A specific ultrastructural stain for arylsulfatase A activity in human cultured fibroblasts

A staining reaction was developed to specifically detect arylsulfatase A activity in the presence of arylsulfatases B and C. Nitrocatechol, generated by all arylsulfatases from the substrate p-nitrocatechol sulfate, can be coupled to produce Hatchett 's brown which reacts with 3,3'-diaminobenzidine to yield an osmiophilic polymer visible under the electron microscope. The reaction was made specific for arylsulfatase A by inhibiting arylsulfatase C activity with low pH and arylsulfatase B activity with pyrophosphate. The specificity was confirmed both by electrophoretic analysis and by patient fibroblasts deficient only in arylsulfatase A activity. Under optimal conditions for preserving structural integrity and enzyme activity, enzyme reaction deposits were found mainly around vesicles. Some of these vesicles were large and heterogeneous (48-330 nm in diameter), distributed randomly within the cytoplasm, but most of the positive-reacting vesicles were uniform in size (86 +/- 18 nm in diameter) and distributed in a peripheral zone about 0.1-0.5 micron wide. These periplasmic vesicles might be partly fused with each other or with the plasma membrane. In conclusion, a specific stain for arylsulfatase A activity suitable for light and electron microscopy and the optimal conditions for structural and enzymatic preservations were developed. Although this enzyme has been considered to be lysosomal in origin, most of the activity was detected in periplasmic vesicles near the cell surface.     

Exchange assay for androgen receptors in the presence of molybdate

Several reports have shown that sodium molybdate stabilizes steroid hormone receptors. We have utilized these observations to develop an exchange assay for the androgen receptor at elevated temperatures. Exchange was found to be complete after 30 min at 30 degrees C. Receptor degradation was negligible during this treatment. Scatchard analysis indicated that the dissociation constant of the androgen receptor was similar both in the absence (Kd = 3.9 nM) and presence (Kd = 2.9 nM) of molybdate. Steroid specificity of the androgen receptor was unaltered by this treatment. The exchange procedure was reproducible, with an interassay variation of 2.45% and intraassay variation less than 10.0%. Using this assay, highest concentrations of androgen binding were measured in androgen target tissues of the rat (Dunning R3327 tumor, prostate and seminal vesicle; 23.37, 20.20 and 19.84 fmol/mg protein respectively). Lower concentrations were observed in other tissues (lung, brain, heart, spleen, liver and kidney; 9.06, 5.63, 3.50, 2.42, 2.33 and 1.36 fmol/mg protein respectively). These results demonstrate that molybdate stabilization of the androgen receptor allows efficient steroid exchange without significant alteration of the receptor's steroid binding properties. Furthermore, this exchange assay can be used to obtain a reasonable measurement of receptor concentrations in different androgen target tissues.     

Ovarian response to active immunization against luteinizing hormone in the cow

Two experiments were conducted to determine whether active immunization against luteinizing hormone (LH) could lead to ovarian cyst development in the cow. In Experiment 1, cyclic beef heifers were randomly assigned to receive bovine LH (bLH) conjugated with ovalbumin (LH-immunized; n=4) or ovalbumin alone (control; n=5). Blood samples were collected at monthly intervals from the LH-immunized heifers to determine antibody titers. Heifers were observed for estrous behavior twice daily. All heifers were slaughtered 4 mo after initial immunization and ovaries examined for follicular status. In Experiment 2, mature dairy cows were immunized with bLH (LH-immunized; n=4) or ovalbumin alone (control; n=3). Weekly blood samples were collected from all cows for 26 wk and ovaries were rectally palpated. Sera from all of the LH-immunized heifers and cows had antibodies to LH. All of the LH-immunized animals stopped cycling 1 mo after immunization. In spite of the fact that serum follicle stimulating hormone levels were unaffected, ovarian cysts could not be found in either the LH-immunized heifers or cows.     

Computer control for continuous cultivation ofSaccharomyces cerevisiae

Summary An on-line respiratory quotient control system has been developed for the continuous cultivation of baker's yeast. This system is based on moving identification of the microbial dynamics. The optimal dilution rate that was selected as the control variable was determined by minimizing a performance index. Without resorting to complicated microbial analysis, a simple and practical moving model is obtained by continually updating the input and output data. The experimental results indicate the satisfactory controllability of the present system and the possible extention of the proposed method to other bioprocesses.     

