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From CellBiology

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Add your own student page to the site.

This is my first time posting on this page! :D

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Hello!

Konnichiwa

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PubMed

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Sushi

This is a picture of two neurons.

Reference

Tomohiro Shimada, Yukiko Yamazaki, Kan Tanaka, Akira Ishihama The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli. PLoS ONE: 2014, 9(3);e90447 PubMed 24603758


This is about prokaryotes. [1]

Puping Liang, Chenhui Ding, Hongwei Sun, Xiaowei Xie, Yanwen Xu, Xiya Zhang, Ying Sun, Yuanyan Xiong, Wenbin Ma, Yongxiang Liu, Yali Wang, Jianpei Fang, Dan Liu, Zhou Songyang, Canquan Zhou, Junjiu Huang Correction of β-thalassemia mutant by base editor in human embryos. Protein Cell: 2017; PubMed 28942539

Manyu Xu, Xiaopeng Zhu, Jinfang Yu, Jinpeng Yu, Sulan Luo, Xinquan Wang Erratum to: The crystal structure of Ac-AChBP in complex with α-conotoxin LvIA reveals the mechanism of its selectivity towards different nAChR subtypes. Protein Cell: 2017; PubMed 28942487

Katterine Salazar, Fernando Martínez, Margarita Pérez-Martín, Manuel Cifuentes, Laura Trigueros, Luciano Ferrada, Francisca Espinoza, Natalia Saldivia, Romina Bertinat, Katherine Forman, María José Oviedo, Antonio J López-Gambero, Christian Bonansco, Ernesto R Bongarzone, Francisco Nualart SVCT2 Expression and Function in Reactive Astrocytes Is a Common Event in Different Brain Pathologies. Mol. Neurobiol.: 2017; PubMed 28942474

Clara Franco-Jarava, Elena Álvarez de la Campa, Xavier Solanich, Francisco Morandeira-Rego, Virgínia Mas-Bosch, Marina García-Prat, Xavier de la Cruz, Andrea Martín-Nalda, Pere Soler-Palacín, Manuel Hernández-González, Roger Colobran Early Versus Late Diagnosis of Complement Factor I Deficiency: Clinical Consequences Illustrated in Two Families with Novel Homozygous CFI Mutations. J. Clin. Immunol.: 2017; PubMed 28942469

Karsten Krieger, Sarah E Millar, Nadine Mikuda, Inge Krahn, Jennifer E Kloepper, Marta Bertolini, Claus Scheidereit, Ralf Paus, Ruth Schmidt-Ullrich NF-κB participates in mouse hair cycle control and plays distinct roles in the various pelage hair follicle types. J. Invest. Dermatol.: 2017; PubMed 28942365


  1. Tomohiro Shimada, Yukiko Yamazaki, Kan Tanaka, Akira Ishihama The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli. PLoS ONE: 2014, 9(3);e90447 PubMed 24603758


Add your signature for Lab attendance.

--Z3421035 (talk) 15:39, 13 March 2014 (EST)

Individual Assessments

Lab 1

Neuronal Cytoskeleton.

Neuronal Cytoskeleton Bruno Pontes, Yareni Ayala, Anna Carolina C Fonseca, Luciana F Romão, Racκele F Amaral, Leonardo T Salgado, Flavia R Lima, Marcos Farina, Nathan B Viana, Vivaldo Moura-Neto, H Moysés Nussenzveig Membrane elastic properties and cell function. PLoS ONE: 2013, 8(7);e67708 PubMed 23844071

© 2013 Pontes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Lab 2

In a recent study done by Christian Kukat and his team, confocal microscopy and stimulated emission depleted (STED) microscopy was used to study mitochondrial DNA (mtDNA) nucleoids. They have found that the nucleoids have a defined and averagely uniform shape and size in mammal and also, they determined that mitochondrial transcription factor A (TFAM) is one of the main components of the nucleoids. This is particularly significant as it helps broaden the understanding of mitochondrial dysfunction. They were able to do so by comparing two microscopy techniques, confocal and stimulated emission depleted microscopy. Confocal microscopy has shown that the two areas, TFAM and mtDNA, overlapped spatially and are present in large aggregates in the nucleoid. Confocal imaging would be use to see the size of the nucleoid, however, Kukat and his team would then use STED to find a more accurate size. It was concluded that confocal microscopy was not able to find various details of the nucleiod with precision in comparison to STED microscopy. However, it did contribute to the the findings of the article as it had a broader view of the mitochondrial nucleoids and allowed for more in depth study of the nucleoids with other techniques. [1]

