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Biomedical subjects

A Aharoni

Publications and source records attributed to A Aharoni.

At least 19 recordsLinked to original sources
PubMed2006

UV disinfection of wastewater effluents for unrestricted irrigation.

Wastewater reuse in arid regions is important for the production of a water resource to be utilised for non-potable purposes and to prevent the environmental transmission of disease-causing agents. This study was conducted to evaluate the effect of water quality on the comparative disinfection efficiency of viruses, bacteria and spores by UV irradiation. Furthermore, the microbial quality of effluent produced by coagulation, high rate filtration (HRF) and either UV irradiation or chlorination was determined. Using low pressure collimated beam, a UV dose of 80 mWs/cm2 was needed to achieve a 3-log10 inactivation of either rotavirus SA-11 or coliphage MS2, whereas over 5-log10 inactivation of E. coli was reached with a dose of only 20 mWs/cm2. B. subtilis inactivation was found to be linear up to a dose of 40 mWs/cm2 and then a tailing up to a UV dose of 120 mWs/cm2 was observed. It is worth noting that effluent turbidity of < 5 NTU did not influence the inactivation efficiency of UV irradiation. Operation of a pilot plant to treat secondary effluent by coagulation, HRF and UV disinfection at a UV dose of 80 mWs/cm2 resulted in the production of high quality effluent in compliance with the Israel standards for unrestricted irrigation (< 10 CFU/100 mL faecal coliform and turbidity of < 5 NTU). Sulphite reducing clostridia (SRC) were found to be more resistant than coliphages and F coliform for UV irradiation. The results of this study indicated that UV disinfection is suitable for the production of effluents for unrestricted irrigation of food crops.

Bacteria↗
PubMed2001

Retinal isomerization in bacteriorhodopsin is controlled by specific chromophore-protein interactions. A study with noncovalent artificial pigments.

It has previously been shown that, in mutants lacking the Lys-216 residue, protonated Schiff bases of retinal occupy noncovalently the bacteriorhodopsin (bR) binding site. Moreover, the retinal-Lys-216 covalent bond is not a prerequisite for initiating the photochemical and proton pump activity of the pigment. In the present work, various Schiff bases of aromatic polyene chromophores were incubated with bacterioopsin to give noncovalent pigments that retain the Lys-216 residue in the binding site. It was observed that the pigment's absorption was considerably red-shifted relative to the corresponding protonated Schiff bases (PSB) in solution and was sensitive to Schiff base linkage substitution. Their PSB pK(a) is considerably elevated, similarly to those of related covalently bound pigments. However, the characteristic low-pH purple to blue transition is not observed, but rather a chromophore release from the binding site takes place that is characterized by a pK(a) of approximately 6 (sensitive to the specific complex). It is suggested that, in variance with native bR, in these complexes Asp-85 is protonated and Asp-212 serves as the sole negatively charged counterion. In contrast to the bound analogues, no photocycle could be detected. It is suggested that a specific retinal-protein geometrical arrangement in the binding site is a prerequisite for achieving the selective retinal photoisomerization.

Aspartic Acid↗
PubMed2001

Nonisomerizable non-retinal chromophores initiate light-induced conformational alterations in bacterioopsin.

The photoactivation of retinal proteins is usually interpreted in terms of C=C photoisomerization of the retinal moiety, which triggers appropriate conformational changes in the protein. In this work several dye molecules, characterized by a completely rigid structure in which no double-bond isomerization is possible, were incorporated into the binding site of bacteriorhodopsin (bR). Using a light-induced chemical reaction of a labeled EPR probe, it was observed that specific conformational alterations in the protein are induced following light absorption by the dye molecules occupying the binding site. The exact nature of these changes and their relationship to those occurring in the bR photocycle are still unclear. Nevertheless, their occurrence proves that C=C or C=NH(+) isomerization is not a prerequisite for protein conformational changes in a retinal protein. More generally, we show that conformational changes, leading to changes in reactivity, may be induced in proteins by optical excitation of simple nonisomerizable dyes located in the macromolecular matrix.

Journal Article↗
PubMed2001

Cytosolic aldolase is a ripening related enzyme in strawberry fruits (Fragaria x ananassa).

