Prolactin as a promoter of initial progression of spontaneous mammary tumors in mice and lack of relationship to age

In a high mammary tumor strain of SHN virgin mice, the percent increase in the number of palpable mammary tumors during 3 weeks after the 1st tumor appearance was significantly retarded by ovariectomy and this retardation was prevented by pituitary grafting associated with the decrease and the increase in the circulating prolactin levels, respectively. A low mammary tumor strain of SLN virgin mice grafted with a single pituitary each at 2 and 5 months of ages developed mammary tumors 4 and 2 months after grafting (6 and 7 months of ages), respectively. However, there was no difference between groups in mammary tumor incidence at any month after mice in each group developed tumors first. Mammary tumor incidences of both groups were significantly higher than that of the untreated control at all ages examined. These findings have demonstrated that prolactin plays a key role in the initial progression of spontaneous mammary tumors of mice besides its prerequisite role in tumor development. They also suggest that there is no intrinsically age-related difference in prolactin effect on mammary tumorigenesis.     

Prostaglandin-sepharose: Application to the purification of bovine lung 15 (S) -hydroxyprostaglandin dehydrogenase

Prostaglandins E₁, F_1α and A₂ were covalently joined to the surface of Sepharose as carboxamide linkages. The insolubilized prostaglandins were shown to function effectively in the purification of 15(S) -hydroxyprostaglandin dehydrogenase.     

Identification of a novel T cell surface disulfide-bonded dimer distinct from the alpha/beta antigen receptor

Hybridomas were prepared from the spleen of a BALB/c mouse immunized with EL-4 T lymphoma cells. One, designated A1, was found to secrete a monoclonal antibody that reacted with two T lymphoma cells of C57BL origin, EL-4 and C6VLB, but not with normal C57BL/6 splenocytes or thymocytes, C57BL/6 T cell clones, or other T or B lymphomas by complement-mediated cytotoxicity or indirect immunofluorescent staining. Monoclonal antibody (MAb) A1 precipitated a protein that migrated at 85 kD under nonreducing and 43 kD under reducing conditions. The fact that the antigen defined by MAb A1 was a disulfide-linked dimer, together with the essentially clone-specific distribution of the reactive epitope, raised the possibility that the antibody defined an epitope of the antigen receptor. However, several additional observations revealed that the antibody defined a distinct and novel T cell surface structure. MAb 124-40, previously shown to react with the antigen receptor of C6VLB cells, reacted with variants of C6VLB that failed to express the A1 epitope. Sequential immunoprecipitation indicated that MAb A1 and MAb 124-40 reacted with distinct molecular species on C6VLB cells. Endoglycosidase digestion showed that the structure reactive with MAb A1 was not derived from that reactive with MAb 124-40 by addition of N-linked oligosaccharide residues. Two-dimensional gel electrophoretic analysis of precipitates obtained from radioiodinated C6VLB cells with MAb 124-40 resolved the alpha and beta subunits of the antigen receptor. Similar analysis of precipitates obtained with MAb A1 revealed only a single basic chain under reducing conditions, although anomalous mobility suggestive of a second, more acidic chain was observed under nonreducing conditions. Two-dimensional maps of tyrosine-containing chymotryptic peptides of the proteins isolated with MAb A1 and MAb 124-40 were completely different, suggesting that the molecules shared no peptides and were distinct in primary structure. Finally, cross-linking studies performed with a cleavable reagent indicated that the A1 molecule, unlike the antigen receptor defined with MAb 124-40, was not associated with additional, T3-like structures on the surface of C6VLB cells. Although the MAb A1 was unreactive with normal cells in cytotoxicity or staining assays, a molecule of the appropriate size was immunoprecipitated in small amounts from lysates of radioiodinated normal spleen and thymus cells. These data indicate that MAb A1 defines a novel disulfide-linked T cell surface molecule distinct from the antigen receptor.     

Nitrile hydratase is a quinoprotein. A possible new function of pyrroloquinoline quinone: activation of H2O in an enzymatic hydration reaction

Nitrile hydratase has been proved to be a quinoprotein with pyrroloquinoline quinone (PQQ) as a prosthetic group. The broad shoulder from 300 to 500 nm in the absorption spectrum of Brevibacterium nitrile hydratase suggested the presence of PQQ. Since PQQ was attached to the enzyme through a covalent linkage, the chromophores were isolated by acid hydrolysis, protease digestion and successive chromatographic separation. The isolated chromophores showed the similar spectroscopic characteristics to those of obtained from the amine oxidase of Aspergillus niger, in which PQQ is covalently linked. The isolated chromophores potently activated apo-D-glucose dehydrogenase (EC 1.1.99.17), supporting the presence of PQQ or a PQQ-like compound in nitrile hydratase. The finding of PQQ in nitrile hydratase strongly suggests a new function of PQQ, i.e., the activation of H2O in the enzymatic hydration reaction.     

Unique cell surface phenotypes of proliferating lymphocytes in mice homozygous for lpr and gld mutations, defined by monoclonal antibodies to MRL/Mp-lpr/lpr T cells

In mice bearing the autosomal recessive gene of either lpr or gld, generalized T-cell proliferation and autoimmunity occurs. The surface antigen profiles of these proliferating cells were analyzed using two-color flow cytometry analysis with two newly established rat monoclonal antibodies (ALP-1, ALP-2) directed to lpr cells. The Lp-1 antigen, defined by ALP-1, is expressed exclusively on approximately one-half of proliferating lpr and gld lymph node cells. The Lp-2 antigen, like B 220, is expressed on 80-90% of lpr and gld lymph node cells, the cells in B-cell lineage and a small population of Ly-2+ T cells from normal mice. Thus, the lpr and gld lymph node cells were classified into three subsets, Lp-1+/Lp-2+, Lp-1-/Lp-2+ and Lp-1-/Lp-2-. After stimulation with Con A or a combination of IL-2 and phorbol ester, a small population of T cells from normal mice became Lp-1+. The same treatment increased Lp-2+/Ly-2+ and induced Lp-2+/L3T4+ T-cell populations. Therefore, it seems likely that these phenotypically unique T cells are generated at some stage during the proliferation and differentiation of certain normal T-cell subpopulations. The aberrant T cells in mice with lpr and gld mutations may even be normal regulatory T cells, if they are not proliferating abnormally.     

