Differential Labeling Patterns Research Articles

The expression of antigens by embryonic neurons and glia in segmental ganglia of the leech Haemopis marmorata

Monoclonal antibodies (mAbs) raised against adult leech nervous systems were screened on embryos of the leech Haemopis marmorata in order to determine when in development specific antigens are first expressed and the order in which they are expressed by different cells or tissues. Three of the mAbs produced by Zipser and McKay (Zipser, B., and R. McKay (1981) Nature 289: 549-554) were screened: Lan3-1, Lan3-5, and Lan3-6. Each mAb shows a different pattern of labeling in the adult leech nerve cord (Zipser, B. (1982) J. Neurosci. 2: 1453-1464). The embryonic stages studied were from 5 days after egg deposition to 30 days (emergence from the cocoon). The pattern of labeling was assayed in whole mounts using horseradish peroxidase-conjugated second antibodies. The principal results are as follows. (1) Antigens recognized by Lan3-5 are first expressed by the glia of the roots of the anterior segmental ganglia at 6 to 7 days, several days later by the interganglionic connective glia, and near the end of embryonic development by ganglionic neurons. An anterior to posterior temporal gradient is observed in the expression of these antigens. In addition, Lan3-5 also labels the protonephridia and nephridia from early development onward. (2) Antigens recognized by Lan3-6 are first expressed by a pair of neurons in each segmental ganglion and later in development by additional neurons. By the time of emergence, however, only about half of the neurons that label in the adult have done so, implying that some neurons express these antigens postembryonically. Labeling with Lan3-6 is first seen in neuronal somata and only later in neuronal processes. (3) Antigens recognized by Lan3-1 and expressed by segmentally specific neurons in ganglia 5 and 6 are not detectable during embryonic development, but are so at early postembryonic stages. Thus, these three mAbs provide an approach to study different aspects of the development of the leech nervous system, specifically the relation between glial and neuronal differentiation and the genesis of segmentally specific phenotypes.

Cell site and time course of DNA synthesis in pancreas after caerulein and secretin.

Pancreatic weight, [3H]-thymidine incorporation into DNA, labeling indices, and total DNA and RNA content were measured in rats treated with vehicle or 1 microgram/kg caerulein, 100 micrograms/kg secretin, or a combination of these peptides injected every 8 h for 1-5 days. Incorporation of [3H]thymidine into DNA increased 12-fold after 2 days of treatment with the combination of peptides. DNA content increased after 3 days and reached a level 1.8 times control after 5 days. Autoradiography showed that two cell types, acinar and an unidentified type, were the sites of increased DNA synthesis. Different patterns of labeling were seen in the two populations: acinar cell labeling indices were increased at 1 and 2 days (20-fold) and then fell; nonacinar cells showed an increase only after 2 days and maintained this increase after 5 days. Potentiation (greater than additive effects) was found when caerulein and secretin were injected together for all measurements except RNA content. These data indicate that DNA synthesis in two cell populations is affected by secretin and caerulein and support the occurrence of potentiation between secretin and caerulein for trophic effects on the exocrine pancreas.

Differential labeling of sensory cell and neural populations in the organ of Corti following amino acid incubations.

Label localization was compared by light and electron microscopic autoradiography in the organ of Corti of the gerbil, following in vivo incubation with one of eight 3H-labeled amino acids. Following incubation with GABA, efferent endings underneath the outer hair cells were heavily labeled, as were all tunnel crossing fibers, many small fibers in the inner spiral bundle and some efferents beneath the inner hair cells. This pattern was absent following incubations with the GABA analog muscimol. Following L- and especially D-aspartic acid incubations, efferent endings under the inner hair cells and small fibers of the inner spiral bundle were preferentially labeled. It is concluded that these differential labeling patterns after GABA and aspartic acid incubations reflect two populations of efferent fibers in the organ of Corti: one terminating primarily upon the outer hair cells and one underneath the inner hair cells. Cochlear hair cells showed the highest level of labeling following incubation with alanine. Inner hair cells were more heavily labeled than outer hair cells following incubations with alanine and glycine. Hair cell labeling was comparable to that of supporting cells for most amino acids. However, for alanine and glycine the labeling of inner hair cells, and for L-aspartic and glutamic acid the labeling of all hair cells, was higher than that of structural cells. Labeling of hair cells was substantially lower than that of structural cells following taurine incubations.

