Dual Regulatory Role of Polyamines in Adipogenesis.

Adipogenesis is a complex process, accompanied by a chain of interdependent events. Disruption of key events in this cascade may interfere with the correct formation of adipose tissue. Polyamines were demonstrated necessary for adipogenesis; however, the underlying mechanism by which they act has not been established.

Expression profiling and biochemical analysis suggest stress response as a potential mechanism inhibiting proliferation of polyamine-depleted cells.

Polyamines are small organic polycations that are absolutely required for cell growth and proliferation; yet the basis for this requirement is mostly unknown. Here, we combined a genome-wide expression profiling with biochemical analysis to reveal the molecular basis for inhibited proliferation of polyamine-depleted cells. Transcriptional responses accompanying growth arrest establishment in polyamine-depleted cells or growth resumption following polyamine replenishment were monitored and compared.

Antizyme affects cell proliferation and viability solely through regulating cellular polyamines.

Antizymes are key regulators of cellular polyamine metabolism that negatively regulate cell proliferation and are therefore regarded as tumor suppressors. Although the regulation of antizyme (Az) synthesis by polyamines and the ability of Az to regulate cellular polyamine levels suggest the centrality of polyamine metabolism to its antiproliferative function, recent studies have suggested that antizymes might also regulate cell proliferation by targeting to degradation proteins that do not belong to the cellular polyamine metabolic pathway. Using a co-degradation assay, we show here that, although they efficiently stimulated the degradation of ornithine decarboxylase (ODC), Az1 and Az2 did not affect or had a negligible effect on the degradation of cyclin D1, Aurora-A, and a p73 variant lacking the N-terminal transactivation domain whose degradation was reported recently to be stimulated by Az1.

The role of polyamines in supporting growth of mammalian cells is mediated through their requirement for translation initiation and elongation.

Polyamines are essential cell constituents whose depletion results in growth cessation. Here we have investigated potential mechanisms of action of polyamines in supporting mammalian cell proliferation. We demonstrate that polyamines regulate translation both at the initiation and at the elongation steps.

Antizyme 3 inhibits polyamine uptake and ornithine decarboxylase (ODC) activity, but does not stimulate ODC degradation.

Azs (antizymes) are small polyamine-induced proteins that function as feedback regulators of cellular polyamine homoeostasis. They bind to transient ODC (ornithine decarboxylase) monomeric subunits, resulting in inhibition of ODC activity and targeting ODC to ubiquitin-independent proteasomal degradation. Az3 is a mammalian Az isoform expressed exclusively in testicular germ cells and therefore considered as a potential regulator of polyamines during spermatogenesis.

Crystallographic and biochemical studies revealing the structural basis for antizyme inhibitor function.

Antizyme inhibitor (AzI) regulates cellular polyamine homeostasis by binding to the polyamine-induced protein, Antizyme (Az), with greater affinity than ornithine decarboxylase (ODC). AzI is highly homologous to ODC but is not enzymatically active. In order to understand these specific characteristics of AzI and its differences from ODC, we determined the 3D structure of mouse AzI to 2.

Yeast antizyme mediates degradation of yeast ornithine decarboxylase by yeast but not by mammalian proteasome: new insights on yeast antizyme.

Mammalian antizyme (mAz) is a central element of a feedback circuit regulating cellular polyamines by accelerating ornithine decarboxylase (ODC) degradation and inhibiting polyamine uptake. Although yeast antizyme (yAz) stimulates the degradation of yeast ODC (yODC), we show here that it has only a minor effect on polyamine uptake by yeast cells. A segment of yODC that parallels the Az binding segment of mammalian ODC (mODC) is required for its binding to yAz.

ODCp, a brain- and testis-specific ornithine decarboxylase paralogue, functions as an antizyme inhibitor, although less efficiently than AzI1.

