Johan Zwaan

Macromolecular events during differentiation of the chicken lens.

The synthesis of lens proteins during differentiation of the chicken lens was studied with antisera to adult lens and to fractions isolated by polyacrylamide gel electrophoresis. δ-Crystallin (FISC) was first detected by immunoelectrophoresis at 60 hr. Lens placode cells showed immunofluorescence at 52 hr, mainly in their basal parts. Autoradiography after exposure to [3H]thymidine showed that their nuclei undergo interkinetic migration. During interkinesis they are located in the basal parts of the cells, but for mitosis the cells contract to the lumen. Following division the daughter cells again elongate and the nuclei return to the retinal side. Thus it is not surprising that δ-crystallin first appears here. At 57 hr the reaction had spread throughout the posterior wall, and at 3 days the epithelium was positive. At 8 days the latter reacted progressively weaker but the fibers remained positive. In 5-week-old chicks and in adults the cortex was negative while the center still showed strong fluorescence. α-Crystallin was detectable by immunoelectrophoresis on the fourth day of incubation. It was first demonstrated with immunofluorescence in centrally located lens fibers at 3ȁ3½ days. At 8 days the epithelium became positive and the fibers lost some fluorescence. This continued until in 5-week-old chicks the lens core was negative. The β-crystallins of the adult chicken consist of 8 or 9 immunologically related proteins. At 7 days the first component was seen immunoelectrophoretically. The pattern became more complicated until it was comparable at 17 days of embryonic life with that of the adult. Fluorecence was first observed in 28-somite embryos in the same location as δ-crystallin. At 3½ days the epithelium began to react and at 4 days the complete lens was positive. The reaction disappeared from the nucleus at a faster rate than that for α-crystallin. γ-Crystallins were not found. Fluorescence was never seen outside the lens. We conclude that crystallin synthesis is restricted to the lens itself and first occurs after placode formation has taken place. The most plausible explanation is that lens differentiation is dependent on differential gene activity at the level of the lens cell nuclei.

Estimation of molecular weights of proteins by polyacrylamide gel electrophoresis

Abstract 1. 1. The vertical polyacrylamide gel electrophoresis method of Raymond, which allows the study of several samples simultaneously, was used to examine the relation between gel concentration and migration rates of several proteins. A linear relation was found between the two in gel concentrations from 5 to at least 10%, but below and above this range the angle of the plotted lines changed. The angles of the main part of the plots for each protein with the vertical as well as the higher gel concentrations at which the breaks in the lines occurred were roughly related to the molecular weights of the proteins. 2. 2. The ratio of the absolute mobilities in two different gel concentrations was linearly related to log molecular weight and therefore allowed a simple molecular weight estimation. 3. 3. On theoretical grounds it is highly probable that this ratio is related to diffusion coefficient or molecular radius rather than to molecular weight. The good results obtained with the electrophoretic method of molecular weight estimation are probably based on the use of “ideal” proteins, having similar partial specific volumes and frictional ratios, as calibration standards.

Cataracts and abnormal proliferation of the lens epithelium in mice carrying the catfr gene.

In mice carrying the dominant gene Cotaract-Fraser (or Shrivelled) pyknosis of the nuclei of the lens fibers is followed by cytoplasmic abnormalities and eventually complete destruction of the main lens fiber mass. The process begins at 14 days of embryonic life and is continuous throughout life. In addition to this degeneration of the fibers, the anterior epithelium becomes abnormal postnatally leading to anterior polar cataracts. The epithelial cells show unusual proliferative activity and form multiple layers of atypical cells. The cell masses may become very large and sometimes seem to infiltrate the remainder of the lens fibers. Occasionally whorls of spindle-shaped cells are present. The anterior capsule is much thicker than normal and irregular clumps of capsule-like material are found in between the epithelial cells. It is possible, that the changes in the anterior epithelium and capsule are secondary to the damage found in the fibers.

The changing cellular localization of α-crystallin in the lens of the chicken embryo, studied by immunofluorescence

Abstract The appearance and distribution of α-crystallin in chicken embryos and in animals after hatching were studied with the indirect fluorescent antibody method. α-Crystallin was isolated by two-dimensional polyacrylamide gel electrophoresis. Antisera were prepared against the purified fraction and against total chicken lens. They were controlled for their specificity in Ouchterlony plates and immunoelectrophoresis. The first fluorescence with total lens antiserum was observed in the morphologically most advanced cells of the lens placode of 23-somite embryos (50–53 hours of incubation). α-Crystallin antisera did not react at this time, indicating that another structural protein was synthesized before α-crystallin appeared. With further development of the lens rudiment the fluorescence gradually spread to more peripheral parts, and at 3 to 3/12 days the entire lens reacted with total lens antiserum. At this time α-crystallin could first be detected in and around the nuclei of a few centrally located fibers. Gradually more peripheral fibers became involved, then the equatorial zone, and finally the anterior epithelium. While some cells reacted very strongly, others were only weakly fluorescent or even remained negative; there resulted a mosaiclike staining pattern. At 8 days of embryonic life all epithelial cells were positive for α-crystallin and the reaction in the fibers became progressively weaker. In the adult lens the center of the lens was negative, the epithelial cells and equatorial zone, with the exception of a few completely negative cells, were strongly positive and the superficial fibers showed an intermediate reaction. The fluorescence was always confined to the lens. It is concluded that lens placode formation is independent from crystallin synthesis. Furthermore, the production of crystallins is restricted to the lens rudiment and lens and does not take place in the optic cup and its derivates or in other ectodermal tissues. α-Crystallin appears relatively late in development and cannot be considered as an essential protein for lens formation.

