31P NMR spectra of an oligodeoxyribonucleotide duplex lac operator-repressor headpiece complex

The interaction of a symmetric lac operator duplex, d(TGTGAGCGCTCACA)2, with the N-terminal 56-residue headpiece fragment of the lac repressor protein was monitored by 31P NMR spectroscopy. The changes in the 31P chemical shifts upon addition of the headpiece demonstrated an end point of two headpiece fragments per symmetric 14-mer duplex with each headpiece binding to the T1pG2pT3pG4pA5 ends of the duplex. The specific phosphate 31P perturbations observed are consistent with those residues implicated in protein binding by previous NMR, molecular biological, and biochemical techniques. Upon complexation, the 31P signals of phosphates G2-A5 showed upfield or downfield shifts (less than 0.2 ppm) while most other residues were unperturbed. The interactions were dependent on ionic strength. The 31P NMR data provide direct evidence for predominant recognition of the 5' strand of the 5'-TGTGA/3'-ACACT binding site.     

Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum.

The zona pellucida of mouse oocytes becomes resistant to chymotrypsin digestion, or "hardened", when spontaneous maturation occurs in serum-free medium (De Felici and Siracusa, Gam Res 1982; 6:107). The hardened zona pellucida is refractory to sperm penetration, thus preventing fertilization. Conversion of the zona pellucida glycoprotein ZP2 to ZP2f by a protease from precociously released oocyte cortical granules appears to be a major contributory factor of zona pellucida hardening (Ducibella et al., Dev Biol 1990; 137:46). Fetal bovine serum (FBS) prevents zona hardening and the ZP2 to ZP2f conversion during oocyte maturation in vitro (Downs et al., Gam Res 1986; 15:115; Ducibella et al., Dev Biol 1990; 137:46). This study was conducted to determine whether fetuin, a major glycoprotein constituent of FBS and a protease inhibitor, could prevent zona pellucida hardening during murine oocyte maturation in serum-free medium. Commercially available preparations of fetuin purified by three different methods were all active in inhibiting zona pellucida hardening in a concentration-dependent manner. Further chromatographic purification of one of these preparations indicated that the activity preventing zona pellucida hardening was associated specifically with fetuin. Fetuin also inhibited the conversion of ZP2 to ZP2f in a concentration-dependent manner during oocyte maturation in serum-free medium. Moreover, oocytes that matured in serum-free medium containing fetuin could be fertilized and could undergo preimplantation development to the blastocyst stage. These results indicate that fetuin, a component of FBS, inhibits zona pellucida hardening during oocyte maturation, and suggest that fetuin acts by preventing the proteolytic conversion of ZP2 to ZP2f by precociously released cortical granules.     

Earliest mechanical evidence of cross-bridge activity after stimulation of single skeletal muscle fibers.

The stiffness of single fibers from frog skeletal muscle was measured by the application of small 2-kHz sinusoidal length oscillations during twitch and tetanic contractions at a range of initial sarcomere lengths. The earliest mechanical signs of activation were a fall in tension (latency relaxation) and a rise in stiffness. The earliest stiffness increase and the earliest tension fall occurred simultaneously at all sarcomere lengths. This suggests a cross-bridge origin for the latency relaxation. The lead of stiffness over tension seen during the rise of tension was substantially established during the latent period. Reducing the size of the twitch by reducing calcium release with D-600 (methoxyverapamil) reduced the latency relaxation and the stiffness development during latency much less than it reduced the twitch tension. For very small twitches the peak of the stiffness response occurred during the latent period and the times of onset of both latency relaxation and stiffness rise were delayed, but remained coincident. This suggests a strong connection between the latency relaxation and the rise of stiffness during the latent period, whereas the connection between these events and positive tension generation appears to be less strong.     

Interaction of fluorescent delta 5,7,9(11),22-ergostatetraen-3 beta-ol with sterol carrier protein-2

