Hypercalciuria: its value as a predictive risk factor for nephrolithiasis in asymptomatic primary hyperparathyroidism?

CONTEXT: The latest guidelines of the 4th International Workshop on Asymptomatic Primary Hyperparathyroidism (aPHPT) reintroduced hypercalciuria (i.e. urinary calcium > 400 mg/day) as criterion for surgery. However, the value of hypercalciuria as a predictor of nephrolithiasis and the correct cut-off values still need to be confirmed. OBJECTIVE: To evaluate the prevalence of silent kidney stones in a large series of patients with aPHPT and the sensibility, specificity and predictive value of different cut-off values of hypercalciuria in identifying patients with nephrolithiasis. DESIGN: One hundred seventy-six consecutive patients with aPHPT were evaluated at our Institution by serum and urinary parameters and kidney ultrasound. RESULTS: Silent nephrolithiasis was found in 38 (21.6%) patients. In the univariate and multivariate model, hypercalciuria was a predictor of nephrolithiasis using the criterion of 400 mg/24 h [(OR 2.30, (1.11-4.82) P = 0.025], 4 mg/kg/bw [OR 2.65, (1.14-6.25) P = 0.023], gender criterion [OR 2.79, (1.15-6.79) P = 0.023] and the cut-off value derived from the ROC analysis [(> 231 mg/24 h) OR 5.02 (1.68-14.97) P = 0.004]. Despite these several predictive criteria, however, hypercalciuria had a low positive predictive value (PPV), ranging from 27.4 to 32.7%. CONCLUSIONS: Hypercalciuria is a predictor of nephrolithiasis, but its PPV is low.

Molecular mechanisms driving Streptococcus mitis entry into human gingival fibroblasts in presence of chitlac-nAg and saliva.

The molecular mechanisms leading to Streptococcus mitis capability of entering oral cells were investigated in a co-culture of S. mitis and Human Gingival Fibroblasts (HGFs) in the presence of saliva. An innovative colloidal solution based on silver nanoparticles (Chitlac-nAg), a promising device for daily oral care, was added to the experimental system in order to study the effects of silver on the bacterial overgrowth and ability to enter non-phagocytic eukaryotic cells. The entry of bacteria into the eukaryotic cells is mediated by a signalling pathway involving FAK, integrin ß1, and the two cytoskeleton proteins vinculin and F-actin, and down-regulated by the presence of saliva both at 3 and 48 h of culture, whereas Chitlac-n Ag exposure seems to influence, by incrementing it, the number of bacteria entering the fibroblasts only at 48 h. The formation of fibrillary extrusion from HGFs and the co-localization of bacteria and silver nanoparticles within the fibroblast vacuoles were also recorded. After longer experimental times (72 and 96 h), the number of S. mitis chains inside gingival cells is reduced, mainly in presence of saliva. The results suggest an escape of bacteria from fibroblasts to restore the microbial balance of the oral cavity.

In vitro activity of Aloe vera inner gel against microorganisms grown in planktonic and sessile phases.

The failure of traditional antimicrobial treatments is becoming a worldwide problem. The use of Aloe vera is of particular interest for its role as curative agent and its efficacy in complementary therapies for a variety of illnesses. This study evaluated the antimicrobial activity of A. vera inner gel against a panel of microorganisms, Gram-positive and -negative bacteria, and Candida albicans. In addition to A. vera inner gel being used in the treatment of peptic ulcers, in dermatological treatments, and wound healing, it was also tested on the sessile phase of clinical Helicobacter pylori strains (including multi-drug-resistant strains) and on planktonic and sessile phase of Staphylococcus aureus/Pseudomonas aeruginosa clinical isolates from venous leg ulcers.A. vera inner gel expresses its prevalent activity against Gram-negative bacteria and C. albicans in respect to Gram-positive bacteria. The results of the A. vera antibiofilm activity showed a decrease of the produced biomass in a concentration-dependent-way, in each analyzed microorganism. The data obtained show that A. vera inner gel has both an antimicrobial and antibiofilm activity suggesting its potential use for the treatment of microbial infections, in particular for H. pylori gastric infection, especially in case of multi-drug-resistance, as well as for an effective wound dressing.

