Differential basic nucleoprotein kinetics in the two kinds of lepidoptera spermatids: Nucleate (eupyrene) and anucleate (apyrene)

Normal lepidopteran males produce two kinds of spermatozoa: nucleate (eupyrene) and anucleate (apyrene). Eupyrene spermatozoa have the usual type of elongate nuclei. But in apyrene spermatids, the nuclei never elongate and the chromatin remains in a telophase-like condition until enucleation occurs. The study of the differential nucleoprotein kinetics of the two types of spermatids, using the fluorescent dye sulfoflavine, shows that: (1) In the elongate eupyrene nuclei, lysine-rich nucleoproteins are replaced by arginine-rich ones, while in the non-elongating apyrene nuclei only lysine-rich nucleoproteins are detected. However, nuclear elongation is not causally related to nucleoprotein transitions as transitions occur in the eupyrene spermatids after nuclear elongation. (2) The replacement of the nucleoproteins occurs in the eupyrene nuclei in a polarized manner. This may be correlated with the heterogeneous ultrastructural configuration of the chromatin fibers in elongating spermatid nuclei, as shown in other insect species. (3) Concomitantly with the eupyrene spermatid nucleoprotein transition, the cytoplasm of the head cyst cell shows an increasing amount of cytoplasmic lysine-rich proteins, while no such a phenomenon occurs in apyrene cysts. This differential pattern distribution may reflact functional differences among the two types of cysts and is probably related to the regulation of the dichotomy in lepidopteran spermatogenesis.     

ELECTRON MICROSCOPY OF THE AMMONIACAL SILVER REACTION FOR HISTONES IN THE ERYTHROPOIETIC CELLS OF THE CHICK

The product of the postformalin ammoniacal silver reaction, which has been claimed to distinguish lysine-rich from arginine-rich histones with the light microscope on the basis of a color difference, was examined in developing erythroid cells of chick bone marrow with the electron microscope. Stem cells and early erythroblasts exhibit no, or little, ammoniacal silver reaction product, while small basophilic erythroblasts, polychromatophilic erythrocytes, and reticulocytes exhibit an increasing amount of reaction product as maturation proceeds. The reaction product is in the form of discrete electron-opaque particles associated with heterochromatin. The ammoniacal silver reaction in the erythroid cell series is interpreted as reflecting either the accumulation of newly synthesized arginine-rich histones or changes in the availability of reactive sites in preformed histones.     

1. Effects of Embedding 2. Experiments with Modifications

Paraffin embedding was found to be satisfactory for brain stained by a modification of the Golgi dichromate-silver method. Nitrocellulose embedding caused fading in a few specimens. Several modifications in which the tissue was impregnated with silver nitrate before treating it with potassium dichromate were investigated. The following one is recommended. Fix pieces of brain 5-6 mm. thick for 2 days in: silver nitrate;0.5%, 90 ml.; formalin, comml. unneutralized (37-40% gas), 10 ml.; pyridine, pure, 0.05-0.1 ml. Mix in the order given and test for pH with brom cresol purple. A pH of 5.5-6.0 is about optimum and the amount of pyridine added can be varied to adjust it. A slight turbidity of the fixing fluid may be disregarded, but precipitation indicates too much alkalinity. Rinse the tissues with distilled water and place them in a mixture of potassium dichromate, 2.5%, 100 ml. and osmic acid, 1%, 1 ml., for 3-5 days. Wash in water, dehydrate with alcohol and embed in soft paraffin for thick sectioning. Greater intensity of staining (but with an increase in precipitate) can be secured by rinsing the blocks after the dichromate treatment and resilvering in a 0.5% solution of silver nitrate for a day or two, then washing, dehydrating and embedding. This modification of the Golgi method was worked out on brain of adult rat, guinea pig, cat and monkey. Results with fetal material were not good. All solutions used were aqueous, and staining was done at room temperature.     

