Essential

Molecular Biology of the Cell 6th editionGarland Publishing, Inc.

A

etc.

M78Figure 11-3 Essential Cell Biology (© Garland Science 2010)

50 µm : 5 nm = 10 m : X

X = 1 mm

2-

2- 2-ME

Urea

HS-CH2-CH2-OH

O=CCH3

CH3

--

NH2+=C

CH3

CH3

--

α β

NH2I1IMSSVQSQQEQLSQSDPSPSPNSCSSFELIDMDAGSLYEPVSPHWFYCKIIDSKETWIPFNSEDSQQLEEAYSSGKGCNGRVVPTDGGRYDVHLGERMRYAVYWDELASEVRRCTWFYKGDKDNKYVPYSESSSQVLEETYMLAVTLDEWKKKLESPNREIIILHNPKLMVHYQPVAGSDGWGSTPTEQGRPRTVKRGVENISVDIHCGEPLQIDHLVFVVHGIGPACDLRFRSIVQCVNDFRSVSLNLLQTHFKKAQENQQIGRVEFLPVNWHSPLHSTGVDVDLQRITLPSINRLRHFTNDTILDVFFYNSPTYCQTIVDTVASEMNRIYTLFLQRNPDFKGGVSIAGHSLGSLILFDILTNQKDSLGDIDSEKDSLNIVMYQGDTPTLEEDLKKLQLSEFFDIFEKEKVDKEALALCTDRDLQEIGIPLGPRKKILNYFSTRKNSMGIKRPAPQPASGANIPKESEFCSSSNTRNGDYLDVGIGQVSVKYPRLIYKPEIFFAFGSPIGMFLTVRGLKRIDPNYRFPTCKGFFNIYHPFDPVAYRIEPMVVPGVEFEPMLIPHHKGRKRMHLELREGLTRMSMDLKNNLLGSLRMAWKSFTRAPYPALQASETPEETEAEPESTSEKPSDVNTEETSVAVKEEVLPINVGMLNGGQRIDYVLQEKPIESFNEYLFALQSHLCYWESEDTVLLVLKEIYQTQGIFLDQPLQI711ICOOH

Ap5A

5

Aβ

PrP

PrP

PrPsc

500

75

PrP

PrP

PrP

PrP

PrPsc PrPc PrPsc

PrPsc β

1997

Figure 4-8 Essential Cell Biology (© Garland Science 2010)

PrpC Prpsc

Prpsc

PrpC PrpSC

PrpC PrpSC

α- α- β-

APP

APP

Figure 10-20 Molecular Biology of the Cell (© Garland Science 2008)

#

DNA

DNA → mRNA →

DNA → mRNA →

2013

autocrine

YY

Question 16-2 Essential Cell Biology (© Garland Science 2010)

Histamine

H1-H4 GPCR

マスト（肥満）細胞�

・ 蕁麻疹・ 喘息・ 花粉症・ アトピー性皮膚炎�

・ ヒスタミン・ LTC4, LTD4・ TXA2・ PGD2・ プロテアーゼ・ IL-5・ TNF-α

メディエーター�

関連病態�

・ 粘膜�・ 皮膚・ 腸管�・ 血管周囲�

存在部位�

抗原

ヒスタミン Leukotriene PAF ThromboxaneIL-5 TNF-α プロテアーゼ

マスト細胞

IgE 受容体IgE

マスト細胞は IgE-抗原に反応し、脱顆粒する

Figure 25-1 Molecular Biology of the Cell (© Garland Science 2008)

Figure 25-2 Molecular Biology of the Cell (© Garland Science 2008)

T

Figure 25-3 Molecular Biology of the Cell (© Garland Science 2008)

Figure 25-6 Molecular Biology of the Cell (© Garland Science 2008) Figure 25-7a Molecular Biology of the Cell (© Garland Science 2008)

Figure 25-7b Molecular Biology of the Cell (© Garland Science 2008) Figure 25-7c Molecular Biology of the Cell (© Garland Science 2008)

Figure 25-2 Molecular Biology of the Cell (© Garland Science 2008)

T

Figure 25-19 Molecular Biology of the Cell (© Garland Science 2008)

Table 25-1 Molecular Biology of the Cell (© Garland Science 2008) Figure 25-21 Molecular Biology of the Cell (© Garland Science 2008)

Figure 25-23 Molecular Biology of the Cell (© Garland Science 2008)

144

THE ANTIBODY MOLECULE B CELLS PRODUCE ANTIBODIES

ANTIBODY SPECIFICITY

Antibodies are proteinsthat bind very tightly totheir targets (antigens).They are produced invertebrates as a defenseagainst infection. Eachantibody molecule ismade of two identicallight chains and twoidentical heavy chains,so the two antigen-binding sites areidentical.

