Similar cell surface antigens on hamster cells transformed by different papovaviruses.

Transformed baby hamster kidney (BHK) cells were tested for surface antigens by an immunocytoadhesion method. The cells were sensitized with rabbit antisera to cell clones transformed by polyoma or by BK virus and then rosetted with erythrocytes coated with antibody to rabbit immunoglobulin. These antisera detected common antigens on BHK cells transformed by either of three papovaviruses, polyoma, BK, or SV40, but apparently not on normal BHK cells.     

Expression of the major AgB histocompatibility complex antigens on different structural components of rat kidney and heart

We have analyzed the expression of the major AgB locus antigens on the parenchymal cell components of rat kidney and heart using (a) heterologous antisera raised against isolated molecules and (b) conventional alloantisera, directed to the serologically detectable AgB and Ia allodeterminants. A modified Staphylococcus aureus Cowan I rosette assay, enabling the characterization of the rosette-forming cell type from stained cytocentrifuged cell smears, was used. The AgB and Ia antigens were detected by both types of antisera on the “passenger” cells of either organ: the anti-Ia reacted especially strongly with a fraction of “passenger” lymphocytes, and anti-AgB also with the “passenger” erythrocytes. All kidney parenchymal components were nearly devoid of both AgB and Ia antigens: only a weak reaction was observed with the vascular endothelial, tubular, or glomerular cells after treatment of either type of antiserum. The heart endothelial cells expressed the AgB and probably some Ia: an intermediary intensive reaction was observed after treatment with heterologous or allospecific anti-AgB, and a weak reaction with anti-Ia. The heart myocardial muscle cells, on the other hand, were nearly devoid of both antigens: no reactivity with heterologous anti-Ia and only a marginal reactivity with heterologous or allospecific anti-AgB was observed.     

Secretory proteins of the lung in rodents: immunocytochemistry

The reactivity of rabbit antisera to rat lung secretory proteins with other rodent species was evaluated by immunocytochemistry. Rabbit anti-rat surfactant apoprotein antiserum reacts with the cytoplasm of rat, mouse, and hamster type II pneumocytes and is specific for these cells. Rabbit antiserum to rat Clara cell secretory proteins stains rat, mouse, and hamster Clara cells. Rabbit antisera specific to the two antigenic types of rat Clara cell antigens were also both reactive with rat, mouse, and hamster Clara cells. An antiserum to the non-serum proteins of hamster lung lavage was also prepared and shown to be specifically reactive with hamster Clara cells. The availability of specific reagents for secretory proteins of rodent lungs is expected to facilitate studies of the respective cell types in various pathologic states.     

Analysis of the specificity of five murine anti-blood group A monoclonal antibodies, including one that identifies type 3 and type 4 A determinants

The specificity of five mouse monoclonal anti-A blood group antibodies (Ab), four of which were produced by immunization with cultured human cancer cells and one with a synthetic antigen, has been determined by examining their reactivity with purified A glycolipids, erythrocyte glycolipids, oligosaccharides, ovarian cyst glycoproteins, and salivary glycoproteins. Two of the antibodies (HT29-36 and CB) reacted with all A variant structures tested and have a broad anti-A reactivity. Ab CLH6 did not agglutinate A erythrocytes and reacted preferentially with the type 1A structure. Ab S12 agglutinated all A1 erythrocytes and reacted best with simple, monofucosyl type 2 A structures, such as Aa-2, Ab-2, and A tetrasaccharide. Ab M2 has a novel, but complex, spectrum of reactivity. It reacts with type 3 and type 4 A chains and not with type 1 and type 2 A chains. It appears to recognize both an external A structure (formula; see text) (I) (found) in type 3 and type 4 chains) and also an internal structure (II) found in type 3 chains. Ab M2 agglutinates all A and AB erythrocytes but does not react with salivary glycoproteins.     

