MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

The structure of 180° supernumerary limbs and a hypothesis of their formation

The results of a detailed analysis of 100 supernumerary limbs generated by 180° ipsilateral rotation (on the same limb stump) of regeneration blastemas is presented. The limbs were analyzed in terms of their position of origin, frequency, cartilage structure by Victoria blue staining, and muscle structure by serial sections. Single, double, or triple supernumeraries can be produced at no unique position of origin, although the posterodorsal quadrant was preferred. Four classes of supernumerary limbs were generated by such operations—normal; double dorsal or double ventral; part normal/part mirror imaged; part normal/part inverted in approximately equal frequencies. After amputation of these supernumeraries the same muscle patterns are faithfully regenerated. A hypothesis to explain the production of these abnormal limbs is proposed based on the observed phenomenon of fusion of supernumerary blastemata, but their regenerative behaviour presents problems for current models of pattern formation. Similar results have been obtained with developing limb buds and the relation between development and regeneration is discussed.     

Electrophysiological evidence for the autoregulation of β-cell secretion by insulin

Electrophysiological studies of cultured rat pancreatic β-cells using intracellular microelectrodes show that exogenous insulin over the range of 0.1–10.0 μg/ml inhibits the electrical activity due to 27.8 mM glucose in a dose-related manner. This inhibitory effect is manifested by a mean increase of the membrane potential from about ?20 to ?30 mV and inhibition of the manner of cells impaled showing spike activity from 60 to less than 10%. The inhibitory influence of insulin is rapid occuring within 5 min for the highest level used. The results provide evidence for a negative feedback role of insulin in regulating its own release.     

Analysis of the effects of cesium ions on potassium channel currents in biological membranes

Cesium ions block potassium channels in biological membranes in a voltage dependent manner. For example, external cesium blocks inward current with little or no effect on outward current. Consequently, it produces a characteristic N-shaped current-voltage relationship. We have modeled this result by single file diffusion of ions in a narrow channel spanning the membrane with a special blocking site in the channel for cesium ions. The model enables us to make detailed comparisons of the effects of cesium on potassium channels in different types of biological membranes.     

Biological responses to overload training in endurance sports

Five subjects undertook 10 days of twice daily interval training sessions on a treadmill followed by 5 days of active recovery. On days 1, 6, 11, and 16 the subjects were required to undertake a test of submaximal and maximal work capacity on a treadmill combined with a performance test consisting of a run to exhaustion with the treadmill set at 18 km.h-1 and 1% gradient. Also on these days a pre-exercise blood sample was collected and analysed for a range of haematological, biochemical and immunological parameters. The subjects experienced a significant fall in performance on day 11 which had returned to pretraining levels on day 16. Serum ferritin concentrations were depressed significantly from pretraining concentrations at the conclusion of the recovery period while the expression of lymphocyte activation antigens (CD25+ and HLA-DR+) was increased both after the training phase and the recovery phase. The number of CD56+ cells in the peripheral circulation was depressed at the conclusion of the recovery period. Several parameters previously reported to change in association with overload training failing to reflect the decrease in performance experienced by subjects in this study, suggesting that overtraining may best be diagnosed through a multifactorial approach to the recognition of symptoms. The most important factor to consider may be a decrease in the level of performance following a regeneration period. The magnitude of this decreased performance necessary for the diagnosis of overtraining and the nature of an "appropriate" regeneration period are, however, difficult to define and may vary depending upon the training background of the subjects and the nature of the preceding training. It may or may not be associated with biochemical, haematological, physiological and immunological indicators. Individual cases may present a different range of symptoms and diagnosis of overtraining should not be excluded based on the failure of blood parameters to demonstrate variation. However, blood parameters may be useful to identify possible aetiology in each separate case report of over-training. An outstanding factor to emerge from this study was the difficulty associated with an objective diagnosis of overtraining and this is a possible reason why there have been new accounts of overtraining research in the literature.     

Cholecystokinin receptor mediated hydrolysis of inositol phospholipids in guinea pig gastric glands

CCK-octapeptide (CCK-8) (EC50 = 0.5 nM), in the presence of Li+, increased 3H-inositol phosphate (IP) accumulation in guinea pig gastric glands prelabeled with 3H-inositol. CCK-8 desulfate, human gastrin I and pentagastrin were much less potent than CCK-8. Antagonists of CCK receptors such as proglumide, dibutyryl-c-GMP and CBZ-Tyr (SO3H)-Met-Gly-Trp-Met-AspNH2 shifted the CCK dose response curve to the right. However, histamine (H1 and H2), cholinergic, substance P and alpha- and beta-adrenergic receptor antagonists had no effect on 3H-IP accumulation induced by CCK. The results suggest that CCK receptor activation in gastric glands leads to an enhanced breakdown of inositol phospholipids which may relate to calcium mobilization and pepsinogen secretion.     