1. Effects of Embedding 2. Experiments with Modifications

Paraffin embedding was found to be satisfactory for brain stained by a modification of the Golgi dichromate-silver method. Nitrocellulose embedding caused fading in a few specimens. Several modifications in which the tissue was impregnated with silver nitrate before treating it with potassium dichromate were investigated. The following one is recommended. Fix pieces of brain 5-6 mm. thick for 2 days in: silver nitrate;0.5%, 90 ml.; formalin, comml. unneutralized (37-40% gas), 10 ml.; pyridine, pure, 0.05-0.1 ml. Mix in the order given and test for pH with brom cresol purple. A pH of 5.5-6.0 is about optimum and the amount of pyridine added can be varied to adjust it. A slight turbidity of the fixing fluid may be disregarded, but precipitation indicates too much alkalinity. Rinse the tissues with distilled water and place them in a mixture of potassium dichromate, 2.5%, 100 ml. and osmic acid, 1%, 1 ml., for 3-5 days. Wash in water, dehydrate with alcohol and embed in soft paraffin for thick sectioning. Greater intensity of staining (but with an increase in precipitate) can be secured by rinsing the blocks after the dichromate treatment and resilvering in a 0.5% solution of silver nitrate for a day or two, then washing, dehydrating and embedding. This modification of the Golgi method was worked out on brain of adult rat, guinea pig, cat and monkey. Results with fetal material were not good. All solutions used were aqueous, and staining was done at room temperature.     

CYTOCHEMICAL AND BIOCHEMICAL DETERMINATIONS OF CHANGES IN NUCLEAR HISTONE CONTENT DURING DIFFERENTIATION OF BARLEY ROOT CELLS

Changes in nuclear histone content in barley root cells have been studied by cytochemical methods for identification of histone subtypes and by conjunction with standard biochemical extraction procedure for various histone fractions and alkaline fast green stainability. The results obtained by the cytochemical methods indicate that the nuclear histones in cell nuclei found in their terminal stages of cellular differentiation or elongation contain histones rich in arginine, whereas the nuclei in meristematic cells contain histones rich in lysine. Cytochemicaly intermediate or transitional types of nuclear histones have been observed in cell nuclei which are undergoing differentiation or elongation and in chromosomes of mitotic nuclei. Information obtained from the conjunction of methods of biochemical extraction procedures for various histone fractions and alkaline fast green stainability indicate that the nuclei in well-differentiated cells contain predominantly histones rich in arginine (f3), whereas the nuclei of meristematic cells contain both very lysine-rich histones (f1) and slightly lysine-rich histones (f2). These results suggest the replacement of lysine-rich histones in the nuclei of meristematic cells by arginine-rich histones during cellular differentiation.     

Hydroxylation of lysyl residues in lysine-rich and arginine-rich histones by lysyl hydroxylase in vitro.

Lysine-rich and arginine-rich histones were examined as substrates for lysyl hydroxylase. Both proteins are known to be rich in lysyl residues, and lysine-rich histone also contains -X-Lys-Gly-sequences, whereas no such sequences are found in the arginine-rich histone. Both histones were found to be hydroxylated by lysyl hydroxylase, and the time courses of the hydroxylation reactions with these substrates were linear for at least 60 min. The Km values observed where 3 - 10(-6)M for heat-denatured lysine-rich histone and 6 - 10(-6)M for heat-denatured arginine-rich histone. Heat-denatured lysine-rich histone was hydroxylated at a higher rate than non-denatured both at 37 and 25 degrees C. No such phenomenon was found, however, when arginine-rich histone was examined as a substrate. Furthermore, at 37 degrees C lysine-rich histone was a better substrate for lysyl hydroxylase then arginine-rich histone, but this relationship was reversed at 25 degrees C. The synthesis of hydroxylysine observed with arginine-rich histone indicates that the lysyl hydroxylase preparation used in these experiments catalyzes the synthesis of hydroxylysine not only in the sequence -X-Lys-Gly-, but also in some other sequences. Certain collagen polypeptide chains are known to contain one hydroxlysyl residue in a sequence other than -X-Lys-Gly-, and the present results may explain this finding.     

Inhibition of mammary gland cyclic AMP-dependent protein kinase by arginine-rich histones.

The cyclic AMP-dependent protein kinase activity from lactating bovine mammary gland efficiently phosphorylates lysine-rich histones but not arginine-rich histones. It is shown that arginine-rich histones in fact inhibit phosphorylation of lysine-rich histones. Polyarginine and a range of low molecular weight cationic molecules are also inhibitors. Inhibition of histone H2b phosphorylation by histones H4 and H3 is competitive with respect to H2b. This inhibition behaviour may be tissue-specific since the protein kinase activity in crude extracts from lactating bovine mammary gland, although heterogeneous, may be completely inhibited (>95%) by arginine-rich histones and polyarginine.     

