putative mechanisms underlying neuronal control of myelinogenesis are discussed.

Animals↗

PubMed1978

[Emptying of the stomach after selective proximal vagotomy (author's transl)].

Cream cheese containing 14C-labelled glutaminic acid was given to patients after proximal selective vagotomy and 14CO2 measured in the expired air in order to test the gastric emptying process. Results do not allow exact quantitative evaluation, however definite changes of gastric emptying for up to 6 month after surgery could be demonstrated.

Carbon Radioisotopes↗

PubMed1979

Effect of extension of infarction on serial CK activity.

The effects of extension of myocardial infarction by reduction of regional myocardial blood flow (RMBF) to an ischemic region on serum CK activity was examined in 14 awake dogs. Initial infarction was effected by occlusion of the distal left circumflex coronary artery (LCCA) and subsequent extension was produced by occlusion of the proximal LCCA 6, 12 or 18 hours after distal occlusion. Extension was verified by serial measurements of RMBF using radioisotope-labeled microspheres before and after proximal occlusion. Serum CK activity increased initially 2-4 hours after distal coronary occlusion and then increased rapidly and reached peak values 12 hours after occlusion. When the infarction was extended at 6, 12 or 18 hours after initial occlusion, CK appearance was immediately reduced in the 6- and 12-hour experiments, but not in the 18-hour experiments. Extension of infarction at each interval caused delayed increases in CK activity beginning 2-5 hours after proximal occlusion, with peak values occurring 12 hours later. The immediate effects of extension of infarction by reducing blood flow on CK activity are a function of whether the infarcted myocardium continued to release CK, e.g., at 6 and 12 hours after occlusion, or CK release was completed, e.g., 18 hours. The immediate effects of extension of infarction were the result of perfusion on myocardium that is infarcted and continues to release CK, and do not necessarily indicate alterations in the extent of myocardial injury. The delayed effects of proximal and distal occlusion on CK activity were comparable, suggesting that delayed and not immediate alterations in CK activity represent extension of infarction.

Acute Disease↗

PubMed1975

Gastric emptying following vagotomy and antrectomy and proximal gastric vagotomy.

Gastric emptying of solid meals labelled with 129Cs was studied in patients for up to one year after vagotomy and antrectomy or after proximal gastric vagotomy. Significant delay was found one month after vagotomy and antrectomy but this had returned to normal by six months. No delay was found after proximal gastric vagotomy. The effect of posture on gastric emptying was also studied in the same subjects. No significant differences were found between gastric emptying in the supine or sitting positions after solid meals.

Duodenal Ulcer↗

PubMed1978

3h-actinomycin-D binding to mitotic chromosomes of Drosophila melanogaster.

The binding of 3H-AMD to the metaphase chromosomes of Drosophila melanogaster has been analyzed after two different periods of exposure to photographic emulsion. The entirely heterochromatic Y chromosome was markedly less labelled than euchromatin and other heterochromatic regions. Moreover, the few grains present on the Y chromosome were clustered in two regions, one localized in the middle Ys and the other in the proximal third of YL. This labelling pattern is not affected by removing histones with a 2-hour treatment with 2N HCl. It is suggested that the specific underlabelling of the Y chromosome reflects a peculiar AT richness.

Animals↗

PubMed1978

Alterations in cell surface membrane components of adapting rat small intestinal epithelium. Studies with lectins after massive proximal jejunoileal resection and jejunoileal transposition.

After proximal resection or transposition of intestinal segments, the small intestinal mucosa of rats was examined with fluorescein-conjugated lectins derived from Ricinus communis and Triticum vulgare (wheat germ). These agglutinins are thought to be specific for the nonreducing end-terminal carbohydrate residues, beta-D-galactose and beta-N-acetyl-D-glucosamine, respectively. After 70% proximal jejunoilealal resection as well as transposition of ileal segments to jejunum, ileal villus cell surface labeling by both lectins was changed to a pattern characteristic of normal rat proximal intestine. Alterations were not detected proximal to the surgical anastomosis after massive resection or in the jejunal segments transposed to ileum. Although the labeling pattern of goblet cell mucin differed in proximal duodenum from the remainder of control small intestine in normal animals, no differences were detected in goblet cell mucin labeling between normal and resected or transposed animals. This study confirms the previously reported differential lectin-labeling patterns of the small intestinal crypt and villus cells and further emphasizes the differences between cell surface carbohydrate and secretory mucins of goblet cells. Furthermore, the changes induced in the cell surface-labeling pattern by massive proximal jejunoileal resection and transposition suggest that cell surface and secretory components may be altered differently during adaptation of the small intestine.

Adaptation, Physiological↗

PubMed1979

Effects of inhibitors in lumen on PAH and urate transport by isolated renal tubules.

Effects of the presence of unlabeled p-aminohippurate (PAH) or urate, probenecid, and phenol red in the lumen on labeled PAH or urate transport by isolated, perfused snake (Thamnophis spp.) proximal renal tubules were studied. Net secretion of labeled urate and luminal membrane permeability to urate were unaffected by the presence of unlabeled urate (up to 0.1 mM) or probenecid (up to 1.0 mM) in lumen only. The data are compatible with movement of urate from cells to lumen during urate secretion by a simple passive process. Net secretion of labeled PAH was rapidly and reversibly depressed to about 25-35% of control when unlabeled PAH (0.05 mM), phenol red (0.05 mM), or probenecid (0.1 mM) was added to the lumen only. During maximum depression of PAH transport, luminal membrane permeability to PAH was reduced by 60-70%. The data suggest that movement of PAH from cells to lumen down an electrochemical gradient during PAH secretion occurs by a readily inhibited, mediated process.

Animals↗