0022-1554/90/$3.30

The Journal

of

Copyright

c

and

Histochemistry

1990

Vol.

Cytochemistry

The Histochemical

by

Society,

Original

IWAKI,2

Department New

AKIKO

ofPatbology,

York,

NY

Received

KUME-IWAKI,

Columbia

University

and JAMES

College

ofPhysicians

for publication

April

28,

1989

and

in revised

form

July

Cnystallins are composed ofsevenal families ofsoluble lens proteins, the most common ofwhich are the a- , -, y-, and 8-crystallins [see Wistow and Piatigorsky (1988), for review]. a-Crystallin is a major of the vertebrate

during

lens

lens

and

differentiation

in

is the first the

mouse

crystalhin

to ap-

(Zwaan,

1983).

a-Crystallin is a multimenic aggregate, composed of the products of two genes, aA and aB, which are located on different chromosomes

(Quax-Jeuken

et al., 1985).

the tissue

specificities

Crystallin

expression

in culture

(Chepelinsky

transgenic

mice

however,

et al., 1988). skeletal analysis central tected

of aA and seems

been

1989;

muscle, revealed

Dubin

Northern

blot

to lens,

1985;

different.

et al.,

and

Iwaki

detected

that aAboth and in

as determined

Okazaki

sciatic nerve, kidney, a more widespread

reactions

demonstrate

aB are completely

et al., 1989;

immunoblots

nervous system, by immunoblotting.

studies

et al., 1985). aB-Crystalhin reported in extra-lenticular

(Ovenbeek

Our

Recent

restricted

et al.,

has recently

and Nagineni,

1985)

Supported Correspondence

U. College

1990

in USA.

E. GOLDMAN

and

Surgeons,

The

12,

1989;

accepted

ney,

Schwann

New

York

State

Psychiatric

Institute,

there

by a Javits

Neuroscience

of P & 5, 630

West

168th

St.,

Dept.

(9A1682).

system,

component

in Alexander’s

of Rosenthal

disease,

and

that some non-lenticular protein (Iwaki et al., of such

diverse

examined

the

histochemistry.

are unknown. functions protein

cellular

than

inclusions and

One

glial

in astrocytes scars,

indicates

large amounts of aB-crystallin strategy

that

may

of this in cells reveal

is to determine the specific cellular in non-lens tissues. In this study, we

distribution

Rather

gans, the expression appears In general, the distribution idative

fibers, gliomas

cells can accumulate 1989). The functions

tissues

clues as to possible localization of this

in some

being

of aB-cnystallin widely

restricted correlates

by immuno-

distributed

to highly well with

in many

specialized high levels

on-

cells. of ox-

function.

Duguid

Materials

in heart,

Materials. A rabbit polyclonal anti-aB-crystallin antibody was raised against aB-crystallin purified from rat cardiac muscle and was affinitypurified (Iwaki et al., 1989). Bovine lens a-crystallin was provided by Dr. A. Spector. Normal lamb serum and normal fetal bovine serum were punchased from Gibco (Grand Island, NY). Horseradish peroxidase-conjugated anti-rabbit IgG goat antiserum was purchased from Vector Labs (Burlingame, CA), anti-myoglobin rabbit antiserum from Dako (Santa Barbara, CA), and Bio-Lyte ampholytes from Bio-Rad Labs(Rockville Center, NY,). Other chemicals were obtained from Sigma (St Louis, MO).

Northern including

is a relatively

Investigator

to: Dr. Toni Iwaki,

1989

reactions

and placenta. distribution,

that

ii,

et al., 1989;

positive

lung, in addition to the organs The densities of the immunoblot

suggest

August

cells of peripheral nerves, glia of the central and decidual cells of the placenta. A dose correlation with markers of oxidative activity suggests that aB-crystallin is expressed in cells that have high levels of oxidative function. (JHistochem Cytochem 38:31-39, 1990) KEY WORDS: Crystallins; hnmunoenzyme technique; Rat; Lens, crystallin; Muscle fiber types; Kidney tubules; Schwann cells; Neuroglia; Placenta. nervous

high

blot skin, deand abun-

vo-diinensional

1

31-39,

expression, tissues (Bhat

dance ofaB-crystallin in striated muscle tissues and that the highest concentration is in cardiac muscle. Our finding that aB-crystallin

2

pp. Printed

in

is a major

Introduction

pear

1,

10032.

aB-Crystallin is a subunit of a-crystallin, a major protein component of the vertebrate lens. Recently, its expression in various extra-lenticular tissues has been demonstrated by both Western and Northern blotting. In this study, the eellular distribution of aB-aystallin in rat organs was exammed in detail using immunohistochemistry Positive reactions were observed in lens, iris, heart, skeletal muscle (type 1 and type 2A fibers), striated muscle in skin and esophagus, Henle’s loop and medullary collecting duct ofthe kid-

component

No.

Artide

Cellular Distribution of aB-Crystallin Non-lenticular Tissues’ IORU

38,

Inc.