Intracellular degradation of hemoglobin transferred into fibroblasts by fusion with red blood cells

Hamster fibroblast protein and rabbit hemoglobin were labelled by incubation of fibroblasts (BHK21) or reticulocytes with [³H]leucine. Alternatively, human or rabbit hemoglobin was labelled by carbamoylation of erythrocytes with K¹⁴CNO. The labelled hemoglobins were introduced into fibroblasts by virus-mediated fusion between the blood cells and fibroblasts. The hemoglobins became uniformly distributed throughout the cytoplasm. Degradation was assessed from release of acid-soluble radioactivity into the medium. Radioactivity from [¹⁴C]-carbamoylhemoglobin was released as carbamoylvaline and homocitrulline, and these compounds were not metabolized or reincorporated by the cells. Intermediate degradation products could not be detected. The degradation of hemoglobin followed first-order kinetics. The half-life of both carbamoylated and native rabbit hemoglobin in hamster fibroblasts was 28 h, and the half-life of carbamoylated human hemoglobin was about 150 h in fibroblasts from hamster (BHK21), mouse (Balb/3T3), and man (MRC 5), corresponding to that of the more stable endogenous proteins. Phenylhydrazine increased the intracellular degradation of carbamoylated human hemoglobin about 13 times, whereas the degradation of endogenous proteins was little affected. Hemoglobin was degraded in homogenates at 31% h^?1 at pH 5 and 0.3% h^?1 at pH 7.4. Phenylhydrazine increased these rates to 45% h^?1 and 9.7% h^?1, respectively. Growing hamster fibroblasts, which are brought into quiescence by serum deprivation or by high culture density, increase the degradation of endogenous protein and of hemoglobin in parallel.     

Glycoprotein biosynthesis by organ cultures of hamster trachea

Conditions have been developed for maintaining hamster tracheas in organ culture for at least 10 days. Secreted glycoproteins labelled with [¹⁴C]glucosamine and [³H]fucose were isolated from the spent medium and digested with papain, and the digest was fractionated on DEAE-Sephadex by stepwise elution with NaCl. The fractions eluted by 0.1 and 0.2 M NaCl and some of the products eluted with 0.4 M NaCl were shown to be derived from epithelial glycoproteins. Glycosaminoglycans were eluted by 0.4 M and by 1.25 M NaCl. Glycopeptides isolated from the epithelium by homogenization, ethanol precipitation and papain digestion, and defined as “intracellular”, gave a very similar profile on DEAE-Sephadex. The 0.1 M glycopeptide peak was the major fraction of epithelial origin from both secreted and “intracellular” material; it labelled extensively with both glucosamine and fucose and had a molecular weight of approx. 5000 (as judged by its elution from Sephadex G-75). This fraction was purified further by chromatography on Sephadex G-75 and DEAE-Sephadex; its amino acid and carbohydrate compositions were determined.     

Immunosorbent method for the detection of A, B, O blood group specificity on CEA preparations.

The detection of A,B,O blood group specificity on some CEA preparations was carried out with anti-A,B,H antisera coupled to sepharose 4B and (¹²⁵I)-labelled antigens. This method was compared to classical FARR's method using ammonium sulfate precipitation of the antigen-antibody complex. In this last procedure, the introduction of adjuvants (excess of γ-globulin or saliva perchloric extract) limits its sensitivity. The use of immunosorbents which allows to eliminate these adjuvants, makes the results more reproducible and the sensitivity higher. Using this method, A blood group determinant was identified in two CEA preparations. Moreover, binding inhibition of labelled A blood group substance to anti-A antiserum by these CEA corroborated this result.     

Attempt to inhibit cytotoxic alloantibody with specific anti-receptor site antiserum

Utilizing a ⁵¹Cr release cytotoxicity inhibition assay, putative anti-receptor site antisera (anti-A RS_B) were assayed for their capacity to inhibit specific alloantibody, a result predicted by the recent work of Ramseier and Lindenman. Utilizing various isogenic strains of rats, anti-RS antisera prepared by injecting appropriate F₁ hybrid animals (A × B) with either lymph node cells (A) or specific alloantiserum (A anti-B) were found to be inactive in this assay. It appears that the Ramseier-Lindenman phenomenon cannot be corroborated using a ⁵¹Cr release inhibition assay.     

