Purification and characterization of a biliverdin-associated protein from the hemolymph of Manduca sexta

A biliverdin binding protein, insecticyanin, has been isolated from the hemolymph of the fourth instar tobacco hornworm Manduca sexta. The protein has been purified to apparent homogeneity by conventional chromatography with a cumulative yield of 40-50%. The protein (Mw 71 600) is composed of three subunits (Mr 23 000). Each subunit binds one biliverdin molecule. Proton magnetic resonance spectroscopy and absorption spectroscopy demonstrate that the bilin is the biliverdin IX gamma isomer.     

Immunohistochemical studies on the localization of cellular retinol-binding protein in rat testis and epididymis

The immunohistochemical localization of cellular retinol-binding protein (CRBP) was studied in rat testis and epididymis. Parallel studies were also carried out on the localization of plasma retinol-binding protein (RBP) and transthyretin (TTR) in testis. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. For RBP and TTR, specific immune staining was found in the interstitial spaces between the seminiferous tubules, and not in the tubules themselves. In contrast, strong specific immune staining for CRBP was found in the seminiferous tubules, with a striking localization within Sertoli cells. Moreover, a distinct cyclic variation of specific staining for CRBP within Sertoli cells was observed during the spermatogenic cycle. This cyclic variation was seen with regard to both the intensity of staining and to the anatomic distribution of CRBP within the Sertoli cells. Within the epididymis CRBP was selectively localized to the proximal portion of the caput epididymidis, with variations in intensity of the staining of the epithelium of the ducts in different histological zones. Specific immune staining for CRBP was very weak or absent in the other portions of the epididymis. These results were confirmed by radioimmunoassay. Vitamin A-deficient rats showed markedly reduced specific immune staining for CRBP in both testes and epididymides, and greatly reduced levels of CRBP in these tissues on radioimmunoassay. These studies on the localization of CRBP provide information concerning the specific cells and anatomic loci within the testis and epididymis where retinol may be playing an important role in sperm formation and maturation.     

Starch-Ampicillin Agar for the Quantitative Detection of Aeromonas hydrophila

Interest in Aeromonas hydrophila as a food-borne and human pathogen is increasing. Isolation media from the clinical laboratory were evaluated for food use and either did not give quantitative recovery of A. hydrophila or did not permit ready differentiation of A. hydrophila from the background microflora. A new medium was developed which permitted quantitative recovery of A. hydrophila from foods. The medium consisted of phenol red agar base (Difco Laboratories), soluble starch (10 g/liter), and ampicillin (10 mg/liter). All foods surveyed contained A. hydrophila. Foods sampled included red meats, chicken, raw milk, and seafood (fish, shrimp, scallops, crab, and oysters). The count of A. hydrophila at the time of purchase ranged from 1 × 10²/g (lower limit of detection) to 5 × 10⁵/g. In most instances, the count of A. hydrophila increased during 1 week of storage at 5°C. The starch-ampicillin agar developed permitted rapid quantitative recovery of A. hydrophila from foods in the presence of very large numbers of competing microflora.     

Increased analgesic potency of morphine and increased brain opioid binding sites in the rat following chronic naltrexone treatment

Implantation of rats with prolonged-release naltrexone pellets increased both morphine's analgesic potency in the tailflick assay and radiolabeled opioid binding in the brain. The increases in both radiolabeled opioid binding and morphine potency were time-dependent. Implantation for 24 hours did not increase binding, whereas increases of approximately 45% were seen following 8 days of implantation. Similarly, morphine's analgesic potency, measured as ED50 values, was increased by 50% following 8 days of exposure to naltrexone while a 24 hour exposure had no significant effect.     

Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. Regulation of glyceraldehyde-3-phosphate dehydrogenase

The effect of insulin on protein biosynthesis was examined in differentiated 3T3-L1 and 3T3-F442A adipocytes. Insulin altered the relative rate of synthesis of specific proteins independent of its ability to hasten conversion of the fibroblast (preadipocyte) phenotype to the adipocyte phenotype. Although more than one pattern of response to insulin was observed, we focused on the induction of a Mr 33,000 protein which was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure of 3T3 adipocytes to insulin throughout differentiation specifically increased GAPDH activity and protein content by 2- to 3-fold as compared to 3T3 adipocytes differentiated in the absence of insulin. These changes in enzyme activity and content could be accounted for by a 4-fold increase in the relative rate of synthesis of GAPDH and a 9-fold increase in hybridizable mRNA levels. Within 2 h of insulin addition to 3T3 adipocytes differentiated in the absence of hormone, hybridizable GAPDH mRNA levels increased 3-fold, and within 24 h GAPDH mRNA levels increased 8-fold, and [35S] methionine incorporation into GAPDH protein increased 5-fold. The increase in GAPDH mRNA and GAPDH biosynthesis could be demonstrated using physiologic concentrations of insulin (0.24 nM), indicating that these effects are mediated through a specific interaction with the insulin receptor. These studies demonstrate that insulin, as the sole hormonal perturbant, can increase the synthesis of certain 3T3 adipocyte proteins by altering the cellular content of a specific mRNA.     

