A homologous radioimmunoassay for guinea pig corticosteroid-binding globulin

A rapid, specific, and sensitive (requiring only 20 fmole of antigen equivalent to 0.007 microliter of serum) radioimmunoassay (RIA) was developed for the measurement of guinea pig corticosteroid-binding globulin (CBG). CBG was purified to homogeneity from guinea pig serum by affinity chromatography and used for immunization, as the standard and as the radiolabeled trace in the RIA. The antiserum to CBG was raised in rabbits. It was judged specific by immunoelectrophoresis and by comparison of RIA values with steroid-binding assay profiles obtained on serum separated on the basis of size and ion-exchange properties. The results of the radioimmunoassays agree with those of a steroid-binding assay run on identical samples. The sensitivity of the assay allows detection of CBG in serial serum samples, other biologic fluids such as milk, and cell culture supernatants.     

Farrowing induction with cloprostenol-xylazine combination

Eighty crossbred, multiparous sows, weighing between 190 and 320 kg, were randomly assigned to the following four treatment groups of 20 sows each: 1) saline-saline, 2) cloprostenol-saline, 3) saline-xylazine and 4) cloprostenol-xylazine. The mean gestation length of each multiparous sow was calculated. Cloprostenol (250 ug/sow, i.m.) or saline was given 3 d prior to the calculated due date at 11:30 a.m. Xylazine (2 mg/kg, i.m.) or saline was given 20 h after either the cloprostenol or previous saline treatment. Cloprostenol-xylazine treated sows had the shortest mean farrowing interval (1.5 +/- 0.3 h) when compared with the rest of the treatment groups (saline-saline:66.0 +/- 8.1, cloprostenol-saline:10.5 +/- 1.9, saline-xylazine:60.6 +/- 5.6 h). Farrowing time, percentage of stillbirths, average birth weight, d-5 and d-21 postbirth weights, number of pigs born, number of pigs born alive, and number of pigs surviving at 5 and 21 d afterbirth were not significantly different among the four groups. This study demonstrated that cloprostenol-xylazine treatment decreases the time to onset of farrowing with less variation than cloprostenol or xylazine alone. Therefore, the use of a cloprostenol-xylazine combination is suggested as an alternative method for inducing farrowing.     

L-Histidinol phosphate aminotransferase from Salmonella typhimurium. Kinetic behavior and sequence at the pyridoxal-P binding site

A coupled assay with alpha-hydroxyglutarate dehydrogenase was used to analyze the kinetic behavior of histidinol phosphate aminotransferase from Salmonella typhymurium. Data obtained from studies of initial velocity, inhibition by products or substrate analogues, isotope exchange rates, and the determination of the equilibrium constant were consistent only with a Ping-Pong Bi Bi mechanism. Variations in inhibition patterns by different substrate analogues indicate that the microenvironment about the pyridoxal phosphate and the pyridoxamine phosphate forms of histidinol phosphate amino-transferase are different, and favor the presence of one active site with partially overlapping substrate-binding subsites for these 2 forms of the enzyme. Histidinol phosphate aminotransferase also catalyzes decomposition of beta-chloro-L-alanine to pyruvate, NH3 and Cl-; no transamination of this substrate occurs and inactivation of the enzyme accompanies this reaction. After reduction of histidinol-P aminotransferase with [3H]NaBH4, carboxymethylation, and tryptic digestion, one major radioactive peptide absorbing at 325 nm was isolated. Its primary structure was determined to be TLSK*AFALAGLR, where K* is the P-pyridoxyllysine residue. Although this peptide is only 30-40% homologous with the corresponding segment reported for other transaminases, all of these peptides are similar in placement of an hydroxyamino acid residue three residues upstream from the lysine residue, and in the cluster of hydrophobic amino acid residues immediately following the lysine residue.     

Pathogenesis and immunity in murine salmonellosis.

Salmonella is traditionally described as a facultative intracellular parasite, and host macrophages are regarded as the primary effector cells in both native and acquired immunity in mouse typhoid. This concept has not been unanimously accepted in the literature. Based on cell culture experiments and electron microscopic examinations of infected tissues, we observed that virulent Salmonella typhimurium is killed within polymorphs and macrophages of guinea pigs and mice. In a systemic disease, the organism propagates primarily in the extracellular locations of sinusoids and tissue lesions and within hepatocytes. Hence, it is more likely to be an extracellular pathogen and its virulence is directly related to its antiphagocytic property. The conspicuous absence of macrophages in the primary lesions of murine salmonellosis disputes the likelihood of their significant role in native resistance to the disease. Acquired cellular immunity is expressed as an enhanced antibacterial activity of macrophages facilitated by cytophilic antibodies rather than as an altered antibacterial action of immune macrophages. It is proposed that acquired immunity in murine salmonellosis is a synergistic manifestation of the innate capacity of polymorphs and macrophages to destroy ingested salmonellae, the activated antibacterial functions of macrophages mediated by cytophilic antibodies, the opsonic and agglutinating actions of antiserum, and the accelerated inflammation associated with delayed hypersensitivity to bacterial antigens. Unlike live attenuated vaccines, nonviable vaccines offer a significant, though not a solid, protection against subsequent challenges.     

