scholarly journals Dynamics of keratin assembly: exogenous type I keratin rapidly associates with type II keratin in vivo

1993 ◽  
Vol 122 (1) ◽  
pp. 123-135 ◽  
Author(s):  
RK Miller ◽  
S Khuon ◽  
RD Goldman
Keyword(s):  
Mammalian Cells ◽  
Close Association ◽  
Immunogold Electron Microscopy ◽  
Type I ◽  
Post Injection ◽  
Type Ii ◽  
Rapid Formation ◽  
Double Label ◽  
Keratin Intermediate Filaments

Keratin intermediate filaments (IF) are obligate heteropolymers containing equal amounts of type I and type II keratin. We have previously shown that microinjected biotinylated type I keratin is rapidly incorporated into endogenous bundles of keratin IF (tonofilaments) of PtK2 cells. In this study we show that the earliest steps in the assembly of keratin subunits into tonofilaments involve the extremely rapid formation of discrete aggregates of microinjected keratin. These are seen as fluorescent spots containing both type I and type II keratins within 1 min post-injection as determined by double label immunofluorescence. These observations suggest that endogenous type II keratin subunits can be rapidly mobilized from their endogenous state to form complexes with the injected type I protein. Furthermore, confocal microscopy and immunogold electron microscopy suggest that the type I-type II keratin spots from in close association with the endogenous keratin IF network. When the biotinylated protein is injected at concentrations of 0.3-0.5 mg/ml, the organization of the endogenous network of tonofilaments remains undisturbed during incorporation into tonofilaments. However, microinjection of 1.5-2.0 mg/ml of biotinylated type I results in significant alterations in the organization and assembly state of the endogenous keratin IF network soon after microinjection. The results of this study are consistent with the existence of a state of equilibrium between keratin subunits and polymerized keratin IF in epithelial cells, and provide further proof that IF are dynamic elements of the cytoskeleton of mammalian cells.

scholarly journals Activation of a mammalian origin of replication by chromosomal rearrangement.

1992 ◽  
Vol 12 (6) ◽  
pp. 2804-2812 ◽  
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Chromosomal Rearrangement ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Replication Initiation ◽  
Type I ◽  
Type Ii ◽  
Direct Demonstration

The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.

scholarly journals Autophagy as a modulator of cell death machinery

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Masayuki Noguchi ◽  
Noriyuki Hirata ◽  
Tsutomu Tanaka ◽  
Futoshi Suizu ◽  
Hiroshi Nakajima ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Survival ◽  
Mammalian Cells ◽  
Critical Parameter ◽  
Type I ◽  
Type Ii ◽  
Critical Question ◽  
A Cell ◽  
Underlying Mechanisms

Abstract The balance between cell death and survival is a critical parameter in the regulation of cells and the maintenance of homeostasis in vivo. Three major mechanisms for cell death have been identified in mammalian cells: apoptosis (type I), autophagic cell death (type II), and necrosis (type III). These three mechanisms have been suggested to engage in cross talk with each other. Among them, autophagy was originally characterized as a cell survival mechanism for amino acid recycling during starvation. Whether autophagy functions primarily in cell survival or cell death is a critical question yet to be answered. Here, we present a comprehensive review of the cell death-related events that take place during autophagy and their underlying mechanisms in cancer and autoimmune disease development.

scholarly journals Activation of a mammalian origin of replication by chromosomal rearrangement

1992 ◽  
Vol 12 (6) ◽  
pp. 2804-2812
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Chromosomal Rearrangement ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Replication Initiation ◽  
Type I ◽  
Type Ii ◽  
Direct Demonstration

The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.