Antibodies are made by a class of white blood cells called Blymphocytes, or B cells. Each resting B cell carries a differentmembrane-bound antibody molecule on its surface that servesas a receptor for recognizing a specific antigen. When antigenbinds to this receptor, the B cell is stimulated to divide and tosecrete large amounts of the same antibody in a soluble form.

RAISING ANTIBODIES IN ANIMALSAntibodies can be made in the laboratory by injecting an animal(usually a mouse, rabbit, sheep, or goat) with antigen A.

Repeated injections of the same antigen at intervals of several weeks stimulate specific B cells to secrete large amounts of anti-A antibodies into the bloodstream.

Because many different B cells are stimulated by antigen A, theblood will contain a variety of anti-A antibodies, each of whichbinds A in a slightly different way.

antigen-binding sites

light chain

heavy chainhinge

5 nm

antigen

heavy chain

light chain

An individual animalcan make billions ofdifferent antibodymolecules, each with adistinct antigen-bindingsite. Each antibodyrecognizes its antigenwith great specificity.

different B cells

Antigen binds toB cell displaying anantibody that fitsthe antigen.

B cell is stimulated to make and secrete more of same antibody.

inject antigen A take blood later

A

A A Atime

amou

nt o

f an

ti-A

anti

bodi

es in

blo

od

ANTIBODIES DEFEND US AGAINST INFECTION

foreignmolecules

viruses bacteria

ANTIBODIES ( ) FORM AGGREGATES

Antibody and antigenaggregates are ingested

by phagocytic cells.

Special proteins inblood kill antibody-

coated bacteriaor viruses.

PANEL 4–3 Making and using antibodies144

THE ANTIBODY MOLECULE B CELLS PRODUCE ANTIBODIES

ANTIBODY SPECIFICITY

Antibodies are proteinsthat bind very tightly totheir targets (antigens).They are produced invertebrates as a defenseagainst infection. Eachantibody molecule ismade of two identicallight chains and twoidentical heavy chains,so the two antigen-binding sites areidentical.

Antibodies are made by a class of white blood cells called Blymphocytes, or B cells. Each resting B cell carries a differentmembrane-bound antibody molecule on its surface that servesas a receptor for recognizing a specific antigen. When antigenbinds to this receptor, the B cell is stimulated to divide and tosecrete large amounts of the same antibody in a soluble form.

RAISING ANTIBODIES IN ANIMALSAntibodies can be made in the laboratory by injecting an animal(usually a mouse, rabbit, sheep, or goat) with antigen A.

Repeated injections of the same antigen at intervals of several weeks stimulate specific B cells to secrete large amounts of anti-A antibodies into the bloodstream.

Because many different B cells are stimulated by antigen A, theblood will contain a variety of anti-A antibodies, each of whichbinds A in a slightly different way.

antigen-binding sites

light chain

heavy chainhinge

5 nm

antigen

heavy chain

light chain

An individual animalcan make billions ofdifferent antibodymolecules, each with adistinct antigen-bindingsite. Each antibodyrecognizes its antigenwith great specificity.

different B cells

Antigen binds toB cell displaying anantibody that fitsthe antigen.

B cell is stimulated to make and secrete more of same antibody.

inject antigen A take blood later

A

A A Atime

amou

nt o

f an

ti-A

anti

bodi

es in

blo

od

ANTIBODIES DEFEND US AGAINST INFECTION

foreignmolecules

viruses bacteria

ANTIBODIES ( ) FORM AGGREGATES

Antibody and antigenaggregates are ingested

by phagocytic cells.

Special proteins inblood kill antibody-

coated bacteriaor viruses.