Two aldolase isoenzymes have been isolated from ripe strawberry fruits (Fragaria x ananassa cv. Camarosa and Elsanta) and partially purified by DEAE anion exchange and Sephacryl size exclusion chromatography. The isoenzymes were identified as class I cytosol and plastid aldolase on the basis of their chromatographic behavior on DEAE-cellulose columns, native molecular weight, pH optimum pattern, Km value for D-fructose-1,6-bisphosphate, tendency to be inactivated by lower pH values and SDS-PAGE subunit determination of 40 and 38 kDa, respectively. Total aldolase activity and distribution of both aldolase isoenzymes was also investigated at different stages of strawberry fruit ripening. Strawberries in the green and white ripening stage showed the same ratio of the two isoenzymes as green leaves with 15 and 8% cytosol aldolase activity, respectively. During strawberry fruit development the overall total aldolase activity decreased until the pink ripening stage and then increased due to a rise of cytosol aldolase yielding up to 75% in red strawberries. A cDNA putatively encoding the cytosolic form of aldolase in strawberry was cloned during the course of this study. Both microarray and RNA gel blot analyses showed that the cytosolic aldolase gene expression is induced during ripening as detected for the cytosolic aldolase enzyme. We suggest that induction of the cytosolic aldolase both at the levels of transcription and translation might be part of a ripening related stress response in the receptacle tissue.

Cloning, Molecular↗
PubMed2001

Non-isomerizable artificial pigments: implications for the primary light-induced events in bacteriorhodopsin.

The primary events in the photosynthetic retinal protein bacteriorhodopsin (bR) are reviewed in light of photophysical and photochemical experiments with artificial bR in which the native retinal polyene is replaced by a variety of chromophores. Focus is on retinals in which the "critical" C13=C14 bond is locked with respect to isomerization by a rigid ring structure. Other systems include retinal oxime and non-isomerizable dyes noncovalently residing in the binding site. The early photophysical events are analyzed in view of recent pump-probe experiments with sub-picosecond time resolution comparing the behavior of bR pigments with those of model protonated Schiff bases in solution. An additional approach is based on the light-induced cleavage of the protonated Schiff base bond that links retinal to the protein by reacting with hydroxylamine. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that in bR the initial relaxation out of the Franck-Condon (FC) state does not involve substantial C13=C14 torsional motion and is considerably catalyzed by the protein matrix. Prior to the decay of the relaxed fluorescent state (FS or I state), the protein is activated via a mechanism that does not require double bond isomerization. Most plausibly, it is a result of charge delocalization in the excited state of the polyene (or other) chromophores. More generally, it is concluded that proteins and other macromolecules may undergo structural changes (that may affect their chemical reactivity) following optical excitation of an appropriately (covalently or non-covalently) bound chromophore. Possible relations between the light-induced changes due to charge delocalization, and those associated with C13=C14 isomerization (that are at the basis of the bR photocycle), are discussed. It is suggested that the two effects may couple at a certain stage of the photocycle, and it is the combination of the two that drives the cross-membrane proton pump mechanism.

Animals↗
PubMed2001

The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.

Fruit ripening is characterized by dramatic changes in gene expression, enzymatic activities and metabolism. Although the process of ripening has been studied extensively, we still lack valuable information on how the numerous metabolic pathways are regulated and co-ordinated. In this paper we describe the characterization of FaMYB1, a ripening regulated strawberry gene member of the MYB family of transcription factors. Flowers of transgenic tobacco lines overexpressing FaMYB1 showed a severe reduction in pigmentation. A reduction in the level of cyanidin 3-rutinoside (an anthocyanin) and of quercetin-glycosides (flavonols) was observed. Expression of late flavonoid biosynthesis genes and their enzyme activities were adversely affected by FaMYB1 overexpression. Two-hybrid assays in yeast showed that FaMYB1 could interact with other known anthocyanin regulators, but it does not act as a transcriptional activator. Interestingly, the C-terminus of FaMYB1 contains the motif pdLNL(D)/(E)Lxi(G)/S. This motif is contained in a region recently proposed to be involved in the repression of transcription by AtMYB4, an Arabidopsis MYB protein. Our results suggest that FaMYB1 may play a key role in regulating the biosynthesis of anthocyanins and flavonols in strawberry. It may act to repress transcription in order to balance the levels of anthocyanin pigments produced at the latter stages of strawberry fruit maturation, and/or to regulate metabolite levels in various branches of the flavonoid biosynthetic pathway.

