scholarly journals Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides.

1985 ◽  
Vol 101 (5) ◽  
pp. 1826-1841 ◽  
Author(s):  
M Hatzfeld ◽  
W W Franke
Keyword(s):  
Type I ◽  
Type Ii ◽  
Different Types ◽  
Electrophoretic Techniques ◽  
Specific Pair ◽  
Chemical Cross Linking ◽  
Selection Of ◽  
Acidic Type

Cytokeratins are expressed in different types of epithelial cells in certain combinations of polypeptides of the acidic (type I) and basic (type II) subfamilies, showing "expression pairs." We have examined in vitro the ability of purified and denatured cytokeratin polypeptides of human, bovine, and rat origin to form the characteristic heterotypic subunit complexes, as determined by various electrophoretic techniques and chemical cross-linking, and, subsequently, intermediate-sized filaments (IFs), as shown by electron microscopy. We have found that all of the diverse type I cytokeratin polypeptides examined can form complexes and IFs when allowed to react with equimolar amounts of any of the type II polypeptides. Examples of successful subunit complex and IF formation in vitro include combinations of polypeptides that have never been found to occur in the same cell type in vivo, such as between epidermal cytokeratins and those from simple epithelia, and also heterologous combinations between cytokeratins from different species. The reconstituted complexes and IFs show stability properties, as determined by gradual "melting" and reassociation, that are similar to those of comparable native combinations or characteristic for the specific new pair combination. The results show that cytokeratin complex and IF formation in vitro requires the pairing of one representative of each the type I and type II subfamilies into the heterotypic tetramer but that there is no structural incompatibility between any of the members of the two subfamilies. These findings suggest that the co-expression of specific pair combinations observed in vivo has other reasons than general structural requirements for IF formation and probably rather reflects the selection of certain regulatory programs of expression during cell differentiation. Moreover, the fact that certain cytokeratin polypeptide pairs that readily form complexes in vitro and coexist in the same cells in vivo nevertheless show preferential, if not exclusive, partner relationships in the living cell points to the importance of differences of stabilities among cytokeratin complexes and/or the existence of extracytokeratinous factors involved in the specific formation of certain cytokeratin pairs.

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.

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.

External PS in Sickle RBC: Relationships among Aminophospholipid Translocase (APLT), Phospholipid Scramblase (PLSCR), Cell Maturity, and Cell Density.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 148-148
Author(s):  
Latorya E. Arnold ◽  
Mary B. Palascak ◽  
Clinton H. Joiner ◽  
Robert S. Franco
Keyword(s):  
Annexin V ◽  
Type I ◽  
Type Ii ◽  
Color Flow ◽  
Low Level ◽  
Aminophospholipid Translocase ◽  
Phospholipid Scramblase ◽  
High Level