Tightly (covalently) bound fatty acids in cell membrane proteins

We find tightly (covalently) bound fatty acids in membrane proteins from human red cells and polymorphonuclear cells and several tissues of the rat. These fatty acids are not removed by exhaustive extraction with organic solvents, by phospholipase A 2 treatment, or by sodium dodecyl sulfate (SDS). The fatty acids are released by refluxing with hot methanolic-HCl. They account for 0.2–6.8% of the total fatty acids. 50–60% of the tightly bound fatty acids are removed by mild alkaline hydrolysis. [ 3H]Palmitic acid is incorporated into lipids and proteins of the red cell membrane. In red cell ghosts incorporation is stimulated by ATP and CoA but maximal incorporation is achieved with [ 14C]palmityl-CoA. The labeling of lipids and proteins by [ 14C]palmityl-CoA is greater in ghosts than in cells, however basal incorporation of [ 3H]palmitic acid into lipids and proteins is greater in cells than in ghosts. [ 3H]Palmitic acid is also incorporated into proteins of polymorphonuclear cells. The incorporation in polymorphonuclear cell proteins is inhibited by cycloheximide but not by actinomycin D. Labeled stearic, linoleic, linoleic, and palmitic acid are incorporated into red cell membrane proteins. These incorporated fatty acids are not removed by exhaustive solvent extraction, by prolonged dialysis or by SDS. On SDS-polyacrylamide gel electrophoresis several protein bands are labeled by all the fatty acids although each fatty acid gives a different pattern of labeling. In all cases, the major protein band which is labeled stains very weakly with Coomassie blue and runs between band 4.2 and 5. Other proteins which have appreciable label are bands 1, 2 and 3. We postulate that fatty acids are covalently attached to certain proteins as a post-translational event and act to direct, insert, and anchor the proteins in cell membranes. A similar postulate has been enunciated by Schmidt and Schlessinger (Cell 17, 813–819 (1979)).

Interaction of the mitochondrial ATPase complex with phospholipids.

The interaction of bovine heart mitochondrial oligomycin-sensitive ATPase (Serrano, R., Kranner, B. L., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) with phospholipids has been examined by labeling the subunits exposed to lipids with photoreactive radioactive phospholipids. A subunit of Mr = 29,000 and some polypeptides in the range of 6,000 to 13,000 daltons were labeled. F1-ATPase subunits did not interact with the photoactive probes. This result is compared with the different pattern of labeling obtained with another mitochondrial ATPase preparation (Galante, Y.M., Wong, S. Y., and Hatefi, Y. (1979) J. Biol. Chem. 254, 12372-12378), which is devoid of the 29,000 component.

Light and electron microscopic autoradiographic approaches to the chemistry of auditory brain stem nuclei

A fresh brain slice preparation has been developed which allows the simultaneous characterization of the anatomical localization of radioactively labeled substances in a variety of different auditory brain stem areas. Labeled substances may include putative neurotransraittcrs, ligands for receptor binding sites and antibodies to specific proteins. Varying incubation conditions can be applied to adjacent slices, avoiding the complication of differences between non‐inbred animals. These slices can be examined at both the light and electron microscopic level. Differential distributions of label in cells and synaptic terminal populations in the cat cochlear nucleus following incubations with putative neurotransmitter tritiated amino acids suggest differences in the chemical properties of at least three classes of terminals in the dorsal cochlear nucleus. Studies are underway to describe the differential labeling patterns in several auditory nuclear groups and to characterize the conditions under which they occur. [Work supported by NIH.]

