Resolution of 4-amino-cyclopentanecarboxylic acid methyl esters using hydrolytic enzymes.

A number of esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) of microbial and mammalian origin were screened for the ability to resolve racemic 4-amino-cyclopentanecarboxylic acid methyl ester derivatives as potential intermediates in the production of carbocyclic nucleosides. Surprisingly, functionalization of the remote amino group had a profound effect on both the rate and enantioselectivity of hydrolysis of the methyl ester. 4-(Benzoylamino)-2-cyclopentenecarboxylic acid, methyl ester (V) with pig liver esterase gave the highest enantioselectivity. The residual ester, which was of the correct absolute stereochemistry [(+) 1S, 4R] for carbocyclic nucleoside synthesis, could be obtained in high optical purity. Optimization of pH, solvent type, and concentration improved the enantioselectivity of the process by a further twofold.     

GENDER-RELATED DIFFERENCES IN GAS EXCHANGE ARE NOT RELATED TO HOST QUALITY IN THE XYLEM-TAPPING MISTLETOE,PHORADENDRON JUNIPERINUM (VISCACEAE)

Male and female individuals of dioecious species often differ in morphology, physiology, growth, and habitat distribution. Where habitat distribution differences have been demonstrated, female plants generally occupy those habitats with greater resource availability (“rich” habitats). Gender-specific habitat preferences are often presumed to be a consequence of greater resource requirements, per gamete, of female reproduction. Previous work has shown that Phoradendron juniperinum, a xylem-tapping dioecious mistletoe that parasitizes Juniperus species in western North America, displays the opposite pattern: males are relatively more numerous than females in richer sites (i.e., branches with relatively high light and low evaporative demand within the host tree). We report here differences in host (“site”) quality and gas-exchange properties between the sexes. To minimize environmental variation, all measurements were made on sunlit foliage between 9:00 a.m. and 2:00 p.m. Males had significantly higher photosynthetic rates (4.0 [SE = 0.2] μmol m^-2 sec^-1) than either females (2.9 [0.3] μmol m^-2 sec^-1) or nonreproductive individuals (3.0 [0.2] μmol m-² sec^-1). Female photosynthetic rates were not statistically different from those of nonreproductive individuals. No concomitant differences in stomatal conductance were observed. Gas exchange data were independently confirmed by significant differences in carbon isotope ratio (δ¹³C). Gender-related differences were not related to host quality as measured by foliar N, foliar δ¹³C, or water potential of the host tree. The fate of the additional photosynthate in males is unknown, but we discuss the possibility that carbon costs of reproduction in males have been underestimated in past work.     

Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), ‘RPRK’, within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, G_αi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.     

Comparison of the Virulence for Mice of Mycobacterium avium and Mycobacterium intracellulare Identified by DNA Probe Test

The virulence of various serovars of Mycobacterium avium and M. intracellulare identified by DNA probe test was compared with each other. We found species- and serovar-dependencies of M. avium complex (MAC) virulence to mice in terms of mortality, incidence of lung lesions and bacterial load in the visceral organs, as follows. First, human- or environment-derived M. intracellulare was more virulent for mice, as compared to M. avium isolated from patients or environmental sources. Second, the virulence of MAC isolates belonging to serovars 1, 8, 9 (M. avium), 14 and 16 (M. intracellulare) is in the order of serovars 16 > 14 > 8 > 1 > 9. These aspects were different from those for MAC virulence to human and bird, swine and cattle.     

Cellobiose versus glucose utilization by the ruminal bacterium Ruminococcus albus.

Cellulose degradation and metabolism in the rumen can be adversely affected by the presence of soluble sugars, but relatively little information is available on substrate preferences of cellulolytic bacteria. When the ruminal bacterium Ruminococcus albus was incubated with a combination of cellobiose and glucose, the organism preferentially utilized the disaccharide. This preference appeared to be related to repression of glucose uptake systems in cellobiose-grown cells. Glucose transport kinetics exhibited low- and high-affinity uptake, and high-affinity transport was apparently driven by ATP hydrolysis. Bacterial yield in continuous culture was as much as 38% greater when the organism was grown on cellobiose versus glucose, and the increased yield could be partially attributed to constitutive cellobiose phosphorylase activity. The maintenance coefficient of glucose-grown cells was significantly greater than that of cells provided with cellobiose (0.225 g of glucose per g of protein per h versus 0.042 g of cellobiose per g of protein per h), and this result suggested that more energy was devoted to glucose uptake. Substrate affinities were examined in carbon-excess continuous culture, and affinities for glucose and cellobiose were relatively low (0.97 and 3.16 mM, respectively). Although R. albus maintained a proton motive force of approximately 60 mV from pH 6.7 to 5.5, growth ceased below pH 6.0, and this inhibition of growth may have been caused by a depletion of ATP at low pH.     

Aldoxime-Forming Microsomal Enzyme Systems Involved in the Biosynthesis of Glucosinolates in Oilseed Rape (Brassica napus) Leaves

Glucosinolates and cyanogenic glucosides are synthesized from amino acids via similar intermediates, N-hydroxyamino acids and aldoximes. Microsomal preparations from young green leaves of oilseed rape catalyze the NADPH-dependent metabolism of homo-phenylalanine and dihomomethionine to the respective aldoximes, precursors of 2-phenylethyl and 3-butenyl glucosinolates. Cytochrome P-450-type enzymes are not involved (in contrast to cyanogenic glucoside biosynthesis), because neither activity was affected by carbon monoxide or other cytochrome P-450 inhibitors. Copper ions and diethyl pyrocarbonate were potent inhibitors of the enzymes, and treatment of microsomes with detergents abolished the overall activity. Two distinct enzyme systems with similar properties appear to be involved, each specific for a particular substrate. One utilizes dihomomethionine and is not active with homophenylalanine or any other amino acid tested, and the other is specific for homophenylalanine. From the characteristics of these enzymes, it seems that these early steps in glucosinolate biosynthesis may be catalyzed by flavin-containing monooxygenases comparable to those found in mammalian tissues and elsewhere. The pathways for the biosynthesis of glucosinolates and cyanogenic glucosides have apparently evolved independently, despite the similar chemical conversions involved.