Award,

NS 17125.

of Pathology,

New York, NY

Columbia 10032.

and

Methods

Gel Electrophotcais

and

Immunobloaing.

Two-dimen-

sional gel electrophoresis was performed according to O’Farrel (1975). For isoelectnic focusing, gels containing 1.6% Bio-Lyte ampholyte, pH 5-8, 0.4% Bio-Lyte ampholyte, pH 3-10, 2% Triton X-100, and 9.2 M ureawere used. Samples containing 10 tg ofbovine a-crystallin were applied in a solution containing 9.5 M urea, 2% Triton X-iOO, and 5% 2-mercaptoethanol. For 3’

Downloaded from jhc.sagepub.com at KAI NAN UNIV on March 21, 2015

32

KUME-IWAKI,

IWAKI,

aIEF

Table

1 . Immunohistochemistry

rat

of

aB-crystallin

D

#{149}

Positive

Eye

A2A

B2B1

Lens

(

cuboidal +

#{247}

body

(

Bi

intestine

None

Kidney

Pars

(1970).

in the presence

Immunoblotting

ofsodium was carried

dodecylsulfate out

according

by transferring

the

to Laemmli proteins

onto

nitrocellulose

paper, blocking with 5% non-fat milk in PBS, and incubatin primary antibody (diluted i:soo in 5% non-fat milk in PBS). The next day the blots were washed in PBS and incubated with horseradish peroxidase-labeled anti-rabbit IgG goat antiserum for 1 hr (diluted 1:2000 in PBS). Blots were then washed with PBS and visualized with 3,3’overnight

diaminobenzidine

DAB/0.01%

tetrahydrochloride

H202/50

(DAB)

mM Tnis-HCI,

reaction

solution

(0.03%

),

+

astrocytes(

±)

cells

1 fibers

muscle

fibers

in

muscularis

+),

+

+)

+

propria

+

recta

of proximal

limbs

medullary Bladder

None

Trachea

None

Lung

None

convoluted

(

tubules

of loops

of Henle

collecting

ducts

( (

+

+

+

),

+),

+

inner

#{247} +

None nodes

None

Thymus

resis was done

(

None

Liver

Lymph

gel electropho-

of ciliary

Striated

Spleen

polyacrylamide

+),

+

+)

+

Schwann Type

thin

13%

epithelium

fibers

+

Esophagus Small

electrophoresis,

), lens

+

of iris (

Heart

nerves muscles

(+

the second-dimensional

+

None

Skin

Figure 1. Two-dimensional analysis (IEF followed by SDS-PAGE) of bovine a-crystallin(10 sg each gel). (Abevs)Coomassie blue-stained gel. (Below)lmmunoblot. The antibody recognized both the primary gene product, aB2, and its phosphorylated form, aBi, as well as several other aB species, but not aAcrystallins.

(+ epithelium

( #{247} + ±) ( + + + ), type 2A fibers ( type 2B fibers (± Striated muscle fibers in the hypodermis (+ Muscle fibers ( + + +)

Peripheral Skeletal

B2

adult

cells

of cuboidal

Oligodendrocytes

Pineal

ing

layer

body Brain

epithelium

), cuboidal

+

a deep

I

S

in

organs’

Organs

S

GOLDMAN

None

Pituitary

None

Adrenal

gland

None

Ovary Uterus

None

Placenta

Decidual

None

(

cells

+

+

+

Criteria ofstaining intensity ofaB.crystallin: ± , a few, weakly positive cells; cells with morphological characteristics of oligodendrocytes are positive (we do not yet know whether all oligodendrocytes express aB.crystallin); + + , the large .1

+

.

some

majority

of

cells

are

positive;

+

+

,

+

all

cells

are

strongly

positive.

pH 7.4).

andTissue Sectioning. Female Sprague-Dawley rats were purfrom ‘1conic Farms (Germantown, NY). The day the vaginal plug

Animals

chased

was detected

sia, adult

is designated

animals

as embryonic

were perfused

day zero.

Under

deep

ether

anesthe-

the left ventricle with PBS, pH 7.4, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, and organs were removed and post-fixed overnight. For the study of rat placentae, pregnant rats were sacrificed by dislocation of the neck and both embryos and placentac were quickly removed. The discoidal placenrae were cut in halfand fixed with the 4% paraformaldehyde solution overnight. Fixed tissues were sequentially equilibrated with 10% sucrose in PBS for 4 hr, 15% sucrose in PBS for 4 hr. 20% sucrose in PBS overnight, and were frozen in an acetone/dry-ice bath. Six- to fourteen-micrometer sections were cut with a cryostat ( - 20’C), mounted on gelatin-coated slides, and air-dried. Immunohistochemistry. munoperoxidase method with