ODC (ornithine decarboxylase), the first enzyme in the polyamine biosynthesis pathway in mammalian cells, is a labile protein. ODC degradation is stimulated by Az (antizyme), a polyamine-induced protein, which in turn is regulated by an ODC-related protein termed AzI (Az inhibitor). Recently, another ODCp (ODC paralogue) was suggested to function as AzI, on the basis of its ability to increase ODC activity and inhibit Az-stimulated ODC degradation in vitro.

Antizyme inhibitor: a defective ornithine decarboxylase or a physiological regulator of polyamine biosynthesis and cellular proliferation.

ODC (ornithine decarboxylase) is a central regulator of cellular polyamine synthesis. ODC is a highly regulated enzyme stimulated by a variety of growth-promoting stimuli. ODC overexpression leads to cellular transformation.

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions.

Antizyme inhibitor (AzI) is a homolog of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis. Antizyme inhibitor retains no enzymatic activity, but exhibits high affinity to antizyme (Az), a negative regulator of polyamine homeostasis. As polyamines are involved in maintaining cellular proliferation, and since AzI may negate Az functions, we have investigated the role of AzI in regulating cell growth.

Mitochondrial localization of antizyme is determined by context-dependent alternative utilization of two AUG initiation codons.

Ornithine decarboxylase-antizyme (Az), a polyamine-induced protein that targets ornithine decarboxylase (ODC) to rapid degradation, is synthesized as two isoforms. Studies performed in vitro indicated that the 29 and 24.5 kDa isoforms originate from translation initiation at two alternative initiation codons.

20S proteasomal degradation of ornithine decarboxylase is regulated by NQO1.

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a very labile protein. ODC is a homodimeric enzyme that undergoes ubiquitin-independent proteasomal degradation via direct interaction with antizyme, a polyamine-induced protein. Binding of antizyme promotes the dissociation of ODC homodimers and marks ODC for degradation by the 26S proteasomes.

Degradation of antizyme inhibitor, an ornithine decarboxylase homologous protein, is ubiquitin-dependent and is inhibited by antizyme.

Ornithine decarboxylase (ODC) is the most notable example of a protein degraded by the 26 S proteasome without ubiquitination. Instead, ODC is targeted to degradation by direct binding to a polyamine-induced protein termed antizyme (Az). Antizyme inhibitor (AzI) is an ODC-related protein that does not retain enzymatic activity yet binds Az with higher affinity than ODC.

Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity.

Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells.

The use of skin delayed-type hypersensitivity as an adjunct test to diagnose brucellosis in cattle: a review.

Brucellosis, caused by bacteria of the genus Brucella, is a contagious disease that causes economic loss to owners of domestic animals due to loss of progeny and milk yield. Because cattle, sheep, goats, and to a lesser extent pigs are considered to be the source of human brucellosis, serological tests have been used to screen domestic animals for antibodies against Brucella. Although the serological tests helped to eradicate brucellosis in many countries, serological tests are not always adequate to detect latent carriers of Brucella.

The efficacy of the skin delayed-type hypersensitivity using a Brucellin prepared from a mucoid strain of Brucella abortus to detect brucellosis.

Eight-hundred-and-ninety-six cattle belonging to herds officially designated Brucella-free, and 190 cattle belonging to infected herds were tested with the skin delayed-type hypersensitivity (SDTH) test, using brucellin (273) prepared from a mucoid strain of Brucella abortus. An increase in skinfold thickness > or = 2 mm was considered a positive SDTH test. The serum agglutination test, complement fixation test and bacteriological examination were used to confirm SDTH test results.

Estimation of the biological activity (potency) of batches of brucellin prepared from a mucoid strain of Brucella abortus.

A study was conducted to determine the repeatability of a procedure used to prepare brucellin from a mucoid strain of Brucella abortus, and to determine the biological activity of those brucellins. The brucellins were standardized to contain 1 mg protein/ml, and their potency was estimated according to the European pharmacopoeia norm for tuberculin. Estimation of the potency was done in cattle that have been sensitized with living or killed brucellae.

