- Email: [email protected]
Further Studies on Nucleic Acid Metabolism in the Lens*
I N J E C T A B L E S I L A S T I C FOR E V I S C E R A T I O N
243
3. This method permits instillation of a DISADVANTAGES more precise amount of material in the 1. It is more difficult and time consuming scleral socket than approximation of size in to handle the material during surgery. a prosthesis. Frequently, a prosthesis of the 2. Wound closures must be more secure exact size is not available in the operating since the liquid material will seep into the room at the time of surgery. With injectable wound edges, preventing healing and leading silicone, the surgeon can always add more to extrusion. material at the end of the operation. Even SUMMARY if reoperation should be necessary, it is The results of our limited experience with simpler to add the injectable silicone than to injectable silicone in evisceration surgery reopen the entire wound to insert a larger have been reported. While this material implant. 4. Better motility and fitting of a specially shows promise in this field of eye surgery, it appears that it may be even more useful molded conformer (and later artificial eye) are possible, if the conformer is pressed in plastic lid surgery. 255 South 17th Street (3). against the globe while the silicone fluid is hardening. This was done in our first case but we are not at all sure that we achieved much or any molding of the scleral wall.
ACKNOWLEDGMENT
W e wish to thank Dr. Charles G. Steinmetz, III, pathologist at Wills Eye Hospital, for his help in reviewing the histologic preparations.
REFERENCES
1. Todd, Ε. M , Sheldon, C. H , Crue, B. L , Pudenz, R. H , and Agnew W . F.: Plastic jackets for certain intracranial aneurysms. J . A . M . A , 179:935-939, 1962. 2. Des Prez, J . D.: Paper presented at the Plastic Research Council in Galveston, April, 1963. 3. Prentiss, R , Booturight, D , Pennington, R., Hohn, W , and Schwartz, M . : Testicular prothesis: Materials, methods and results. J . Urol, 90 :208-210, 1963. 4. Conway, H , and Goulian, D.: Experience with an injectable silastic R T V as a subcutaneous prosthetic material: A preliminary report. Plast. & Reconstruct. S u r g , 31:294-302, 1963. 5. Blocksma, R.: Correction of velvopharvngeal insufficiency. Plast. & Reconstruct. S u r g , 31:268-274, 1963.
FURTHER
STUDIES
ON NUCLEIC ACID
SIDNEY LERMAN, AND
M.D,
Y'HESKEL Rochester,
The lens is known to contain at least three apparently distinct fractions of R N A ; solu ble, microsomal and albuminoid. The com-
METABOLISM
SEYMOUR A. Neiv
SA'AT,
PH.D.,
M.S.
York
position and relative concentration of these three fractions in rat and dogfish lenses have been reported in previous communica tions. The purpose of the present investi gation was to characterize further the al buminoid R N A fraction, to determine whether messenger R N A was present in the lens and to study the rate of transfer of one of the component bases of R N A across the isolated dogfish lens capsule. 1,2
* From the Division of Ophthalmology, Depart ment of Surgery, and the Department of Bio chemistry, University of Rochester School of Medicine and Dentistry. This work was supported by U.S.P.H.S. research grant B-3081 and by Fightfor-Sight fellowships (Zigman and Sa'at). A por tion of this study was performed at the Marine Bilogical Laboratory, Woods Hole, Massachusetts, supported in part by an M.B.L.-A.E.C. grant.
ZIGMAN,
IN T H E L E N S *
S I D N E Y
244
MATERIALS
L E R M A N ,
AND
S E Y M O U R
Z I G M A N
METHODS
A N D
Y ' H E S K E L
A.