Numerical abundance of myxomycetes (myxogastrids) in soils in the West of England

Abstract A most-probable-number technique was used to quantify the abundance of myxomycetes (myxogastrids) in soil samples taken from 23 'Sites of Special Scientific Interest' and one other site in the West of England. Associated organisms and soil conditions were also recorded. Sixteen of the 24 sites yielded myxomycete plasmodium-forming units (PFUs) in numbers averaging 230 cm⁻³ fresh soil. Where detectable in samples of grassland soil, the numbers ranged from 20–2750 cm⁻³, in woodland soil from 20–800 cm⁻³, and in sand dunes from 80–400 cm⁻³. Repeated sampling revealed changing numbers at single sites. The abundance of PFUs was correlated positively with numbers of soil amoebae, ciliates and nematodes, and with levels of magnesium and soil density, and negatively with organic matter content and levels of NH₄-nitrogen and phosphate. Each PFU probably corresponded to one or several uninucleate cells in the soil rather than to a plasmodium.     

Bronchodilatory properties of 2-decarboxy-2-hydroxymethyl prostaglandin E1

We evaluated in a double-blind study the bronchodilatory properties of 2-decarboxy-2-hydroxymethyl prostaglandin E₁ (PGE₁-carbinol), described recently as a nonirritant bronchodilator in animals. Fifteen asthmatic patients received by inhalation single doses of 1, 10, and 30 μg PGE₁-carbinol, 55 μg PGE₂, and placebo (10% ethanol in normal saline, which was also used as diluent for the PGs). Such pulmonary function tests as forced expiratory volume in 1 second, forced vital capacity, and maximal expiratory flow were monitored during 2 hours following inhalation of each compound. 10 and 30 μg PGE₁-carbinol produced significant but short-acting bronchodilation, similar to that caused by 55 μg PGE₂. One-third of the patients reported mild cough and throat irritation during and shortly after inhalation of 30 μg PGE₁-carbinol or 55 μg PGE₂. Placebo and 1 μg PGE₁-carbinol produced minimal side effects, but neither agent caused bronchodilation. In an adjunctive, unblinded trial, the same patients received 400 μg fenoterol. Fenoterol caused greater bronchodilation 15 and 30 minutes after inhalation than did the PGs in the double-blind study.     

The pathogenesis of trehalose dimycolate-induced interstitial pneumonitis: III. Evidence for a role for T lymphocytes

Trehalose dimycolate, a glycolipid component of the cell walls of mycobacteria, induces interstitial pneumonitis and alveolar hemorrhages in C57BL/6 and C57BL/10 mice. Homozygous nude (nu/nu) mice of these backgrounds are not susceptible to this form of pulmonary injury. However, after administration of T-lymphocyte-enriched spleen cell preparations from syngeneic donors, homozygous nude mice become susceptible to trehalose dimycolate. The observations suggest that production of pulmonary lesions by this mycobacterial component is dependent on T lymphocytes. While the mechanisms are still under study, we propose that trehalose dimycolate can function as an activator of T lymphocytes and that products of activated T cells are responsible for production of the pulmonary lesions.     

Effects of glucagon on the redox states of cytochromes in mitochondria in situ in perfused rat liver

The effects of glucagon on the respiratory function of mitochondria in situ were investigated in isolated perfused rat liver. Glucagon at the concentrations higher than 20 pM and cyclic AMP (75 microM) stimulated hepatic respiration, and shifted the redox state of pyridine nucleotide (NADH/NAD) in mitochondria in situ to a more reduced state as judged by organ fluorometry and beta-hydroxybutyrate/acetoacetate ratio. The organ spectrophotometric study revealed that glucagon and cyclic AMP induced the reduction of redox states of cytochromes a(a3), b and c+c1. Atractyloside (4 micrograms/ml) abolished the effects of glucagon on these parameters and gluconeogenesis from lactate. These observations suggest that glucagon increases the availability of substrates for mitochondrial respiration, and this alteration in mitochondrial function is crucial in enhancing gluconeogenesis.