The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

Novel Method for Detection of Butanolides in Streptomyces coelicolor Culture Broth, Using a His-Tagged Receptor (ScbR) and Mass Spectrometry

γ-Butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.     

Pleiotropic mutations in Serratia marcescens which increase the synthesis of certain exocellular proteins and the rate of spontaneous prophage induction

Summary Mutants of S. marcescens HY have been isolated which produce between five and one hundred times more exocellular nuclease than does the parental strain. These nuclease-superactive (nuc ^su) mutants are highly pleiotropic: they produce more exocellular marcescin A and lipase than the wild-type and their ability to inactivate penicillin G is increased. Furthermore, all nuclease-superactive mutants if lysogenic for the heteroimmune phages Kappa and/or y show spontaneous induction rates for both prophages 10 to 200 fold greater than the corresponding wild-type. Nuc ^su mutants of independent origin synthesize nuclease and marcescin A in approximately proportional amounts although the corresponding structural genes do not seem to be part of a single operon because some bacteriocin-superactive mutants were isolated which showed an increase of the synthesis of marcescin A only. Nuclease-defective (nuc) mutants are all of the non-pleiotropic type. Three hypotheses to explain the effects of the nuc ^su mutation at the molecular level are discussed and some evidence in support of one of these hypotheses (gene-dosage effect) is presented in an accompanying paper (Timmis and Winkler, 1973).     

Phytochrome A regulates red-light induction of phototropic enhancement in Arabidopsis.

Phytochrome A (phyA) and phytochrome B photoreceptors have distinct roles in the regulation of plant growth and development. Studies using specific photomorphogenic mutants and transgenic plants overexpressing phytochrome have supported an evolving picture in which phyA and phytochrome B are responsive to continuous far-red and red light, respectively. Photomorphogenic mutants of Arabidopsis thaliana that had been selected for their inability to respond to continuous irradiance conditions were tested for their ability to carry out red-light-induced enhancement of phototropism, which is an inductive phytochrome response. We conclude that phyA is the primary photoreceptor regulating this response and provide evidence suggesting that a common regulatory domain in the phyA polypeptide functions for both high-irradiance and inductive phytochrome responses.     

The cytomegalovirus enhancer: a pan-active control element in transgenic mice.

In an effort to identify widely active positive regulatory elements, we have examined the action of the cytomegalovirus enhancer-promoter in transgenic mice. These elements activated expression in 24 of 28 tissues tested. The greatest expression was observed in the heart, kidney, brain, and testis. Maximum expression further localized to specific cells within the heart and kidney.     

Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.