A dual role for ß1 integrin in an in vitro Streptococcus mitis/human gingival fibroblasts co-culture model in response to TEGDMA.

AIM: To evaluate the effect of TEGDMA on human gingival fibroblasts (HGFs) in vitro co-cultured with Streptococcus mitis, focusing on the signalling pathways underlying cell tissue remodelling and inflammatory response processes. METHODOLOGY: ß1 integrin expression was evaluated by means of imaging flow cytometry. The Western blot technique was used to investigate the expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), matrix metalloproteinase 9 (MMP9) and 3 (MMP3). RT-PCR was performed to quantify nuclear factor-kb subunits (Nf-kb1, ReLa), IkB kinase ß (IkBkB), cyclooxygenase II (COX-2) and tumour necrosis factor-α (TNF-α) mRNA levels. Statistical analysis was performed using the analysis of variance (anova). RESULTS: When HGFs are co-cultured with S. mitis, ß1 integrin intensity, phosphorylated PKC (p-PKC), activated ERK (p-ERK), IkBkB mRNA level and MMP9 expression increased (for all molecules P < 0.05 HGFs versus HGFs co-cultured with S. mitis). A higher level of MMP3 in HGFs treated with TEGDMA was recorded (P < 0.05 HGFs versus HGFs exposed to TEGDMA). COX-2 inflammatory factor mRNA level appeared higher in HGFs exposed to 1 mmol L(-1) TEGDMA (P < 0.01 HGFs versus HGFs exposed to TEGDMA), whereas TNF-α gene expression was higher in HGFs co-cultured with S. mitis (P < 0.05 HGFs versus HGFs co-cultured with S. mitis). CONCLUSIONS: ß1 integrin triggered the signalling pathway, transduced by p-PKCα and involving ERK 1 and 2 and MMPs. This pathway resulted in an unbalanced equilibrium in tissue remodelling process, along with inflammatory response when HGFs are exposed to bacteria or biomaterial alone. On the contrary, the TEGDMA/S. mitis combination restored the balance between extracellular matrix deposition and degradation and prevented an inflammatory response.

NF-kB mediated down-regulation of collagen synthesis upon HEMA (2-hydroxyethyl methacrylate) treatment of primary human gingival fibroblast/Streptococcus mutans co-cultured cells.

PURPOSE: In vitro studies have evidenced the cytotoxic effect of HEMA (2-hydroxyethyl methacrylate), the most common component of dental resin-based restorative material, which is released within the oral cavity, on eukaryotic cells such as gingival fibroblast and epithelial cells. However, since the presence of microorganisms within the oral cavity cannot be excluded and little is known about the interactions occurring between eukaryotic cells and the human oral microbiota, our attention has been addressed to investigate the effect of 3 mM HEMA on the molecular mechanisms driving the response of human gingival fibroblasts (HGFs) co-cultured with Streptococcus mutans. METHODOLOGY: HGF/S. mutans co-culture has been set up in our lab, and upon HEMA treatment, S.mutans and HGF cells' viability and adhesion along with type I collagen gene and pro-collagen I, Bax, Bcl2, nuclear factor kB (NF-kB), IkBα, pIkBα protein expression by PCR, Western blotting and ELISA assays have been investigated. RESULTS: HEMA treatment determines a significant decrease of type I collagen protein production, even in the presence of S. mutans, in parallel to a decrease of cell viability and adhesion, which seem to be regulated by NF-kB activation. In fact, when SN50, NF-kB-specific pharmacological inhibitor, is added to the culture, cell proliferation along with collagen synthesis is restored. CONCLUSION: The modulation exerted by S. mutans on the cytotoxic effect of HEMA suggests that within the oral cavity, the eukaryotic/prokaryotic cell interactions, maintaining the balance of the environment, allow HEMA to perform its adhesive and bonding function and that the use of a co-culture system, which simulates the oral cavity organization, improves the knowledge concerning the biocompatibility of this dental material.

Sexual distress, sexual dysfunction and relationship quality in women with systemic sclerosis: correlation with clinical variables.

