Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Acridinium ester conjugated to lectin as chemiluminescent histochemistry marker

Abstract

Cell differentiation/dedifferentiation includes changes in oligosaccharide composition and distribution in the cell surface glycoconjugates. Lectins have been used as auxiliary tools in histopathological diagnosis of mammary, uterus and brain pathologies. Acridinium ester (AE) conjugated to biomolecules has been employed in chemiluminescent analytical applications. This work aimed to use a lectin, concanavalin A (Con A), conjugated to AE as a chemiluminescent histochemistry tool. Biopsies of normal and infiltrating duct carcinoma (IDC) of mammary tissues were treated by a Con A–AE derivative. Photon emission, observed during the breakage of the chemical bound between Con A and AE, was quantified, expressed in relative light units (RLU) and correlated to the labelling of the normal and transformed tissues. The results demonstrated that RLU presented a linear relationship with the labelled tissue area in the range 0.125–1.0?cm² (r=0.98). Furthermore, RLU was much higher for the IDC (1283.920×10³±220.621×10³) than the normal tissue (2.565×10³±0.247×10³), namely, about 500 times higher. The Con A–AE conjugation efficiency, differential staining of normal and IDC tissues, and quantification of results contribute to a decrease in the subjectivity in routine histopathological diagnoses and indicate that acrydinum ester can join other lectin marker to be used in histochemistry.     

Effects of flask sealing and growth regulators on in vitro propagation of neem (Azadirachta indica A. Juss.)

In vitro propagation of neem (Azadirachta indica A. Juss.) may offer an efficient alternative to seed propagation of this species. For optimization of in vitro propagation, different basal salt formulations, growth regulators, and culture container sealants (polytetrafluoroethylene hydrophobic membranes [PTFE]) were evaluated. Nodal segments cultured on Murashige and Skoog (MS) medium showed the highest shoot formation per explant (1.67). Explants cultured in flasks containing MS medium with 0.5 mg L⁻¹ benzyladenine, 0.5 mg L⁻¹ kinetin, and 0.05 mg L⁻¹ naphthaleneacetic acid, and sealed with two PTFE membranes, produced the highest number of shoots (4.04). In contrast, explants cultured in flasks without membranes showed leaf chlorosis and senescence. For plant recovery, regenerants were acclimatized in a substrate of coconut fiber and eucalyptus bark (1:1) and showed 80% survival. Our results indicated that the use of flasks with vents was beneficial for in vitro propagation of this important plant.     

Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.     

The part played by the mucopolysaccharides in the form birefringence of the Collagen

We stressed in our wourk the necessity of knowing the details of the interaction between the acid mucopolysaccharides and the collagen. Employing the appropriate methods we carried out the measurements of birefringence of strictly limited parts of collagen bundles. The measurements were carried out before and after the collagen bundles were subjected to the action of testicular hyaluronidase.The statistic study of the data we arrived have revealed that the testicular hyaluronidase which removes the acid mucopolysaccharides causes a decrease of form birefringence.These results are discussed on the regard to former research with done by the author and the conclusions may be drawn that the acid mucopolysaccharides play a part in the form birefringence of collagen bundles, and therefore have a macromolecular orientation.The author is indebted to Prof. Lucien Lison who has done the statistical processing.     

Regional Brain Atrophy and Functional Connectivity Changes Related to Fatigue in Multiple Sclerosis

Fatigue is one of the most frequent symptoms in multiple sclerosis (MS), and recent studies have described a relationship between the sensorimotor cortex and its afferent and efferent pathways as a substrate of fatigue. The objectives of this study were to assess the neural correlates of fatigue in MS through gray matter (GM) and white matter (WM) atrophy, and resting state functional connectivity (rs-FC) of the sensorimotor network (SMN). Eighteen healthy controls (HCs) and 60 relapsing-remitting patients were assessed with the Fatigue Severity Scale (FSS). Patients were classified as fatigued (F) or nonfatigued (NF). We investigated GM and WM atrophy using voxel-based morphometry, and rs-FC changes with a seed-based method and independent component analysis (ICA). F patients showed extended GM and WM atrophy focused on areas related to the SMN. High FSS scores were associated with reductions of WM in the supplementary motor area. Seed analysis of GM atrophy in the SMN showed that HCs presented increased rs-FC between the primary motor and somatosensory cortices while patients with high FSS scores were associated with decreased rs-FC between the supplementary motor area and associative somatosensory cortex. ICA results showed that NF patients presented higher rs-FC in the primary motor cortex compared to HCs and in the premotor cortex compared to F patients. Atrophy reduced functional connectivity in SMN pathways and MS patients consequently experienced high levels of fatigue. On the contrary, NF patients experienced high synchronization in this network that could be interpreted as a compensatory mechanism to reduce fatigue sensation.     

