Pathophysiology and Nutritional Approaches in Polycystic Ovary Syndrome (PCOS): A Comprehensive Review.

PURPOSE OF REVIEW: Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder in women of reproductive age worldwide. This disease causes menstrual, metabolic, and biochemical abnormalities such as hyperandrogenism, oligo-anovulatory menstrual cycles, polycystic ovary, hyperleptinemia, insulin resistance (IR), and cardiometabolic disorders, often associated with overweight or obesity and visceral adiposity. RECENT FINDINGS: The etiology and pathophysiology of PCOS are not yet fully understood, but insulin seems to play a key role in this disease. PCOS shares an inflammatory state with other chronic diseases such as obesity, type II diabetes, and cardiovascular diseases; however, recent studies have shown that a healthy nutritional approach can improve IR and metabolic and reproductive functions, representing a valid therapeutic strategy to ameliorate PCOS symptomatology. This review aimed to summarize and collect evidence about different nutritional approaches such as the Mediterranean diet (MedDiet) and the ketogenic diet (KD), as well as bariatric surgery and nutraceutical supplementation as probiotics, prebiotics, and synbiotics, among the others, used in patients with PCOS.

Microvascular Blood Flow Improvement in Hyperglycemic Obese Adult Patients by Hypocaloric Diet.

The present study was aimed to assess the changes in skin microvascular blood flow (SBF) in newly diagnosed hyperglycemic obese subjects, administered with hypocaloric diet. Adult patients were recruited and divided in three groups: NW group (n=54), NG (n=54) and HG (n=54) groups were constituted by normal weight, normoglycemic and hyperglycemic obese subjects, respectively. SBF was measured by laser Doppler perfusion monitoring technique and oscillations in blood flow were analyzed by spectral methods under baseline conditions, at 3 and 6 months of dietary treatment. Under resting conditions, SBF was lower in HG group than in NG and NW ones. Moreover, all subjects showed blood flow oscillations with several frequency components. In particular, hyperglycemic obese patients revealed lower spectral density in myogenic-related component than normoglycemic obese and normal weight ones. Moreover, post-occlusive reactive hyperemia (PORH) was impaired in hyperglycemic obese compared to normoglycemic and normal weigh subjects. After hypocaloric diet, in hyperglycemic obese patients there was an improvement in SBF accompanied by recovery in myogenic-related oscillations and arteriolar responses during PORH. In conclusion, hyperglycemia markedly affected peripheral microvascular function; hypocaloric diet ameliorated tissue blood flow.

A new transformation-regeneration procedure in the model legume Lotus japonicus: root explants as a source of large numbers of cells susceptible to Agrobacterium-mediated transformation.

We describe herein a simple and efficient transformation procedure for the production of transgenic Lotus japonicus plants. In this new procedure, dedifferentiated root explants, used as starting material, are the source of a large number of cells that are competent for the regeneration procedure, with a high susceptibility to Agrobacterium infection. The application of this protocol resulted in a tenfold increase in the number of transformants produced by a single plant in comparison to the widely used hypocotyl transformation procedure. Furthermore, our procedure allowed the use of intact plants stored for a long time at 4 degrees C, thus providing a potential continuous supply of explants for transformation experiments. The overall time of incubation under tissue culture conditions required to obtain a plant transferable into soil is 4 months. The transgenic nature of the transformants was demonstrated by the detection of beta-glucuronidase (GUS) activity in the primary transformants and by molecular analysis. Stable transformation was indicated by Mendelian segregation of the hygromycin selectable marker and of the gusA activity after selfing of the transgenic plants.

Positive-negative selection for homologous recombination in Arabidopsis.

In plants gene knock-outs and targeted mutational analyses are hampered by the inefficiency of homologous recombination. We have developed a strategy to enrich for rare events of homologous recombination in Arabidopsis using combined positive and negative selection. The T-DNA targeting construct contained two flanking regions of the target alcohol dehydrogenase gene as homologous sequences, and neomycin phosphotransferase and cytosine deaminase as positive and negative markers, respectively. A root explant transformation procedure was used to obtain transgenic calli. Among 6250 transformants isolated by positive selection, 39 were found to be resistant to negative selection as well. Of these 39, at least one had undergone homologous recombination correlated with a unidirectional transfer of information. Although the ADH locus was not changed, our data demonstrate that a homologous recombination event can be selected by positive negative selection in plants.

Functional characterization of an ammonium transporter gene from Lotus japonicus.