A delayed rectifier conductance in type I hair cells of the mouse utricle

1996 ◽  
Vol 76 (2) ◽  
pp. 995-1004 ◽  
Author(s):  
A. Rusch ◽  
R. A. Eatock
Keyword(s):  
Hair Cells ◽  
Time Course ◽  
Dissociation Constants ◽  
Resting Potential ◽  
Delayed Rectifier ◽  
Type I ◽  
Type Ii ◽  
Voltage Range ◽  
Average Maximum

1. Membrane currents of hair cells in acutely excised or cultured mouse utricles were recorded with the whole cell voltage-clamp method at temperatures between 23 and 36 degrees C. 2. Type I and II hair cells both had delayed rectifier conductances that activated positive to -55 mV. 3. Type I, but not type II, hair cells had an additional delayed rectifier conductance (gK,L) with an activation range that was unusually negative and variable. At 23-25 degrees C, V(1/2) values ranged from -88 to -62 mV in 57 cells. 4. gK,L was very large. At 23-25 degrees C, the average maximum chord conductance was 75 +/- 65 nS (mean +/- SD, n = 57; measured at -54 mV), or approximately 21 nS/pF of cell capacitance. 5. gK,L was highly selective for K+ over Na+ (permeability ratio PNa+/PK+:0.006), but unlike other delayed rectifiers, gK,L was significantly permeable to Cs+ (PCs+/PK+:0.31). gK,L was independent of extracellular Ca2+. 6. At -64 mV, Ba2+ and 4-aminopyridine blocked gK,L with apparent dissociation constants of 2.0 mM and 43 microM, respectively. Extracellular Cs+ (5 mM) blocked gK,L by 50% at -124 mV. Apamin (100 nM) and dendrotoxin (10 nM) has no effect. 7. The kinetic data of gK,L are consistent with a sequential gating model with at least two closed states and one open state. The slow activation kinetics (principal time constants at 23-25 degrees C:600-200 ms) had a thermal Q10 of 2.1. Inactivation (Q10:2.7) was partial at all temperatures. Deactivation followed a double-exponential time course and had a Q10 of 2.0. 8. At 23-25 degrees C, gK,L was appreciably activated at the mean resting potential of type I hair cells (-77 +/- 3.1 mV, n = 62), so that input conductances were often more than an order of magnitude larger than those of type II cells. If these conditions hold in vivo, type I cells would produce unusually small receptor potentials. Warming the cells to 36 degrees C produced parallel shifts in gK,L's activation range (0.8 +/- 0.3 mV/degrees C, n = 8), and in the resting potential (0.6 +/- 0.3 mV/degrees C, n = 4). Thus the high input conductances were not an artifact of unphysiological temperatures but remained high near body temperature. It remains possible that in vivo gK,L's activation range is less negative and input conductances are lower; the large variance in the voltage range of activation suggests that it may be subject to modulation.

scholarly journals Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei

2006 ◽  
Vol 189 (3) ◽  
pp. 807-817 ◽  
Author(s):  
Narisara Chantratita ◽  
Vanaporn Wuthiekanun ◽  
Khaemaporn Boonbumrung ◽  
Rachaneeporn Tiyawisutsri ◽  
Mongkol Vesaratchavest ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Vaccine Development ◽  
Colony Morphology ◽  
Phenotypic Switching ◽  
Type I ◽  
Type Ii ◽  
Altered Expression ◽  
Replication Fitness

ABSTRACT Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42°C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.

Dye coupling between rat striatal neurons recorded in vivo: compartmental organization and modulation by dopamine

1994 ◽  
Vol 71 (5) ◽  
pp. 1917-1934 ◽  
Author(s):  
S. P. Onn ◽  
A. A. Grace
Keyword(s):  
Intravenous Administration ◽  
D2 Receptor ◽  
Type I ◽  
Type Ii ◽  
Dye Coupling ◽  
Striatal Neurons ◽  
Receptor Stimulation ◽  
D2 Agonist ◽  
Response Profile