Differences in incorporation of axonally transported protein in regenerating motor and sensory axons

Different patterns of labeling were obtained in regenerating rat sciatic nerve after incorporation of labeled, fast-transported protein by motor or sensory axons. In motor axons incorporation of label occurred principally at the distal end of the regenerating fibers, whereas in sensory axons incorporation occurred uniformly along the entire length of nerve between the original site of injury and the most rapidly growing tips. This difference is not a consequence of the different types of axonal trauma used to provoke regeneration but may reflect a less synchronous outgrowth of sensory axons after injury.

Methylation of DNA in target and non‐target organs of the rat with methylbenzylnitrosamine and dimethylnitrosamine

The sites of labelling of DNA with [14C]methyl groups from methylbenzylnitrosamine and dimethylnitrosamine were studied in rat oesophageal epithelium and liver. All four combinations of tissue and carcinogen were studied. Tissues were labelled in vitro and the DNA contained therein purified and hydrolysed (pH 1, 37 degrees C) to free purines and apurinic acid. Quantitative analysis was performed with the aid of thin-layer chromatography. The apurinic acid and 7-methylguanine fractions were found to be extensively labelled. Smaller amounts of radioactivity were found in O6-methylguanine and some of the methylated adenines. The same carcinogen produced different patterns of labelling is oesophageal and liver DNA. The proportion of O6-methylguanine to 7-methylguanine was higher when the methylating agent was a carcinogen specific for the organ.

Iodination of calsequestrin in the sarcoplasmic reticulum of rabbit skeletal muscle: a re-examination.

The exposed proteins of sarcoplasmic reticulum (SR) vesicles from skeletal muscle were iodinated with the use of Sepharose 4B-bound lactoperoxidase, so that the location of the proteins in the membrane could be determined. It was found that the pattern of protein labelling could be modified simply by changing the constituents of the incubation media. This implies that the position or configuration of a particular protein in the membrane can be altered by the local environment. When the reaction was performed in the presence of 25 mM tris-maleate, pH 7 �0, alone, the Ca2+ pump ATPase (molecular weight 105000) and calsequestrin (47000) were both heavily labelled, suggesting they are at least partially exposed on the outer surface of the membrane. By contrast the high affinity calcium-binding protein (55000) was not labelled. However, when the vesicles were iodinated under conditions that were suitable for ATPase activity and Ca2+ accumulation, namely in the presence of 25 mM tris-maleate, pH 7 �0, 5 mM ATP, 5 mM Mg2+ and 0�05 mM Ca2+, a different pattern of labelling was obtained. No labelling of calsequestrin was observed whereas the extent of labelling of the Ca2+ pump ATPase remained about the same. The inclusion of anyone of the additives mentioned was effective in inhibiting the iodination of calsequestrin in the SR vesicle. When added alone, Ca2+ was more effective than Mg2+ in preventing labelling of calsequestrin. Half-maximal inhibition was observed at concentrations of approximately 0�05 mM Ca 2+ and 0�2-0�3 mM Mg2+ . Although much reduced, significant labelling of calsequestrin was observed even in the presence of 5 mM ATP. Investigations with partially purified calsequestrin revealed that the iodination of calsequestrin was the same in both the presence and absence of 1 mM Ca2 +. Therefore the reduction in label observed in intact SR vesicles probably represents a change in the location of calsequestrin within the membrane, rather than inhibition by Ca2+ of the iodination sites of the protein itself.

Synthesis of rat liver mitochondrial proteins after the administration of a nonlethal dose of cycloheximide.