Arginine-rich nucleoprotein transition occurs in the two size classes of spermatozoa of Drosophila subobscura males

The normal male of Drosophila subobscura displays polymegaly, which is the presence of two sizes of spermatozoa in the same testis. It is still unknown whether both kinds of sperm are able to fertilize the egg. An indicator of normal functioning of Drosophila spermatozoa is the replacement of the somatic histones by sperm-specific arginine-rich nucleoproteins during spermiogenesis. The appearance of these arginine-rich nucleoproteins in the two kinds of sperm was investigated using the fluorescent dye sulfoflavine, which stains basic proteins at pH 8. In the spherical nuclei of early spermatids of Drosophila subobscura the somatic histones fluoresced strongly, but fluorescence could not be detected in later stages when the spermatid nuclei were elongating. After elongation, however, the nuclei of both kinds of sperm, long and short, fluoresced brightly again, due to the presence of sperm-specific arginine-rich nucleoproteins. Half of the cysts of both types contained spermatid nuclei with aberrant fluorescent pattern including 5–9% of both cyst types which do not undergo histone transition at all. These results indicate that both sperm types may be functional.     

Cytophotometric studies of nucleic acids and basic proteins during somatic and germinal differentiation of the interstitial cells of Hydra cauliculata (Hyman 1938)

Cytophotometric studies were made of the interstitial cells of Hydra during their differentiation into cnidoblasts and spermatozoa. DNA, RNA and basic proteins were measured by ultraviolet, Feulgen and alkaline Fast Green methods. Interstitial cells were found to contain approximately twice as much Feulgen-stained DNA as spermatozoa but an amount equal to that of the cnidoblast. DNA values for spermatogenic cells obtained by UV measurements were comparatively greater than those obtained using the Feulgen reaction, but were approximately equivalent after treatment of the cells with RNase. The Fast Green-histone/Feulgen-DNA ratio increased during the differentiation of both the cnidoblast and the spermatozoon. A relative increase in the lysine-rich histone fractions appeared to be largely responsible for these changes. During the terminal stages of spermatogenesis, however, the lysine-rich histones were replaced by more arginine-rich varieties.     

Evaluation of protocols used in 2-d electrophoresis for proteome analysis of young rice caryopsis

In order to obtain a high-resolution electrophorogram of rice young panicle proteome, we evaluated various protocols commonly used in two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) of proteins, including gel staining protocol, pH range of immobilized pH gradient (IPG) strips and sample loading quantity. Results showed that a silver staining protocol using sensitized solution containing glacial acetic acid, sodium acetate and sodium thiosulfate (reported by Heukeshoven and Dernick in 1988) and a Coomassie Brilliant Blue staining method using solution containing G-250, ammonium sulfate and phosphoric acid (reported by Pink et al in 2010) demonstrated the superior staining effect. In addition, we also showed that higher resolution was achieved when IPG gel strip with pH range of 5-8 was used, compared to that with pH range of 4-7. Finally, the optimal loading quantity was determined as 130 μg using the 17 cm-long nonlinear IPG strip with pH 5-8 in combination with the silver nitrate staining protocol. The evaluated results would be helpful in proteome analysis of young rice caryopsis.     

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. II. Metabolism and binding of histones.

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.     

Histone-acetylating enzyme of brain

1. Acetylation of histones by an enzyme system derived from rat brain and liver (histone acetylase) was studied by using [1-(14)C]acetyl-CoA as the acetyl group donor. 2. The activity of this enzyme was largely confined to the nucleus. 3. Histone-acetylating activity of cerebral nuclei purified by centrifugation through 1.9m-sucrose was not altered by the presence of the cytoplasmic fraction. 4. Cerebral nuclei from adult rats exhibited greater histone-acetylating activity than did the corresponding preparation from newborn animals. 5. Nuclear acetylating activity was higher in brain than in liver of adult rats but not in newborn animals. 6. The partially purified enzyme from cerebral nuclei, prepared by ammonium sulphate fractionation of an acetone-dried powder, specifically catalysed histone acetylation. 7. Polylysine, protamine, serum albumin and gamma-globulin were not enzymically acetylated by this preparation. 8. Soluble acetylating preparations from both brain and liver nuclei were more active towards arginine-rich F3 and slightly lysine-rich F2a and F2b histone fractions than towards the lysine-rich F1 fraction. 9. Enzymic acetylation of chromatin-bound proteins was much less extensive than that of free histones. 10. The high histone acetylase activity in mature brain may reflect the importance of this process in the genetic control of cerebral function.     