1. The presence of dye coupling between striatal neurons was investigated using in vivo intracellular recording and dye injection in adult rats. In 17% of the cases in which a single striatal neuron was injected with Lucifer yellow, more than one labeled neuron was recovered. In control rats, this dye coupling was observed only between single pairs of medium spiny neurons and only when the neuron injected exhibited the Type II response profile as defined by paired-pulse stimulation of corticostriatal afferents. 2. After intravenous administration of the D1/D2 agonist apomorphine at a behaviorally effective dose (i.e., 0.1–0.3 mg/kg), an increase in the incidence (from 17% to 82% of injected cells) and extent (from 2 cells to 3–7 cells labeled per injection) of dye coupling was observed. This effect was mediated by D2 receptor stimulation because administration of the D2 agonist quinpirole caused similar alterations in the incidence and extent of dye coupling (66% coupled). In contrast, administration of the D1 agonist SKF 38393 or the D1 antagonist SCH 23390 did not result in any significant alteration in dye coupling. 3. In control rats, the entire somatodendritic regions of dye-coupled neurons were found to be localized within single matrix compartments of the striatum. However, after intravenous administration of apomorphine or quinpirole, clusters of dye-coupled neurons were found to extend across the patch/matrix boundary. Moreover, dye coupling was observed after injecting cells exhibiting either the Type I or the Type II response profile. 4. In response to D2 receptor stimulation, both the extent and the pattern of coupling between striatal neurons is altered, resulting in direct coupling between neurons that are otherwise functionally and anatomically segregated in the control animal.

scholarly journals LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor

2019 ◽  
Vol 47 (12) ◽  
pp. 6369-6385
Author(s):  
Jia-Yi Fan ◽  
Qian Huang ◽  
Quan-Quan Ji ◽  
En-Duo Wang
Keyword(s):  
Tertiary Structure ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Type I ◽  
Type Ii ◽  
Specific Domain ◽  
Recognition Mechanism ◽  
Variable Loop

Abstract Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

scholarly journals Fucoidan from Ascophyllum nodosum Suppresses Postprandial Hyperglycemia by Inhibiting Na+/Glucose Cotransporter 1 Activity

Marine Drugs ◽  
2020 ◽  
Vol 18 (9) ◽  
pp. 485
Author(s):  
Xindi Shan ◽  
Xueliang Wang ◽  
Hao Jiang ◽  
Chao Cai ◽  
Jiejie Hao ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Leptin Receptor ◽  
Specific Binding ◽  
Fasting Blood Glucose ◽  
Postprandial Hyperglycemia ◽  
Oral Glucose ◽  
Type I ◽  
Type Ii ◽  
Everted Gut Sac

We previously demonstrated that fucoidan with a type II structure inhibited postprandial hyperglycemia by suppressing glucose uptake, but the mechanism remains elusive. Here, we aimed to assess whether the effect of glucose absorption inhibition was related to the basic structure of fucoidans and preliminarily clarified the underlying mechanism. Fucoidans with type II structure and type I structure were prepared from Ascophyllumnodosum (AnF) or Laminariajaponica (LjF) and Kjellmaniellacrassifolia (KcF), respectively. The effects of various fucoidans on suppressing postprandial hyperglycemia were investigated using in vitro (Caco-2 monolayer model), semi-in vivo (everted gut sac model), and in vivo (oral glucose tolerance test, OGTT) assays. The results showed that only AnF with a type II structure, but not LjF or KcF with type I structure, could inhibit the glucose transport in the Caco-2 monolayer and everted gut sac models. A similar result was seen in the OGTT of Kunming mice and leptin receptor-deficient (db/db) mice, where only AnF could effectively inhibit glucose transport into the bloodstream. Furthermore, AnF (400 mg/kg/d) treatment decreased the fasting blood glucose, HbA1c, and fasting insulin levels, while increasing the serum glucagon-like peptide-1 (GLP-1) level in obese leptin receptor-deficient (db/db) mice. Furthermore, surface plasmon resonance (SPR) analysis revealed the specific binding of AnF to Na+/glucose cotransporter 1 (SGLT1), which indicated the effect of AnF on postprandial hyperglycemia could be due to its suppression on SGLT1 activity. Taken together, this study suggests that AnF with a type II structure can be a promising candidate for hyperglycemia treatment.

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