Cellular nucleus analysed by confocal imaging with fluoresence.[2]


Copyright © 2003, The National Academy of Sciences

Lab 3

This article explores one of the mechanisms that the mitochondria uses to transport proteins or preproteins through its outer and inner membranes. From other research articles, it has been found that there are protein complexes embedded in the membranes which aid the transportation of charged preproteins. There are differing groups for the inner and outer membranes. One such group of complexes are called the TIM complexes, which contain various subgroups with the main ones being TIM17, TIM23 and TIM44. These proteins help the preprotein cross the inner membrane. TIM17 and TIM23 have been recognised as integral proteins which span across the inner membrane and are said to be structural members. TIM44 is loosely associated with TIM23 and has been discovered to have a matrix heat shock protein 70 (mtHsp70) binding area. Dekker and his team identified that a complex, of approximately 90K containing TIM17 and TIM23, was the major preprotein import site. Through various techniques, they were able to suggest that the import channel is able to hold a translocating chain. [3]

This next article investigates the cleaving mechanism used to separate preproteins, such as Oxal, from TOM complexes in order to transport them through the TIM complexes and into the cytosol of the mitochondria. It has been identified that the inner membrane potential and the matrix heat shock protein 70 (mtHsp70) enable the release of Oxal from the TOM complexes in the outer membrane. The article suggest that there is a close interaction between TOM complexes and translocases, such as the TIM complexes, of the inner membrane. One of the experiments done by Frazier and her team, examined whether mtHsp70 was only required for the transportation of Oxal or was it involved in other preprotein transport across the inner membrane of the mitochondria. They found that the mtHsp70 was required to release Oxal from the TOM complex in the outer membrane. Through various experiments, they concluded that the mitochondria has more than one pathway for translocation of hydrophobic proteins.[4]

The following article explores the structural organisation of the TIM17.73 complex. It identifies the sub-components of the complex, which are TIM17, TIM23 and TIM44 and states that these components are found in equimolar amounts. TIM44 is said to be a peripheral protein and therefore is assoicated with the matrix side of the membrane and the TIM17.73 complex. Through immunodepletion, Moro and his team were able to identify that all TIM23 complexed together with TIM17 to form the TIM17.73 complex. TIM44 was found to associated with this complex at the matrix side of the inner membrane. [5]

In this last article, it was noted that various compounds, such as steroid hormones, are transported into the mitochondrial cytosol though various proteins within the outer and inner membranes. One such hormone which was investigated was cholesterol. A complex called the transduceosome which is where steroidogenic proteins interact with outer mitochondrial membrane translocator proteins and voltage-dependent anion channels. It has been suggested that the cholesterol transfer happens through the specialised interaction sites between the inner and outer membranes, which are composed of voltage-dependent anion channels and the adenine nucleotide translocase of the inner membrane. In this article, Rone and her team set out to identify the mechanisms in which cholesterol is transferred from the outer membrane to the inner membrane of the mitochondria where the cytochrome P450 enzyme, CYP11A1, is located. Through various experimental technqiues, they were able to conclude that several mitochondrial and cytosolic proteins are needed to aid the transportation of cholesterol from the outer membrane to the inner membrane. The outer membrane protein, translocator protein, is needed to bind and separate the cholesterol, the mitochondrial-targeted steroidogenic acute regulatory protein is required to initiate the transportation of cholesterol into the inner membrane. The outer membrane voltage-dependent anion channel anchors the translocator protein and the steroidogenic acute regulatory protein. Finally, the transfer of cholesterol CYP11A1 occurs after the formation of the AAA domain-containing protein 3A, which forms a bridge, connecting the outer membrane to inner membrane. [6]


Uploading image

Import of Oxal through inner mitochondrial membrane.[7]

