MTPA: a crustacean metallothionein that affects hepatopancreatic mitochondrial functions

Metallothioneins are cysteine-rich proteins, with a high capacity to bind metallic ions, and for which a precise biological role has not been established. Here we investigated the effects of MTPA, a metallothionein from the lobster Panulirus argus, on mitochondrial oxygen consumption and ROS production. An HPLC-RP-ESI-MS analysis of recombinant MTPA showed that despite its extra Cys, MTPA binds 6 Zn²⁺ per molecule akin to other crustacean metallothioneins with 18 Cys. The extra Cys is not involved in zinc binding, since its side-chain would be oriented to the outside of the molecule according to a preliminary model of the tridimensional structure of MTPA. MTPA-Zn²⁺₆ is imported into the hepatopancreatic mitochondria intermembrane space and inhibits mitochondrial oxygen consumption, increasing thereby ROS production. Nevertheless, the stimulation of ROS production by MT-bound Zn²⁺ is weaker compared to equivalent amounts of free Zn²⁺, suggesting that MTPA protects against oxidative stress. This constitutes the first report on metallothioneins effects on mitochondrial function in invertebrates and agrees with the results described for mammals, suggesting a connection between metallothioneins and energy metabolism.     

Changes in lipids containing long- and very long-chain polyunsaturated fatty acids in cryptorchid rat testes

The aim of the present study was to examine the effects of experimental cryptorchidism on rat testicular phospholipids and neutral lipids that contain long-chain (C(18)-C(22)) and very long-chain (VLC) (C(24)-C(32)) polyunsaturated fatty acids (PUFA). The weight of the cryptorchid testis was nearly half that of the contralateral control at postsurgical Days 7-10 owing to the depletion of germ cells. Concomitantly, the amounts of major glycerophospholipids (GPL) and sphingomyelin (SM) per testis decreased. Both these lipids lost their characteristic long-chain and very long-chain PUFA, notably 22:5n-6 and 28:4n-6, respectively, which suggests that these species are linked to the membranes of germ cells. In contrast, the amounts and concentrations of triglycerides (TG; triacylglycerols and 1-O-alkyl-2,3-diacylglycerols) and cholesterol esters (CE) increased several fold in the surviving cells (mainly Sertoli cells) in the cryptorchid testis. All these neutral lipids, but especially CE, accumulated large amounts of the major PUFA of the testis, 22:5n-6, as well as pentaenes with longer carbon chains (i.e., 24:5n-6 in TG and 28:5n-6 in CE). This accretion suggests that neutral lipids may store preformed PUFA coming from dying germ cell GPL and also VLCPUFA no longer needed as a source of PUFA destined to assemble new germ cell GPL. The lipid adjustments observed in cryptorchidism suggest a possible role for Sertoli cell CE in the turnover and conservation of PUFA within seminiferous tubules.     

Unfolding kinetics of beta-lactoglobulin induced by surfactant and denaturant: a stopped-flow/fluorescence study

The beta-->alpha transition of beta-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine beta-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the alpha-helical content increases, leading to the so-called alpha-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I(1)) with mostly native secondary structure but loose tertiary structure appears between the native (beta) and alpha-states; in GnHCl, another intermediate (I(2)) appears between states beta and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species.     

Breakdown of self-incompatibility in a natural population of Petunia axillaris (Solanaceae) in Uruguay containing both self-incompatible and self-compatible plants

 Many members of the Solanaceae display a type of gametophytic self-incompatibility which is controlled by a single multiallelic locus, called the S-locus. From our previous survey of more than 100 natural populations of Petunia axillaris (a solanaceous species) in Uruguay, we had found that the majority of the populations of subspecies axillaris were comprised of virtually all self-incompatible individuals. The rest were ”mixed populations” which contained mostly self-incompatible and some self-compatible individuals. In this study, we examined the self-incompatibility behavior and determined the S-genotypes of 33 plants raised from seeds obtained from one such mixed population, designated U1. We found that 30 of the 33 plants (designated U1–1 through U1–33) were self-incompatible and a total of 18 different S-alleles were represented. To determine the S-genotypes of the three self-compatible plants (U1–2, U1–16, and U1–22) and the possible causes for the breakdown of their self-incompatibility, we carried out reciprocal crosses between each of them and each of the 18 S-homozygotes (S ₁ S ₁ through S ₁₈ S ₁₈ ) obtained from bud-selfed progeny of 14 of the 30 self-incompatible plants. For U1–2 and U1–16, we also carried out additional crosses with U1–25 (with S ₁ S ₁₃ genotype) and an S ₁₃ S ₁₅ plant (obtained from a cross between an S ₁₃ -homozygote and an S ₁₅ -homozygote), respectively. Based on all the pollination results and analysis of the production of S-RNases, products of S-alleles in the pistil, we determined the S-genotypes of U1–2, U1–16, and U1–22, and propose that the breakdown of self-incompatibility in these three plants is caused by suppression of the production of S₁₃-RNase from the S ₁₃ -allele they all carry. We have termed this phenomenon ”stylar-part suppression of an S-allele” or SPS. Received: 25 September 1998 / Revision accepted: 22 December 1998     

Intestinal microflora and the interaction with immunocompetent cells

The intestinal mucosal surface is colonised by the comensal microflora that attains very high numbers of bacterial cells in the distal intestine, more specifically in the colon. At the same time these extensive areas are the interface with the external environment, through which most pathogens initiate infectious processes in mammals. Intestinal mechanisms of defense need to discriminate accurately between comensal, symbiotic microflora, and exogenous pathogens. Today we do not fully understand the essence of the mechanism of discrimination but, probably, innate as well as adaptive immune responses participate in this process. We have explored , in in vitro models, the capacity of mucosal immunocompetent cells to discriminate amongst signals delivered by different types of bacteria. We have found at least two different patterns of innate response to gram-negative and gram-positive bacteria, and within this last group big differences are observed between species. We have only wo rked with non-pathogenic bacteria in what may represent the modulation of the physiological host status. The understanding of these modulatory functions could render a unique possibility for the use of food-borne bacteria to prevent or correct intestinal problems associated with food allergy, inflammatory bowel disease, and autoimmunity.