S A ' A T
placed on dry ice for one or Iwo minutes. The RA A from the capsular fraction was separated into soluble and particulate frac tions and extracted by hot TCA ; the remain ing lens matter was treated as already out lined for the intact lens. The experiments with the isolated lens capsules were performed with a verv simple diffusion chamber consisting of glass tubing tightly sealed with a portion of fresh ante rior or posterior dogfish lens capsule im mersed in Elasmobranch Ringers Solution. The possibility of leakage was ruled out by periodic checks of the media inside and out side the tubes employing 1.0% solutions of hemoglobin and cobalt as indicators. C uracil was employed as the test substance. The methods employed to determine the base ratios of the capsular RNA and the distri bution of C ' uracil in the bases and nucleo tides derived from acid hydrolysates of al buminoid, microsomal and soluble RNA from the dogfish lens have been described in earlier communications. ' T
Albuminoid RNA was extracted from fresh pooled dogfish lenses and from frozen rabbit lenses according to methods previously described. It was determined to be over 95 /c pure by spectral assay, micro-orcinol and micro-indole measurements, and the ex tent of protein contamination was checked by the Lowry method. " Approximately onepercent solutions were run in the ultracentrifuge at 56,100 rpm at 20°C in order to determine their sedimentation coefficients. The assay for messenger RNA was per formed by means of pulse-labelling experi ments utilizing fresh dogfish, rabbit and rat lenses. These lenses were incubated in Elasmobranch Ringers solution (dogfish lenses) or Eagle's medium containing added glutamate (rabbit and rat lenses) for 5, 15 and 60 minutes in most of the experiments or for 15, 45 and 120 minutes in one experi ment. C uracil (50 microcuries) was added at the beginning of each incubation to the tubes containing the dogfish lenses and a similar amount of C adenine was added to the rat and rabbit lens incubates. Suitable controls were run with each experiment in which the conditions were the same except that these lenses were incubated under 9 5 % nitrogen and 5.0% CO- in an ice bath. The experimental lenses were all incubated under 9 5 % oxygen and 5.0% C 0 and the tem perature was maintained at 37°C for the mammalian lenses and 22°C for the marine lenses. The various RNA fractions (solu ble, microsomal and albuminoid) were iso lated by differential centrifugation and sub jected to hot TCA extractions as previously described. ' After sufficient washings in cold TCA (until the final wash had no activity), the fractions were washed with ether, plated and counted in an end-window gas flow counter. In some experiments the capsules were carefully removed at the end of the incubation periods. In order to facilitate re moval of the capsule without leaving a dis integrated lens behind, these lenses were first 1
c
3
5
14
14
1 1
1 1
14
2
1
1 6
RESULTS
The results of the ultracentrifugation ex periments on dogfish and rabbit lens albumi noid RNA revealed a single peak with a sedimentation coefficient of 7.4s and 7.3s respectively. This would indicate that albu minoid RNA is a relatively small molecule with a molecular weight which would ap proximate the values reported for soluble RNA (less than 50,000). The results of the pulse labelling experi ments on the dogfish lens are shown in Fig ures 1 and 2. In the RNA fractions derived from the whole lens incubates there is a peakin the specific activity of both the microso mal and albuminoid RNA after five minutes followed by a steady decline over the re maining 55 minutes (fig. 1 ) . The soluble fraction appears to become labelled very rap idly but continues to increase throughout the incubation period. If the capsule is removed from these lenses, the initial peak in activity
NUCLEIC A C I D M E T A B O L I S M
IN L E N S
245
1200 5000-
1000
0 Fig.
10
1 (Lerman,
20
Zigman
experiment
30 40 MINUTES and
with
Sa'at). C "
50
60 0
20
40
Fig.
2
beling
The base ratios of the dogfish capsular RNA are shown in Table 1. They are simi lar to the values previously reported for al buminoid and microsomal RNA. The rate 2
80
100
120
MINUTES
Pulse-labeling
uracil.
in the microsomal and albuminoid RNA fractions disappears and is now present only in the particulate RNA fraction derived from the capsule (fig. 2 ) . Both the soluble fractions derived from the lens matter and the capsule show a continuous rise in spe cific activity over the two-hour period of incubation. The results of the pulse labelling experiments with the mammalian lenses were inconclusive because the resultant activity was too close to background.
