Change of speciation of Cu(II) in growth medium due to uptake of ammonia byPseudomonas testosteroni during growth

Growth ofPseudomonas testosteroni in a medium containing 1mm Cu(II) causes a color change from blue to green. The spectrum of the supernatant solution from the blue culture shows an absorption at 660 nm, identical to that of 1mm [Cu(II)] in the medium. The green supernatant solution shows a UV absorption, which tails into the visible and so is responsible for the green color, and ad-d absorption at 720 nm. The absorption at 660 nm for the blue supernatant solution is probably due to [Cu(NH₃)₃(H₂O)₃]²⁺. Growth of the organism causes loss of ammonia and a speciation change to [Cu(NH₃)₂(H₂O)₄]²⁺, with a shift in absorption maximum from 660 to 720 nm. These conclusions are based upon the spectra of known aquaammine complexes of Cu(II) and calculations of the speciation of Cu(II) before and after growth. Change in metal speciation owing to nutrient uptake by an organism does not appear to have been recognized previously.     

Leucasin, a triterpene saponin from Leucas nutans.

A new saponin, leucasin, has been isolated from Leucas nutans and characterized on the basis of chemical investigation and spectroscopic studies as 3-O-[β- -glucopyranosyl(1→2)β- -glucopyranosyl]2,3β-dihydroxylup-20(29)-ene. Lupeol palmitate, sitosterol and stigmasterol were also isolated.     

Leucasin, a triterpene saponin from Leucas nutans

A new saponin, leucasin, has been isolated from Leucas nutans and characterized on the basis of chemical investigation and spectroscopic studies as 3-O-[β- -glucopyranosyl(1→2)β- -glucopyranosyl]2α,3β-dihydroxylup-20(29)-ene. Lupeol palmitate, sitosterol and stigmasterol were also isolated.     

Macrophylloside, a flavone glucoside from Primula macrophylla

A new flavone glucoside macrophylloside has been isolated from the whole plant of Primula macrophylla and its structure was determined by spectroscopic methods as 2′-hydroxy-7-O-β- -glucopyranosyloxyflavone. Sitosterol glucoside was also isolated for the first time from this plant.     

Exploiting the chemical synthesis of RNA.

A number of nucleotide phosphoramidites are now available that permit the chemical synthesis of RNA, modified RNA and RNA-DNA chimeric oligonucleotides. Since the chemical strategy allows the introduction of a particular modification at any given site in a nucleotide polymer, very subtle and specific questions regarding structure-function relationships in RNA may be addressed.     

Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus. Purification, properties and immunological cross-reactivity

The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91,000) composed of 45-kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L-phenylalanine (inhibitor of both activities) or L-tyrosine (activator of the dehydratase activity) during gel filtration. Chorismate mutase activity followed Michaelis-Menten kinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was a relatively poor non-competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate-saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate-binding sites. L-Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L-Phenylalanine inhibited the dehydratase activity, and the substrate-saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L-phenylalanine gave Hill plots having a slope of -2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate-saturation curves were changed to near-hyperbolic form. The happ values decreased with increase in ionic strength. Antibodies raised against the purified P protein showed cross-reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross-reaction was superior with P protein from Neisseria gonorrhoeae than with that from the more closely related Escherichia coli.     

The effects of fetal exposure to 1,2-dibromo-3-chloropropane on adult male reproductive function

Several drugs have been shown to cross the placental barrier and affect the fetal testis causing a reduction in testosterone with a resultant impairment of sexual differentiation and an ultimate problem in adult sexual function. In this study, pregnant female rats were treated with 25 mg/kg of the pesticide 1,2-dibromo-3-chloropropane (DBCP). Treatment began on Days 14.5, 16.5, or 18.5 and continued through Day 19.5 of gestation. Some animals were killed on Day 20.5 of intrauterine life and fetal intratesticular testosterone was measured. All other animals were allowed to deliver, and the males were raised to adulthood. At adulthood, body, testis, prostate glands and seminal vesicle weights were recorded. Intratesticular testosterone and luteinizing hormone (LH) receptors were measured. Male and female sexual behavior was quantified and the volume of the sexually dimorphic nucleus of the preoptic area of the hypothalamus was calculated. The histological appearance of the testis was also examined. Treatment for 6 days during fetal life with DBCP decreased intratesticular testosterone by 50% compared to controls at 20.5 days of gestation. At adulthood, all male rats treated during fetal life had a reduced body weight that was correlated with the duration of exposure. Adult testis weight was reduced to 75% of controls as a result of 2 days of fetal exposure to DBCP, whereas 4 and 6 days of exposure during fetal life reduced testis weight by greater than 90%. LH receptors and intratesticular testosterone, in the adults treated during fetal life, were also dramatically reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple phosphorylation sites of rat liver glycogen synthase

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.     

Estimation of the stability of globular proteins

The interpretation of ΔG (the free energy change for the reaction, globular conformation ? randomly coiled conformation, in the absence of denaturant), in terms of the free energies of transfer of various parts of the protein molecule from water to denaturant solution, is unsatisfactory because the latter are assumed to be identical to the transfer-free energies of similar groups attached to smaller model compounds. We have made empirical adjustments to transfer-free energy theory that make possible linear extrapolation of the free energy of denaturation of a protein from transition region to zero denaturant concentration. The modified theory, used to analyze the denaturation of proteins by guanidine hydrochloride and urea, allowed us to calculate reasonable values for Δα, the average change in accessibility to solvent of the component groups of protein.     

Endogenous production of prothymocyte differentiating activity by phytohemagglutinin-stimulated T-cell-depleted human marrow

The PHA responsiveness of marrow T-cell precursors remains a matter of controversy. We have investigated the capacity of human marrow to proliferate under phytohemagglutinin (PHA) stimulation following extensive removal of mature T cells by complement-dependent cytotoxicity with MBG6 and RFT8 monoclonal antibodies. PHA-induced thymidine uptake by marrow cells occurred with a peak on Days 6-8 of incubation instead of Day 3 for PBL. This peak was observed 48 hr earlier in the presence of PHA-stimulated T-depleted marrow cell supernatants. These supernatants can also promote the growth of mature T-cell colonies from MBG6-, RFT8-, T11-, T3- marrow. However, full colony development requires exogenous interleukin 2 (IL-2). IL-2 could be detected in marrow supernatants but only at very low levels and beyond Days 3 and 4. In contrast Days 1-6 marrow supernatants were equally effective in promoting MBG6-RFT8- marrow cell responsiveness to PHA. We conclude that marrow T-cell precursors are not PHA responsive and that PHA induces the production by marrow non-T cells of a prothymocyte-differentiating activity (PTDA); PTDA can differentiate marrow T-cell progenitors into PHA-responsive T cells; following activation by PHA, these cells undergo limited proliferation induced by IL-2 endogenously released from de novo differentiated T cells. It is suggested that this mechanism may account for extrathymic differentiation of the T-cell lineage in heavily irradiated marrow transplantation recipients.