0.3%

H2O2

through

Staining was carried out by the indirect im(Nakane and Pierce, 1967). After pre-incubation

in absolute

methanol

for

30 mm

at room

temperature

to inhibit endogenous peroxidase, sections were incubated sequentially in a diluent (10% lamb serum/10% fetal bovine serum in PBS) for 30 mm; anti-rat aB-crystallin rabbit antibody (12#{174} in diluent) at 4C for 40 hr; PBS for 10 mm; horseradish peroxidase-labeled goat anti-rabbit IgG (12#{174} in PBS) at room temperature for 2 hi; PBS for 10 mm. The sections were then incubated with DAB reaction solution. The sections were dehydrated, mounted, and examined with an Olympus BH-2 photomicroscope equipped

with the

Nomarski

optics.

affinity-purified

crystallin

(Iwaki

Skeletal

of aB-crystallin

Controls

using

aB-crystallin et al.,

1989)

pre-immune

antibody failed

after

to show

any

Musde Fiber Classification. To compare in skeletal muscles with the muscle

rabbit

serum

absorption tissue

or using

with

lens

a-

staining. the

staining

pattern

fiber types, fresh gastrocnemius muscles were removed from an adult female rat and frozen in an acetone/dry-ice bath without fixation. Ten-micrometer sections were consecutively cut and mounted on gelatin-coated slides. Two sets of serial sections were stained with immunohistochemistry using the anti-aB-crystallin antibody and the anti-myoglobin antiserum (12#{174}in a diluent). Next, consecutive sections were stained by the myosin ATPase method, pH 9.4 (Round et xi., 1980)and by the NADH-tetrazolium reductase(diphosphopyridine nucleotide diaphorase) reaction (Farber et al., 1956a,b), which was modifled as follows: the sections were incubated in 10 mg of nitroblue tetrazohum

and

mm

at 37’C.

8 mg

of NADH

in 10 ml of 0.2

M Tris

buffer,

pH

7.4,

for 30

Results The degree

affinity-purified ofspecificity

antibody

used

for aB-cnystallin

dimensional electrophoresis (Figure The distribution of aB-crystallin

Downloaded from jhc.sagepub.com at KAI NAN UNIV on March 21, 2015

in this

study

showed

on the immunoblot 1). in adult

female

a high after

rats

two-

is sum-

CELLULAR

DISTRIBUTION

OF aB-CRYSTALLIN

33

which

a striated

tions

C,

5”

Skeletal

3a and

could

be observed

ofgastrocnemius

muscle

showed

determined

NADH-tetrazolium bic (type 1) fibers ..-

by the myosin

neductase and about

ATPase

(b) a relatively

high

content

ofthe

myosin

ATPase

reaction

Ofthe

type

2A fibers,

97%

the

reaction

tivity

:

1

H-

and

(c) inhibition

and

Kaiser,

showed

type

1 and

positive

the

crystallin

type

staining

than

__

were the

(not

shown).

observed

and placenta.

Weak

positive

skin, reactions

tem. Details of the cellular organ are as follows.

reactions

esophagus, were

were

observed

peripheral

seen

nerve,

in the central

distributions

upper

esophagus

sys-

in each

(Figures

No

rat lens was strongly were

anterior cuboidal lens, the cuboidal of the ciliary nite positive nerve

were

positive

observed

epithelium epithelium

with

between

the aB-crystallin the

staining

of the

and that of lens fiber cells. Besides ofthe iris and that ofthe basal layer No defiin the optic

2).

positive.

2A fibers.

muscle

fibers

showed

a diffuse

staining

of

Connective

negative. a mosaic

diffuse and 3a and

aB-

tissues

of

Rat extraocular pattern of aB-

homogeneous

in the

musculanis

reactions propnia

of

of aB-crystallin for immunohisto-

in

3c).

Because Western blotting revealed the presence rat abdominal skin, we used abdominal skin chemistry. hypodermis

There is a thin layer of striated muscle fibers in the between hair roots and sweat glands. Muscle fibers in positive

reactions,

but no other

cell types

stained

3d).

Peripheral

Nerves

Sciatic nerves and trigeminal nerves were examined. of these nerves showed diffuse cytoplasmic positive

non satellite

cells

were

fibroblasts, ganglion,

Schwann reactions

cells (Fig-

and penineunial neither ganglion

stained.

Central

Nervous

Astrocytes

in optic

the subpial in cerebrum,

System nerves

glial limitans cerebellum,

were positive,

especially

pattern,

against

cell processes

near

(Figure 3f). Positive cells were also seen and brainstem. Most ofthe positive cells

were located in white matter and displayed small round profiles with a few thin processes (Figures 3g and 3h). By morphological criteria, the majority ofthese appeared to be oligodendrocytes. However, further studies with glial cell markers tive identification. Some subpial astrocytes

Heart All cardiac

ac-

antibody.

intensity

body showed positive reactions (Figure reaction was seen in retina, but astrocytes

strong pattern

Skin

cells

differences

revealed positive

parallels that of NADH-tetrazotype 1 fibers showed stronger

ure 3e). Axons, myelin, endoneunial cells were negative. In the tnigeminal

Eye Adult

muscle

the

but

there is a selec2A fibers. Since

in eye, kidney,

nervous

of aB-crystallin

Therefore, 1 and type

2A fibers,

In contrast,

in cardiac

this layer all showed

1. Strong

the

1970).