Enzymatic degradation of polar lipids in Vigna unguiculata leaves and influence of drought stress

¹⁴C-labelled polar lipids (monogalactosyl-diacylglycerol [MGDG], digalactosyl-diacylglycerol [DGDG], phosphatidylcholine [PC] and phosphatidylglycerol [PG]), purified from Vigna unguiculata leaves, were used as substrates to study the lipolytic activities of Vigna unguiculata leaf extracts. Analysis of the radioactive degradation products revealed the presence of at least three enzyme activities contributing to the hydrolysis of the four main leaf membrane lipids: Lipolytic acyl hydrolase (LAH) activities responsible for the deacylation of galactolipids and phospholipids, phospholipase D (PLD, EC 3.1.4.4) activity which gives rise to phosphatidic acid, and as suggested by the presence of diacylglycerols in minor quantities after phospholipid hydrolysis, phosphatidate phosphohydrolase (PAP, EC 3.1.3.4) and/or phospholipase C (PLC, EC 3.1.4.3.) activity. Under the conditions described in the present paper, the presence of phospholipase A (PLA₁, EC 3.1.1.3 and PLA₂, EC 3.1.1.4) activities remains hypothetical, due to the absence of lysophospholipids. LAH and PLD were partially soluble and partially associated with the membranes. When Vigna unguiculata plants were submitted to drought, the enzymatic degradation of galactolipids and phospholipids increased. The stimulation of lipolytic activities was greater in the drought-sensitive cultivar of Vigna unguiculata (cv. 1183) than in the drought-tolerant (cv. EPACE-1) one. In cv. 1183, MGDG- and DGDG-LAH activities in the membrane fractions were dramatically stimulated at a rather moderate water deficit (?0.75 MPa). A sharp increase in membrane phospholipolytic activities was also observed at mild drought stress (?1.2 MPa). In contrast, in cv. EPACE-1, the stimulation of lipolytic activities was less drastic and occurred at lower leaf water potentials (below ?1.2 MPa for galactolipases, and below ?1.4 MPa for phospholipases). Our results confirm the presence in leaves of higher plants of a very active LAH acting on galactolipids, whereas PLD is the main enzyme responsible for the degradation of phospholipids, particularly when plants are submitted to drought stress. The differences in stimulation of lipolytic activities between the two Vigna cultivars was in accordance with the different levels of membrane lipid degradation shown previously and could explain their different capacity to sustain drought.     

Distinct lung microbial community states in patients with pulmonary tuberculosis

An improved understanding of the lung microbiome may lead to better strategies to diagnose, treat, and prevent pulmonary tuberculosis(PTB). However, the characteristics of the lung microbiomes of patients with TB remain largely undefined. In this study, 163 bronchoalveolar lavage(BAL) samples were collected from 163 sputum-negative suspected PTB patients. Furthermore, 12 paired BAL samples were obtained from 12 Mycobacterium tuberculosis-positive(MTB+) patients before and after negative conversion following a two-month anti-TB treatment. The V3–V4 region of the 16 S ribosomal RNA(rRNA) gene was used to characterize the microbial composition of the lungs. The results showed that the prevalence of MTB in the BAL samples was 42.9%(70/163) among the sputum-negative patients. The α-diversity of lung microbiota was significantly less diverse in MTB+ patients compared with Mycobacterium tuberculosis-negative(MTB–) patients. There was a significant difference in β-diversity between MTB+ and MTB– patients. MTB+ patients were enriched with Anoxybacillus, while MTB– patients were enriched with Prevotella, Alloprevotella, Veillonella, and Gemella. There was no significant difference between the Anoxybacillus detection rates of MTB+ and MTB– patients. The paired comparison between the BAL samples from MTB+ patients and their negative conversion showed that BAL negative-conversion microbiota had a higher α-diversity. In conclusion, distinct features of airway microbiota could be identified between samples from patients with and without MTB. Our results imply links between lung microbiota and different clinical groups of active PTB.     

Establishment and in vitro morphogenesis of sapucaia explants (Lecythidaceae)

Lecythis pisonis Cambess, popularly known as sapucaia, has great economic and socio-environmental potential. The objective of this study was to evaluate the establishment and in vitro morphogenesis of L. pisonis under the effect of disinfecting agents, plant growth regulators, and thermal stress. The study was divided into three experiments: (i) development of the disinfection protocol by testing different concentrations and times of exposure to sodium hypochlorite (NaOCl) and different concentrations and methods of amoxicillin application, (ii) in vitro budding induction by testing different concentrations of 6-benzylaminopurine (BAP) or kinetin (KIN) supplemented to Woody Plant Medium (WPM) and Murashige and Skoog (MS) culture media, and (iii) in vitro formation from plantlets by analyzing different concentrations of indole-3-butyric acid (IBA) with different exposure times to a thermal stress of 40°C. The disinfection of stem segments was effective using 3% NaOCl and 3.0 g L⁻¹ amoxicillin solution. MS culture medium supplemented with 0.25 mg L⁻¹ BAP induced more shoots in vitro. One milligram per liter IBA promoted greater rooting in vitro, and it is not necessary for thermal stress tolerance.