Water-soluble hippocampal proteins of the rat in early postnatal ontogeny

Using ion exchange chromatography on DEAE-cellulose, studies have been made on protein extracts obtained at low ionic strength from hippocampal slices of rats within the 1st month of their postnatal life. Significant changes in fractional composition were found. To the end of the 2nd week, a reliable decrease in the relative content of low-acidic fractions was observed together with the increase in the content of those fractions which were eluted by a buffer with a higher molarity. In 3-week animals, the relative content of weak-acidic fractions decreases by 1 1/2 times, the difference being even more significant in adult animals. It is suggested that the observed redistribution of fractions within protein spectra is due to the appearance of the brain specific proteins.     

An immunohistochemical study of early embryogenesis in the clawed toad Xenopus laevis by using monoclonal antibodies to intermediate filament proteins

Distribution of cytokeratin epitopes was studied in X. laevis embryos at stages 10-25 using 5 monoclonal antibodies against proteins of the human and rat keratin filaments. Specific staining was observed in chorda, outer layers of ectoderm and presumptive epidermis (late gastrula), and inner layer of presumptive epidermis. The cells of the stained zone (presumptive epidermis) were compressed while the cells of unstained zone (presumptive neuroectoderm) were extended tangentially.     

Surface and internal antigens of rat spermatozoa distinguished using monoclonal antibodies

Fluorescent antibody labeling techniques are frequently used to investigate the topography of antigens on spermatozoa. It is generally assumed that these procedures detect molecules only on the sperm surface but we now show that this assumption is not always valid. Using monoclonal antibodies that recognize either surface or internal antigens we demonstrate how spurious conclusions can be made, and we suggest simple procedures for assigning the position of an antigen to the cell surface or to an intracellular organelle. Antibodies against plasma membrane antigens should stain 100% of normal intact spermatozoa, but this proportion should be greatly reduced if the spermatozoa have previously been demembranated. If ? 100% of spermatozoa are stained but the proportion increases following permeabilization, then the possibility should be considered that the antigens are intracellular. We conclude that assignment of an antigen to a regional domain on the sperm surface using fluorescent antibody techniques should be validated by a demonstration that the antigen is actually located on the cell surface.     

Erythroblast antigen expression in murine hematopoietic and nonhematopoietic cells. 2. An immunofluorescent study in pre- and postnatal ontogeny using monoclonal antibodies

The expression of erythroblast antigen (Ag-Eb) in cell membranes during pre- and postnatal mouse development was studied by immunofluorescence using the monoclonal antibody MAE-15. Ag-Eb was detected in embryonic liver, spleen, epithelia of intestine, various glands and skin, as well as in extraembryonic tissues (yolk sac and trophoblast). In pregnant mice positive immunofluorescence was observed in placenta and on the surface of decidual cells in uterus. In adult non-pregnant mice Ag-Eb expression was detected not only in membranes of erythroid cells, but also in non-hemopoietic tissues, such as epithelia of various glands, intestine, kidney and testis, brain endothelium, basal layer of epidermis, and intercalated discs of the heart muscle. A possible role of Ag-Eb in processes of cell transport is discussed.     

Changes in the renal corpuscles during early postnatal ontogeny of the rat (stereologic analysis)

Kidneys of 60 white rats at the age of 1-45 days of postnatal life have been investigated. Increase in size of the renal corpuscles during the developmental process is accompanied with certain changes in the form and variability of volumes in the structures studied. During early postnatal period, against the background of linear increase in the size of the renal corpuscles, the parameters of variability in volumes, form and in the total number of the renal corpuscles in the organ change non-linear. Stabilization moment of the total number (11-14 days) on the 8th-10th day outgoes that for the parameters of the form and variability of the renal corpuscle volumes.     

Analysis of class I MHC antigens in the rat by monoclonal antibodies

Monoclonal antibodies (mAb) were made against class I MHC antigens of the i (mAb 42,70,39) and u (mAb 68-D) haplotypes in the rat by using specific strain combinations in order to obtain reagents for identifying the products of the RT1.An, RT1.Au, and RT1.Eu loci. These antibodies were hemagglutinating only; were IgG except for mAb 68-D3, which had a defective heavy chain; reacted identically with MHC-congenic strains and with their inbred donor strains; and precipitated class I MHC antigens. Strain distribution, sequential immunoprecipitation, and peptide mapping studies were used to define the specificities of the mAb, and the assignments were checked by comparing the specificities of the mAb with those of haplotype-specific alloantisera. The specificities were the following: mAb 42, An; mAb 68-D, Au; mAb 70, Eu; and mAb 39, an antigen encoded by a locus different from A and E. This new locus was designated RT1.F, and the allele detected by mAb 39, as Fa. The serologic data place RT1.F between RT1.A and RT1.D. The plasma membranes of DA.1I(BI) lymphocytes contain comparable amounts of An, Eu, and Fa antigens but express them on the cell surface in the order An much greater than Eu greater than Fa.     