immunoreactions,

reductase

type

aB-crystallin immunoreactivity hum reductase. In general,

(Figure

muscle,

the

ofmyoglobin,

the endomysium and penimysium were muscles and tongue muscle also showed

Figure 2. Immunohistochemical localization ofaB-crystallin in a rat eye using the indirect method. (a) Strong positive reactions are seen in the lens (L) and cuboidalepithelium ofthe iris(l), and weak reaction in the deep layer of cuboidal epithelium of the ciliary body (CB). The cornea (C) and the neural retina (A) are negative. The dark line of the innermost surface of the neural retina is due to differential interference contrast of the relatively thick specimen and does not represent a positive reaction. (b) High magnification of the iris. The cytoplasm of cuboidal cells shows a diffuse positive reaction. Bars: a = 180 sm; b = 10 sm.

in Table

and

at pH 4.5 (Brooke

of NADH-tetrazolium

in both

crystallin

S

method

reaction (Figure 4). All of the aerohalf of the anaerobic (type 2) fibers

only 3% oftype 2B fibers were positive. tive expression of aB-crystallin in type

skeletal

mo-

distribution of aBto the muscle fiber

were positive for aB-crystallin (Table 2). Rat type 2 fibers were funthen subdivided into two groups (2A and 2B). Type 2A fibers are characterized by (a) a strong reaction ofNADH-tetrazolium reduc-

.

tase,

heart,

a heterogeneous

of positive muscle fibers. The in each fascicle was examined relative

classification

manized

see-

saledistribution crystallin

Lb

in longitudinal

3b).

Muscles

Cross-sections

CB

peniodicity

(Figures

and

brainstem

showed

Downloaded from jhc.sagepub.com at KAI NAN UNIV on March 21, 2015

weak

positive

are required for definiin cerebral hemispheres

reactions,

but

Bergman

glia

.5

H

-S-S

.:

.

. S

:-

#{149}

d --

.,.

S

-S

.

\

‘

S,..,

S S

S

f.

,

. ‘

S

,.

S.

‘:

S

.-

.

.-

,

.

)

T”

.

.

.

‘?

....5T-i-S

‘S

S

‘

I

.S,,..f

4

#{149}‘

/

S

,/

F

:

,‘#{149},

c

1,

: p.

)S

.t

S

tw,

5,1.

I

,,

‘

‘I

t,

:

‘I.

:

+,#{231}:.

\ . S.

.

1

,

S

;‘

‘

,g.’ ,

. S

#{149}SQ___ #{149}

S.

-

I

h

S

Figure a Immunostaining of rat tissues with anti-uB-crystallin antibody. (a) Heart; longitudinal section of the cardiac muscle fibers. (b) Heart; cross-section. All cardiac muscle fibers are stained. (C) Esophagus; note diffuse staining ofthe muscularis propria. (d) Abdominal skin; the thin layer of striated muscle fibers beneath the root of hair (H) is positive. (a) Cross-section of sciatic nerve; the cytoplasm of Schwann cells is strongly positive, but myelin (arrowheads) and axon (arrows) are negative. (f) Long cytoplasmic processes of astrocytes and the glial limiting membrane of the optic nerve are weakly positive. (g) Small round glial cells in midbrain, probably oligodendrocytes, are positive. (h) Positive cells are mainly seen in the white matter (W) and a few positive cells in granular layer (G) of cerebellum. Bars: a,b - 38 Mm; c,d = 140 Rm; a = 14 tm; f-h = 36 tm.

34

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CELLULAR

DISTRIBUTION

OF aB-CRYSTALLIN

35

Figure 4. Serial sections of gastrocnemius stained with(a)myosin ATPaSe reaction (pH 9.4), (b) Immunostaining for aB-crystallin, and (C) NADH-tetrazolium reductase reaction. Type 1 fibers are marked by 1”. Bar = 100 tm.

:-‘

rA.

.

S

#{163}4.*t#L4

eSS

F’

-:

Table 2. Histochemical rat gastrocnemius

profiles

of muscle

fiber

types

in

Table

3.

Distribution

of aB-crystallin

NADPH-tetrazolium rat kidne/

reductase

and

in the

aB-crystallin-

Total

number

positive

number

(%)

Type

1 fibers

177

177 (100)

Type

2 fibers

838

434 (52)

2A

fibers

434

423

2B fibers

404

Structure

Zone

Cortex

in the cerebellum populations with

were

not stained.

in hemispheres,

the

(97)

We did not find

brainstem,

or cerebellum

that

reacted

antibody.