60
of
the
the
(Lerman,
experiment lens
Zigman with
capsules
and
C "
were
Sa'at).
uracil
in
removed
at
Pulse-la
which the
some end
of passage of C uracil through the isolated anterior dogfish lens capsule was 3.2 X 10"" millimicromoles per hour compared with 5.0 X 10"° millimicromoles per hour through the posterior lens capsule (table 2 ) . This effect is illustrated in Figure 3 in which the slope of the activity accumulating within the dif fusion tube is plotted for the anterior and posterior lens capsule. The activity of the isotope in the outside media decreases over the 24-hour period of observation while there is a much more marked increase in activity within the diffusion tube when posterior 14
TABLE 1 B A S E RATIOS OF R X A FROM DOGFISH CAPSULES Moles Guanylic
of
incubation.
c /C
Ratio
Adenylic
Cytidylic
Uridylic
Acid
Acid
Acid
Acid
Pyrimidines
30
20
27
23
1.0
Purines
to
Of 6-Amino 6-Keto
to
Bases
0.9
S I D N E Y
246
L E R M A N ,
S E Y M O U R
Z I G M A N
20,000
A N D
Y ' l I E S K E L
A.
S A ' A T
pated a more rapid rate of isotope transfer through the anterior rather than the poste rior lens capsule. The dogfish lens is similar to the mammalian lenses in that it has a single layer of subcapsular epithelium along its anterior surface which extends slightly beyond either equator. If one were to invoke a transport process for the entry of nucleic acid metabolites into the lens, the necessary energy requirements would most likely be more concentrated where the lenticular epithelium was present (anterior capsule). When the lens capsule is removed, the epithelial layer comes off with it, and yet the results of these experiments indicate that more C uracil gets through the posterior as compared with the anterior capsule (almost twice as much). This result may simply be due to the fact that the physi ologic state of the lens capsule has been sufficiently altered to change its properties. 14
HOURS Fig.
3
(Lermaii,
the
accumulation
and
posterior
lens
Zigman
of
C'
4
and
uracil
capsules
Sa'at).
across
the
Slope
of
anterior
(dogfish).
capsule is employed as compared with ante rior capsule.
A second possibility is that the anterior capsule with its underlying epithelium accu mulates a large amount of isotope during the experimental procedure while the posterior capsule (which has no epithelial cells associ ated with i t ) permits free passage of C ' uracil by means of simple diffusion. In sev eral experiments where C leucine was em ployed as the test substance, a similar result was obtained. In these experiments the ad dition of ouabain or cyanide to the outside media or lowering the temperature to 5°C resulted in a significant increase in the rate of penetration of this isotope through the anterior lens capsule but there was no effect on the posterior capsule. 1
DISCUSSION
The results of these experiments indicate that albuminoid RNA has a low molecular weight approximating that of the soluble RNA fraction. Since R Ν Aase does not ap pear to be present in the dogfish lens and the duponol extraction procedure does not break down RNA, the low molecular weight for albuminoid RNA appears to be a valid result. This observation correlates well with other studies in our laboratory which would indicate that albuminoid RNA actually de rives from soluble RNA."" The presence of messenger RNA in the lens is to be expected since this organ has a high and complex protein content ( 3 0 - 3 5 % ) and must have the necessary template in order to synthesize the proper protein frac tions. The results of the isolated lens capsule experiments are, at first glance, somewhat disconcerting since one might have antici R
11
14
12
These experiments indicate that metabolic inhibitors allow for an increased rate of passage of certain substances through the isolated anterior lens capsule containing a contiguous epithelial cell layer. That is, an inhibition of cellular metabolism allows for more isotopes to get through, while in the normal state the presence of epithelial cells either inhibits or traps this substance. It is also possible that these isotopes penetrate by means of simple diffusion which would ac-
N U C L E I C
A C I D
M E T A B O L I S M
count for the slower rate of transfer across the anterior capsule since there is actually a second cell layer to be traversed here as com pared with the posterior capsule. Studies are
I N
247
L E N S
now in progress to elucidate this phenomenon further, 260 Crittenden
Boulevard
(20).
REFERENCES 1. L e r m a n ,
S . : Cataracts:
2.
S , Zigman,
Lerman,
Chemistry,
S , Burton,
lation a n d composition o f R N A . 3.
Mejbaum
\ V . : Ueber
Ceriotti,
( j . : A
Invest. O p h t h ,
die Bestimmung
saure. Ztschr. Physiol. C h e m , 4.
Mechanisms and Therapy.
M , and Fontaine,
Springfield, 111, T h o m a s ,
J . : Nucleic
acid
metabolism
1964, chap. 2.
in the lens:
I.
Iso
2:617, 1963.
kleiner
Pentosemengen
Insbesodere
in D e r i v a t e n
der Adenyl-
258:117, 1939.
microchemical
determination
of
desoxvribonucleic
acid.