Maintenance of Brucella abortus-free herds: a review with emphasis on the epidemiology and the problems in diagnosing brucellosis in areas of low prevalence.

This review covers some epidemiological aspects that allow Brucella to survive, spread, and maintain itself in the environment. Because the success of maintaining Brucella-free herds is determined by the efficiency of the serological tests to detect a single infected animal the limitations of the traditional serological tests are emphasized. Serological tests cannot differentiate between cattle infected with Brucella and cattle infected with microorganisms that serologically cross-react with B.

The sensitizing effect of a Brucella abortus antigen in cattle after repeated intradermal inoculations.

An antigen prepared from a mucoid strain of B. abortus was repeatedly injected intradermally into cattle to determine whether it sensitizes cattle so that they test false positive with the skin delayed-type hypersensitivity (SDTH) test. Cattle (n = 14) that were experimentally inoculated.

A comparison of the potency of several Brucella allergens used to detect brucellosis in cattle.

The potency of Brucella allergens prepared from a smooth Brucella abortus strain S-99, mucoid strain Leewarden, rough strain 45/20, and rough Brucella melitensis strain B-115 was assessed. The potency of these allergens was compared with that of a standard allergen prepared from smooth Brucella abortus S-99 that efficiently detected bovine brucellosis in other studies. Eight cattle experimentally inoculated with Brucella abortus 544 were tested with the allergens 4 and 10 weeks after infection, and again 8 months after infection.

Serological response of cattle to Brucella allergen after repeated intradermal applications of this allergen.

A study was conducted to determine whether an allergen that has been prepared from a mucoid strain of Brucella abortus triggers a serum antibody response that interferes with the interpretation of serologic tests results. Fifteen cattle seronegative for Brucella antigen were tested with the SDTH test several times. Blood samples were collected weekly and tested with the serum agglutination test and complement fixation test.

Production of Brucella allergens and evaluation of their biological activity in a guinea-pig bio-assay.

A study was conducted to evaluate the biological activity of Brucella allergens extracted with hydrochloride or trichloroacetic acid. Smooth and mucoid Brucella abortus cells and the medium in which brucellae were propagated were used to prepare the allergens. The biological activity of the allergens was estimated in guinea-pigs sensitized with Brucella abortus strain 544.

A unified pathway for the degradation of ornithine decarboxylase in reticulocyte lysate requires interaction with the polyamine-induced protein, ornithine decarboxylase antizyme.

Recent studies have provided convincing evidence to add to a number of earlier observations suggesting that the rapid intracellular degradation of mammalian ornithine decarboxylase (ODC) is further accelerated by the action of ornithine decarboxylase antizyme (ODC-Az), a polyamine-induced protein. However, the mechanism whereby ODC-Az exerts its effect in this proteolytic process is mostly unknown. Here, by using reticulocyte-lysate-based synthesis and degradation systems, we demonstrate that interaction of ODC-Az with ODC results in two related outcomes: (a) ODC is inactivated as a result of its monomerization, and (b) ODC degradation is dramatically accelerated.

Specificity of the skin delayed-type hypersensitivity test in brucellosis free cattle tested with a Brucella allergen.

A study was conducted to determine possible nonspecific skin delayed-type hypersensitivity (SDTH) test reactions in cattle tested with a Brucella allergen. Cattle (n = 14) experimentally inoculated with microorganisms known serologically to cross-react with Brucella and cattle (n = 549) from Brucella free herds were tested serologically and with the SDTH test. The increase in skinfold thickness at the injection site of the allergen was measured to the nearest mm with calipers 48 hours after injection.

Involvement of the 20S proteasome in the degradation of ornithine decarboxylase.

Eukaryotic cells have been shown to contain two high-molecular-mass proteases of 700 kDa and 1400 kDa (20S and 26S proteases, respectively). It has been suggested that the 20S protease, also known as proteasome, may constitute the catalytic core of the 26S protease. While the role of the free 20S protease in intracellular protein degradation is unclear, the 26S protease is implicated in the degradation of ubiquinated proteins.