Inner

outer

stripe

medulla;

and

single papilla, of Henle begin tex (zone

1) and

terdigitate

of outer zone

medulla;

4, inner

the outer

in a saw-tooth

1 are glomeruli outer

stripe

This

zone

2 contains

pars

recta

zone

medulla.

patterns in the rat zone 1, cortex; zone

3, inner The

stripe

rat kidney

zone 4. The majority of the thick abruptly at the junction of zones

The the

stripe

Inner

To facilitate the description of the localization kidney, the following terminology will be used:

zone

stripe

any neuronal

Kidney

2,

and

(zone

stripe

ofthe

outer

medulla

The

predominant

proximal

and

distal

2) is a conspicuous and

proximal

has

(zone

zone

a

medulla

_

,

, moderate

unstained in aB.crystallin staining; + + , dark

There

scnibed

However, from the

reaction

as the decidua

6). In particular,

in the uterus

mouse.

up principally

strong

at the early

with

cells of

well-

prolifera-

portion of the placenta, which of the endometnium and is de-

basalis,

showed

positive

reactions

a positive were seen

reaction

(-)

(+) (- ) (- )

(+) ( -) ( -)

+

)

+

(

(- ) (- ) +

(

staining,

+

(

)

+

+

-) -)

+)

(+

)C

red or pink

+)

+

( (

in

reaction;

NADPH-TR

staining. reductase

is given

C

of the

blastic giant fetal portion

(Figure

in the decidual

compact

decidua,

an

cells of the fetal of the placenta.

area

portion.

that

There

borders

the

tropho-

was no reaction

in the

Discussion ticular tibody

the maternal stromal cells

(-)

(

(

+)

in

Placenta

was no positive

phase. is derived tive

(+

(-)

b The source of the histochemistry of NADPH-tetrazolium in Stemberg et xl. (1956). Progressively darker reaction in distal third.

Recently,

and

(+)

(-)

2) in-

delineated brush borders. The zonal structures in rat kidney are illustrated in Figure 5 and a detailed distribution of aB-cnystallin in the various zones is presented in l#{224}ble3 and Figure 5.

Uterus

NADPH-TR1

tubules.

in rat and tubules

a +

only

elements

convoluted

is made

convoluted

of outer

ascending limbs 3 and 4. The con-

fashion.

no glomeruli

of the

Glomeruli Proximal convoluted tubule Distal convoluted tubule Pars recta of proximal convoluted tubule Thick ascending limb Collecting duct Thin limb Thick ascending limb Collecting duct Thin limb Collecting duct

11 (3)

Outer

aB-Crystallin

we demonstrated

aB-crystallin

tissues by Northern against aB-crystallin

purified

antibody

homogenates placenta distribution

reacted

of heart, by Western

only

skeletal blotting.

ofaB-crystallin

em and Northern

blotting

Downloaded from jhc.sagepub.com at KAI NAN UNIV on March 21, 2015

expression

in extra-len-

blotting and produced a polyclonal an(Iwaki et al., 1989). This affinitywith muscle, The

a 22 KD sciatic

consistency

by immunohistochemistry further

supports

protein

nerve, between

in total kidney,

and

the

tissue

and by West-

this immunohistochem-

Figure 5. Schematic representation of a rat kidney (left) and immunocytochemical localization of aB-crystallin in tissue sections (rIght). Positive reactions are seen in the (a) pars recta of proximal convoluted tubules, outer stripe; (b) the thin limbs of loops of Henle, inner stripe; (C) loops of Henle in a border zone between inner stripe and inner medulla; (d) loops of Henle in inner half of inner medulla and the inner medullary collecting ducts, also shown by oblique lines in the scheme. Bar = 100 rim.

36

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CELLULAR

OF rzB-aYSTALLIN

DISTRIBUTION

37

from

embryonic

recent

quail

evidence

twitch

oxidative

fibers

(type

the

all cardiac This

,

.

‘..

.

S

,‘5D

S...

,

‘#{149} S

.

;

(

;.

-

%

-S..

fibers

is consistent

with

1 fibers,

expressed

in slow-

showed

the fact that

the so-called type

a positive

red fibers.

heart The

of a muscle

fiber

2B).

reaction

to

is composed

determination is believed

to be

on the nature of the particular motor neuron that inthe motor unit (Salmons and Sn#{233}ter, 1976). Thus, the exof aB-crystallin in muscle fibers presumably depends on

physiological

characteristics

of innervating

neurons

and

may

by denervation and reinnervation. This would provide model for the regulation of aB-crystallin expression

in muscle. In brain,

.

;,4’.

was selectively

muscle

histochemical

be changed an attractive

.

-..S

muscle

fibers (type 1)and fast-twitch oxidative-glycolytic 2A), but not in fast-twitch glycolytic fibers (type

type

dependent nervates pression

‘

some

pinealocytes

study. in skeletal

of a specific

,

Although

glandular

in our

of only

.-

of the

crystallin

uB-crystallin.

C

in cell culture. many

and other vertebrates evolved from pineal photorecepthe adult rat pineal did not show any reaction for aB-

In contrast,

.

that

ofhumans ton cells,

uB-Crystallin

-.:

pinealocytes

suggests

positive

reactivity

was mainly

found

in small

round

cells that bore the appearance of oligodendrocytes. A few astrocytes were also labeled, principally those near the pial surface. We

I(

“sd

.

S,.v’

performed

double

brain sections tein (GFAP),

S

“/

:

have

‘‘

“

and aB-crystallin. The with the GFAP antibody,

:i.’