Calcium Enhances In Vitro Free Radical-Induced Damage to Brain Synaptosomes, Mitochondria, and Cultured Spinal Cord Neurons

Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal gamma-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 microM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.     

Demonstration of T-cell-dependent and T-cell-independent components of 8-mercaptoguanosine-mediated adjuvanticity

The contribution of T cells to potentiation of humoral immunity by the C8-substituted guanine ribonucleosides and the origin of the increased numbers of antigen-responsive B cells generated consequent to their action have been investigated. Augmentation of the antigen-specific antibody response by these nucleosides, exemplified by 8-mercaptoguanosine (8MGuo), can be separated into T-cell-dependent and T-cell-independent components, both by use of the T-cell tropic immunosuppressive agent, cyclosporin A, and by experiments using separated populations of T and B cells. Augmentation of adjuvanticity by T cells is hypothesized to involve a B-cell subpopulation not otherwise subject to the action of 8MGuo. This subpopulation could potentially arise by either of two mechanisms, one whereby preexisting antigen-specific B cells undergo clonal expansion, and one in which cells not normally participating in the response are recruited in the absence of clonal expansion. Although the former mechanism makes a minor contribution to adjuvanticity, the latter mechanism appears to be the dominant one, insofar as models in which 8MGuo-induced proliferation fails to occur (such as after irradiation, or in the SJL mouse) nonetheless exhibit strong adjuvant effects. Analysis of precursor frequency of antigen-specific B cells indicates that for each mature, antigen-responsive B cell present in adult murine spleen, an average of four additional cells can be recruited by the conjoint actions of antigen and 8MGuo. One group subject to such recruitment is the immature antigen-specific B cell, whose degree of functional maturity is accelerated in the presence of antigen and 8MGuo.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Effects of pentobarbital anesthesia on tonic luteinizing hormone secretion in the ewe: evidence for active inhibition of luteinizing hormone in anestrus

In the ewe, seasonal anestrus appears to result from two effects of inhibitory photoperiod: 1) estradiol gains the capacity to suppress luteinizing hormone (LH) pulse frequency and hence becomes a potent inhibitor of tonic LH secretion and 2) a steroid-independent decrease in LH pulse frequency occurs in ovariectomized ewes. In this study, we have obtained evidence, using pentobarbital anesthesia, that both these actions of photoperiod reflect the activation, in anestrus, of an inhibitory neural system. Administration of pentobarbital to intact anestrous ewes produced a dramatic, 3-fold increase in LH pulse frequency during the 6 h of anesthesia. In contrast, during the breeding season, pentobarbital inhibited LH pulse frequency in luteal phase animals. There was also a seasonal variation in the effects of pentobarbital in ovariectomized ewes. During the breeding season this drug again suppressed LH secretion, inhibiting both LH pulse amplitude and frequency. In anestrus, pentobarbital also suppressed pulse amplitude, but it produced a transitory increase (lasting 3 h) in pulse frequency. To account for the stimulatory actions of pentobarbital, we propose that in anestrus, but not the breeding season, LH pulse frequency is held in check by a set of estradiol-sensitive inhibitory neurons. Further, we suggest that these neurons are activated by inhibitory photoperiod and account for both the steroid-dependent and steroid-independent actions of photoperiod.     

Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii

Candida yeasts rapidly form peroxisomes of simple function and composition when grown on methanol. Because the induction is both massive and rapid, this system may be useful for a detailed dissection of peroxisomal biogenesis. We report procedures to express peroxisomal proteins in cells and spheroplasts of Candida boidinii to stabilize peroxisomes in a lysate of spheroplasts and to obtain an enriched peroxisomal fraction. With these techniques we have been able to study the assembly of alcohol oxidase, a homo-octomeric flavoprotein, into the organelle in vivo. The primary translation product of alcohol oxidase comigrates on sodium dodecyl sulfate-polyacrylamide gels with the mature subunit. Pulse-chase experiments indicate that the newly synthesized monomer of alcohol oxidase has a half-life of about 20 min in intact cells and 13 min in spheroplasts before conversion to octomer. The monomer first appears in a high speed supernatant of a lysate of spheroplasts and then chases into a purified peroxisomal fraction before or during its octomerization. There is no detectable intermediary organelle involved in this process.