On Choosing Among Several Experimental Treatments and a Control

The two classical selection approaches in comparing experimental treatments with a control are combined to form an integrated approach. In this integrated approach, there is a preference zone (PZ) and an indifference zone (IZ), and the concept of a correct decision (CD) is defined differently in each of these zones. In the PZ, we are required to select the best treatment for a correct decision (CD₁) but in the IZ, we define any selected subset to be correct (CD₂) if it contains the best treatment among all the experimental treatments and the controlled treatment. We propose a single-stage procedure R to achieve the selection goals CD₁ and CD₂ simultaneously with certain probability requirements. It is shown that both the probability of a correct decision under PZ, P(CD₁ | PZ), and the probability of a correct decision under IZ, P(CD₂ | IZ), satisfy some monotonicity properties and the least favorable configuration in PZ and the worst configuration in IZ are derived by these properties. We also derive formulas for the probabilities of correct decision and provide a brief table to illustrate the magnitude of the procedure parameters and the common sample sizes needed for various probability requirements and configurations.     

Selecting the Best Population,Provided It Is Better than a Control: The Unequal Variance Case

Bechhofer and Turnbull (1978) proposed two procedures to compare k normal means with a standard and the procedures guarantee that (1) with probability at least P₀* (specified), no category is selected when the best experimental category is sufficiently worst than the standard, and (2) with probability at least P₁* (specified), the best experimental category is selected when it is sufficiently better than the second best and the standard. For the case of common known variance, they studied a single-stage procedure. For the case of common unknown variance, they studied a two-stage procedure. Under the same formulation of Bechhofer and Turnbull (1978) and for the same selection goals (1) and (2) described above, Wilcox (1984a) proposed a procedure to the case of unknown and unequal variances, and supplied a table of the necessary constants to implement the procedure. This paper considers the case of unknown and unequal variances for the same formulation of Bechhofer and Turnbull, and Wilcox, but assumes that μ₀ is an unknown control. A two-stage procedure is proposed to solve the problem. A lower bound of the probability of a correct selection is derived and it takes the same form as the double integral appeared in Rinott (1978) which was used for the lower bound of the probability of a correct selection for a different selection goal.     

Two-Stage Procedures for Comparing Treatments with a Control: Elimination at the First Stage and Estimation at the Second Stage

We consider the problem of comparing a set of p₁ test treatments with a control treatment. This is to be accomplished in two stages as follows: In the first stage, N₁ observations are allocated among the p₁ treatments and the control, and the subset selection procedure of Gupta and Sobel (1958) is employed to eliminate “inferior” treatments. In the second stage, N₂ observations are allocated among the (randomly) selected subset of p₂(≤p₁) treatments and the control, and joint confidence interval estimates of the treatment versus control differences are calculated using Dunnett's (1955) procedure. Here both N₁ and N₂ are assumed to be fixed in advance, and the so-called square root rule is used to allocate observations among the treatments and the control in each stage. Dunnett's procedure is applied using two different types of estimates of the treatment versus control mean differences: The unpooled estimates are based on only the data obtained in the second stage, while the pooled estimates are based on the data obtained in both stages. The procedure based on unpooled estimates uses the critical point from a p₂-variate Student t-distribution, while that based on pooled estimates uses the critical point from a p₁-variate Student t-distribution. The two procedures and a composite of the two are compared via Monte Carlo simulation. It is shown that the expected value of p₂ determines which procedure yields shorter confidence intervals on the average. Extensions of the procedures to the case of unequal sample sizes are given. Applicability of the proposed two-stage procedures to a drug screening problem is discussed.     