‘

Figure 6. Rat placenta(gestational day 17). (a)The strong positive reaction with aB-crystallin antibody in the decidualtissueofthe maternal portion (D) sharply delineates the negative reaction of the trophoblastic giant cells in the fetal porlion (F). (b) Serial section is stained by hematoxylin and eosin. Bar = 100 tm.

and

However,

the

cellular

staining

iris by several a lens Mullen

investigators

pattern

is quite

has

(Clayton

protein fraction enriched cells ofseveral vertebrate

been et al.

reported ,

in retina

1968).

in a-crystallin species (Moscona

and

An antiserum

to

bound to retinal et al., 1985), and

cell

line

with both uB-crystallin

(U-373MG)

imal

convoluted

reported

cells.

This

phenomenon

expression

oflens

has been

proteins

predispose

them

to the lentoid

ton

1986;

Moscona

et al., In reptiles

the skin diminutive expression

and

other

(lens-like

and lower

termed

in non-lens Unset,

tnansdifferentiation. eye cells in culture

structures) 1983;

vertebrates,

Clayton the pineal

formation et al.,

may

of all crystallin

classes

in putative

bution

positive

subdivisions

The

fibers

1988). consists

which

of(a)

an initial

thick

of the

prox-

and

also often (b) a thin

sharply

Similar

referred

descending

(d) a thick ascending portions of(a), (b), and

for aB-crystallin.

using

(Iwaki

a continuation tubule,

in the kidney nephron.

are formed

is therefore

portion, structures,

reactions

rat and human

to decidual

lens cells diffrrentiated

Rosenthal

proximal

by Western

The

regional

delineates

zonal

distinctions

oxidative

enzymes

distri-

the segmental have

been

(Sternberg

et

al., 1956). In particular, the histochemical localization of NADPHtetrazolium reductase resembles that ofaB-crystallin, with the exception of reaction in the proximal convoluted tubule (l#{224}ble3).

1979). of

and

of the

in the

demonstrated

normal showed

and Corbin, 1988). Therelittle aB-crystallin they are ofthe protein in patholog-

represents

by histochemistry

onal,

lies at or near

tubule

ofaB-crystallin

(Clay-

surface, where it functions as a photoreceptor organ eye-like structure. Watanabe et al. (1985) demonstrated

when

and Corbin, in the kidney which

recta

readily

(c) a thin ascending Among these tubular

adult rat and in part of the retinal pigmented the rat fetus (embryonic day 17; unpublished bnyo retina has a potential for differentiation fiber

especially

portion

portion, portion.

been

1989; Goldman astrocytes have large amounts

Goldman of Henle

to as the pars

(c)showed

The

has

blotting (Iwaki et al., fore, although normal capable ofaccumulating

In contrast to their around brain infarcts

react with

GFAP and aB-crystallin (unpublished in rat astrocytes and a human glioma

positivity In culture,

anti-mouse a-cnystallin antibody labeled both MUllen cells and astrocytes in the cat retina (Lewis et al. , 1988). We have detected positive reactions to aB-crystallin in the iris and ciliary body of the epithelial cells of data). The chick emin culture into lens

did not stained

strong data).

et al., 1989; The loop

unique

round cells in white matter but some subpial astrocytes

of human acidic proof astrocytes,

data). astrocytes

descending

rather complicated. In ocular tissue, a-crystallin

staining fibnillary protein

both antibodies (unpublished counterparts, some reactive

ical conditions, ical study.

immunocytochemical

using antibodies against glial the major intermediate filament

phase

in which

placentae

the stromal

cells.

The uterine

were negative

Downloaded from jhc.sagepub.com at KAI NAN UNIV on March 21, 2015

stromal

for aB-crystailin.

cells were strongly positive. tial to produce aB-crystallin mation.

have

cells ofthe

been

as hemochoni-

classified

endometnium

have changed

cells at the early proliferative At the same

Therefore, stromal in undergoing

time,

the decidual

cells have the potena decidual transfor-

38

IWAKI,

The

localization

to be similar ned

out

patterns

to those

histochemical

in peripheral

in skeletal

muscle

oxidative

enzymes.

of some staining

nerves,

heart,

of aB-crystallin

tent

with

tive

astrocytes

the striking

placenta.

crystalhin-positive

strong data).

increase

astrocytes

seems

enzyme

Therefore,

the

cells in extra-lenticular

activity

of

appears

of aB-

the exception of despite its ac-

tive metabolism.

is an enzyme

It is possible

that

aB-crystallin

for enzymes involved in oxidative activity. crystallin species (taxon-specific crystallins)

on

In fact, sevhave recently

been found to be enzymes or to be related to enzymes metabolic pathways (Hendniks et al., 1988; Wistow

of common et al., 1987,

1988). For example, p-crystallin, a major protein offrog lens, shows about 50% protein sequence identity with human aldehyde reducta.se and rat lens aldose reductase, NADPH-dependent oxidoreductases

(Carper

et al.

ditol:NADP

1987).