To assess the rate of sexual distress, sexual dysfunction and relationship quality and their association with clinical variables in women with systemic sclerosis (SSc), 102 sexually active women with SSc were recruited. Sexual distress, sexual dysfunction and dissatisfaction with relationship quality were investigated by Female Sexual Distress Scale Revised (FSDS-R), Female Sexual Function Index (FSFI) and Dyadic Adjustment Scale (DAS), respectively. The patients underwent medical examinations and nailfold videocapillaroscopy (NVC). Of the 102 patients, 37 (36%) reported sexual distress with FSDS-R score >11, 45 (44%) had sexual dysfunction with FSFI score <19 and 49 (48%) were not satisfied with relationship quality with DAS score <100. There was a negative correlation (p<0.001, R= -0.30) between FSDS-R and FSFI. No correlation was found between FSDS-R and DAS. FSFI showed a positive correlation with DAS (p<0.0001, R= 0.36). Age correlated negatively (p<0.05, R= -0.26) with FSFI, while FSDS-R and DAS did not correlate (p>0.05) with age. SSc women with digital ulcers (DU) had a reduction of FSFI and DAS compared with women without DU. In patients with late capillaroscopic pattern, mean value of FSFI was significantly lower than the other two capillaroscopic patterns. DAS decreased with progression of capillaroscopic damage. In a high percentage of women with SSc FSDS-R was increased, while FSFI and DAS were reduced. Age correlated negatively with FSFI, while skin score showed a negative correlation with DAS. Digital vascular damage negatively influenced FSFI and DAS.

In vitro activity of Aloe vera inner gel against Helicobacter pylori strains.

UNLABELLED: Aloe barbadensis Miller (Aloe vera) is a herbal remedy widely used for a variety of illnesses; A. vera leaf extracts have been promoted for detoxification, cure constipation, help flush out toxins and wastes from the body, promote digestion and are used in the treatment of peptic ulcer for cytoprotective action. The aim of this study was to evaluate the antibacterial activity of A. vera inner gel against both susceptible and resistant Helicobacter pylori strains isolated in Abruzzo region, Italy. The inner gel of leaves of a 5-year-old plant of A. vera was extracted, homogenized and tested from 800 to 1.56 mg ml(-1) against 14 clinical strains and one reference strain of H. pylori using the broth microdilution methodology. Furthermore, the sample of A. vera was investigated for the chemical fingerprint of anthraquinones. The inhibitory concentrations of A. vera inner gel were similar to the bactericidal ones, with values ranging from 6.25 to 800 mg ml(-1) . Fifty per cent of the detected strains, independently of their susceptibility profile, were inhibited in their growth at 100 mg ml(-1) . Aloe vera inner gel expresses antibacterial properties against H. pylori and, therefore, in combination with antibiotics, could represent a novel strategy for the treatment of the infection of H. pylori, especially in cases of multiresistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates that the Aloe vera inner gel expresses antibacterial properties against both susceptible and resistant Helicobacter pylori strains. These findings may impact on the antimicrobial resistance phenomenon of H. pylori, proposing the A. vera inner gel as a novel effective natural agent for combination with antibiotics for the treatment of H. pylori gastric infection.

pPKC α regulates through integrin ß 1 human gingival fibroblasts/Streptococcus mitis adhesion in response to HEMA.

AIM: To investigate in coculture of human gingival fibroblasts (HGFs) and Streptococcus mitis, the molecular mechanisms driving the response to 2-hydroxyethyl methacrylate (HEMA) in terms of eukaryotic/prokaryotic cell adhesion, signal transduction and apoptosis. METHODOLOGY: The clinical strain S. mitis DS12, cultured in Trypticase soy broth was added to HGFs, obtained from fragments of healthy marginal gingival tissue and cultured in DMEM, treated with 3 mmol L(-1) 2-hydroxyethyl methacrylate (HEMA) for 48 h and processed for microscopic, western blotting and flow cytometric analyses. RESULTS: 2-hydroxyethyl methacrylate (HEMA) treatment increased the adhesion between S. mitis and HGFs, which seemed to be mediated by the PKC α/integrin ß 1 signalling system, improved by the presence of saliva. It also reduced the viability and the adhesion of HGFs to polypropylene substrate in terms of procollagen I and MMP3 expression. The presence of saliva and S. mitis reduced the number of necrotic HGFs and upregulated the expression of both procollagen I and MMP3. CONCLUSIONS: These results shed more light on the biological and molecular events occurring in vitro in a coculture model that mimics the environment of the oral cavity with HEMA treatment. The key role played by oral bacteria and saliva in preventing inflammatory and toxic processes that occur in vivo in human gingival fibroblasts upon the release of dental material monomers is confirmed.