Amino Acid Sequence↗
PubMed2000

Bacteriorhodpsin experiences light-induced conformational alterations in nonisomerizable C(13)=C(14) pigments. A study with EPR.

The mechanism by which bacteriorhodopsin is activated following light absorption is not completely clear. We have detected protein conformational alterations following light absorption by retinal-based chromophores in the bacteriorhodopsin binding site by monitoring the rate of reduction-oxidation reactions of covalently attached spin labels, using EPR spectroscopy. It was found that the reduction reaction with hydroxylamine is light-catalyzed in the A103C-labeled pigment but not in E74C or M163C. The reaction is light-catalyzed even when isomerization of the C(13)=C(14) bond of the retinal chromophore is prevented. The reverse oxidation reaction with molecular oxygen is effective only in apomembrane derived from the mutant A103C. This reaction is light-accelerated following light absorption of the retinal oxime, which occupies the binding site. The light-induced acceleration is evident also in "locked" bacteriorhodopsin in which isomerization around the C(13)=C(14) bond is prevented. It is evident that the chromophore-protein covalent bond is not a prerequisite for protein response. In contrast to the case of the retinal oxime, a reduced C=N bond A103C-labeled pigment did not exhibit acceleration of the oxidation reaction following light absorption. Acceleration was observed, however, following substitution of the polyene by groups that modify the excited state charge delocalization. It is suggested that protein conformational alterations are induced by charge redistribution along the retinal polyene following light absorption.

Bacteriorhodopsins↗
PubMed2000

The application of DNA microarrays in gene expression analysis.

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.

Biotechnology↗
PubMed2000

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

Acyltransferases↗
PubMed1999

Assessment of endometrial receptivity for gestation in patients undergoing in vitro fertilization, using endometrial thickness and the endometrium-myometrium relative echogenicity coefficient.

OBJECTIVE: To evaluate the outcome of in vitro fertilization (IVF) treatment in relation to the sonographic parameters of the endometrium. DESIGN AND METHODS: Seventy-five patients with no uterine pathology (age 31.1 +/- 5.4 years) treated in our IVF clinic for various indications were assessed during 75 cycles in which good-quality (grades 1 and 2) embryos were transferred. Controlled ovarian stimulation was achieved by the long protocol (gonadotropin releasing hormone agonist and gonadotropins). The bilayered endometrial thickness (BET), estradiol, luteinizing hormone and progesterone serum levels were measured in 272 tests. A special computer program was used to measure endometrial echogenicity relative to myometrial echogenicity. The gray-level data were analyzed on the basis of the midsagittal sonographic uterine image. Endometrium-myometrium relative echogenicity coefficient (E/M REC) values were computed and displayed graphically along the anteroposterior axis of the endometrial layers in the upper part of the uterine cavity. The area under the E/M REC curve within the BET limits was defined as the relative echogenicity area (REA) and was used as a measure of endometrial echogenicity. Each cycle was sampled in six time segments representing desensitization, follicular and luteal phases. Assigning the day of ovum pick-up as day 0, the time segments of each cycle were: first, day -20 to day -11; second, day -10 to day -6; third, day -5 to day -2; fourth, day 0; fifth, day +7 to day +14; sixth, day +15 to day +21. RESULTS: A total of 276 embryos were transferred (3.68 +/- 1.01 per cycle), of which 223 were of good quality (2.97 +/- 1.51 per cycle). An intrauterine pregnancy was diagnosed in 29 patients. All patients in this study had a BET of > 5 mm in the third and the fourth time segments. There was no significant difference in BET and REA between pregnant and non-pregnant patients tested in the first to the fifth time segments of the IVF cycles. Both BET and REA measured in the sixth time segment were significantly higher in pregnant compared to non-pregnant patients. CONCLUSIONS: Our results suggest that the proposed sonographic assessment of the endometrium shows no benefit in characterization of uterine receptivity in IVF patients with a reactive endometrium. High BET and REA values can indicate pregnancy during the sixth time segment, when the decidualization of the endometrium is well established.

Adult↗
PubMed1998

Prospective randomized comparison between an open and closed vaginal cuff in abdominal hysterectomy.