Cell population kinetics in the chicken lens primordium during and shortly after its contact with the optic cup

Cell population kinetics were studied in the lens rudiment of the chicken embryo during the period of contact with the optic cup and the start of crystallin synthesis. Data were compared with those for uninduced head ectoderm. Labeling with thymidine-3H or Colcemid treatment were used. The 1-hour labeling index of lens at 36 or 50 hours of incubation and of ectoderm at 36 hours was about 0.6. All 3 tissues had the same cell cycle, i.e., a total generation time of about 8.5–9 hours, a S-phase of about 5 and a G2 of 3.5 hours. Mitosis took 20 minutes and G1 was very short, perhaps of the order of 10 minutes or less. The potential population doubling times determined from the mitotic index and duration and from the Colcemid index were about equal to the total generation time found in the labeling experiments. Together with the high 1 hour labeling index this led us to believe that few, if any, cells had dropped out of the cell cycle in lens or ectoderm during the period studied. Calculation of the growth fraction which had a value of about 1 confirmed this view. We showed that loss of cells from the rudiment did not occur. In fact, a small fraction may have been added to the population; this, however, can not have influenced the results. We conclude that neither induction nor the onset of crystallin synthesis are reflected in drastic changes in cellular replication.

Electrophoretic studies on the heterogeneity of the chicken lens crystallins

The number of crystallins demonstrable in the lenses of various species by zone electrophoretic techniques is in general larger than the number of fractions revealed by immunological methods. To investigate the basis of this phenomenon the crystallins of the chicken were studied with two-dimensional polyacrylamide gel electrophoresis, immunoelectrophoresis and immunodiffusion. A method was developed that allowed molecular weight estimations on the basis of polyacrylamide gel patterns. Individual proteins were identified by immunodiffusion after they were punched out of the polyacrylamide gel. The chicken lens contained 17 fractions; 9 were identified as β -crystallins. Most of them had a molecular weight of around 55,000, but they had different electrophoretic mobilities. They showed reactions of partial or complete immunological identity. Hence, all β -crystallins share some superficial antigenic groupings but they may differ in other parts of their polypeptide chain(s). Other crystallins or their subfractions also showed physicochemical heterogeneity combined with immunological homogeneity.

Mitotic activity in the lens rudiment of the chicken embryo before and after the onset of crystallin synthesis

Summary1.Previous work has shown that the lens rudiment of the chicken embryo starts producing crystallins in stage 14 at the beginning of invagination (Ikeda and Zwaan, 1966). To establish if this appearance of organ-specific proteins is coupled with changes in cell replication, the mitotic activity in the anlage was determined by Colcemid treatment in stages 10 to 17. No significant differences in activity were noted between the center and the periphery of the organ in the stages studied or between stages of development.2.We conclude that cessation of cell replication and the onset of crystallin production are not directly linked.3.A mitotic duration of 24 min and a total cell cycle time of 15 h were calculated. The latter value does not agree with data from earlier studies using autoradiography after3H-thymidine application. Possible reasons for the discrepancy are discussed and caution is urged in the interpretation of cell cycle data obtained by treatment with mitotic inhibitors.Zusammenfassung1.Wie in vorhergehenden Arbeiten gezeigt werden konnte, beginnt die Linsenanlage des Hühnchens mit der Cristallinproduktion im Stadium 14 mit beginnender Einstülpung (Ikeda and Zwaan, 1966). Um zu prüfen, ob die Entstehung organspezifischer Proteine mit Veränderungen der Zellvermehrung gekoppelt ist, wurde die mitotische Aktivität in der Anlage durch Colcemidbehandlung in den Stadien 10–17 ermittelt. Signifikante Aktivitätsunterschiede zwischen dem Zentrum und der Peripherie des Organs oder zwischen den untersuchten Entwicklungsstadien wurden nicht gefunden.2.Hieraus wird geschlossen, da\ Beendigung der Zellreplikation und Beginn der Cristallinproduktion nicht direkt zusammenhängen.3.Als Mitosedauer wurden 24 min und als Gesamtzeit des Zellzyklus wurden 15 Std errechnet. Letzterer Wert stimmt nicht mit den Daten aus früheren Untersuchungen (Autoradiographie nach3H-Thymidin) überein. Mögliche Ursachen dieser Diskrepanz werden diskutiert, und es wird darauf hingewiesen, da\ bei der Beurteilung von Zellzyklus-Daten nach Behandlung mit Mitosegiften Vorsicht geboten ist.

Detection of protein-bound sulfhydryl groups after agar gel electrophoresis

Abstract A procedure is described for the visualization of the sulfhydryl groups of small quantities of proteins after separation by agar gel micro electrophoresis.

The immunologic specificity of human lens proteins.

Human lens extract was examined by electrophoretic and immunochemical methods. Paper electrophoresis failed to give any separation of the protein fractions, but electrophoresis in polyacrylamide gel resulted in the appearance of five distinct bands. The agar diffusion as well as the immunoelectrophoretic methods showed the presence of seven precipitin bands, indicating that the human lens contains a minimum of seven antigenic components. When the species specificity of the human lens was examined, it was found that its antigenic composition greatly resembled that of the monkey lens, except for minor differences of quantitative nature. From all other vertebrates, however, it differed by the presence of one species specific component, which is characterized by a high electrophoretic mobility. When the tissue specificity of the human lens was examined, cornea, retina, iris, aqueous humor and vitreous body were found to have a number of antigenic components in common with the lens. Despite the fact that these intraocular lens antigens are capable of reacting with lens antibodies in vitro, no evidence is presently available suggesting a similar reaction in vivo.