Lab 4

TIM17 is an essential component in the TIM23 complex which mediates protein translocation across the double membrane of the mitochondria. One antibody that has been created for the TIM17 protein is the TIM17 antibody [C1C3]. It also has various names such as TIMM17A. The antibody is derived from rabbit and is a polyclonal antibody and is reactive with humans. The concentration given in a vial of antibody product is 1mg/ml, however it was suggested that it should be diluted 1:500 to 1:3000 fold in order for it to work with Western Blotting. Genetex

A secondary antibody that can be used with the TIM17 antibody C1C3 is the ABC-elite reagent, which can be used with any biotinylated primary or secondary antibody. Vectorlabs

TIM17 antibody has been used in a research which involves the investigation of the TIM complex. The antibody was used to precipitate the protein so that it can be identified through immunoblotting.[8]

Lab 6

Exercise 1

Graph of Undifferentiated B35 cells.PNG

Lab 7/8

Group 1 - Phagocytosis

Introduction

• Good brief introduction of phagocytosis

• External links may need to be edited a bit and given a suitable title

Receptors

• Some abbreviations may need to be explained for example FcγRI and etc.

• Complement receptor abbreviations should be more clearer, i.e. maybe complement receptors (CRs) have various types such as C3b or C3bi

• If possible, expand more on receptors as to what their structure is

Mechanism of phagocytosis

• Definition of phagocytosis should be in the introduction, and since it already was, it doesn't need to be defined again

• When comparing two different types of models, it would be good to have 2 pictures. So having 1 picture about one model is a good start. Also, name the pictures 'Figure 1' since it is being referred to in the text as fig 1

• It also might be useful to add subheadings underneath this section .E.g. "Zipper model" and "Trigger model"

• Good detailed description of the mechanism; however, there are some bits of information that didn’t flow, for example, the sentence from Exocytosis to the next sentence of Engulfment.

• Also, some of the numbered referencing doesn’t seem to work (i.e. some were superscripted and hyperlinked but others weren’t)

Diseases

• In-text referencing numbers would be more suitable if placed right after the information rather than a sentence underneath

• Chronic granulomatous disease - how is the activity of neutrophils impaired?

• Chediak Higashi Syndrome - Maybe add a few more details like particular gene sequence if possible and a brief overview how that gene causes hyperactivity in phagocytosis

References

• Was organised quite poorly; there are 2 reference sections when there should only be one reference section at the end

• References: some of the references double up in the reference list. There should be a link on the side that helps with editing and adjusting the references so that it doesn't double up

Still a few headings that need to be filled out

Overall, good solid information presented! However, formatting could be more thoroughly edited.


Group 3 - Nucleocytoplasmic Transport

Introduction + Complexes

• Images describing the structure of the transport proteins, models and nuclear envelope would be useful

• Abbreviations should be written out in full first otherwise readers won't be clear on what the abbreviations mean, for example 'FG-Nups' - underneath nuclear pore complex models. There was an explanation to what FG-Nups were further down in the project, but it would’ve been more useful if the readers knew what FG-Nups were, before reading about it in nuclear pore complexes

• Definition, structure and function of RanGTP and RanGDP aren't very clear

Quite a few empty headings that need to be filled out

• If possible, expand more on the diseases relating to the Nup nuclear porins, i.e. specific details

• Also, formation of images underneath the image heading should be aligned and fixed

References: some of the references double up in the reference list. There should be a link on the side that helps with editing and adjusting the references so that it doesn't double up

Overall, the project has a good basic format and brief introduction to the topic. It can be improved with the addition of more detailed and specific information relating to structure and function and also images to aid with description.


Group 4 - Neuron Soma to processes

Introduction

• Lack sufficient information in the introduction at this stage. Different types of neurons shouldn’t really be mentioned unless the transport mechanisms in these neurons differ from each other. If they are very similar, it should be generalised

• Good overview of the Fast and Slow transport; would be helpful to add diagrams to help explain the 'Stop and Go' model

Motor proteins

• Good use of a table to compare kinesins and dyeins; however, diagrams would be very helpful

• Abbreviations should be explained so readers will know what they are reading about, for example, KIF1A and KIF1Bβ

• Still lacks a lot of information underneath quite a few headings, but it seems as though you guys are heading on the right track

• Images are present at the bottom of the page, but they would be more helpful if they were formatted to be underneath their corresponding heading

References

• two particular references in the list was referenced incorrectly so it doesn't show up properly

Overall, the lack of information makes it slightly hard to give more constructive feedback, however, from the headings and subheadings, with dot points underneath show that there was consideration put into what information was to be written there and so far, the guidelines seem straightforward enough.