Helicobacter pylori biofilm: a protective environment for bacterial recombination.

AIMS: The aim of this work was to investigate the interaction between two Helicobacter pylori strains in promoting genetic transfer, when grown in the biofilm mode. METHODS AND RESULTS: Biofilms produced by H. pylori 9/10 (A), H. pylori 15/4 (B) and their mixture (C) were studied for biomass production and cell viability. The genetic heterogeneity of 45 clones, coming from mature biofilm of co-cultured H. pylori strains was studied by both RAPD and cagA (EPIYA motifs)/vacA virulence genes analysis. Helicobacter pylori A, B and C developed a well-structured biofilm without significant differences in viability. No significant differences were recorded between A and B biomass measurement, whereas C biofilm expressed a significant (P < 0.001) higher adhesive capability when compared with A and B biofilms. C-clones DNA-fingerprintings showed an high genetic heterogeneity (mean similarity value = 0.528). The 60% of C-clones displayed vacA allelic combination s1i1m1m2 associated with cagA EPIYA motif pattern P1P2P3P3P3. CONCLUSIONS: Biofilms developed by multiple H. pylori strains are more complex than those associated with single strains. Such condition might promote the genetic exchange favouring the generation of more virulent strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The 'biofilm niche' represents a successful strategy and a suitable environment for promoting bacterial population persistence by recombination events.

Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour.

AIMS: This study detected and characterized the extracellular DNA (eDNA) in the biofilm extracellular polymeric substance (EPS) matrix of Helicobacter pylori and investigated the role of such component in the biofilm development. METHODS AND RESULTS: Extracellular DNA was purified and characterized in a 2-day-old mature biofilm developed by the reference strain H. pylori ATCC 43629, the clinical isolate H. pylori SDB60 and the environmental strain H. pylori MDC1. Subsequently, the role of eDNA in the H. pylori biofilm was evaluated by adding DNase I during biofilm formation and on mature biofilms. Extracellular DNA was detected in the 2-day-old EPS biofilm matrix of all analysed H. pylori strains. The DNA fingerprintings, performed by RAPD analysis, on eDNA and intracellular DNA (iDNA), showed some remarkable differences. The data obtained by microtitre biofilm assay as well as colony forming unit count and CLSM (confocal laser scanning microscopy) qualitative analysis did not show any significant differences between the DNase I-treated biofilms and the corresponding not treated controls both in formation and on mature biofilms. CONCLUSIONS: In this study, we provide evidence that eDNA is a component of the EPS matrix of H. pylori biofilm. The different profiles of eDNA and iDNA indicate that lysed cells are not the primary source of eDNA release, suggesting that other active mechanisms might be involved in this process. Moreover, the biomass assay suggests that eDNA may not be the main component of biofilm matrix, suggesting that it could be primarily involved in other mechanisms such as recombination processes, via transformation, contributing to the wide genomic variability of this micro-organism defined as a 'quasi-species'. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of eDNA in H. pylori biofilm can contribute to the active dynamic exchange of information aimed to reach the best condition for the bacterial survival in the host and in the environment.

Effect of 2-hydroxyethyl methacrylate on Streptococcus spp. biofilms.

AIMS: The effect of different concentrations of 2-hydroxyethyl methacrylate (HEMA) was evaluated on biofilm formation and preformed biofilm of Streptococcus mitis, Streptococcus mutans and Streptococcus oralis, alone or combined to each other. METHODS AND RESULTS: Twofold serial dilution of HEMA ranged from 12 to 0·75 mmol l(-1) was added to Streptococcal broth cultures and mature biofilms in 96-well-microtitre plates to evaluate bacterial biomass and cell viability. HEMA affected the Streptococcal population in a strain-specific way producing few significant effects. A reduction on biofilm formation and a detachment of preformed biofilm was recorded in Strep. mitis ATCC 6249, whereas in mixed cultures, the monomer expressed a general aggregative effect on mature biofilms. A reduction in cell viability was also recorded in an HEMA-concentration-dependent way in each experimental condition studied. CONCLUSIONS: These results suggest that the HEMA prevalent effects are both the reduction of bacterial adhesion to a polystyrene surface and the increase in dead cells also characterized by an aggregative status. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the potential effect of HEMA, released from resin-based materials, on oral bacteria may furnish information for surveillance of the risk reduction in secondary caries via hindering biofilm generation.