Silver staining for selective detection of histones in polyacrylamide gels

A simple silver-staining technique was developed for selective visualization of histones in polyacrylamide gels. The specificity of the stain was confirmed using a variety of protein mixtures and isolated histones. The staining procedure requires a relatively short time to perform (2.5-3 h), and the sensitivity to lysine-rich histones is comparable to that of the conventional Coomassie blue stain (about 0.1 microgram per band). A possible mechanism for the selective staining was deduced from a comparison with the widely used ultrasensitive silver staining.     

METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C¹⁴-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.     

Chromatin-associated protein phosphokinases of rat ventral prostate. Characteristics and effects of androgenic status.

Protein phosphokinase activity endogenous to rat ventral prostate chromatin was assayed by using edphosphophosvitin as an exogenous substrate. For maximal activity of the kinase reaction, the presence of 200 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol was essential. Two apparent pH optima were observed, a broad one between pH 7 and 7.4, and one at pH 7.89. At pH 7.4 the apparent Km for 31% dephosphophosvitin was 0.3 mg per ml. With respect to ATP, two apparent Km values (0.04 and 0.41 mM) were found. The kinase activity was minimal toward exogenous histones when used as substrates (3% for lysine-rich and 0.3% for arginine-rich (f3) histones, compared with dephosphophosvitin controls). The protein phosphokinases were not significantly stimulated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) when histones used as substrate. With dephosphophosvitin as substrate, cyclic AMP produced a small inhibition (5 to 15%). Orchiectomy of adult rats resulted in a rapid decline in the chromatin-associated protein phosphokinase activity assayed using optimal experimental condition described above. At 9 hours postorchiectomy, a 30% decline in the activity was observed; this was further reduced to about 50% of the control by 18 hours. This decrease in the kinase activity (e.g. at 9 hours postorchiectomy) appears to precede measurable changes in the protein and RNA complements of chromatin. Testosterone replacement following orchiectomy abolished this decline in the chromatin-associated activity. The chromatin-associated protein phosphokinase activity toward lysine-rich and arginine-rich histones was also sensitive to androgenic status of the animals and declined rapidly postorchiectomy. The results suggest the presence of multiple and androgen-sensitive protien phosphokinases associated with rat ventral prostate chromatin, which may modulate the phosphorylation of nuclear nonhistone phosphoproteins with changing gene action mediated by testosterone in this target tissue.     

A Reliable and Sensitive Method to Localize Terminal Degeneration and Lysosomes in the Central Nervous System

After reconsidering the physico-chemical mechanisms involved in the so-called degeneration methods for the demonstration of axons and nerve terminals, the method of Eager was fundamentally modified in order to stabilize the staining process. This resulted in a simple and reliable method which stains degenerating terminals and lysosomes with a high degree of selectivity and sensitivity. Frozen sections 30 to 50μm thick are prepared from material fixed with formaldehyde by cardiac perfusion. The staining procedure consists of 5 steps: 1) alkaline pretreatment (pH 13), 2) silver impregnation, 3) washing, 4) development at pH 5.0-5.5 monitored by an indicator, and 5) washing in acetic acid. Possible faults can be easily detected by their specific effects on the staining results. Primary submicroscopic silver precipitates are localized selectively in the osmiophilic parts of lysosomes and those degenerating presynaptic elements that are surrounded by glial processes. In degenerating axons, precipitates originating from mitochondria can usually be distinguished from terminal degeneration by their different size, shape, or characteristic arrangement. Nonspecific staining is restricted to glial fibrils, erythrocytes, and single cell nuclei. Dark field illumination can be applied routinely and television image analysis can be used for quantitative evaluation because of low background staining.     