NH(4)(+) is the main product of symbiotic nitrogen fixation and the external concentration of combined nitrogen plays a key regulatory role in all the different step of plant-rhizobia interaction. We report the cloning and characterization of the first member of the ammonium transporter family, LjAMT1;1 from a leguminous plant, Lotus japonicus. Sequence analysis reveals a close relationship to plant transporters of the AMT1 family. The wild type and two mutated versions of LjAMT1;1 were expressed and functionally characterized in yeast. LjAMT1;1 is transcribed in roots, leaves and nodules of L. japonicus plants grown under low nitrogen conditions, consistent with a role in uptake of NH(4)(+) by the plant cells.

The Rhizobium leguminosarum glnB gene is down-regulated during symbiosis.

Symbiotic nitrogen fixation involves the development, on the legume plant root, of specialised organs called nodules, within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. The glnB gene encodes a signal transduction protein (P(II)) which is a component of the bacterial nitrogen regulation (Ntr) system and an essential regulator of ammonium assimilation. We demonstrate that in Rhizobium leguminosarum the glnB promoter is strongly regulated by nitrogen and NtrC, but still shows a significant level of activity in conditions of nitrogen excess. Expression of genes involved in nitrogen assimilation has been shown to be absent in nitrogen-fixing bacteroids, and, in agreement with this, we find that the glnB promoter is down-regulated during bacteroid differentiation at a time coincident with the arrest of bacterial division in the nodule. This pattern is common to other bacterial genes involved in nitrogen assimilation and it is noteworthy that the zone where the glnB promoter is active is coincident with the region in which NtrC is expressed.

The soybean ENOD40(2) promoter is active in Arabidopsis thaliana and is temporally and spatially regulated.

The ENOD40 gene is induced early during Rhizobium-legume symbiosis and has probably a primary role in the nodule organogenesis. In this paper we show that the 1.7 kb 5'-flanking region of the GmENOD40(2) is able to drive the expression of a gusA-int marker in transgenic Arabidopsis thaliana. The promoter activity is developmentally regulated and the major activity is detected in the root and in the stigma.

Characterisation of the bacteriophage T4 comC alpha 55.6 and comCJ mutants. A possible role in an antitermination process.

We have performed a new screen for T4 mutants (comC) that overcome the phage growth restriction caused by the Escherichia coli rho/tabC mutants. We show that one such mutant (comCJ) identifies a different gene from that identified by canonical comC mutants. We compare the regulation of T4 prereplicative transcription in a rho/tabC mutant infected by T4 wild-type, by a canonical comC mutant (comC alpha 55.6) and by comCJ. The transcription rates of the two prereplicative genes 39 and 43 is depressed in a T4 wild-type infected tabC host mutant. When comC alpha 55.6 and/or comCJ single and double mutants are the infecting phages, transcription of genes 39 and 43 is resumed to different extents; in particular, in the double mutant infections there appears to be a synergistic effect on transcription. Furthermore, we find that the comC alpha 55.6 phage mutant affects the transcription rate of the gene rIIA in a wild-type host.

Enhancement of somatic intrachromosomal homologous recombination in Arabidopsis by the HO endonuclease.

The HO endonuclease promotes gene conversion between mating-type alleles in yeast by a DNA double-strand break at the site of conversion (the MAT-Y/Z site). As a first step toward understanding the molecular basis of homologous recombination in higher plants, we demonstrate that expression of HO in Arabidopsis enhances intrachromosomal recombination between inverted repeats of two defective beta-glucuronidase (gus) genes (GUS- test construct). One of these genes has the Y/Z site. The two genes share 2.5 kb of DNA sequence homology around the HO cut site. Somatic recombination between the two repeats was determined by using a histochemical assay of GUS activity. The frequency of Gus+ sectors in leaves of F1 plants from a cross between parents homozygous for the GUS- test construct and HO, respectively, was 10-fold higher than in F1 plants from a cross between the same plant containing the GUS- test construct and a wild-type parent. Polymerase chain reaction analysis showed restoration of the 5' end of the GUS gene in recombinant sectors. The induction of intrachromosomal gene conversion in Arabidopsis by HO reveals the general utility of site-specific DNA endonucleases in producing targeted homologous recombination in plant genomes.

Arabidopsis mutants deficient in T-DNA integration.

Arabidopsis thaliana mutants originally isolated as hypersensitive to irradiation were screened for the ability to be transformed by Agrobacterium transferred DNA (T-DNA). One of four UV-hypersensitive mutants and one of two gamma-hypersensitive mutants tested showed a significant reduction in the frequency of stable transformants compared with radioresistant controls. In a transient assay for T-DNA transfer independent of genomic integration, both mutant lines took up and expressed T-DNA as efficiently as parental lines. These lines are therefore deficient specifically in stable T-DNA integration and thus provide direct evidence for the role of a plant function in that process. As radiation hypersensitivity suggests a deficiency in repair of DNA damage, that plant function may be one that is also involved in DNA repair, possibly, from other evidence, in repair of double-strand DNA breaks.

Termini and telomeres in T-DNA transformation.