Anti-adhesive and pro-apoptotic effects of 2-hydroxyethyl methacrylate on human gingival fibroblasts co-cultured with Streptococcus mitis strains.

AIM: To evaluate and observe the cellular reactions that occur during the interaction/integration between 2-hydroxyethyl methacrylate/host tissue/microbial environment, in a co-culture of human gingival fibroblasts (HGF) and Streptococcus mitis strains. METHODOLOGY: Streptococcus mitis were cultured with strains in the presence of 3 mmol L(-1) HEMA for 48 h and 72 h. Cytotoxicity was evaluated by the trypan blue dye exclusion test. Apoptosis was evaluated by TUNEL analysis. Adhesion was evaluated by immunofluorescence and western blot analyses. Quantitative analyses of the results were acquired by Qwin Plus 3.5 and QuantityOne I-D analysis software, respectively. The statistical significance of the results was evaluated using t-tests and linear regression tests. RESULTS: The trypan blue dye test revealed 47.3% and 46.5% of dead fibroblasts after 48 and 72 h HEMA treatment, respectively, while bacterial viability was not influenced by the presence of HEMA and fibroblasts. The expression of pro-collagen I, involved in fibroblast adhesion, in untreated samples ranged from 12.49% to 6.91% of the positive area after 48 and 72 h, respectively, dropping to below 2% of the positive area in the other experimental conditions. Unlike the trypan blue test, co-cultured samples treated with HEMA showed 20% and 25% versus 17% and 21% (after 48 and 72 h, respectively) of apoptotic cells. CONCLUSIONS: The evidence for HEMA toxicity and anti-adhesive effects against eukaryotic cells was reduced in the presence of bacteria, suggesting that dental resins should be well polymerized to avoid the spread of toxic monomers within the mouth.

Characterization of an Helicobacter pylori environmental strain.

AIMS: To investigate the main genotypic virulence markers and the phenotypic features of an environmental Helicobacter pylori strain, named MDC1. METHODS AND RESULTS: The H. pylori MDC1 genotypic status was evaluated by PCR amplification. The mosaicism in vacA alleles was expressed by the s1m1 allelic combination, as found in strains which are strong vacuolating cytotoxin producers; the number of cagA variable EPIYA motifs displayed P1P2P3P3 pattern and the iceA1 was recorded between the iceA allelic types and the babA2 gene found in strains causing more severe disease. The biofilm formation was evaluated on a polystyrene surface in static conditions by scanning electron microscopy and confocal scanning laser microscopy. Helicobacter pylori MDC1 displayed a dense mature biofilm with cells in a coccoid morphology persistent in time in which the expression of the luxS gene, related to the quorum-sensing signalling, was always detected. CONCLUSIONS: Helicobacter pylori MDC1 strain had the main virulence markers closely related to gastric pathogenesis and displayed a well-structured biofilm which allowed this bacterium to be more protected in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The persistence of the environmental virulent H. pylori strain in a clustered state suggests a long-term survival of this bacterial community outside of the host, enabling the bacterial transmission with important clinical repercussions.

Liposome-induced exfoliation of graphite to few-layer graphene dispersion with antibacterial activity.

Few-layer graphene aqueous dispersions are obtained by exploiting liposomes as effective exfoliating agents for graphite. Raman measurements evidence the presence of non-oxidized double layer graphene as well as amphiphilic phospholipid molecules organized in bilayers in the samples. TEM analyses confirmed that the obtained homogeneous graphene nanosheets are embedded in the liposomal bilayer. The as-prepared graphene aqueous dispersion is stable for days and demonstrates significant antibacterial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) strains, with a reduction in the growth of S. aureus and E. coli as high as 60 and 78%, respectively.

The universal ancestor was a thermophile or a hyperthermophile.