Development of in vivo somatic mutation system using antibody against hemoglobin Preparation and use of an anti-hemoglobin antibody for identifying C57BL/6 red cells in artificial mixture of DBA/2 and C57BL/6 red cells

A cellular specific-locus mutation test is described for detecting mutant cells in mammals. The test is based upon the use of specific anti-C57BL/6J mouse hemoglobin antibody that binds hemoglobin “single” (hemoglobin s, present in C57BL/6J mouse) and not hemoglobin “diffuse” (hemoglobin d, present in DBA/2J mouse). Attempts to purify such antibody from pony and rabbit antisera through cross-absorption were unsuccessful. Immunization of LP/J mouse with C57BL/6J hemoglobin produced antiserum that reacted with s hemoglobin but not with d hemoglobin. In a fluorescent antibody technique, this antibody was found to label fixed red blood cells from C57BL/6J mice but not from DBA/2J mice. In a mixture of C57BL/6J and DBA/2J red cells, the C57BL/6J cells could be differentiated by their bright fluorescence from the non-fluorescent DBA/2J cells. Reconstruction experiment with artificial mixtures of DBA/2J and C57BL/6J cells showed that s hemoglobin bearing cells could be detected in DBA/2J red cells at frequencies as small as 0.4×10^?6. Thus, the system is sensitive enough to detect d → s mutation in DBA/2J mice. Amino acid comparison of the globin chains of s and d hemoglobins shows that our antibody can probably detect mutations leading to a substitution of serine or proline by alanine at β²⁰ position and/or a substitution of threonine by alanine at β¹³⁹ position.     

Presence of Sarcoma Genome in a “Non-infectious” Mammalian Virus

Virus particles, antigenically related to mouse sarcoma virus, can be induced from hamster tumour cells. They are “defective” in their failure to replicate in or transform hamster, mouse or rat cells, although they contain viral-specific RNA and, with “helper” mouse leukaemia virus, show sarcomagenic activity.     

Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells.

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polyoma virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.     

Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polymo virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.     

Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

Effects of a DNA vaccine in an animal model of Alternaria alternata sensitivity

Sensitivity to the fungus Alternaria is associated with asthma persistence and severity. Current therapeutic options for treating Alternaria-induced airway inflammation are limited. In this study, Brown Norway rats are used to study the effectiveness of a DNA-based vaccine delivered to the airway in attenuating the response to a major Alternaria allergen, rAlt a 2. Compared to untreated sensitized animals, or animals receiving an “out-of-frame” DNA-based vaccine, animals treated with “in-frame” DNA vaccine showed an attenuation in specific IgE antibody titers to rAlt a 2, an increase in IgG^2b (a Th1 response), a reduction in spontaneous IL-13 release by peribronchial lymph node cell suspensions, and an attenuation in the decrease in total lung capacity 72 h post-allergen challenge. Further, histopathologic examination of the lung tissues revealed reduced pulmonary inflammation post-allergen challenge in the DNA-vaccine-treated compared to sensitized, untreated animals. We conclude that a DNA-based vaccine delivered to the airway significantly influences the immunologic, pulmonary physiologic, and histological alterations induced by challenge with a major Alternaria allergen, rAlt a 2, in sensitized animals.     

The isochore patterns of mammalian genomes and their phylogenetic implications

The compositional distributions of high molecular weight DNA fragments from 20 species belonging to 9 out of the 17 eutherian orders were investigated by analytical CsCl density gradient centrifugation and by preparative fractionation in Cs₂SO₄/BAMD density gradients followed by analysis of the fractions in CsCl. These compositional distributions reflect those of the isochores making up the corresponding genomes. A “general distribution” was found in species belonging to eight mammalian orders. A “myomorph distribution” was found in Myomorpha, but not in the other rodent infraorders Sciuromorpha and Histricomorpha, which share the general distribution. Two other distributions were found in a megachiropteran (but not in microchiropteran, which, again, shares the general distribution) and in pangolin (a species from the only genus of the order Pholidota), respectively. The main difference between the general distribution and all other distributions is that the former contains sizable amounts (6–10%) of GC-rich isochores (detected as DNA fragments equal to, or higher than, 1.710 g/cm³ in modal buoyant density), which are scarce, or absent, in the other distributions. This difference is remarkable because gene concentrations in mammalian genomes are paralleled by GC levels, the highest gene concentrations being present in the GC-richest isochores. The compositional distributions of mammalian genomes reported here shed light on mammalian phylogeny. Indeed, all orders investigated, with the exception of Pholidota, seem to share a common ancestor. The compositional patterns of the megachiropteran and of Myomorpha may be derived from the general pattern or have independent origins.     

Thymus-derived lymphocyte-dependent rejection of syngeneic papovavirus (SV40) and methylcholanthrene tumors in inbred hamsters.

