Inheritance of hydronephrosis in the inbred mouse strain DDD

The mode of inheritance of hydronephrosis was investigated by crossing inbred DDD mice having a high incidence of hydronephrosis and C57BL/6 mice having normal kidneys. In the males, incidences of hydronephrosis in F1 animals were intermediate between the two parental strains at a rate of 32.6% in (DDD x C57BL/6)F1 and 23.4% in reciprocal F1. The same tendency was observed in F2 male animals. In BCF1 males, the number of affected mice was higher in (C57BL/6 x DDD) F1 x DDD (72.4%) than in (DDD x C57BL/6)F1 x C57BL/6 (11.1%). A few affected mice were found among the females of hybrids F1, F2 and BCF1. These results suggested that hydronephrosis in the DDD strain of mice was controlled by polygenes, and that male hormones may have some effect on the occurrence of hydronephrosis.     

Properties of three creatine kinases MM from porcine skeletal muscle

The properties of three fractions (FI, FII, and FIII) of porcine creatine kinase MM, which have been isolated by isoelectric focusing, were compared. Sugars were not detected in them. Their carboxyl-terminal sequences were identical and were determined to be -Thr-Lys by digestion with carboxypeptidases A and B. Immunodiffusion and competitive radioimmunoassay could not differentiate the three fractions from one another. Their amino-terminal sequences revealed that they had different primary structures. At residue 1, although all the three fractions had Pro, FI and FIII had an additional amino acid, Ser. At residue 23, only FI had Leu in addition to Ser, the amino acid common to the three fractions. These results indicate that differences among the three fractions of porcine creatine kinase MM are based on differences in the primary structures of the subunits in their dimer structures, and confirm the conclusion that FII is a homodimer and FI and FIII are heterodimers, which was reported in the preceding paper [Takasawa, T. & Shiokawa, H. (1983) J. Biochem. 93, 383-388].     

Endotoxic properties of chemically synthesized lipid A analogs. Studies on six inflammatory reactions in vivo, and one reaction in vitro

Biological activities of two groups of synthesized lipid A analogs, the counterpart of biosynthetic precursor, Lehmann's Ia type, 406, and E. coli lipid A type, 506, as well as their non-phosphorylated, and mono-phosphorylated analogs were investigated. The activities employed included four bone marrow cell reactions in mice, mice skin reaction, leukocytes migration in rabbits' cornea, and hemagglutination. Compound 406 and 506 elicited bone marrow reactions in mice and hemagglutination of mouse RBC, although 406 failed to elicit hemorrhage and necrosis also in mice skin. Compound 406 did not elicit corneal reaction in rabbits. The results suggest that for elicitation of this reaction and mice skin reaction, acyloxyacyl structure is required. Cytotoxicity and thromboplastin production of four bone marrow reactions had been reported by us to be endotoxic reactions, since these had not been elicited by peptidoglycan of Lactobacillus and Staphylococcus (1981) and 300 series synthesized analogs (1984) which did not have endotoxic structures. From these results, it seems that these two marrow reactions and hemagglutination require, as does the limulus test, the lipid A part structure as is present in 406.     

Copulatory behavior unaccompanied by ovulation in the Japanese monkey (Macaca fuscata)

Copulatory behavior unrelated to conception is sometimes observed in some non-human primates including the Japanese monkey. In the present study, the authors examined whether a mature follicle or a newly formed fresh corpus luteum was observed in the ovaries of female Japanese monkeys which displayed the copulatory behavior unrelated to conception. Post-conception copulatory behaviors were observed in three out of four females usually kept in individual cages in an air-conditioned room, and in two out of three females without infants kept in an outdoor group cage. However, neither a mature Graafian follicle nor a fresh corpus luteum formed newly after conception was observed in any of these females by laparoscopic examinations conducted immediately after termination of the copulatory behavior. In females with infants born in the preceding birth season, copulatory behaviors were observed in three out of four females kept in the outdoor group cage, and in two out of four females in a free-ranging troop. Ovulation was confirmed in one case out of the three kept in the outdoor group cage, but neither a mature follicle nor a newly formed corpus luteum was observed in the remaining four females. These findings suggest that copulatory behavior in the Japanese monkey is not always controlled by the development of a follicle or ovulation in the ovary.     