Charge variants of tubulin,tubulin S,membrane-bound and palmitoylated tubulin from brain and pheochromocytoma cells

Isoelectric focusing (IEF) of only approximately 1 microg of rat brain tubulin yields 27-30 distinct charge variants in the pH range of 4.5-5.4 with band separations of 0.01-0.02 pH units as detected by silver staining. Variants can be efficiently transferred from the immobilized gradient strip to polyvinylidene difluoride (PVDF) membranes for reaction with monoclonal antibodies. C-terminal-directed antibodies to alpha- and beta-tubulin yield patterns similar to N-terminal-directed antibodies. Removal of the acidic C-termini with subtilisin to form tubulin S increases the pI values by approximately 1 pH unit, leads to a loss in the isoelectric distinction between the alpha- and beta-tubulin variants seen by N-terminal-directed antibodies, and abolishes reactions with all beta-variants and all but three alpha variants by C-terminal-directed antibodies (TU-04 and TU-14). Many, but not all, of the variants are substrates for autopalmitoylation of rat brain tubulin. The distribution of isoelectric variants differs between cytoplasm and membrane fractions from PC12 pheochromocytoma cells. A potential role for different variants is suggested.     

Experimental studies of mechanisms involved in methods demonstrating axonal and terminal degeneration

Factors influencing the consistency and specificity of the staining of neuronal degeneration products were studied in brain sections by varying systematically the composition of solutions used in the steps which are common to the degeneration methods. The formation of nuclei of metallic silver was determined either by physical development of 110Ag, after dissolving reducible silver by acetic acid. In degenerating axons metallic silver nucleic are formed by their own reducing groups in the first (acid) and in the second (alkaline) impregnating bath. The first impregnation turned out to be sufficient to produce complete staining of degenerating axons. The reducing capacity of normal axons and myelin can be suppressed by oxidation or by lowering the pH of the impregnating solution. Degenerating axon terminals are not able to reduce silver ions in either of the impregnating baths. Rather, the metallic silver nuclei initiating their staining are formed in the Nauta reducer by interaction of its reducing agent (formol) with silver ions which had been trapped in the tissue during the impregnation. Thus the nuclei are enlarged to microscopic visibility by a nonstandardized physical developer coming about from the Nauta reducer and the silver ions transferred with the sections. In this reaction catalytic sites in degenerating terminals as well as ammonium ions and the alkali reserve of the tissue play an important role. On the basis of the present results it was possible to stabilize the conditions for staining degenerating axons and degenerating axons terminals in two separate staining procedures detailed in following papers.     

Protein kinases in rat testes: evidence for different fractions of the enzyme.

Protein kinase activity of rat testis homogenate was separated into five fractions by means of pH 4.8 acidification and DEAE-cellulose chromatography. The five fractions showed a peculiar pattern of activity and cAMP dependency with the substrates used: casein, protamine, histone mixture, arginine-rich histone, lysine-rich histone, and phosvitin. The casein-sepharose substrate affinity column separated two fractions from the pH 4.8 precipitate. Peak number one phosphorylates histone preferently and is cAMP-dependent, while peak number tow has a strong affinity toward casein as substrate and is non cAMP-dependent.     

Experimental Studies of Mechanisms Involved in Methods Demonstrating Axonal and Terminal Degeneration

Factors influencing the consistency and specificity of the staining of neuronal degeneration products were studied in brain sections by varying systematically the composition of solutions used in the steps which are common to the degeneration methods. The formation of nuclei of metallic silver was determined either by physical development or ¹¹⁰Ag, after dissolving reducible silver by acetic acid. In degenerating axons metallic silver nuclei are formed by their own reducing groups in the first (acid) and in the second (alkaline) impregnating bath. The first impregnation turned out to be sufficient to produce complete staining of degenerating axons. The reducing capacity of normal axons and myelin can be suppressed by oxidation or by lowering the pH of the impregnating solution. Degenerating axon terminals are not able to reduce silver ions in either of the impregnating baths. Rather, the metallic silver nuclei initiating their staining are formed in the Nauta reducer by interaction of its reducing agent (formol) with silver ions which had been trapped in the tissue during the impregnation. Thus the nuclei are enlarged to microscopic visibility by a nonstandardized physical developer coming about from the Nauta reducer and the silver ions transferred with the sections. In this reaction catalytic sites in degenerating terminals as well as ammonium ions and the alkali reserve of the tissue play an important role. On the basis of the present results it was possible to stabilize the conditions for staining degenerating axons and degenerating axon terminals in two separate staining procedures detailed in following papers.