I.
Biol.
Chem,
198:297,
1952. 5.
Lowry,
O. H ,
6.
N . J , Farr,
Rosebrough,
reagent. J .Biol. C h e m ,
folin phenol
Lernian,
S , Devi, A , and Banerjee.
m e t a b o l i s m in t h e r a t lens. Invest. O p h t h , 7. L e r m a n , Ophth, 8.
S , and Zigman,
2:626,
Zigman.
S , Burton,
Zigman,
R . J . : Protein
measurement
with
the
R . : T h eeffect
o f ionizing radiation
on protein
and carbohydrate
1 :95, 1962.
S . : Nucleic acid
metabolism
in t h e l e n s :
I I I . Effect
o f x-radiation.
Invest.
1963. M , Fontaine
nuclease activity a n d R N A turnover. 9.
A . L , and Randall,
193:265, 1951.
S , and Lerman,
J . and Lerman
Invest. O p h t h ,
S . : Properties
S . : Nucleic acid metabolism
in t h e l e n s :
I I . Ribo-
2:621, 1963.
o f a cold
precipitable
protein
( C P P ) o f t h e lens.
Exper.
E y e R e s , in print. 10. tein
Zigman, ( C P P )
11.
S , Burton,
M , Munro,
o f t h e lens. I n
J , and Lerman
S . : Metabolic studies
o f the cold precipitable
pro
preparation.
Lerman,
S . : Metabolic
Lerman,
S , Zigman,
pathwavs
in e x p e r i m e n t a l
sugar
a n d radiation
cataract.
Phvsiol.
R e v ,
Ian.
1965. 12. (b)
albuminoid
R N A .
S , and Sa'at, Υ . Α . : Studies
Biol. B u l l ,
BACTERIAL
on the dogfish
lens:
( a ) membrane
permeabilitv,
125:383, 1963.
FLORA OF
AND PREOPERATIVE DONOR
TREATMENT
CORNEAS*
HAL J . ROLLINS, J R . . M . D . , A N D F . W . STOCKER, M . D .
Durham,
North
Postoperative infection in keratoplasty as with other types of ocular surgery is a seri ous complication. One possible source of such infection is the donor material which in most cases is obtained after death. Paton reported that approximately 50% of the eyes received in eye-banks show positive bacterial cultures. Other investigators have shown as high as 85.3% positive cultures from cadaver eyes. It seemed of interest to see what bacteria are cultured from the donor eyes as received 1
Carolina
from the eye-bank and then to determine if any postoperative infections could be corre lated with the preoperative findings. Also, an evaluation of our present method of pre operative treatment of the donor eyes could be made.
2
* From of the
the Division
Medicine,
o f Ophthalmology,
University
McPherson
Hospital.
o f
North
School
Carolina,
and
MATERIALS
A N D METHODS
One hundred donor eyes received from the eye-bank in sterile moist containers and stored at + 4 ° C were used for this study. Cultures for bacterial organisms were set up in all and for fungi in some of the cases. An applicator moistened in brain-heart in-
243
3. This method permits instillation of a DISADVANTAGES more precise amount of material in the 1. It is more difficult and time consuming scleral socket than approximation of size in to handle the material during surgery. a prosthesis. Frequently, a prosthesis of the 2. Wound closures must be more secure exact size is not available in the operating since the liquid material will seep into the room at the time of surgery. With injectable wound edges, preventing healing and leading silicone, the surgeon can always add more to extrusion. material at the end of the operation. Even SUMMARY if reoperation should be necessary, it is The results of our limited experience with simpler to add the injectable silicone than to injectable silicone in evisceration surgery reopen the entire wound to insert a larger have been reported. While this material implant. 4. Better motility and fitting of a specially shows promise in this field of eye surgery, it appears that it may be even more useful molded conformer (and later artificial eye) are possible, if the conformer is pressed in plastic lid surgery. 255 South 17th Street (3). against the globe while the silicone fluid is hardening. This was done in our first case but we are not at all sure that we achieved much or any molding of the scleral wall.
ACKNOWLEDGMENT
W e wish to thank Dr. Charles G. Steinmetz, III, pathologist at Wills Eye Hospital, for his help in reviewing the histologic preparations.