,

The latter

oxidoreductase;

concentrations ripheral nerves,

in the epithelium renal papillae,

of the

vesicles

seminal

teins (Ingolia and (Nene et al., 1986). inhibitor Such

(Sharma

has been

site egg antigen cessive proteolysis

aldose

reductase

is found

(al-

in significant

of the lens, Schwann islets of Langerhans,

cells of peand cells

1973).

to the ubiquitous

small

et al.,

1987;

postulated

‘The and

heat-shock

pro-

Ortwerth,

to be potentially

and also in heat while proteins

shock,

when

1982). for a para-

useful it could

are partially

prevent

unfolded.

ex-

It is not

clear that the concentrations of the protein in most cells reach a level at which proteolytic inhibition would be significant, with the possible exception in non-lenticular tion

and

ofthe lens. Further examination of aB-crystallin cells, including studies of its subcellular localiza-

regulation,

function

of this

are

now

important

for a clearer

idea

of the

protein.

Acknowledgments We thank bovine sions,

Drs A. Spector, lens

and

Literature

a-crystallin,

R. Cb:esa,

andM.

Drs A. P Hays

E. Corbin

and

E. Randailfor

kind?

MH, Kaiser Arch

fr D4gatifor technical

helpful

the discus.

assistance.

Neurol

KK

(1970):

Muscle

oflens-specific

protein

tissues. Biochem fiber

types:

a-crystallin

Biophys Res Com-

how many

and what

23:369

AB, King CR, Z.elenka of the chloramphenicol

with

its relationship

The presence

transdiff#{232}rentiation

of to

20:137

I, de Pomerai Dl (1979): in retina cells and their 282:628

Relationship between cryscapacity to re-differentiate

Dubin RA, aB-crystallin

Wawrousek EF, PiatigorskyJ (1989): gene is not restricted to the lens.

Expression Mol Cell

of the murine Biol 9:1083

Duguid

JR.

modulated Acad

Rohwer

Sci USA

nucleotide Histochem

E, Sternberg

and 4:254 WH,

oxidative

ing methods

Isolation

of cDNAs

ofa cDNA

triphosphopynidine

of scrapie-

library.

Dunlap

nucleotide

CE (1956b):

enzymes.

III. Evaluation

for diphosphopyridine

nucleotide

dine nucleotide diaphorase Cytochem 4:284 RL (1962):

Proc Natl

The

Histochemical

studies

cytochemistry 21:471

localization

of tetrazolium

diaphorase,

and the succindehydrogenase of normal

stain-

tniphosphopyri-

system.J

and

J

diaphorase.

Histochem

reactive

J

astrocytes.

Exp Neurol

Gabbay KH (1973): tes. N Engl J Med

The

sorbitol

pathway

and

the complications

W, MuldersJWM,

Isolation 130:569

ofa

major

Bibby MA, Slingsby

protein

lens c-crystallin and lactate dehydrogenase gene product with two distinct functions.

Ingolia

EA (1982):

Craig

are related to each other Sci USA 79:2360

Four

small

component

C, Bloemendal

WW (1988): Duck tical: a single-copy Sci USA 85:7114 TD,

of diabe-

288:831

GoldmanJE, Corbin E (1988): Rosenthal fibers. Am J Pathol Hendriks

B (1988):

hybridization

WH, Dunlap CE (1956a): Histochemical localization enzymes. I. Tetrazolium stains for diphosphopynidine

diaphorase Cytochem

of specific

Friede

Seed

85:5738

Farber E, Sternberg of specific oxidative

Farber

RG,

RNAs by subtractive

of

H, deJong

B4 are idenProc Natl Acad

heat shock proteins a-crystallin. Proc Natl Acad

Drosophila

and to mammalian

Iwaki T, Kume-Iwaki A, Liem RKH, GoldmanJE expressed in non-lenticular tissues and accumulates

(1989):

aB-crystallin

in Alexander’s

is

disease

brain. Cell 57:71 Laemmli UK (1970): Cleavage of the head of bacteriophage

of structural T4. Nature

proteins 227:680

during

the

assembly

Lewis GP, Erickson PA, Kaska DD, FisherSK (1988): An immunocytochemical comparison ofM#{252}ller cells and astrocytes in the cat retina. Exp Eye Res 47:839 Moscona AA, Fox L, SmithJ, Degenstein L (1985): Antiserum to lens antigens immunostains Muller glia cells in the neural retina. Proc Nail Acad Sci USA 82:5570

Nakane PK, Pierce GBJr (1967): Enzyme-labeled and application for the localization of antigens.

antibodies: J Histochem

changes expression

preparation Cytochem

14:929

Nene V. Dunne DW, Johnson KS, Taylor DW, Cordingley JS (1986): Sequence and expression of a major egg antigen from Schistosoma mansoni. Homologies to heat shock proteins and alpha-crystallins. Mol Biochem Parasitol 21:179

Carper D, Nishimura C, Shinohara T, Dietzchold B, Wistow G, Craft C, Kador P, Kinoshita JH (1987): Aldose reductase and p-crystallin belong to the same protein superfamily as aldehyde reductase. FEBS Lett 220:209 Chepelinsky expression