Lab 9

Hypothesis:

Isolated cells grown in optimal tissue culture conditions will most likely undergo apoptosis once placed in a foreign environment.

Aim:

To determine whether the cell undergoes an intrinsic or extrinsic apoptotic pathway by observing the amount of caspase 8 activated.

Method:

Human adherent cells are used for the tissue culture. They are washed carefully and loose cells are isolated. Adherent cells are gently spun and labelled with FAM green fluorescence from the FLICATM kit before trypsinization. Cells are incubated with the FLICA at 37oC for roughly 60 minutes before washing. After the addition of apoptosis wash buffer, cells are placed on ice and protected from light. Observations are made by the analysis of cells with a flow cytometer.

Click ‘Protocol’ tab in the following link for more detailed instructions.

http://www.immunochemistry.com/products/apoptosis-assays/caspase-assay-kits/green-flica-caspase-8-assay-kit.html Suppliers: Immunochemistry Technologies

Resource: Green FLICA™ Caspase 8 Assay Kit

Flow Diagram:

Apoptosis flow diagram.PNG

Results:

If there is a high fluorescence reading observed, that means that there is a high amount of caspase 8 present and therefore, leads to an extrinsic apoptotic pathway. However, if there is a low fluorescence reading observed, there is less caspase 8 and hence, there is no extrinsic pathway activated.


  1. Christian Kukat, Christian A Wurm, Henrik Spåhr, Maria Falkenberg, Nils-Göran Larsson, Stefan Jakobs Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA. Proc. Natl. Acad. Sci. U.S.A.: 2011, 108(33);13534-9 PubMed 21808029
  2. Naoki Itano, Shu-ichi Okamoto, Dongxian Zhang, Stuart A Lipton, Erkki Ruoslahti Cell spreading controls endoplasmic and nuclear calcium: a physical gene regulation pathway from the cell surface to the nucleus. Proc. Natl. Acad. Sci. U.S.A.: 2003, 100(9);5181-6 PubMed 12702768
  3. P J Dekker, F Martin, A C Maarse, U Bömer, H Müller, B Guiard, M Meijer, J Rassow, N Pfanner The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44. EMBO J.: 1997, 16(17);5408-19 PubMed 9312000
  4. Ann E Frazier, Agnieszka Chacinska, Kaye N Truscott, Bernard Guiard, Nikolaus Pfanner, Peter Rehling Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space. Mol. Cell. Biol.: 2003, 23(21);7818-28 PubMed 14560025
  5. F Moro, C Sirrenberg, H C Schneider, W Neupert, M Brunner The TIM17.23 preprotein translocase of mitochondria: composition and function in protein transport into the matrix. EMBO J.: 1999, 18(13);3667-75 PubMed 10393182
  6. Malena B Rone, Andrew S Midzak, Leeyah Issop, Georges Rammouz, Sathvika Jagannathan, Jinjiang Fan, Xiaoying Ye, Josip Blonder, Timothy Veenstra, Vassilios Papadopoulos Identification of a dynamic mitochondrial protein complex driving cholesterol import, trafficking, and metabolism to steroid hormones. Mol. Endocrinol.: 2012, 26(11);1868-82 PubMed 22973050
  7. Ann E Frazier, Agnieszka Chacinska, Kaye N Truscott, Bernard Guiard, Nikolaus Pfanner, Peter Rehling Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space. Mol. Cell. Biol.: 2003, 23(21);7818-28 PubMed 14560025
  8. F Moro, C Sirrenberg, H C Schneider, W Neupert, M Brunner The TIM17.23 preprotein translocase of mitochondria: composition and function in protein transport into the matrix. EMBO J.: 1999, 18(13);3667-75 PubMed 10393182