REFERENCES
1. Todd, Ε. M , Sheldon, C. H , Crue, B. L , Pudenz, R. H , and Agnew W . F.: Plastic jackets for certain intracranial aneurysms. J . A . M . A , 179:935-939, 1962. 2. Des Prez, J . D.: Paper presented at the Plastic Research Council in Galveston, April, 1963. 3. Prentiss, R , Booturight, D , Pennington, R., Hohn, W , and Schwartz, M . : Testicular prothesis: Materials, methods and results. J . Urol, 90 :208-210, 1963. 4. Conway, H , and Goulian, D.: Experience with an injectable silastic R T V as a subcutaneous prosthetic material: A preliminary report. Plast. & Reconstruct. S u r g , 31:294-302, 1963. 5. Blocksma, R.: Correction of velvopharvngeal insufficiency. Plast. & Reconstruct. S u r g , 31:268-274, 1963.
FURTHER
STUDIES
ON NUCLEIC ACID
SIDNEY LERMAN, AND
M.D,
Y'HESKEL Rochester,
The lens is known to contain at least three apparently distinct fractions of R N A ; solu ble, microsomal and albuminoid. The com-
METABOLISM
SEYMOUR A. Neiv
SA'AT,
PH.D.,
M.S.
York
position and relative concentration of these three fractions in rat and dogfish lenses have been reported in previous communica tions. The purpose of the present investi gation was to characterize further the al buminoid R N A fraction, to determine whether messenger R N A was present in the lens and to study the rate of transfer of one of the component bases of R N A across the isolated dogfish lens capsule. 1,2
* From the Division of Ophthalmology, Depart ment of Surgery, and the Department of Bio chemistry, University of Rochester School of Medicine and Dentistry. This work was supported by U.S.P.H.S. research grant B-3081 and by Fightfor-Sight fellowships (Zigman and Sa'at). A por tion of this study was performed at the Marine Bilogical Laboratory, Woods Hole, Massachusetts, supported in part by an M.B.L.-A.E.C. grant.
ZIGMAN,
IN T H E L E N S *
S I D N E Y
244
MATERIALS
L E R M A N ,
AND
S E Y M O U R
Z I G M A N
METHODS
A N D
Y ' H E S K E L
A.
S A ' A T
placed on dry ice for one or Iwo minutes. The RA A from the capsular fraction was separated into soluble and particulate frac tions and extracted by hot TCA ; the remain ing lens matter was treated as already out lined for the intact lens. The experiments with the isolated lens capsules were performed with a verv simple diffusion chamber consisting of glass tubing tightly sealed with a portion of fresh ante rior or posterior dogfish lens capsule im mersed in Elasmobranch Ringers Solution. The possibility of leakage was ruled out by periodic checks of the media inside and out side the tubes employing 1.0% solutions of hemoglobin and cobalt as indicators. C uracil was employed as the test substance. The methods employed to determine the base ratios of the capsular RNA and the distri bution of C ' uracil in the bases and nucleo tides derived from acid hydrolysates of al buminoid, microsomal and soluble RNA from the dogfish lens have been described in earlier communications. ' T
Albuminoid RNA was extracted from fresh pooled dogfish lenses and from frozen rabbit lenses according to methods previously described. It was determined to be over 95 /c pure by spectral assay, micro-orcinol and micro-indole measurements, and the ex tent of protein contamination was checked by the Lowry method. " Approximately onepercent solutions were run in the ultracentrifuge at 56,100 rpm at 20°C in order to determine their sedimentation coefficients. The assay for messenger RNA was per formed by means of pulse-labelling experi ments utilizing fresh dogfish, rabbit and rat lenses. These lenses were incubated in Elasmobranch Ringers solution (dogfish lenses) or Eagle's medium containing added glutamate (rabbit and rat lenses) for 5, 15 and 60 minutes in most of the experiments or for 15, 45 and 120 minutes in one experi ment. C uracil (50 microcuries) was added at the beginning of each incubation to the tubes containing the dogfish lenses and a similar amount of C adenine was added to the rat and rabbit lens incubates. Suitable controls were run with each experiment in which the conditions were the same except that these lenses were incubated under 9 5 % nitrogen and 5.0% CO- in an ice bath. The experimental lenses were all incubated under 9 5 % oxygen and 5.0% C 0 and the tem perature was maintained at 37°C for the mammalian lenses and 22°C for the marine lenses. The various RNA fractions (solu ble, microsomal and albuminoid) were iso lated by differential centrifugation and sub jected to hot TCA extractions as previously described. ' After sufficient washings in cold TCA (until the final wash had no activity), the fractions were washed with ether, plated and counted in an end-window gas flow counter. In some experiments the capsules were carefully removed at the end of the incubation periods. In order to facilitate re moval of the capsule without leaving a dis integrated lens behind, these lenses were first 1