Abstract External phosphatidylserine (PS) is present on some sickle RBC and may contribute to thrombogenesis, endothelial adhesion, and shortened RBC lifespan. Phospholipid scramblase (PLSCR) disrupts phospholipid (PL) asymmetry by causing nonspecific PL equilibration across the membrane. Aminophospholipid translocase (APLT) maintains PL asymmetry by returning externalized PS to the inner membrane leaflet. It has been proposed that both APLT inhibition and PLSCR activation are required for PS externalization. Sickle RBC with low level external PS (Type I PS+) are present in cells of all densities and include some reticulocytes. Sickle RBC with high external PS (Type II PS+) are primarily found in the dense fraction. Type II cells are thought to be more important because: the high level of external PS should have greater consequence; high level external PS occurs primarily in pathologically dehydrated sickle RBC; and low level external PS appears to be physiological in immature RBC. We have previously shown that dense, dehydrated sickle RBC, including the small number of dense transferrin receptor positive (TfR+) reticulocytes, have markedly inhibited APLT. In the current studies, we examined the relationships among external PS, APLT, PLSCR, and density in mature RBC and TfR+ reticulocytes using 3-color flow cytometry. APLT and PLSCR activities were assayed using fluorescent PL analogues (NBD-PS and NBD-PC, respectively), and expressed as the fraction of probe internalized. External PS was measured with Annexin V-PE and TfR+ reticulocytes were identified with anti-TfR-PE/Cy5. PS+ cells had lower APLT activity compared to PS- cells that did not reach significance for n=3 (NBD-PS internalization fraction for PS-: 0.586±0.053; Type I PS+: 0.517±0.158, Type II PS+: 0.523±0.033). PS- sickle RBC had a uniformly low PLSCR activity similar to normal RBC (NBD-PC internalization fractions ∼ 0.1). In mature sickle RBC, PLSCR was more active in PS+ cells (PS-: 0.097±0.096; Type I PS+: 0.163±0.070, Type II PS+: 0.248±0.043; n=3; PS- vs Type I PS+: p=0.06; PS- vs Type II PS+: p=0.04; Type I versus Type II: p=0.03). TfR+ reticulocytes had increased APLT and PLSCR activity compared to mature sickle RBC, but there was no apparent relationship between PLSCR and external PS. Since dense sickle RBC had markedly inhibited APLT, we evaluated the relationship between dehydration and APLT activity. Dehydration of AA RBC from an MCHC of 35.6±2.2 to 49.2±2.0 g/dL inhibited APLT (from 0.484±0.068 to 0.301±0.076; n=7, p= 0.01). Dehydration of SS RBC from an MCHC of 34.8±3.5 to 50.1±3.9 g/dL also inhibited APLT (from 0.460±0.060 to 0.361±0.047; n=3, p=0.006), but not as low as in SS RBC dehydrated in vivo (0.222±0.036 at 44.7±5.6 g/dL; n=4, p=0.007 vs. SS RBC dehydrated in vitro). Rehydration of AA and SS RBC that had been dehydrated in vitro reversed APLT inhibition. However, APLT activity was not reversed upon rehydration of sickle RBC dehydrated in vivo. In summary, our data show that: many dense sickle RBC with significantly inhibited APLT are PS-, indicating that APLT inhibition alone does not result in PS externalization; dehydration contributes to, but is not entirely responsible for, the APLT inhibition seen in dense sickle RBC; and PS+ sickle RBC have increased PLSCR activity.

scholarly journals Type I and Type II Interferons Inhibit the Translation of Murine Norovirus Proteins

2009 ◽  
Vol 83 (11) ◽  
pp. 5683-5692 ◽  
Author(s):  
Harish Changotra ◽  
Yali Jia ◽  
Tara N. Moore ◽  
Guangliang Liu ◽  
Shannon M. Kahan ◽  
...  
Keyword(s):  
Viral Replication ◽  
Small Animal ◽  
Type I ◽  
Type Ii ◽  
Murine Norovirus ◽  
Human Noroviruses ◽  
Interferon Responses ◽  
Replication Intermediates

ABSTRACT Human noroviruses are responsible for more than 95% of nonbacterial epidemic gastroenteritis worldwide. Both onset and resolution of disease symptoms are rapid, suggesting that components of the innate immune response are critical in norovirus control. While the study of the human noroviruses has been hampered by the lack of small animal and tissue culture systems, our recent discovery of a murine norovirus (MNV) and its in vitro propagation have allowed us to begin addressing norovirus replication strategies and immune responses to norovirus infection. We have previously demonstrated that interferon responses are critical to control MNV-1 infection in vivo and to directly inhibit viral replication in vitro. We now extend these studies to define the molecular basis for interferon-mediated inhibition. Viral replication intermediates were not detected in permissive cells pretreated with type I interferon after either infection or transfection of virion-associated RNA, demonstrating a very early block to virion production that is after virus entry and uncoating. A similar absence of viral replication intermediates was observed in infected primary macrophages and dendritic cells pretreated with type I IFN. This was not due to degradation of incoming genomes in interferon-pretreated cells since similar levels of genomes were present in untreated and pretreated cells through 6 h of infection, and these genomes retained their integrity. Surprisingly, this block to the translation of viral proteins was not dependent on the well-characterized interferon-induced antiviral molecule PKR. Similar results were observed in cells pretreated with type II interferon, except that the inhibition of viral translation was dependent on PKR. Thus, both type I and type II interferon signaling inhibit norovirus translation in permissive myeloid cells, but they display distinct dependence on PKR for this inhibition.