PANEL 4–3 Making and using antibodies

IgM

IgG

IgM

Figure 25-25 Molecular Biology of the Cell (© Garland Science 2008) Figure 25-26 Molecular Biology of the Cell (© Garland Science 2008)

抗原

ヒスタミン Leukotriene PAF ThromboxaneIL-5 TNF-α プロテアーゼ

マスト細胞

IgE 受容体IgE

マスト細胞は IgE-抗原に反応し、脱顆粒する

ADCC (Antibody-Dependent-Cellular-Cytotoxicity CDC Complement-Dependent Cytotoxicity :

• 

• 

• 

PD-1, PD-L1

Figure 25-43 Molecular Biology of the Cell (© Garland Science 2008)

B

Y Y

Y

Y

Y Y

Y

Y Y

Y

Y

B

Y Y

Y

Y

Y YY Y

Y

YY Y

Y

Y

Y YY Y

Y

Y

Y Y

Y

Y

Y YY Y

Y

Y

Y Y

Y

Y

Y YY Y

Y

Y

Y Y

Y

Y

Y YY Y

Y

Y

Y Y

Y

Y

Y YY Y

Y

Y

145

USING ANTIBODIES TO PURIFY MOLECULES

MONOCLONAL ANTIBODIES USING ANTIBODIES AS MOLECULAR TAGS

M A A

A

A A A A

A

E P F N O R D

S C

J L B K

Q G H

MA

AA

E P

F

NOR D

SCJ

J

L

B

B

KQG H

IMMUNOPRECIPITATION IMMUNOAFFINITY COLUMN CHROMATOGRAPHY

mixture of molecules

add specific anti-A antibodies

collect aggregate of A molecules andanti-A antibodies by centrifugation

mixture of molecules

discard flow-through wash collect pure antigen A

washelute

antigen A

bead coated with anti-A antibodies

column packed with these beads

Large quantities of a single type of antibodymolecule can be obtained by fusing a B cell(taken from an animal injected with antigen A)with a tumor cell. The resulting hybrid cell divides indefinitely and secretes anti-A antibodies of a single (monoclonal) type.

Tumor cell fromcell culture dividesindefinitely butdoes not makeantibody.

B cell from animalinjected with antigenA makes anti-Aantibody but doesnot divide forever.

FUSE ANTIBODY-SECRETINGB CELL WITH TUMOR CELL

Hybrid cellmakes anti-Aantibody anddividesindefinitely.

couple to fluorescent dye,colloidal gold particle, orother special tag

specific antibodiesagainst antigen A labeled antibodies

Fluorescent antibody binds toantigen A in tissue and isdetected by fluorescence in a light microscope. The antigen here ispectin in the cell walls of a sliceof plant tissue.

Gold-labeled antibody bindsto antigen A in tissue and is detected in an electron micro-scope. The antigen is pectinin the cell wall of a singleplant cell.

Antigen A isseparated fromother moleculesby electrophoresis.

Incubation with thelabeled antibodiesthat bind to antigen Aallows the position of theantigen to be determined.

Note: In all cases, the sensitivity canbe greatly increased by using multiplelayers of antibodies. This “sandwich” method enables smaller numbers of antigen molecules to be detected.

antigen

Labeled second antibody (blue) binds to first antibody (black).

C KNR

A

A

A

A

BA

A

MIC

ROSC

OPI

C D

ETEC

TIO

NBI

OCH

EMIC

AL

DET

ECTI

ON

etc

J

cellwall

Fab

抗体フォーマットの変換

Bivalent Mini-Antibody(Fab_dHLX_MH)

SDS-PAGESDS

Western blot

DetergentSDSTriton X-100Nonidet P-40

Triton X-100

Tween20

n-

Sodium Dodecyl Sulfate

SDS-PAGE

50

37

kDa

NPP6

NPP6

MBP

*

*

**

**

* ** Coronal section Magnified view

0.2 mm 5 µm 5 µm

X NMR

Chapter 10 Analyzing Genes and Genomes

Copyright © Garland Science 2010 Copyright © Garland Science 2010

Chapter 10 Analyzing Genes and Genomes

Copyright © Garland Science 2010

GPCR

X

A

A

PCR

Cytomegalo virus SV40

A

PCR: Polymerase Chain Reaction

RT-PCR: Reverse Transcriptase PCR

mRNAcDNA

mRNA A

× ×

RNA

PCR RT-PCR cDNA

cDNA

RNA

PCR RT-PCR cDNA

cDNA

X

GFP GFP

GFP

mcherry

DNA分子の変性と再生を利用した手法

mRNA

DNA

in situmRNA DNA

#CRISPR TALEN

,

GPCR

T