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure)

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N₆ medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

Phloem Mobility of Xenobiotics: II. Bioassay Testing of the Unified Mathematical Model

Two bioassays were used to test phloem mobility of selected xenobiotic compounds: (a) excised bean leaf assay; (b) rooted bean leaf assay. Compounds assayed were N-alkylpyridiniums with systematic variation in octanol-water partition coefficients (log K_ow), substituted benzoic acids with about the same log K_ow value but variable acidities. Results of the assays strongly conform, quantitatively, to the predictions of the unified mathematical model. Results also indicate that the membrane permeability value of a compound, which depends directly on log K_ow value, is the overriding factor in determining phloem mobility. When the weak acid functionality of a compound confers increased phloem mobility, it does so principally by making the log K_ow value, and consequently the membrane permeability of the compound more optimal.     

Carrier detection and prenatal diagnosis of alpha-thalassemia of Southeast Asian deletion by polymerase chain reaction

Summary Alpha-thalassemia of Southeast Asian deletion (-- SEA/) is very common in Southeast Asia. Homozygosity of this genotype is the major cause of Hb Bart's hydrops fetalis in Taiwan. With polymerase chain reaction using three oligonucleotide primers bridging the common deletion breakpoint, a DNA fragment of 194 basepairs (bp) was amplified in chromosomes with the-- SEA determinant and a DNA fragment of 287 bp was amplified in chromosomes without this deletion. In our pilot study including 8 normal subjects, 20 obligate carriers, and 11 homozygotes of the deletion, all the genotypes were determined and then confirmed by Southern blotting and DNA hybridization with globin gene probe. For prenatal diagnosis, 55 at-risk pregnancies were collected. Chorionic villus sampling was done in 51 cases and early amniocentesis was done in 4 cases. Fourteen cases (25.5%) were diagnosed as normal, 25 (45.5%) as heterozygotes, and 16 (29%) as homozygotes of -- SEA. All of the diagnoses were also confirmed as aforementioned. With polymerase chain reaction, the determination of the -- SEA deletion is straightforward and is much quicker and easier than with conventional Southern blotting and DNA hybridization. In areas with a high prevalence of -- SEA deletion, this method provides a rapid tool for carrier detection and prenatal diagnosis.     

The immediate-early growth response in regenerating liver and insulin-stimulated H-35 cells: comparison with serum-stimulated 3T3 cells and identification of 41 novel immediate-early genes.

Liver regeneration provides a unique system for analysis of mitogenesis in intact, fully developed animals. Cellular immediate-early genes likely play an important role in cell cycle regulation and have been extensively studied in mitogen-stimulated fibroblasts lymphocytes but not in liver. We have begun to characterize the immediate-early growth response genes of mitogen-stimulated liver cells, specifically, regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells, and to address differences in growth response between different cell types. Through subtraction and differential screening of cDNA libraries from regenerating liver and insulin-treated H-35 cells, we have extensively characterized 341 differentially expressed clones and identified 52 immediate-early genes. These genes have been partially sequenced and subjected to Northern (RNA) blot analysis, and 41 appear to be novel. Surprisingly, two-thirds of these genes are also expressed in BALB/c 3T3 cells, but only 10 were identified in previous studies of 3T3 cells, and of these, 6 include well-known genes like jun and fos, and only 4 are novel. Approximately one-third of the immediate-early genes identified in mitogen-stimulated liver cells or serum-stimulated NIH 3T3 cells are expressed in a tissue-specific fashion, indicating that cell type-specific regulation of the proliferative response occurs during the immediate-early period. Our findings indicate that the immediate-early response is unusually complex for the first step in a regulatory cascade, suggesting that multiple pathways must be activated. The abundance of immediate-early genes and the highly varied pattern of their expression in different cell types suggest that the tissue specificity of the proliferative response arises from the particular set of these genes expressed in a given tissue.     

Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro

We used a predominantly diploid Chinese hamster cell line to test a number of naturally occurring and synthetic estrogens for their ability to arrest cells at metaphase, their potential for allowing anaphase recovery, and their capability of inducing aneuploid progeny. The chemicals employed included diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol. We also tested progesterone, estrone and testosterone in this regard. Only estrogens and their synthetic analogs caused mitotic arrest and aneuploidy, while progesterone, estrone and testosterone did not cause mitotic disturbances. Among the estrogens, DES was the most effective arrestant on a comparative molar basis, whereas dienestrol was most potent over a wide range of concentrations. Estriol was the least potent as an arrestant but was an effective inducer of aneuploidy. The addition of a metabolic activator (S9) did not alter the ability of DES to arrest mitosis. Following the removal of the drugs, cells were able to quickly reorganize a spindle apparatus and enter anaphase. Diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol caused significant increase in aneuploidy within a narrow range of high concentrations in recovering cell populations. Aneuploidy was induced in a non-random manner. Immunofluorescence studies with anti-tubulin antibody indicate that estrogens may have a mechanism of mitotic arrest similar to that of colchicine and colcemid, viz inhibiting the polymerization of tubulin to form microtubules. These data suggest that the interaction between estrogens and microtubules may mediate the induction of aneuploidy in somatic cells. Aneuploidy induction by DES and similar compounds may be related to their carcinogenic potential.