Development of geniculocortical axon arbors in a primate

1990 ◽  
Vol 5 (3) ◽  
pp. 291-309 ◽  
Author(s):  
S. L. Florence ◽  
V. A. Casagrande
Keyword(s):  
White Matter ◽  
Visual Experience ◽  
Striate Cortex ◽  
Growth Cones ◽  
Type I ◽  
Type Ii ◽  
Distinctive Features ◽  
Over Time

AbstractThe main objective of the present study was to describe the postnatal development of magnocellular and parvocellular LGN axons within the primate striate cortex. For this purpose, we bulk labeled axons in neonatal prosimians (galagos) in vivo or in vitro at regular intervals from birth (PO) to 12 weeks after birth by injecting horseradish peroxidase (HRP) into white matter anterior to the striate cortex. Filled axons within layer IV were reconstructed, quantitatively analyzed, and compared to a population of adult axons described previously (Florence & Casagrande, 1987).Our results show that although axons are morphologically immature at birth, they are restricted to the upper (IVα) and lower (IVβ) tiers of layer IV of the striate cortex as in adults. In adults, we referred to the presumed magnocellular LGN axons terminating in IVα as type I and the presumed parvocellular axons terminating in IVβ as type II. We used the same convention for developing axons.From birth to 3 weeks postnatal, type I and II axon classes are more variable in appearance than adult counterparts, and are not morphologically class distinct. As axons mature, parent axon shafts increase in caliber, arbors become smaller and more radial, and other immature features (e.g. spikes, protrusions, growth cones) are less evident. Both arbor classes mature slowly and some still exhibit immature features (e.g. growth cones) as late as 12 weeks postnatally. Although arbors do not show class-distinctive features until late in development, each class does show some unique maturational trends. Type I arbors are only slightly larger than adult counterparts at birth, whereas type II arbors are dramatically larger. Type I arbors increase in branch complexity with age, whereas type II arbors simply show a shift in complexity toward the center of the arbor with decreasing size over time. These growth trends suggest that magnocellular and parvocellular pathways to cortex could be differentially vulnerable to the manipulation of postnatal visual experience.

Erectile dysfunction in the type II diabetic db/db mouse: impaired venoocclusion with altered cavernosal vasoreactivity and matrix

2008 ◽  
Vol 294 (5) ◽  
pp. H2204-H2211 ◽  
Author(s):  
Ian P. Luttrell ◽  
Mei Swee ◽  
Barry Starcher ◽  
William C. Parks ◽  
Kanchan Chitaley
Keyword(s):  
Erectile Dysfunction ◽  
Erectile Function ◽  
Leptin Receptor ◽  
Science Studies ◽  
Type I Diabetes ◽  
Type I ◽  
Mrna Levels ◽  
Type Ii

