Efficient access to all four stereoisomers of phenylalanine cyclopropane analogues by chiral HPLC

Bonded polysaccharide‐derived chiral stationary phases were found to be useful for the preparation of the four stereoisomers of the cyclopropane analogue of phenylalanine (c₃Phe) as well as for the direct determination of the enantiomeric purity of c₃Phe derivatives by HPLC. Three chiral stationary phases, consisting of cellulose and amylose derivatives chemically bonded on allylsilica gel, were tested. The mixed 10‐undecenoate/3,5‐dimethylphenylcarbamate of cellulose, 10‐undecenoate/3,5‐dimethylphenylcarbamate of amylose and 10‐undecenoate/p‐methylbenzoate of cellulose were the starting polysaccharide derivatives for CSP‐1, CSP‐2, and CSP‐3, respectively. Using mixtures of n‐hexane/chloroform/2‐propanol as mobile phase on a semi‐preparative column (150 mm × 20 mm ID) containing CSP‐2, we separated about 1.7 g of racemic cis‐methyl 1‐tert‐butoxycarbonylamino‐2‐phenylcyclopropanecarboxylate (cis‐ 6 ) and 1.2 g of racemic trans‐methyl‐1‐tert‐butoxycarbonylamino‐2‐phenylcycloprop‐anecarboxylate (trans‐ 6 ) by successive injections. Chirality 11:583–590, 1999. © 1999 Wiley‐Liss, Inc.     

Zinc hyperaccumulation substitutes for defense failures beyond salicylate and jasmonate signaling pathways of Alternaria brassicicola attack in Noccaea caerulescens

The hypothesis of metal defense as a substitute for a defective biotic stress signaling system in metal hyperaccumulators was tested using the pathosystem Alternaria brassicicola–Noccaea caerulescens under low (2 µM), medium (12 µM) and high (102 µM) Zn supply. Regardless the Zn supply, N. caerulescens responded to fungal attack with the activation of both HMA4 coding for a Zn transporter, and biotic stress signaling pathways. Salicylate, jasmonate, abscisic acid and indoleacetic acid concentrations, as well as biotic stress marker genes (PDF1.2, CHIB, LOX2, PR1 and BGL2) were activated 24 h upon inoculation. Based on the activation of defense genes 24 h after the inoculation an incompatible fungal–plant interaction could be predicted. Nonetheless, in the longer term (7 days) no effective protection against A. brassicicola was achieved in plants exposed to low and medium Zn supply. After 1 week the biotic stress markers were even further increased in these plants, and this compatible interaction was apparently not caused by a failure in the signaling of the fungal attack, but due to the lack of specificity in the type of the activated defense mechanisms. Only plants receiving high Zn exhibited an incompatible fungal interaction. High Zn accumulation in these plants, possibly in cooperation with high glucosinolate concentrations, substituted for the ineffective defense system and the interaction turned into incompatible. In a threshold‐type response, these joint effects efficiently hampered fungal spread and, consequently decreased the biotic stress signaling.     

HSP70 is part of the synaptonemal complex in Eimeria tenella

In the current study the expression and ultrastructural localization of heat shock protein 70 (HSP70) was analyzed by immunogold labelling of surface spreads of meiotic chromosomes from Eimeria tenella oocysts. The authors used a previously reported method that overcomes the difficulties of the resistance of Eimeria oocysts to disruption and permits the release of intact meiotic chromosomes. HSP70 was localized at the ultrastructural level using an anti-HSP70 monoclonal antibody in combination with a secondary antibody coupled to colloidal gold. Synaptonemal complexes (SCs) were visualized by means of the surface spreading technique to study both HSP70 expression and the consequences of the lack of HSP70 in the behaviour of the eimerian chromosomes during meiosis. For that purpose E. tenella oocysts were treated with quercetin, a flavonoid that is known to inhibit the synthesis of HSP70. The results showed a close association of HSP70 with the lateral elements (LEs) of the SCs. That association began at the time that SCs were formed and persisted until disassemble. Comparison between distribution of immunogold label over the SCs from non-treated and treated oocysts revealed a decreasing number of gold particles as the concentration of quercetin increased. The current results demonstrated three dose-dependent effects of the quercetin treatment of Eimeria oocysts: a reduction in the HSP70 synthesis; defects in SC formation or desynapsis, and inhibition of sporulation. HSP70, as a structural component of the SCs, may be involved in SC functions such as chromosomal pairing, recombination, or disjunction.     

Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.     

Malformations of the epididymis, incomplete regression of the mesonephric tubules and hyperplasia of Leydig cells in canine persistence of Müllerian duct syndrome

Persistence of the Müllerian duct syndrome (PMDS) is a rare form of pseudohermaphroditism characterized by the presence of uterus and oviducts in otherwise normally differentiated SRY-positive 78 XY canine males. Undescended testicles are also common. We report a case of a male PMDS dog with a uterus and bilateral cryptorchidism. The dog had an incomplete regression of the mesonephric tubules. As a consequence of this an abnormally enlarged head of the epididymis was observed. In addition, an extreme reduction in size of both the body and the tail was found. Microscopic examination of both testicles revealed bilateral hyperplasia of Leydig cells. The progesterone blood level was measured by ELISA and was found to be abnormally high (3.18 ng/ml) compared to that of normal male dogs (lower than 1 ng/ml). Three months after surgical removal of the internal genitalia, the serum progesterone, testosterone and oestradiol levels were normal for a castrated male dog.