OBJECTIVE: To compare an operative and postoperative course of open vaginal cuff hysterectomy and closed vaginal cuff hysterectomy, and to correlate the length of stay, febrile morbidity and the incidence of pelvic fluid collections to the type of surgery. PARTICIPANTS: One-hundred women scheduled for hysterectomy were prospectively randomized into two groups that underwent either a closed or an open vaginal cuff technique. RESULTS: The open vaginal cuff technique took on average 19% more time than the closed vaginal cuff operation (P < 0.05, t-test). The incidence and size of pelvic fluid collections was significantly higher after the closed vaginal cuff hysterectomy than after the open technique (P < 0.01, t-test). However, the postoperative length of stay, febrile morbidity and the rate of complications were similar. CONCLUSIONS: Both techniques of hysterectomy produced a similar postoperative course despite the fact that the closed vaginal cuff technique resulted in a higher incidence of pelvic fluid collections. Therefore considering a shorter operation time for the closed vaginal cuff hysterectomy, this technique seems slightly preferable.

Adult↗
PubMed1998

Endometrium-to-myometrium relative echogenicity coefficient. A new sonographic approach for the quantitative assessment of endometrial echogenicity.

A computer program was developed to assess the endometrial echogenicity relative to the myometrial one, based on the gray-level processing of the midsagittal uterine image. The endometrial region of interest was specified within the upper part of the uterine cavity. The adjacent area of the myometrium was used to determine the reference brightness. The endometrial region of interest was analyzed along the anteroposterior uterine axis, as a set of thin strips directed parallelly to the midcavitary line. The endometrial/myometrial relative echogenicity coefficient (E/M REC) was computed for each strip and displayed graphically as a function of the distance from the midcavitary line. The area under the E/M REC curve within the limits of the total endometrial width was defined as total area (TA) and was used as a measure of the endometrial echogenicity. This parameter was assessed in 9 patients during their normal ovulatory cycles and in 29 IVF-treated patients with mechanical infertility. TA has a significant linear increase during the days of the ovulatory cycles. TA was found in high correlation with log(estradiol). TA can be used reliably for sonographic endometrial dating in ovulatory cycles.

Endometrium↗
PubMed1998

Differences between Twin Umbilical Coiling Indices Correlate to Differences in Twin Weight and Doppler Indices.

> Objective: The aim of our study was to evaluate sonographic and Doppler detectable differences in umbilical coiling index and fetoplacental circulation of discordant twins. Study Design: Doppler blood flow studies in 13 pairs of concordant and 20 pairs of discordant twins were performed from umbilical artery, middle cerebral artery, inferior vena cava, and ductus venosus. Flow studies were compared and correlated with the antenatal sonographic coiling index and the actual umbilical cord length, number of vascular helices, and birth weight. All studies were performed within 72 h before delivery. Pulsatility index (PI) values were calculated for the arteries and preload index (PLI) values for the veins. The umbilical coiling index (CI) was calculated using sonographic longitudinal views of cord vessels from several segments antenatally and by dividing the total number of helices by cord length (cm) postnatally. Discordancy was defined as a more than 20% intrapair actual birth weight difference. For all these index values the intertwin differences (Delta values) were calculated by subtracting the values obtained in the larger twin with those of the smaller twin. Results: The mean +/- SD intertwin difference in umbilical coiling index was 27.4 +/- 10.5% in the antepartum period and 28.9 +/- 10.0% after birth. Regression analysis showed a significant linear trend (r = 0.77, P < 0.001) between intertwin birth weight difference (DeltaBW) and intertwin coiling index difference (DeltaCI). A good correlation was found between DeltaCI and DeltaPLI in the ductus venosus (r = 0.63, P < 0.05), DeltaPLI in the inferior vena cava (r = 0.51, P < 0.005), and DeltaPI in the middle cerebral artery (r = 0.44, P < 0.05). Conclusions: Intertwin difference in antepartum umbilical coiling index can be determined by ultrasound and correlates well with: 1) the actual difference in coiling indices at birth, 2) the intertwin birth weight difference and 3) the intertwin Doppler flow characteristics in the fetal cerebral and venous circulation.

Journal Article↗
PubMed1997

Fusion peptides derived from the HIV type 1 glycoprotein 41 associate within phospholipid membranes and inhibit cell-cell Fusion. Structure-function study.