A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties.

We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers.     

Structure and conformation of the duplex consensus acceptor exon:intron junction d[(CpTpApCpApGpGpT). (ApCpCpTpGpTpApG)] deduced from high-field 1H-NMR of non-exchangeable and imino protons

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and 1H-1H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D2O:H2O mixtures. Assignment of the non-exchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5' regions exist predominantly in the S form (2'endo-3'exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2'exo-3'endo). The contiguous bases A5-G6 (adjacent to the junction) and A15-G16 are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.     

Nutritional requirements of two flower spiroplasmas and honeybee spiroplasma.

A chemically defined medium (CC-494) was used to study the nutritional requirements of three spiroplasmas representing three distinct serogroups: flower spiroplasmas [Spiroplasma floricola and FS (SR-3)] and honeybee spiroplasma [HBS (AS-576)]. Glucose, fructose, and mannose were utilized by all three spiroplasmas. In addition, the honeybee spiroplasma could ferment trehalose, FS (SR-3) could ferment sucrose, and S. floricola could ferment trehalose, sucrose, and raffinose. The three spiroplasmas varied greatly in their requirements of amino acids for growth. S. floricola was the only strain that utilized arginine. HBS (AS-576) required at least one purine and one pyrimidine base (either free base or ribonucleoside) for growth, while both flower spiroplasmas grew with only one base in the medium. Oleic acid, cholesterol, and bovine serum albumin were essential to all three spiroplasmas. Palmitic acid, which was nonessential, promoted growth significantly.     

Purification and characterization of yeast topoisomerase I

Yeast topoisomerase I (Mr = 76,000) has been purified to 80% homogeneity using a combination of ion exchange, gel filtration, and DNA-cellulose chromatography. The enzyme was characterized with respect to its ability to relax supercoiled DNA and to catenate nicked circular DNA. Yeast topoisomerase I will remove both positive and negative turns in DNA supercoils in the absence of ATP and magnesium ion. The products of the catenating activity of the enzyme were examined on agarose gels and in the electron microscope. These analyses indicate that yeast topoisomerase I will generate large catenated DNA networks which appear to rearrange to multimeric linear structures upon long incubation time.     

Polypeptide structure of DNA polymerase I from Saccharomyces cerevisiae

DNA polymerase I of the yeast Saccharomyces cerevisiae has been purified to near homogeneity. The enzyme sediments under high salt conditions as a band at 7.4 S and two polypeptides of Mr = 140,000 and 110,000 are resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both polypeptides react with rabbit anti-yeast DNA polymerase I serum and can be shown to be enzymatically active by renaturation in situ after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This high molecular weight form of yeast DNA polymerase I is very sensitive to inhibition by aphidicolin. The biochemical properties of the enzyme and inhibitors that may aid in distinguishing yeast DNA polymerases I and II are also described.     

Increase in brain 125I-cholecystokinin (CCK) receptor binding following chronic haloperidol treatment, intracisternal 6-hydroxydopamine or ventral tegmental lesions

Specific 125I-CCK receptor binding was significantly increased in brain tissue taken from guinea pig or mouse following chronic (2-3 week) daily administration of haloperidol (2-3 mg/kg/day). Scatchard analysis indicated the increase in CCK binding was due to an increased receptor number (B max) with no change in affinity (Kd). In guinea pigs, the increased CCK binding was observed in the mesolimbic regions and frontal cortex, but not in striatum, hippocampus nor posterior cortex. In mice, however, the increases occurred in both pooled cerebral cortical-hippocampal tissue, and in the remainder of the brain. Enhanced CCK receptor binding was also observed in membranes prepared from whole brain of mice one month following intracisternal injection of 6-hydroxydopamine. Additionally, an increase in CCK binding was observed in mesolimbic regions and frontal cortex, but not striatum or hippocampus, of guinea pigs 3 weeks after an unilateral radiofrequency lesions of the ipsilateral ventral tegmentum. The present studies demonstrate that three different procedures which reduce dopaminergic function in the brain enhance CCK receptor binding. The data provide further support for a functional interrelationship between dopaminergic systems and CCK in some brain regions and raise the possibility that CCK may play a role in the antipsychotic action of neuroleptics.