SummaryFollowing in vivo treatment of rats with a nonlethal dose of cycloheximide (2 mg/kg body wt), analysis of the newly synthesized liver mitochondrial polypeptides by SDS-PAGE system showed: (i) sublethal levels of cycloheximide did transiently suppress synthesis and/or incorporation of large cytoplasmically synthesized proteins into mitochondria; (ii) synthesis and/or incorporation of this material was stimulated during the recovery phase. The differential labeling patterns of these mitochondrial proteins observed in vivo during cycloheximide treatment substantiate the cooperative nature of the cytoribosomal protein synthetic system to the formation of functional mitochondrion observed with mammalian cells in culture.Fig. 3. Radioactivity profiles of soluble proteins extracted with acidic buffers from control and cycloheximidetreated (2 and 24 hr) mitochondria. For details see the legends to Figs. 1 and 2.Fig. 4. Radioactivity profiles of soluble proteins extracted with alkaline buffers from control ...

Localization of late replicating DNA and nuclear membrane

We investigated whether the specific localization of DNA replicator sites at the nuclear membrane at the end of the S phase, found in dividing animal cells and in the plantHaplopappus gracilis, applied also to other plants. We found that nuclei labelled at the nuclear periphery were observed in plants where the chromocenters are localized at the nuclear membrane. In other plants, where the chromocenters are scattered throughout the nucleus, a different pattern of labelling is observed where the silver grains are restricted to a number of distinct sites, distributed in a fashion similar to that of the chromocenters. We believe that these nuclei were replicating the chromocentric heterochromatin and so were therefore at the end of the S phase. The specific patterns of the distribution of DNA replicator sites at the end of the S phase make it possible to distinguish nuclei which are in the late S phase and thus define a specific stage of the S phase, during which only heterochromatin replication occurs.

Perinuclear DNA and heterochromatin

We have analyzed how and to what measure DNA which replicates specifically at the nuclear membrane at the end of S phase (perinuclear DNA) correlates with heterochromatin. We found that, although perinuclear DNA can be distinguished in autoradiographs in two different patterns of labelling, these appear to constitute the same heterochromatic sites. Furthermore, since perinuclear DNA is only replicated in 7% of the whole S phase, while DNA in heterochromatin amounts to about 25–30% of all chromatin, we concluded that perinuclear DNA only occurs in a small fraction of all heterochromatin.

Synthesis of DNA and RNA in the quiescent center of the primary root ofCalystegia soldanella

The results are reported of autoradiographic analyses accomplished on roots ofCalystegia soldanella fed with a 2,5 mCi/l solution of3H-thymidine and a 10mCi/l solution of3H-uridine. Two regions of the meristem were investigated: cap-initial (CI) and quiescent center (QC). It is shown, in agreement with the literature data, that cap initials and quiescent center cells display a different pattern of labelling. Within the quiescent center the presence of 4 groups of cells is suggested, each group being characterized by some morphological differentiation and/or by a typical pattern of3H-thymidine and3H-uridine incorporation. Based on the labelling pattern the suggestion is advanced that some cells of QC undergo selective replication of particular DNA sequences which are active in transcription. The present observations demonstrating the heterogeneity of quiescent center are discussed in relation toTorrey's (1972) view that the quiescent center might be a site of a specialized biosynthesis of a hormone essential to cell division.

RNA synthetic activities of sertoli cells in the mouse testis.

Electron microscope autoradiographic studies of RNA synthesis in Sertoli cells of the adult mouse, using [‘H]uridine as a precursor, show a random distribution of grains in the nucleoplasmic area and a localized product on the nucleolar fibrillar component at early labeling times (15 mm). The condensed chromatin bodies associated with the nucleolus seem to have a nucleolar organizing function. At later labeling times (3 h) certain interchromatmn granules occurring in clusters in the nucleoplasm become partially labeled. After 7-8 days, the disappearance of label from Sertoli cells, contrasting with the longer persistence in spermatocytes, suggests a rapid turnover of RNA in Sertoli cells. Differential labeling patterns and structural changes in Sertoli cells cytoplasm are related to variations in functional activities presumably required for normal spermatogenesis and/or spermiogenesis. Sertoli cells are found in the seminiferous epithelium of several mammalian species. Though various functions have been attributed to these cells, it is generally accepted that Sertoli cells provide mechanical support for germinal cells. Moreover, they participate in the release of sperm into the lumen of the seminiferous tubules (VitaleCalpe and Burgos, 1970), dispose of cell debris by a phagocytic mechanism (Clegg and Macmillan, 1965; Kierszenbaum, 1970), and play an important role in the transfer of nutrients and metabolites between the intertubular space and the lumen of the tubules, acting as “bridge” cells (Vilar et al., 1962). It is also known that Sertoli cells constitute a unique stable cell population in the seminiferous epithelium. Whereas spermatogonia, spermatocytes, and sper