Cytoskeletal changes in the endothelial cells of the rat aorta in the early periods of postnatal ontogeny]

The cytoskeleton of endothelial cells is a modulator of all the cell reactions. The formation of a definitive structure of the bearing-contracting apparatus of the rat aorta endothelium is finished in the postnatal development (up to the age about 3 months after birth), passing through some qualitative changes. Using the transmission and scanning electron microscopy of detergent extracting preparations, the structuring pattern (saturation) of the aorta endothelial cell cytoskeleton of newborn animals. From 10 days to 1 month after birth, the most important period takes place within peripheral dense microfilament bundles are formed responsible for the cell monolayer integrity for the contractility of cell boundaries (it is most important in recombination of endothelial monolayer in the processes of cell proliferation and vessel growth) and also for the integrity of longitudinal bundles of microfilaments, i.e. fibres of tightening. The increase in anisotropy of cytoskeleton frame during its maturation evidences on the establishment of orientation of microfibril bundles, whose main function being the opposition to haemodynamic loading.     

Rapid diagnosis of cytomegalovirus in immunocompromised patients using monoclonal antibodies that detect early viral antigens

A technique was applied to detect early fluorescent antigens (DEFA) of cytomegalovirus (CMV) using the E13 monoclonal antibodies in 52 immuno-compromised patients hospitalized in the Nephrology Institute of Havana. Of the 75 urine or blood (buffy coat) samples taken, 15 were found positive to CMV. Using classical diploid human fibroblast isolation technique, 12 CMV strains were isolation of previously detected positive samples by DEFA. In addition, CMV was isolated from one sample reported to be negative by DEFA. A coincidence of 80% was found between both techniques. With the ELISA test, all the sample studied have IgG antibodies to CMV.     

Characterization of baboon testicular antigens using monoclonal anti-sperm antibodies

Sperm antigens that appear during spermatogenesis in the baboon were identified by using three monoclonal antibodies generated in culture from mice immunized with baboon caudal epididymal spermatozoa. Antibodies BSA1 and BSA2 recognize trypsin-sensitive 84,000 and 45,000 dalton determinants that are restricted to the tail and anterior acrosomal regions of the sperm, respectively, as determined by Western blot and immunofluorescence techniques. The tail antigen absent in 2- and 3-yr-old baboon testes first appears in spermatid cells at about 4 yr of age. In contrast, the acrosomal antigen recognized by BSA2 is present in 3-yr-old primitive testicular germ cells. In the mature testis, the 45,000 molecular weight determinant is predominantly localized in the nucleus of late pachytene spermatocytes and round spermatid cells as observed via the avidinbiotin immunoperoxidase method. Antibody BSA3 reacted only with sailidase-treated sections of adult testis. This trypsin-resistant determinant, not expressed on testicular sperm, is recognized by antibody BSA3 only on epididymal sperm, thus indicating a post-testicular sperm modification.     

An immunohistochemical study of placenta-specific alpha 2-microglobulin in human ontogeny]

Tissues of adult man and fetus were studied by indirect immunoperoxidase technique using affinity-purified antibodies against placenta-specific alpha 2-microglobulin (APM-2). APM-2 proved to be synthesized mucous membrane cells of uterus during secretory phase of the menstrual cycle. APM-2 was also found in atretic follicle of fertile woman ovary as well as in I order oocytes from the fetus ovary (26 weeks). In men APM-2 is synthesized in seminal vesicles. APM-2 was found both in cytotrophoblast and in syncytiotrophoblast as shown by study of chorionic villi.     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

The experience of maternity exerts a significant effect on the shaping of behavior in rat progeny in early postnatal ontogeny]

Maturation of specific behavioral reactions were studied in 13-24-day-old rats born by females with prior maternal experience (group A) and maternally naive females (group B). The rats of group B showed later eyes opening and earlier maturation of grooming and manipulation than the group A rats. The development of behavioral reactions was found to be related with eye opening in the rats of group A and maturation of manipulation in group B rats. The results suggest that the maternal influence is a significant environmental factor, which affects the early postnatal development of rat pups.     

The surface antigens of Rickettsia prowazekii studied with monoclonal antibodies]

R. prowazekii antigens have been tested with the use of monoclonal antibodies (McAb) to different epitopes of the microorganism. As revealed in these tests, McAb B4/4 and A-3/D, active against species-specific thermolabile antigen, interact with protein having a molecular weight of 90-120 KD. McAb C5/2, active against thermostable group antigen common with that of Rickettsia typhi, interact with LPS-like antigen having a molecular weight of 30 KD. Ultrastructural immunochemical studies have revealed that both R. prowazekii antigens are located on surface structures of rickettsiae, such as the microcapsule and cell wall.     