c
3
5
14
14
1 1
1 1
14
2
1
1 6
RESULTS
The results of the ultracentrifugation ex periments on dogfish and rabbit lens albumi noid RNA revealed a single peak with a sedimentation coefficient of 7.4s and 7.3s respectively. This would indicate that albu minoid RNA is a relatively small molecule with a molecular weight which would ap proximate the values reported for soluble RNA (less than 50,000). The results of the pulse labelling experi ments on the dogfish lens are shown in Fig ures 1 and 2. In the RNA fractions derived from the whole lens incubates there is a peakin the specific activity of both the microso mal and albuminoid RNA after five minutes followed by a steady decline over the re maining 55 minutes (fig. 1 ) . The soluble fraction appears to become labelled very rap idly but continues to increase throughout the incubation period. If the capsule is removed from these lenses, the initial peak in activity
NUCLEIC A C I D M E T A B O L I S M
IN L E N S
245
1200 5000-
1000
0 Fig.
10
1 (Lerman,
20
Zigman
experiment
30 40 MINUTES and
with
Sa'at). C "
50
60 0
20
40
Fig.
2
beling
The base ratios of the dogfish capsular RNA are shown in Table 1. They are simi lar to the values previously reported for al buminoid and microsomal RNA. The rate 2
80
100
120
MINUTES
Pulse-labeling
uracil.
in the microsomal and albuminoid RNA fractions disappears and is now present only in the particulate RNA fraction derived from the capsule (fig. 2 ) . Both the soluble fractions derived from the lens matter and the capsule show a continuous rise in spe cific activity over the two-hour period of incubation. The results of the pulse labelling experiments with the mammalian lenses were inconclusive because the resultant activity was too close to background.
60
of
the
the
(Lerman,
experiment lens
Zigman with
capsules
and
C "
were
Sa'at).
uracil
in
removed
at
Pulse-la
which the
some end
of passage of C uracil through the isolated anterior dogfish lens capsule was 3.2 X 10"" millimicromoles per hour compared with 5.0 X 10"° millimicromoles per hour through the posterior lens capsule (table 2 ) . This effect is illustrated in Figure 3 in which the slope of the activity accumulating within the dif fusion tube is plotted for the anterior and posterior lens capsule. The activity of the isotope in the outside media decreases over the 24-hour period of observation while there is a much more marked increase in activity within the diffusion tube when posterior 14
TABLE 1 B A S E RATIOS OF R X A FROM DOGFISH CAPSULES Moles Guanylic
of
incubation.
c /C
Ratio
Adenylic
Cytidylic
Uridylic
Acid
Acid
Acid
Acid
Pyrimidines
30
20
27
23
1.0
Purines
to
Of 6-Amino 6-Keto
to
Bases
0.9
S I D N E Y
246
L E R M A N ,
S E Y M O U R
Z I G M A N
20,000
A N D
Y ' l I E S K E L
A.
S A ' A T
pated a more rapid rate of isotope transfer through the anterior rather than the poste rior lens capsule. The dogfish lens is similar to the mammalian lenses in that it has a single layer of subcapsular epithelium along its anterior surface which extends slightly beyond either equator. If one were to invoke a transport process for the entry of nucleic acid metabolites into the lens, the necessary energy requirements would most likely be more concentrated where the lenticular epithelium was present (anterior capsule). When the lens capsule is removed, the epithelial layer comes off with it, and yet the results of these experiments indicate that more C uracil gets through the posterior as compared with the anterior capsule (almost twice as much). This result may simply be due to the fact that the physi ologic state of the lens capsule has been sufficiently altered to change its properties. 14
HOURS Fig.