LH (1986):

and

Moscona AA, Linser P (1983): Developmental and experimental in retinal glia cells: cell interactions and control of phenotype and stability. Curr Top Dcv Biol 18:155

Cited

Bhat SP, Nagineni CN (1989): aS subunit is present in other ocular and non-ocular mun 158:319 Brooke

McDermottforprov:ding

and

Clayton RM, Thomson tallin mRNA expression into lens cells. Nature

Neuropathol

Craig, 1982) and to a schistosome egg antigen a-Crystallin has also been shown to have trypsin-

activity

activity

the

(Gabbay,

are related

a-Crystallins

enzyme,

EC 1.1.1.21),

Dcv Bioi

Top

Bower DJ, Errington

to be related

to high levels of oxidative enzyme activities, with liver, which did not contain detectable aB-crystallin a co-factor enal other

crystallins

of the

in neac-

distribution

tissues

RM, JeannyJ-C,

extralenticular lens. Curr

to be consis-

GOLDMAN

Clayton RM, CampbellJC, Truman DES (1968): A re-examination organ specificity of lens antigens. Exp Eye Res 7:11 Clayton

reductase

All aB-crystallin-positive

in oxidative

1962).

appear also car-

reactions for NADH-tetrazoFurthermore, the pattern

in reactive

(Fniede,

kidney

We have

for NADH-tetrazolium

and

cells in those tissues also showed hum reductase (unpublished expression

and

KUME-IWAKI,

PS, PiatigorskyJ acetyltransferase

flanking sequences of the murine aA-crystallin lens epithelia. Proc Natl Acad Sci USA 82:2334

(1985): Lens-specific gene promoted by 5’

gene in explanted

chicken

O’Farrell proteins.

PH (1975): High resolution J Biol Chem 250:4007

two-dimensional

Okazaki K, Yasuda K, Kondoh H, OkadalS(1985): sible for tissue-specific expression of a chicken lens cells. EMBO J 4:2589

Overbeek

PA, Chepelinsky

Downloaded from jhc.sagepub.com at KAI NAN UNIV on March 21, 2015

AB, KhillanJS,

electrophoresis DNA sequences

a-crystalhin

PiatigorskyJ,

gene

Westphal

of

responin mouse

H(1985):

CEUIILAR

DISTRIBUTION

39

OF aB-CRYSTALLIN

Lens-specific expression and developmental regulation ofthe bacterial amphenicol acetyltransf#{232}rase gene driven by the munine nA-crystallin motor in transgenic mice. Proc Nail Acad Sci USA 82:7815 Quax-Jeuken

Y, Quax

W, van

Rens

Complete structure ofthe aB-crystallin tron distribution in the two nonlinked Sci USA 82:5819

G, Khan

PM,

Bloemendal

gene: conservation a-crystallin genes.

chlor-

pro-

hydrogenase

,

teinase

H (1985):

ofthe exon-inProc NatI Acad

Wabe

JM, Matthews histochemical method J 12:707

Y, Jones DA (1980): A quick, simple and reliable for ATPase in human muscle preparations. Histochem

e

Sharma KK, Olesen PR, Ortwerth BJ (1987): of trypsin by a-crystallin. Biochem Biophys Sternberg ofspecific tide and

The

activity binding

in the and

trans-

inhibition

Acta 915:284

WH, Farber E, Dunlap CE (1956): Histochemical localization oxidative enzymes: II. Localization ofdiphosphopyridine nucleotniphosphopyridine nucleotide diaphorases and the succinde-

GJ, Lietrnan

PiatigorskyJ

H, Tamamaki

(1988):

hydrogenase 326:622 Wistow

16:251

T, Williams

LA, t-crystallin/a-enolasc:

protein.

GJ, MuldersJWM, as a structural GJ, PiatigorskyJ

sion ofbroteins

Stapel

J Cell

SO, de Jong WW, Horwitz one gene encodes both an enBiol 107:2729

deJong WW (1987): The enzyme lactate deprotein in avian and crocodilian lenses. Nature (1988):

for a highly

zj

Lens

specialized

crystallins:

tissue.

the evolution and Rev Biochem

Annu

(1983): The appearance ofa-crystallin in relation in the embryonic mouse lens. Dcv Biol 96:173

Downloaded from jhc.sagepub.com at KAI NAN UNIV on March 21, 2015

pro-

Yasujima M, Nojyo Y, Ueda Y, Okada quail pineals as revealed by cell cul-

N,

ofembryonic

zyme and a lens structural Wistow

Salmons 5, Sr&er FA (1976): Significance of impulse formation of skeletal muscle type. Nature 263:30

K, Aoyama

(1985): Oculopotency studies. Cell Differ

‘

Wistow Round

in the kidney. J Histochem Cytochem 4:266 BJ (1982): Activation and release of a trypsin-like bovine lens a-crystallin. Exp Eye Res 34:659

system

Orrth from

expres-

57:479

to cell cycle phase