Heterogeneity of rat macrophages recognized by monoclonal antibodies: an immunohistochemical and immunoelectron microscopic study

Three monoclonal antibodies, designated RM-1, TRPM-1, and TRPM-2, were raised against rat peritoneal macrophages. By the immunoperoxidase method, antigens recognized by these antibodies were distributed throughout most tissue and free macrophages examined, including those of splenic red pulp, lymphatic sinus, connective tissue, and peritoneal cavity, as well as Kupffer cells of liver and alveolar macrophages. The numbers of positive cells were different for each antibody. RM-1 and TRPM-1 were also reactive with interdigitating cells (IDCs) in the thymus-dependent area and with Langerhans cells in the skin, whereas TRPM-2 failed to demonstrate IDCs in thymic medulla and Langerhans cells. The reactions of each antibody were observed by immunoelectron microscopy in the different ultrastructural compartments of the cells. RM-1 recognized a cell surface antigen; reaction products for TRPM-1 were found on a part of the cell membrane and in the cytoplasmic vacuoles; and those of TRPM-2 were present along the nuclear envelope and intracytoplasmic vacuoles. These antibodies seem to be useful not only for the detection of macrophages in tissue sections but also for investigation of macrophage heterogeneity in different tissues.     

Identification of mobile and fixed antigens on the plasma membrane of rat spermatozoa using monoclonal antibodies

We have used monoclonal antibodies to study the mobility and distribution of three different antigens on the cell surface of rat spermatozoa. We classified two of the antigens (designated 2B1 and 2D6) as 'mobile', since when detected by indirect immunofluorescence they were situated over the entire sperm flagellum and were susceptible to antibody-induced patching. Patching was critically dependent upon antibody concentrations and was much reduced at 4 degrees C. Patching of the 2B1 antigen was not induced by the 2B1 monoclonal antibody alone. Thus, 2B1 antibody labelled directly with fluorescein bound with a uniform distribution over the sperm flagellum, but this uniform fluorescence was made patchy on subsequent incubation in an unlabelled second antibody layer of anti-mouse IgG anti-serum. By 'Western blotting', the 2B1 antigen was found to be located to a 40 kD molecular weight polypeptide. The remaining 'fixed' antigen (designated 1B6) was not susceptible to antibody-induced patching, and was restricted to a discrete domain on the post-acrosomal region of the sperm surface. We discuss the relationship between mobility of sperm surface antigens and their segregation to discrete domains on the plasma membrane.     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

In the preceding paper it was suggested that the tumour localisation of 125I-labelled syngeneic rat monoclonal antibodies (mAbs) may be limited in immunocompetent hosts by the presence of competing endogenous serum antibodies. In syngeneic congenitally athymic (nu/nu) and cyclosporin-A-treated rats (both of which fail to mount immune responses to tumour antigens) increased uptake of mAbs in tumour tissue was obtained compared with that in immunocompetent animals. However, in the case of IgG2b and IgG1 mAbs, this appeared to be due primarily to enhanced "non-specific" localisation mediated by Fc binding, since it was abolished by the use of F(ab')2 fragments with two out of three mAbs tested. Normal tissue distribution was also influenced by host immune status: in nu/nu rats the uptake of IgG2b mAbs in the spleen was up to fivefold higher than that previously found in normal animals and the levels in liver were also increased. This effect was not seen in cyclosporin-A-treated hosts, suggesting that the reticuloendothelial system of congenitally athymic animals contains cells with enhanced IgG2b-FcR activity. This hypothesis was strengthened by the observation that splenic uptake was reduced by either the use of F(ab')2 fragments, or prior "blockade" of Fc receptors by "cold competition" with excess unlabelled IgG2b mAbs. This blockade could not be effected by mAbs of any other isotype or by IgG2b F(ab')2 fragments. The former manoeuvre resulted in higher tumour specificity ratios but usually at the expense of reduced levels of tumour associated radiolabelled mAb. The latter was found to increase "absolute" tumour localisation by up to 35%. In an attempt to characterise further and compare the Fc receptor activity of intratumour and intrasplenic host cells. The distribution of IgG2b mAbs was assayed in 3-week, 8-week and 12-week-old rats. We were able operationally to distinguish the activity of these two categories of cells, suggesting that they represent either different lineages or differentially activated subpopulations: the splenic IgG2b binding was fully expressed in weanling nu/nu rats whereas the FcR activity of cells infiltrating MC24 sarcoma was limited in 3-week-old compared with 8-12-week-old hosts. A further difference was apparent in the subclass "preference" of FcR binding: in immunodeprived rats both IgG1 and IgG2b mAbs were able to bind to tumour-infiltrating host cells, but uptake of IgG1 mAbs in the spleen was always low and not reduced further by the use of F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)