Characterization of Cysteine Proteases Functioning in Degradation of Dynorphin in Neuroblastoma Cells: Evidence for the Presence of a Novel Enzyme with Strict Specificity Toward Paired Basic Residues

Two dynorphin-degrading cysteine proteases, I and II, were extracted with Triton X-100 from neuroblastoma cell membrane, isolated from accompanying dynorphin-degrading trypsin-like enzyme by affinity chromatography on columns of soybean trypsin inhibitor-immobilized Sepharose and p-mercuribenzoate-Sepharose, and separated by ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose and TSK gel DEAE-5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes, were inhibited by p-chloromercuribenzoate, N-ethylmaleimide, and high-molecular-weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E-64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1-17) at the Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1-17) at the Lys11-Leu12 bond and the Leu12-Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg-Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme.     

Two Types of Apoptotic Cell Death of Rat Central Nervous System-Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 m M DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.     

Somatic mosaicism of expanded CAG repeats in brains of patients with dentatorubral-pallidoluysian atrophy: cellular population-dependent dynamics of mitotic instability.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disease caused by unstable expansion of a CAG repeat in the DRPLA gene. We performed detailed quantitative analysis of the size and the size distribution (range) of the expanded CAG repeats in various regions of the CNS of eight autopsied patients with DRPLA. Expanded alleles (AE) showed considerable variations in size, as well as in range, depending on the region of the CNS, whereas normal alleles did not show such variations, which indicates the occurrence of somatic mosaicism of AE in the CNS. The AE in the cerebellar cortex were consistently smaller by two to five repeat units than those in the cerebellar white matter. Moreover, the AE in the cerebral cortex were smaller by one to four repeat units than those in the cerebral white matter. These results suggest that the smaller AE in the cerebellar and cerebral cortices represent those of neuronal cells. The ranges of the AE in the cerebral cortex, cerebral white matter, and cerebellar white matter showed considerable variation ranging from 9 to 23 repeat units, whereas those in the cerebellar cortex showed little variance and were approximately 7 repeat units. The ranges of the AE in the cerebral cortex, cerebral white matter, and cerebellar white matter were much broader in patients with higher ages at death than they were in patients with lower ages at death, raising the possibility that the range of AE increases with time, as the result of mitotic instability of AE.     

Transmembrane signal transduction by the Escherichia coli osmotic sensor, EnvZ: intermolecular complementation of transmembrane signalling

Summary
The Escherichia coli regulatory proteins, EnvZ and OmpR, are crucially involved in expression of the outer membrane proteins OmpF/OmpC in response to the medium osmolarity. The EnvZ protein is presumably a membrane-located osmotic sensor (or signal transducer), which exhibits both kinase and phosphatase activities specific for the OmpR protein. To examine the functional importance of the membrane-spanning segments (named TM1 and TM2) of EnvZ molecules in transmembrane signalling, a set of EnvZ mutants, each having amino acid substitutions within the membrane-spanning regions, was characterized in terms of both their in vivo phenotype and in vitro catalytic activities. One of them, characterized further, has an amino acid change (Pro-41 to Ser or Leu) In TM1, and appeared to be defective in its phosphatase activity but not in its kinase activity. This EnvZ mutant conferred a phenotype of OmpF⁻/OmpC-constitutive. For this EnvZ(P41S or P41L) mutant, a set of intragenic suppressors, each exhibiting a wild-type phenotype of OmpF⁺/OmpC⁺, was isolated. These suppresor mutants were revealed to have an additional amino acid change within either TM1 or TM2. Furthermore, they exhibited restored phosphatase activity (i.e., both kinase⁺ and phosphatase⁺ activities). It was further demonstrated that one of the suppressors, EnvZ(Arg-180 to Trp in TM2), was able to suppress the defects in both the in vivo phenotype and the in vitro catalytic activities caused by EnvZ(P41S), through intermolecular complementation. These results are best interpreted as meaning that an intimate intermolecular interaction between the membrane–spanning segments of EnvZ is crucial for transmembrane signalling per se in response to an external osmotic stimulus.