Treatment of specifically sensitized MHA hamster lymphoid cells with rabbit antisera specific for hamster thymus-derived lymphocytes, in the presence of C, eliminated those cells capable of inhibiting the growth of syngeneic SV40 and methylcholanthrene tumors in vivo. Thymectomized, lethally-irradiated, bone marrow-reconstituted hamsters, shown to be devoid to T cell function, were, after attempted specific sensitization to the two syngeneic tumor cell lines, unable to reject either tumor by direct challenge in vivo. In addition, lymphocytes from such animals were incapable of inhibiting the growth of either tumor cell line in normal syngeneic recepients in the tumor cell neutralization assay. These data strongly support the conclusion that specifically sensitized thymus-derived lymphocytes are required for the rejection of syngeneic SV40 and methylcholanthrene tumors in inbred hamsters.     

Three new blood groups of rhesus monkeys

Three new blood group systems, called “T,” “U,” and “V,” have been identified in the rhesus monkey (Macaca mulatta). Each system consists of a single antigenic factor (blood group) detected by a monospecific alloimmune reagent that agglutinates erythrocytes. The antisera that detect these blood groups were obtained following a series of alloimmunizations and absorption fractionizations of the resulting antisera to produce operationally monospecific typing reagents. Analyses of family data indicated that each blood group was controlled by an autosomal dominant gene and that each system was independent of previously defined systems. With the addition of these new blood groups, we can identify 16 different blood group systems and well over one hundred million possible phenotypes in this species.     

The transfer of purine metabolites via the medium rather than through cell contacts

When the medium in which mouse B82 cells had been grown for 24 h with [³H]hypoxanthine was given to HGPRT⁻ Chinese hamster cells (CHW-1102), the acid-insoluble fraction of these cells became radioactive. When the medium in which mouse B82 cells had been grown for 24 h without [³H]hypoxanthine was given to CHW-1102 cells at the same time as [³H]hypoxanthine was added, the acid-insoluble fraction of the cells did not become radioactive. This indicates that CHW-1102 cells acquire from the B82 medium ³H material and not the ability to utilize hypoxanthine. Very little radioactivity was found in the acid-insoluble fraction of the B82 medium and when the medium was given to CHW-1102 cytoplasms, they did not become labelled. These results suggest that ³H purine metabolites (and not ³H nucleic acids) are responsible for the radioactivity in the CHW-1102 cells. Such ³H metabolites were also present in the medium of mouse L929 cells, but were absent in the medium of Chinese hamster (V79), mouse (A9), Syrian baby hamster kidney (BHK) and human fibroblasts. The cells were judged to be free of mycoplasma by three different criteria. This exchange of metabolites through the medium is referred to as contact-independent metabolite transfer (CIMT).     

Localization of nucleic acids in the nucleoli of oocytes and early embryos of mouse and hamster: An autoradiographic study

Mouse preovulatory oocytes, zygotes, parthenogenetically activated pronuclear oocytes, and early embryos, as well as hamster zygotes, were analyzed, by autoradiography, for the distribution of either “maternal” or newly synthesized RNAs. Early mouse embryos were also examined for the distribution of newly replicated DNA. Special attention was attributed to NLBs in oocytes or to NPBs in early embryos. In mouse oocytes, [5-³H]uridine radioactivity accumulated (after a 2-hr pulse) in vitro, in addition to other nuclear compartments, in the central compact material of the NLBs. There was no cytoplasmic labeling. In all parthenogenetic pronuclear embryos developed from similarly labeled oocytes, this label was distinctly detectable in the central compact material of the NPBs; less intensive labeling was seen in the nucleoplasm and cytoplasm. On the contrary, the central compact part of the mouse NPB did not show labeling in DNA after a continuous culture with [6-³H]thymidine. In mouse and hamster pronuclear zygotes, convincing evidence was obtained for a lack of any newly synthesized nucleic acids in the compact material of NPBs using 4- to 10-hr culture with [8-³H]adenosine. Based on these data, it was shown that the NLBs of oocytes or NPBs of early embryos probably contain RNAs synthesized during the last stages of antral follicle oocyte differentiation. This unique pathway of RNAs in the oocyte—embryo system may explain the specific morphology of both oocyte and early embryo “nucleoli.” © 1995 Wiley-Liss, Inc.