Nitrile hydratase of Pseudomonas chlororaphis B23. Purification and characterization

Nitrile hydratase of Pseudomonas chlororaphis B23 was completely stabilized by the addition of 22 mM n-butyric acid. The enzyme was purified from extracts of methacrylamide-induced cells of P. chlororaphis B23 in eight steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular mass of about 100 kDa and consists of four subunits identical in molecular mass (approximately 25 kDa). The enzyme contained approximately 4 mol iron/mol enzyme. The concentrated solution of highly purified nitrile hydratase had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 720 nm. A loss of enzyme activity occurred in parallel with the disappearance of the absorption in the visible range under a variety of conditions. The enzyme catalyzed stoichiometrically the hydration of nitrile to amide, and no formation of acid and ammonia were detected. The enzyme was active toward various aliphatic nitriles, particularly, nitriles with 3-6 carbon atoms, e.g. propionitrile, n-butyronitrile, acrylonitrile and cyclopropyl cyanide, served as the most suitable substrates.     

Purification of Sarcophaga (fleshfly) lectin and detection of sarcotoxins in the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina.

An established cell line originating from a Sarcophaga peregrina (fleshfly) embryo, NIH-Sape-4, was found to synthesize mRNAs for Sarcophaga lectin and sarcotoxin IA, but not those for storage protein or 25 kDa protein. These four proteins are known to be synthesized in the fat-body of third-instar larvae, and the two former in particular are known to participate in the defence mechanism of this insect and to be induced in response to injury of the body wall. Thus the embryonic cell line NIH-Sape-4 synthesizes certain defence proteins constitutively. This cell line will be useful for large-scale purification of Sarcophaga lectin, since 50 micrograms of purified Sarcophaga lectin could be obtained from about 400 ml of culture medium.     

Longitudinal changes of dominance rank among the females of the Koshima group of Japanese monkeys

The changes of dominance rank among female Japanese monkeys of the Koshima group over a period of 29 years from 1957 were studied. The dominance rank order was relatively stable in the early population growing phase, while large scale-changes of dominance rank order occurred successively in the phase of population decrease brought about by the severe control of artificial feeding after 1972. Nevertheless, the rank order of several females of the highest status was stable. Furthermore, the reproductive success of these highest status females was high (Mori, 1979a;Watanabe et al., in prep.). Divergence of the dominance rank order fromKawamura's rules (Kawamura, 1958) was observed in the following respects: (1) Some females significantly elevated their rank depending on the leader males. (2) If mothers died when their daughters were still juveniles or nulliparous, the dominance rank of some of these offspring females was significantly lower than the mother's one. However 55% of daughters which lost their mothers at a young age inherited the mother's rank. (3) Dominance among sisters whose mother had died when at least one of the daughters was under 6 years old followed the rule of youngest ascendancy in 60% (Kawamura, 1958), and in 80% when both of the daughters were nulliparous at the mother's death. The mean rate of aggressive interactions for each female with subordinates to her was calculated by dividing the total aggressive interactions between the female in question and her subordinates by the number of subordinate females to the female in question. A female which showed a high rate of aggressive interactions with her subordinates was categorized as an “Attacker”, and a female showing a lower rate was categorized as a “Non-attacker”. Similarly, categories of “Attacked”, and “Non-attacked” were distinguished by using the rate of aggressive interactions with dominant females. Several females which were once categorized in one category in a year were repeatedly categorized in the same category over different years. The “Attacked” tended to be females of higher rank, and “Non-attackers” tended to be females of lower rank. “The second-higher-status females”, were “Attacked”, and their rank was unstable. In particular, females of lower rank within the lineage of the highest rank suffered this kind of severe status. Most of the daughters of these females showed a sharp drop of rank, and died when they were still at a young age, i.e. “the second-higher-status females” displayed low fitness. “Non-attackers” were significantly “Non-attacked”; i.e. they were females which showed a non-social attitude. Females which underwent a drop of rank tended to be “Non-attackers”. The most important factor which determined the females' rank was the memory of their dominance relations under the influence of their mother [dependent rank (Kawai, 1958)] in their early life during development. This finding corresponds well with the results in baboons obtained byWalter (1980); the target females of aggressive interactions by adolescent females were determined by the rank of the mothers when these adolescent females were born.     

Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization

Nitrilase was purified from an extract of isovaleronitrile-induced cells of Rhodococcus rhodochrous J1 in seven steps. In the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous by polyacrylamide electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 78 kDa and consists of two subunits identical in molecular mass. The purified enzyme exhibits a pH optimum of 7.6 and a temperature optimum of 45 degrees C. The enzyme catalyzed stoichiometrically the hydrolysis of benzonitrile to benzoic acid and ammonia, and no formation of amide was detected. The enzyme required thiol compounds such as dithiothreitol, L-cysteine or reduced glutathione to exhibit maximum activity. The enzyme was specific for nitrile groups attached to an aromatic or heteroaromatic ring, e.g. benzonitrile, 3-chlorobenzonitrile, 4-tolunitrile, 2-furonitrile and 2-thiophenecarbonitrile. The comparison of the properties of the enzyme with other nitrilases and nitrile hydratases has been also discussed.