A T-DNA vector for plant transformation has been constructed in which the cloning site is located 9 bp from the right-border (RB) end and 27 bp from the left-border (LB) end. In this vector cloned DNA homologous to plant chromosomal sequences is located at the T-DNA termini, and will thus be exposed by even limited exonucleolysis in planta. The arabidopsis ADH (alcohol dehydrogenase) locus was mobilized from Agrobacterium, and integration into the recipient genome was studied. Despite the terminal location of ADH homology in this vector, the T-DNA integrated essentially at random in the Arabidopsis genome rather than at the endogenous ADH locus. T-DNA integration was blocked, however, when Arabidopsis telomeric sequences were added to the construct at each end of the ADH homology. Thus the predominant mode by which incoming T-DNA is integrated into the continuity of chromosomal DNA involves free DNA ends, but, in contrast to modes of recombination such as gap repair, does not involve extensive terminal DNA sequence homology.

The Rhizobium leguminosarum biovar phaseoli glnT gene, encoding glutamine synthetase III.

Plasmid pGE203 contains the Rhizobium leguminosarum biovar phaseoli glnT locus. Glutamine synthetase III (GSIII) was purified from a glutamine auxotrophic strain of Klebsiella pneumoniae carrying this plasmid. Sequencing of a 2.4-kb fragment containing the glnT locus reveals an open reading frame of 435 amino acids (aa), whose first eight aa are identical to those determined from pure GSIII by direct aa sequencing, thus confirming that glnT indeed codes for GSIII activity. The comparison of the GSIII aa sequence with the reported sequence of GSs from other organisms shows a significant degree of homology. Since the three-dimensional structure of GS from Salmonella typhimurium is known, a three-dimensional model of GSIII was built by homology.

Activation of the Rhizobium leguminosarum glnII gene by NtrC is dependent on upstream DNA sequences.

The cloning and sequence determination is reported of the DNA region of Rhizobium leguminosarum coding for glutamine synthetase II (GSII). An open reading frame (ORF) encoding 326 amino acids was defined as the glnII gene on the basis of its similarity to other glnII genes and the ability of a DNA fragment carrying this ORF to complement the glutamine auxotrophy of a Klebsiella pneumoniae glnA mutant. We find that the glnII gene in R. leguminosarum is transcribed as a monocistronic unit from a single promoter, which shows structural features characteristic of rpoN (ntrA)-dependent promoters. In K. pneumoniae, such promoters require the ntrC and rpoN (ntrA) gene products for transcription. The intracellular level of glnII mRNA changes when R. leguminosarum is grown on different nitrogen sources, as expected for regulation by the nitrogen regulatory system. Promoter deletion analysis has shown that an extensive upstream DNA sequence (316 bp) is essential for in vivo activation of the glnII promoter in different biovars of R. leguminosarum. This DNA region requires a wild-type ntrC gene for activity and includes two conserved putative NtrC-binding site sequences. The results conclusively show that transcription from the R. leguminosarum glnII promoter is fully dependent on positive control by NtrC protein and on an upstream activator sequence (UAS).

Phenotype of a Rhizobium leguminosarum ntrC mutant.

A Tn5 insertion mutant, strain CFN2012, of Rhizobium leguminosarum biovar phaseoli devoid of glutamine synthetase II (GSII) activity was analysed. It was shown to contain Tn5 within an 11-kb BamHI DNA fragment, which was isolated (pSM261) from the wild-type strain and, when introduced into strain CFN2012, was shown to complement the absence of GSII activity. The DNA sequence of the corresponding region from the wild-type allele revealed the presence of an ntrC regulatory gene, and restriction analysis indicated that the mutant allele carried the Tn5 insertion within it. Further analysis of strain CFN2012 indicated that this mutant has reduced levels of the PII regulatory protein and that, in contrast to ntrC mutants of other Rhizobiaceae, it grows on nitrate as the sole nitrogen source.

Transcriptional analysis of the glnB-glnA region of Rhizobium leguminosarum biovar viciae.

We report that the glnB and glnA genes of Rhizobium leguminosarum biovar viciae are preceded by promoters located upstream of each gene. We find the presence of a glnB-glnA and a glnA mRNA whose intracellular concentration changes two- to three-fold when R. leguminosarum is grown on different nitrogen sources. Primer extension analysis shows unique transcriptional initiation sites upstream of glnB and glnA. The glnB promoter is rpoN(ntrA)-dependent, while the glnA promoter does not contain a typical consensus sequence for previously described promoters. In Klebsiella pneumoniae the glnB promoter requires active ntrC and ntrA genes and a DNA fragment containing 53 nucleotides upstream of the transcription initiation site shows full promoter activity, thus indicating that no NtrC binding sites are necessary for this activation in the glnB upstream region.