The number of men with type II diabetes-associated erectile dysfunction (ED) continues to grow rapidly; however, the majority of basic science studies has examined mechanisms of ED in animal models of type I diabetes. In this study, we first establish an in vivo mouse model of type II diabetic ED using the leptin receptor mutated db/ db and wild-type control BKS mouse. Furthermore, we hypothesized that dual mechanistic impairments contribute to the impaired erectile function in the type II diabetic mouse, altered vasoreactivity, and venoocclusive disorder. In vivo erectile function was measured as intracavernosal pressure (ICP) normalized to mean arterial pressure (MAP) following electrical stimulation of the cavernosal nerve. Venoocclusion was assessed by the maintenance of elevated in vivo ICP following intracorporal saline infusion. Vasoreactivity of isolated cavernosum in response to contractile and dilatory stimulation was examined in vitro by myography. Collagen and elastin content were evaluated by quantification of hydroxyproline and desmosine, respectively, as well as by quantitative PCR and histological analysis of isolated cavernosum. Erectile function was significantly decreased in db/ db vs. BKS mice in a manner consistent with impairments in venoocclusive ability and decreased inflow. Heightened vasoconstriction and attenuated dilation in cavernosum of db/ db vs. BKS mice suggest an overall lowered relaxation ability and thus impaired filling of the cavernosal spaces. A decrease in desmosine and hydroxyproline as well as lowered mRNA levels for tropoelastin, fibrillin-1, and α1(I) collagen were detected. These vasoreactive and sinusoidal matrix alterations may alter tissue compliance dispensability, preventing the normal expansion necessary for erection.

Three Types of Hereditary Antiterombin III Deficiency

1979 ◽  
Author(s):  
I. Nagy ◽  
H. Losonczy
Keyword(s):  
Antithrombin Iii ◽  
Clinical Signs ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Basic Activity ◽  
At Iii ◽  
And Function

The authors detected in the last seven years 15 patients with hereditary antithrombin III/AT III/ abnormality. All of them had typical clinical signs of recurrent arterious and venous thromboembolie. The abnormality inherited as an autosomal trait. Three types of the abnormality could be observed. In Type I both quantity and function of AT III were extremely decreased. In type II AT III is normal in quantity but abnormal in function. In Type III AT III is quantitatively normal and also its function seems normal as far as its basic activity is concerned /activity measured in absence of heparin/, but its abnormality becomes manifest in the presence of heparin in vitro/and also in vivo/. 5 of the patients belonged to Type I, 4 to Type II and 6 to Type III. In 60 examined family members of the 15 patients an abnormal AT III could be observed in 44, clinical signs in 23.The examination of AT III activity in the presence of a given amount of heparin ia of great importance in recognition of the different types of antithrombin III abnormalities.

scholarly journals A yeast-based drug discovery platform to identify Plasmodium falciparum type II NADH dehydrogenase inhibitors

2021 ◽  
Author(s):  
Yu Cao ◽  
Chen Sun ◽  
Han Wen ◽  
Mengfei Wang ◽  
Pan Zhu ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Specific Inhibitor ◽  
Type I ◽  
Type Ii ◽  
Protein Activity ◽  
Asexual Blood Stage

Conventional methods utilizing in vitro protein activity assay or in vivo parasite survival to screen for malaria inhibitors suffer from high experimental background and/or inconvenience. Here we introduce a yeast-based system to facilitate chemical screen for specific protein or pathway inhibitors. The platform comprises several isogeneic Pichia strains that only differ in the target of interest, so that a compound which inhibits one strain but not the other is implicated in working specifically against the target. We used Plasmodium falciparum NDH2(PfNDH2), a type II NADH dehydrogenase, as a proof of principle to show how well this works. Three isogenic Pichia strains harboring respectively exogeneously introduced PfNDH2, its own complex I (a type I NADH dehydrogenase), and PfNDH2 with its own complex I were constructed. In a pilot screen of more than2000 compounds, we identified a highly specific inhibitor that acts on PfNDH2. This compound poorly inhibit the parasites at the asexual blood stage, however, is highly effective in repressing oocyst maturation in the mosquito stage. Our results demonstrate that the yeast cell based screen platform is feasible, efficient, economical and with very low background noise. Similar strategies could be extended to the functional screen for interacting molecules of other targets.