The fusion domain of human immunodeficiency virus (HIV-1) envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N terminus of the transmembrane glycoprotein (gp41). A V2E mutant has been shown to dominantly interfere with wild-type envelope-mediated syncytium formation and virus infectivity. To understand this phenomenon, a 33-residue peptide (wild type, WT) identical to the N-terminal segment of gp41 and its V2E mutant were synthesized, fluorescently labeled, and characterized. Both peptides inhibited HIV-1 envelope-mediated cell-cell fusion and had similar alpha-helical content in membrane mimetic environments. Studies with fluorescently labeled peptide analogues revealed that both peptides have high affinity for phospholipid membranes, are susceptible to digestion by proteinase-K in their membrane-bound state, and tend to self- and coassemble in the membranes. In SDS-polyacrylamide gel electrophoresis the WT peptide formed dimers as well as higher order oligomers, whereas the V2E mutant only formed dimers. The WT, but not the V2E mutant, induced liposome aggregation, destabilization, and fusion. Moreover, the V2E mutant inhibited vesicle fusion induced by the WT peptide, probably by forming inactive heteroaggregates. These data form the basis for an explanation of the mechanism by which the gp41 V2E mutant inhibits HIV-1 infectivity in cells when co-expressed with WT gp41.

Amino Acid Sequence↗
PubMed1997

Detection of changes in pairwise interactions during allosteric transitions: coupling between local and global conformational changes in GroEL.

A protein engineering approach for detecting and measuring local conformational changes that accompany allosteric transitions in proteins is described. Using this approach, we can identify interactions that are made or broken during allosteric transitions. The method is applied to probe for changes in pairwise interactions in the chaperonin GroEL during its ATP-induced allosteric transitions. Two pairwise interactions are investigated: one between subunits (Asp-41 with Thr-522) and the other within subunits (Glu-409 with Arg-501). We find that the intraring intersubunit interaction between Asp-41 and Thr-522 changes little during the allosteric transitions of GroEL, indicating that the hydrogen bond between these residues is maintained. In contrast, the intrasubunit salt bridge between Glu-409 and Arg-501 becomes significantly weaker during the ATP-induced allosteric transitions of GroEL. Our results are consistent with the electron microscopy observations of an ATP-induced hinge movement of the apical domains relative to the equatorial domains.

Adenosine Triphosphate↗
PubMed1996

Inter-ring communication is disrupted in the GroEL mutant Arg13 --> Gly; Ala126 --> Val with known crystal structure.

The crystal structures of the chaperonin GroEL Arg13 --> Gly; Ala126 --> Val double mutant, without and in complex with ATP gamma S, have been determined at atomic resolution. Here, we show that the double mutation Arg13 --> Gly; Ala126 --> Val disrupts negative co-operativity between GroEL rings, with respect to ATP, but has little effect on the positive co-operativity within each ring. Our results help to explain why the double mutation facilitated the crystallization of GroEL and why breaking of dyad symmetry between rings is not observed in crystal structures of this mutant. Our results may also help to explain why the observed structural differences between the GroEL double mutant and its ATP gamma S-bound form are small.

Adenosine Triphosphate↗
PubMed1996

Isolation and characterization of the highly phosphorylated repeat domain of distinct heavy neurofilament subunit (NF-H) isoforms.

1. Recent examination of the hypothesis that distinctly phosphorylated NF-H isoforms exist in different types of neurons revealed that the extent of phosphorylation of the heavy neurofilament polypeptide of bovine ventral root motor neurons is markedly higher than that of dorsal root neurons. 2. In the present study we employed endoproteinase ASP-N for isolating the Lys-Ser-Pro (KSP)-rich domain of NF-H, which contains most of the NF-H phosphorylation sites. 3. Treatment of NF-H with ASP-N endoproteinase results in a cascade of products, the last of which is a polypeptide with apparent molecular weight of 120 kDa. Amino terminal sequence and amino acid composition analysis revealed that this fragment contains the KSP-rich domain of NF-H. 4. Treatment of ventral and dorsal root NF-H with ASP-N endoproteinase and analysis of the phosphoserine contents of the resulting 120 kDa fragments revealed that the 120 kDa fragment of ventral root NF-H is significantly more phosphorylated than that of dorsal root NF-H. 5. These findings show that the difference in extent of phosphorylation of ventral and dorsal root NF-H is due at least partly to the KSP-rich domain of NF-H.

Amino Acid Sequence↗