Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 1. Effect of exogenous sterols

Chinese hamster ovary cells grown in medium containing low-density lipoprotein (LDL) express high acyl coenzyme A:cholesterol acyltransferase (ACAT) activity as measured by an [3H]oleate pulse. Removal of LDL from the medium causes rapid inactivation of ACAT activity; the t1/2 for the initial inactivation rate is 0.8 h. Preincubation with protein synthesis inhibitors (cycloheximide or emetine) for 2 h or longer lengthens the t1/2 for the initial inactivation rate to approximately 2.1 h. When LDL is removed for more than 10 h, the cells contain only 3% of the original ACAT activity. Cycloheximide under this condition causes an 8-fold increase in ACAT activity; the increase approaches a maximum in 6-8 h. The extent of ACAT activation by cycloheximide inversely depends on exogenous sterol present in the medium; LDL diminishes the activation, while cationized LDL or 25-hydroxycholesterol completely abolishes the activation. Adding LDL back to the sterol-free medium causes a 40-70-fold increase in ACAT activity; however, the activation of LDL is not further augmented if the cells are pretreated with cycloheximide. The above observations are qualitatively confirmed by ACAT assays in vitro with cell homogenates. LDL or cycloheximide has no effect on the rates of 3H-labeled triglyceride and 3H-labeled polar lipid synthesis. Efflux of prelabeled cholesterol from cells is cycloheximide-insensitive. Rates of degradation of [3H]-leucine-pulse-labeled total protein in cells grown with or without LDL are identical. The above results imply the existence of at least one specific short-lived factor that directly or indirectly inhibits ACAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenylalanine ammonia-lyase immobilized in microcapsules for the depletion of phenylalanine in plasma in phenylketonuric rat model

Microencapsulation of the enzyme phenylalanine ammonia-lyase was developed for in vivo depletion of systemic phenylalanine in phenylketonuric rats. Compared to normal rats, systemic phenylalanine blood levels in phenylketonuric rats was increased by 15-20-fold. Daily oral administration of 1 unit of phenylalanine ammonia-lyase-loaded artificial cells to phenylketonuric rats lowered the systemic phenylalanine level to 58% +/- 18% (mean + S.D.) in 7 days (P less than 0.010), while 5 units lowered the systemic phenylalanine level to 25% +/- 8%. 5 units of the immobilized enzyme lowered the systemic phenylalanine level to normal levels within 6 days. Phenylketonuric treated rats showed no signs of abnormal behavior and weight loss compared to phenylketonuric non-treated rats. The immobilized enzyme within artificial cells is therefore protected against low gastrointestinal pH and proteolytic enzymes.     

Syngeneic monoclonal internal image anti-idiotopes as prophylactic vaccines

A syngeneic monoclonal anti-idiotope that behaves as an internal image of the mammalian reovirus type 3 cellular attachment protein (viral hemagglutinin) was used in the syngeneic host for the induction of a prophylactic anti-viral antibody response. These studies were performed without the aid of co-stimulation by viral antigens. The high stringency of this system enables us to define the maximum constraints on the use of anti-idiotopes as anti-viral vaccines. We have used the murine BALB/c monoclonal IgM anti-idiotope 87.92.6 to study the idiotope and antigen specificity, kinetics, dose dependence, adjuvant, carrier, and valency requirements of anti-idiotope-induced anti-viral antibody responses. These studies show that the production of high titer neutralizing antibody requires a lengthy (60 day) immunization protocol, which includes the use of adjuvant and multivalent anti-idiotope, and is dependent on anti-idiotope concentrations of greater than 50 micrograms. When administered in this manner anti-idiotope can stimulate serotype-specific antibody responses across species barriers at levels comparable with those obtained after inoculation with virus. The practical efficacy of these reagents and procedures is documented by the ability of maternal immunization with anti-idiotope to confer complete protection in neonates from a potentially lethal reovirus type 3 viral infection.     

Mechanism of bleomycin: evidence for 4'-ketone formation in poly(dA-dU) associated exclusively with free base release

Incubation of poly(dA-[3'-3H]dU), poly(dA-[5'-3H]dU), or poly(dA-[5'-3H]dT) under a variety of conditions with activated bleomycin resulted in the production of free nucleic acid base, base propenal, and a small amount of 3H2O. Adjustment of the terminated reaction mixture to pH 10 and incubation at 95 degrees C resulted in a time-dependent increase in 3H2O to an amount equal to the amount of free base. If the terminated reaction mixture was incubated with NaBH4 prior to the heat and alkaline treatment, the release of 3H2O was significantly inhibited. These results are consistent with the generation by activated bleomycin of a 4'-ketone yielding free base, with the exchange of the 3'- and 5'-hydrogens by enolization and with the alkaline-induced strand scission occurring from this intermediate.     

中国人ε珠蛋白基因5′侧序列中多态性Hinc Ⅱ位点的进一步研究

本文进一步研究了我国不同民族的正常个体以及β地中海贫血患者θ珠蛋白基因5′侧序列中的多态性HincⅡ位点及其遗传性质。在广西壮族正常个体和β地中海贫血纯合子中,该多态性位点的发生频率均为75％,与正常汉族人测得值相近。家系分析资料表明,该多态性位点完全按照孟德尔规律进行遗传。     

Evidence for cAMP-independent inhibition of S-phase DNA synthesis by prostaglandins

Two prostaglandins, prostaglandin E1 (PGE1) and prostaglandin B1 (PGB1), block S-phase DNA synthesis in synchronous cultured baby hamster kidney (BHK) cells. The prostaglandin inhibition of DNA synthesis does not appear to require elevated levels of cAMP. In BHK-21 cells that have been "desensitized" to prostaglandin stimulation of adenylate cyclase and, therefore, have control levels of cAMP, PGE1 retains its inhibitory effect on the incorporation of tritiated thymidine into DNA. When BHK cells are exposed to PGB1 (a prostaglandin that does not elicit a cAMP response), DNA synthesis is also blocked. In nonsynchronous cells exposed for 1 h to PGE and then incubated for 1 h with PGE removed, a rebound of DNA synthesis occurs, therefore providing evidence that a transient rise of cAMP in itself is not capable of causing a cascade of reactions that block the synthesis of DNA. In addition, the concentration of PGE required for inhibition of DNA synthesis is significantly less than that required for cAMP generation. Addition of 1 x 10(-8) M PGE to BHK cells can be shown to significantly inhibit DNA synthesis within 30 min, with half-maximal inhibition seen at 3 x 10(-7) M PGE. Cyclic AMP levels for controls were 4.9 +/- 0.2 and 4.6 +/- 0.1 for 1 x 10(-6) M PGE1. These findings suggest that the prostaglandins can act independently of cAMP at physiological concentrations; and, therefore, it is possible that prostaglandins have a physiological role in the control of cell growth during S-phase.     

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.