Abstract 119: PRMT5 Is A New Epigenetic Regulator In Cardiovascular Disease

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Tobias S Merkel ◽  
David Hassel ◽  
Marcus Krüger ◽  
Dieter Weichenhan ◽  
Christoph Plass ◽  
...  
Keyword(s):  
Arginine Methylation ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Type I ◽  
Type Ii ◽  
Cardiac Phenotype ◽  
Epigenetic Regulator

Protein arginine methylation is an abundant posttranslational modification involved in various cellular events such as DNA repair, splicing, and transcription. Protein arginine methyltransferases (PRMTs) catalyze the symmetric and asymmetric dimethylation of arginines and can be subdivided into two distinct families: type I enzymes catalyze the formation of asymmetric dimethylation whereas type II enzymes enhance the addition of symmetric methyl groups. Here, we report evidence that PRMT5 a type II enzyme serves as an important epigenetic regulator in myocardial disease. We identified PRMT5 via mass spectrometry as an interactant of class IIa histone deacetylases (HDACs). Additional cardiac interaction partners of PRMT5 were identified by a human cDNA library based yeast-two-hybrid screen. Thereby we elucidated Interleukin enhancer binding factor 3 (ILF3) as a putative interacting protein. ILF3 is a member of the NFAT family. NFAT signalling is known to be a potent driver of cardiomyocyte hypertrophy. Accordingly, we detected a 40% decrease in the size of neonatal rat ventricular myocytes in response to siRNA mediated knockdown of prmt5 . A dominant-negative version of PRMT5 is capable of interfering with the Calcineurin-NFAT axis and blocks NFAT activation. In vivo knockdown of endogenous prmt5 in the zebrafish led to a distinct cardiac phenotype with altered morphology and impaired function. To further elucidate prmt5 function in vivo we use mice with floxed prmt5 alleles. As a loss of prmt5 is lethal in the ES cell stadium we use an inducible α myosin heavy chain Mer-Cre-Mer system. Knockout mice display a complete loss of PRMT5 in cardiomyocytes. In line with our in vitro studies these mice are viable and show no phenotype under baseline conditions. Our work in progress investigates the hypothesis that class IIa HDACs serve as a scaffold to recruit PRMT5 to the DNA, forming a complex with ILF3 to block NFAT activity and the pro-hypertrophic gene program.

scholarly journals Differential regulation of cellular activities by GTPase-activating protein and NF1.

1993 ◽  
Vol 13 (4) ◽  
pp. 2497-2503 ◽  
Author(s):  
N al-Alawi ◽  
G Xu ◽  
R White ◽  
R Clark ◽  
F McCormick ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Biological Effects ◽  
Type I ◽  
Type Ii ◽  
Gtpase Activity ◽  
Ras Proteins ◽  
Ras Gtpase ◽  
Intrinsic Gtpase Activity

The regulation of the GTPase activity of the Ras proteins is thought to be a key element of signal transduction. Ras proteins have intrinsic GTPase activity and are active in signal transduction when bound to GTP but not following hydrolysis of GTP to GDP. Three cellular Ras GTPase-activating proteins (Ras-gaps) which increase the GTPase activity of wild-type (wt) Ras but not activated Ras in vitro have been identified: type I and type II GAP and type I NF1. Mutations of wt Ras resulting in lowered intrinsic GTPase activity or loss of response to cellular Ras-gap proteins are thought to be the primary reason for the transforming properties of the Ras proteins. In vitro assays show type I and type II GAP and the GAP-related domain of type I NF1 to have similar biochemical properties with respect to activation of the wt Ras GTPase, and it appears as though both type I GAP and NF1 can modulate the GTPase function of Ras in cells. Here we report the assembling of a full-length coding clone for type I NF1 and the biological effects of microinjection of Ras and Ras-gap proteins into fibroblasts. We have found that type I GAP, type II GAP, and type I NF1 show markedly different biological activities in vivo. Coinjection of type I GAP or type I NF1, but not type II GAP, with wt Ras abolished the ability of wt Ras to induce expression from an AP-1-controlled reporter gene. We also found that serum-stimulated DNA synthesis was reduced by prior injection of cells with type I GAP but not type II GAP or type I NF1. These results suggest that type I GAP, type II GAP, and type I NF1 may have different activities in vivo and support the hypothesis that while type I forms of GAP and NF1 may act as negative regulators of wt Ras, they may do so with differential efficiencies.

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