By exploiting the correlation between the optimal growth temperature of organisms and a thermophily index based on the propensity of amino acids to enter thermophile/hyperthermophile proteins, an analysis is conducted in order to establish whether the last universal common ancestor (LUCA) was a mesophile or a (hyper)thermophile. This objective is reached by using maximum parsimony and maximum likelihood to reconstruct the ancestral sequences of the LUCA for two pairs of sets of paralogous protein sequences by means of the phylogenetic tree topology derived from the small subunit ribosomal RNA, even if this is rooted in all three possible ways. The thermophily index of all the reconstructed ancestral sequences of the LUCA belongs to the set of the thermophile/hyperthermophile sequences, thus supporting the hypotheses that see the LUCA as a thermophile or a hyperthermophile.

The late stage of genetic code structuring took place at a high temperature.

The correlation between the optimal growth temperature of organisms and a thermophily index based on the propensity of amino acids to enter more frequently into (hyper)thermophile proteins is used to conduct an analysis aiming to establish whether genetic code structuring took place at a low or a high temperature. If the number of codons attributed to the various amino acids in the genetic code constitutes an estimate of the mean amino acid composition of proteins produced when the genetic code was definitively structured, then the thermophily index can also be associated to the genetic code. This value and the sampling of the variable thermophily index of different alignments of protein sequences from mesophile, thermophile and hyperthermophile species make it possible to establish, with an extremely high statistical confidence, that the late stage of genetic code structuring took place in a hyperthermophile (or thermophile) 'organism'. Moreover the 95% confidence interval of the temperature at which the genetic code was fixed turned out to be 91+/-24 degrees C. These observations seem to support the hypothesis that the origin of life might have taken place at a high temperature.

Second codon positions of genes and the secondary structures of proteins. Relationships and implications for the origin of the genetic code.

The nucleotide frequencies in the second codon positions of genes are remarkably different for the coding regions that correspond to different secondary structures in the encoded proteins, namely, helix, beta-strand and aperiodic structures. Indeed, hydrophobic and hydrophilic amino acids are encoded by codons having U or A, respectively, in their second position. Moreover, the beta-strand structure is strongly hydrophobic, while aperiodic structures contain more hydrophilic amino acids. The relationship between nucleotide frequencies and protein secondary structures is associated not only with the physico-chemical properties of these structures but also with the organisation of the genetic code. In fact, this organisation seems to have evolved so as to preserve the secondary structures of proteins by preventing deleterious amino acid substitutions that could modify the physico-chemical properties required for an optimal structure.

The origin of protein synthesis: on some molecular fossils identified through comparison of protein sequences.

Sequence data, even if only marginally significant, and evolutionary arguments suggest that a similarity may exist between class II aminoacyl-tRNA synthetases and proteins involved in the nonribosomal biosynthesis of peptide antibiotics, and more in general, those belonging to the family of adenylate-forming enzymes. If correct, this hypothesis of homology may imply that the first peptide syntheses might have occurred on phosphopantetheine molecules in a thioester world and/or on a variant of the coenzyme A (CoA) in an RNA world. Therefore, peptide synthesis probably evolved on tRNA-like molecules from the CoA (or a variant CoA molecule) that had the potential for nucleotide extension, that made possible the evolution to the current mechanism of protein synthesis. Our hypothesis on the existence of such homology implies that a series of evolutionary steps such as the existence of a primitive catalytic domain with poor substrate specificity towards both (amino acids + ATP + pre-CoA (and/or CoA)) and (amino acids + ATP + tRNA-like) molecules may have occurred. Therefore, the pre-CoA (and/or CoA) and the tRNA-like molecules were able to use this enzyme ambiguity for a certain period, thus giving weight to the scheme of evolutionary transitions mentioned above.

On the relationships between the genetic code coevolution hypothesis and the physicochemical hypothesis.

This paper analyzes the relationships between the genetic code coevolution hypothesis and the physicochemical hypothesis by means of a comparative study of the precursor-product amino acid pairs on which the former hypothesis is based. Even if the coevolution between the biosynthetic relationships of amino acids and the organization of the genetic code is not questioned in this paper, the results and the arguments used lead us to believe that the selective pressures considered essential by the physicochemical postulates, played a more active role than that of the precursor-product relationships in defining the allocation of these amino acids in the genetic code. It is furthermore pointed out that the two evolutionary hypothesis might be aspects of the same selective pressure, and thus difficult to differentiate.