The fluorescent sterol delta 5,7,9(11)-dehydroergostatetraen-3 beta-ol (dehydroergosterol) was used as an analogue of cholesterol to examine the molecular interaction of purified rat liver sterol carrier protein-2 (SCP-2) with sterol. The binding of dehydroergosterol to SCP-2 was evidenced by light scatter and by fluorescence polarization, lifetime, limiting anisotropy, and rotational relaxation time of dehydroergosterol. In addition, energy transfer efficiency from SCP-2 tryptophan to dehydroergosterol was 96%, indicating that the apparent distance, R, between the SCP-2 tryptophan (energy donor) and the dehydroergosterol (energy acceptor) was 13.7 A. Scatchard binding analysis of light scatter, lifetime, and energy transfer data all indicated a 1:1 molar stoichiometry with Kd = 1.2, 1.6, and 1.3 microM, respectively. SCP-2 enhanced the activity of microsomal acyl-CoA:cholesterol acyltransferase through transfer of [3H]cholesterol from donor palmitoyloleoyl phosphatidylcholine/cholesterol small unilamellar vesicles to rat liver microsomes containing the enzyme. A recently developed fluorescence assay utilizing dehydroergosterol fluorescence polarization (Nemecz, G., Fontaine, R. N., and Schroeder, F. (1988) Biochim. Biophys. Acta 948, 511-521; Nemecz, G., and Schroeder, F. (1988) Biochemistry 27, 7740-7749) was applied to examine the effect of SCP-2 on sterol exchange. In the absence of SCP-2, two spontaneously exchangeable sterol domains were observed in palmitoyloleoyl phosphatidylcholine/sterol (65:35 molar ratio) small unilamellar vesicles. SCP-2 enhanced the rate of exchange of the faster exchanging domain 2-fold. The transfer rate of the more slowly exchangeable sterol domain and the fraction of cholesterol represented by each domain were not affected. These results demonstrate the utility of dehydroergosterol to probe SCP-2 interactions with sterols and are indicative of a physiological role for SCP-2 as a soluble sterol carrier.     

Genetically determined chromosome instability syndromes

Spontaneously increased chromosomal instability is well documented in the three autosomal recessive diseases, Fanconi's anemia (FA), Bloom's syndrome (BS), and ataxia telangiectasia (AT). Other conditions have been reported to be associated with chromosomal breakage. Some are still single observations: in Werner's syndrome only fibroblasts are affected, and systemic sclerosis may not be an inherited disease. Various aspects of FA, BS, and AT are discussed which have emerged since recent reviews have been published. The differential diagnosis in FA has become more important than it was in the past. Proven heterogeneity in FA demands definition of what to name FA and FA variants. The analysis of cancer frequencies and types in FA and AT lacks important clues. This should stimulate all of us to mutual exchange of data and creation of registries not only of patients and follow-ups, but also of characterized cell strains. A synopsis of results from cell and cytogenetic studies demonstrates similarities and differences in detail of the general phenomenon of chromosomal instability which FA, BS, and AT share. Results from biochemical studies at the DNA level together with cytogenetic findings indicate different but still undefined failures in DNA metabolism or DNA repair mechanisms due to the different genes. A new approach to analyzing the impairment of DNA repair in FA is briefly described. DNA related enzymes are produced in the cytoplasm and have to be transported to the nucleus. The subcellular distribution of topoisomerase activity was found to be unusual in three placentas of FA patients. Other DNA enzymes were distributed normally. Thus, a specific mechanism for movement of the enzyme through the nuclear membrane seems to be defective.     

Psychological treatment of essential hypertension: A controlled comparison of meditation and meditation plus biofeedback

Twenty-one patients with essential hypertension were randomly allocated to eight 1-hour sessions of meditation training, meditation plus biofeedback-aided relaxation, or a no-treatment control group. Statistically significant falls in systolic and diastolic blood pressure occurred after both training programs, although overall reductions in blood pressure were not significantly greater in either program than in the control group. Meditation plus biofeedback-aided relaxation produced falls in diastolic blood pressure earlier in the training program than did meditation alone. All patients practiced meditation regularly between training sessions: The amount of practice did not correlate with the amount of blood pressure reduction after training. On questionnaire measures of psychological symptoms and personality, sex differences emerged, with females showing significant abnormalities in hostility scores and males showing significantly raised levels of somatopsychic symptoms. In females, outward-directed hostility fell significantly and assertiveness increased after training, but in males, somatopsychic symptoms were unchanged.The author is grateful for financial support from the research fund of St. George's Hospital, London, where this project was conducted, and from the Society for Psychosomatic Research. He is indebted to Dr. Chandra H. Patel for invaluable help in conducting the meditation and biofeedback training.     

The identification of unbranched enzyme kinetic mechanisms by computer analysis of qualitative information: Logic of the method

The paper describes the logic of a computer method for identifying unbranched enzyme kinetic mechanisms on the basis of observed initial velocity and product inhibition patterns (Cleland, 1963). The method establishes initially an acceptable order of reactant addition and release, proceeds to list all the mechanisms consistent with that order and the data, and finally determines which of these can also explain data which require either non-competitive or dead-end inhibition.