Pattern analysis of long arm grain count as a criterion of homology in eight cases of Bp.

The variation in grain count between regions of the long arms of the B group chromosomes is examined. The longer, later-labelling pair (4 s) show a different pattern of labelling to the shorter, earlier-labelling pair (5 s). It is concluded that pattern analysis may be a better criterion of homology than either length measurement or total grain count.

An autoradiographic study of haemolymph protein uptake by the tissues of the fifth instar locust

The fate of haemolymph proteins during the fifth instar of Locusta has been investigated autoradiographically using tritiated blood protein. The fate of [ 3H]-leucine was also studied. All tissues took up labelled blood protein at all stages of development. Pericardial cells were the most active tissue in protein uptake throughout the instar. The amount of label in oenocytes, fat body, and epidermis was high at the beginning of the instar, decreased after 136 hr, then rose once more after 200 hr. Low levels of activity were detected in the cuticle at the beginning of the instar. The new cuticle was evident after 200 hr and showed extensive labelling. A different pattern of labelling was evident after injection of labelled leucine. Once again label was evident in all tissues. The labelling was heavier than that observed in blood protein injected animals. During the first 100 hr of the instar, most tissues were heavily labelled. From 100 to 150 hr the level of uptake decreased. There was a second rise in incorporation of amino acid between 150 and 160 hr. The level of incorporation then decreased once more and remained low until the end of the instar. The new cuticle was the only tissue heavily labelled at the end of the instar. The significance of these results and the possible methods of protein uptake are discussed.

Studies on Cassava, Manihot utilissima. II. Biosynthesis of Asparagine‐14C from 14C‐labelled Hydrogen Cyanide and its Relations with Cyanogenesis

AbstractSeeds and seedlings of Manihot utilissima were analysed for cyanogenic glycosides und free amino acids, with special reference to valine and isoleucine which serve as precursors of the aglycone moieties of linamarin and lotaustralin. Seeds contained traces of valine and isoleucine but no glycosides, whereas seedlings contained high concentrations of these amino acids and glycosides. Illumination of seedlings led to a steep increase in the concentration of glycosides followed by a decrease without excretion of detectable HCN. Seeds accumulated asparagine, while seedlings accumulated both asparagine and glutamine in the storage and transport of nitrogen. Seedlings incorporated 13.2 per cent of label from valine‐14C(U) and 2.4 per cent of label from isoleucine‐14C(U)into linamarin and lotaustralin, respectively. In both cases, appreciable amounts of label were also incorporated into asparagine. 49 per cent of label from H14CN was incorporated inio asparagine in which ca. 98 per cent of total radioactivity was located in the amide‐carbon atom. The different patterns of labelling which occurred during the assimilation of H14CN and 14CO2 showed that cyanide metabolism did not proceed via CO2, and that M. utilissima contains an efficient enzyme‐system which catalyses the conversion on high concentrations of HCN into asparagine, which subsequently enters different metabolic pools involved with respiration, protein and carbohydrate syntheses. Cyanogenesis in M. utilissima appears lo be directly influenced by available pools of valine and isoleucine, and the metabolism of HCN released from linamarin and lotaustralin by the action of linamarase may be directly related to respiratory and synthetic processes by way of the incorporation of HCN as a unit into asparagine.