3
(Lermaii,
the
accumulation
and
posterior
lens
Zigman
of
C'
4
and
uracil
capsules
Sa'at).
across
the
Slope
of
anterior
(dogfish).
capsule is employed as compared with ante rior capsule.
A second possibility is that the anterior capsule with its underlying epithelium accu mulates a large amount of isotope during the experimental procedure while the posterior capsule (which has no epithelial cells associ ated with i t ) permits free passage of C ' uracil by means of simple diffusion. In sev eral experiments where C leucine was em ployed as the test substance, a similar result was obtained. In these experiments the ad dition of ouabain or cyanide to the outside media or lowering the temperature to 5°C resulted in a significant increase in the rate of penetration of this isotope through the anterior lens capsule but there was no effect on the posterior capsule. 1
DISCUSSION
The results of these experiments indicate that albuminoid RNA has a low molecular weight approximating that of the soluble RNA fraction. Since R Ν Aase does not ap pear to be present in the dogfish lens and the duponol extraction procedure does not break down RNA, the low molecular weight for albuminoid RNA appears to be a valid result. This observation correlates well with other studies in our laboratory which would indicate that albuminoid RNA actually de rives from soluble RNA."" The presence of messenger RNA in the lens is to be expected since this organ has a high and complex protein content ( 3 0 - 3 5 % ) and must have the necessary template in order to synthesize the proper protein frac tions. The results of the isolated lens capsule experiments are, at first glance, somewhat disconcerting since one might have antici R
11
14
12
These experiments indicate that metabolic inhibitors allow for an increased rate of passage of certain substances through the isolated anterior lens capsule containing a contiguous epithelial cell layer. That is, an inhibition of cellular metabolism allows for more isotopes to get through, while in the normal state the presence of epithelial cells either inhibits or traps this substance. It is also possible that these isotopes penetrate by means of simple diffusion which would ac-
N U C L E I C
A C I D
M E T A B O L I S M
count for the slower rate of transfer across the anterior capsule since there is actually a second cell layer to be traversed here as com pared with the posterior capsule. Studies are
I N
247
L E N S
now in progress to elucidate this phenomenon further, 260 Crittenden
Boulevard
(20).
REFERENCES 1. L e r m a n ,
S . : Cataracts:
2.
S , Zigman,
Lerman,
Chemistry,
S , Burton,
lation a n d composition o f R N A . 3.
Mejbaum
\ V . : Ueber
Ceriotti,
( j . : A
Invest. O p h t h ,
die Bestimmung
saure. Ztschr. Physiol. C h e m , 4.
Mechanisms and Therapy.
M , and Fontaine,
Springfield, 111, T h o m a s ,
J . : Nucleic
acid
metabolism
1964, chap. 2.
in the lens:
I.
Iso
2:617, 1963.
kleiner
Pentosemengen
Insbesodere
in D e r i v a t e n
der Adenyl-
258:117, 1939.
microchemical
determination
of
desoxvribonucleic
acid.
I.
Biol.
Chem,
198:297,
1952. 5.
Lowry,
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FLORA OF
AND PREOPERATIVE DONOR
TREATMENT
CORNEAS*
HAL J . ROLLINS, J R . . M . D . , A N D F . W . STOCKER, M . D .
Durham,
North
Postoperative infection in keratoplasty as with other types of ocular surgery is a seri ous complication. One possible source of such infection is the donor material which in most cases is obtained after death. Paton reported that approximately 50% of the eyes received in eye-banks show positive bacterial cultures. Other investigators have shown as high as 85.3% positive cultures from cadaver eyes. It seemed of interest to see what bacteria are cultured from the donor eyes as received 1
Carolina
from the eye-bank and then to determine if any postoperative infections could be corre lated with the preoperative findings. Also, an evaluation of our present method of pre operative treatment of the donor eyes could be made.
2
* From of the
the Division
Medicine,
o f Ophthalmology,
University
McPherson
Hospital.
o f
North
School
Carolina,
and
MATERIALS
A N D METHODS
One hundred donor eyes received from the eye-bank in sterile moist containers and stored at + 4 ° C were used for this study. Cultures for bacterial organisms were set up in all and for fungi in some of the cases. An applicator moistened in brain-heart in-