Isolation of a carbon disulfide utilizing <Emphasis Type="Italic">Thiomonas</Emphasis> sp. and its application in a biotrickling filter

The carbon disulfide (CS₂)-oxidizing bacterium Thiomonas sp. WZW was enriched and isolated using activated sewage sludge as inoculum. Growth of Thiomonas sp. WZW was observed on CS₂, thiosulfate, dimethylsulfide (DMS), dimethyldisulfide (DMDS), and H₂S. No growth occurred on dimethylsulfoxide, methanol, acetate, and on complex media with glucose, yeast extract, or tryptone. DMDS-grown cells respired CS₂, DMS, and DMDS, while thiosulfate-grown cells did not respire CS₂. Chemostat cultures growing on thiosulfate could be rapidly adapted to growth on CS₂. Growth was observed between pH 6 and 8. The K _s values for CS₂, thiosulfate, and sulfide of CS₂-grown cells were between 5 and 10 μM. CS₂ was inhibitory above 0.3 mM. A lab-scale biotrickling filter with lava stone as carrier material for treatment of CS₂-polluted air was inoculated with Thiomonas sp. WZW. A rapid start up (95% removal in 1 week) was obtained at an inlet CS₂ concentration of 2 cmol l⁻¹ and an initial space velocity (SV) of 54 h⁻¹. Subsequent thiosulfate addition for a week during start up increased the removal to 99%. The step-wise increase of SV to 130 h⁻¹ and a CS₂ concentration to 3 μmol l⁻¹ resulted in a stable performance with a removal efficiency of 95%. Feeding mixtures of volatile sulfur compounds showed simultaneous conversion of H₂S, CS₂, dimethyldisulfide (DMDS), and DMS, with a preference in this order.     

Subset Selection for Exponential Populations: Determination of the Selection Constant

Given are k(≧2) exponential populations differing only in their location parameter. One wishes to choose the best one, that is the population with the largest value of the location parameter. A possible method for solving this problem is to select a subset of the k populations of size at least one which includes the best population with a required confidence P*(k^?1 ≤ P* ≤1). In this paper the required selection constant is determined for different values of k and P*. Also an approximation for the selection constant is derived. A comparison with the exact results is made.     

Subset Selection of an Almost Best Treatment

A generalized goal using subset selection is discussed for the location parameter case. This goal is to select a non-empty subset from a set of k (k ≥ 2) treatments that contains at least one ε-best treatment with confidence level P*. For a set of treatments an ε-best treatment is defined as a treatment with location parameter on a distance less than or equal to ε(ε ≥o) from the best treatment, where best is defined as largest value of the location parameter. The efficiency of subset selection of an ε-best treatment relative to subset selection of the best treatment is investigated and is computed for some values of k and the confidence level for the Normal case as well as for the Logistic case.     

Novel eubacteria able to grow on carbon disulfide

Four eubacterial strains able to grow on carbon disulfide (CS₂) as sole energy substrate were isolated from soil and leaves of the CS₂-producing tree Quercus lobata. Three of the isolates (strains KS1, KS2, and KL1) were gram-negative, facultatively methylotrophic, and heterotrophic, and capable of growth on a wide range of inorganic and organic sulfur compounds. Biochemical and physiological properties differed slightly among the three strains, but all are proposed to be novel thiobacillus species. Growth yields on CS₂ in batch and chemostat culture ranged from 3.3 g dry wt/mol CS₂ (batch) to a maximum growth yield (Y_max) of 11.1 g dry wt/mol (chemostat). Chemostat data for two of the strains growing, autotrophically on thiosulfate gave Y_max values of 7.4 and 7.1 g dry wt/mol, which fall within the range observed with thiobacilli. The three new Thiobacillus strains had DNA containing 39.8 (KS2), 47.8 (KS1), and 50.5 (KL1) mol% G+C. All three were unusual in being able to grow not only on thiosulfate (aerobically or with denitrification), but also on CS₂, carbonyl sulfide and methylated sulfides as sole energy substrates, and one was unique in being able to grow also on substituted thiophenes. They are the first organisms described to be capable, of anaerobic growth with denitrification on CS₂. The fourth isolate (strain KL2) was gram-positive non-motile and nonspore-forming, with 39.0 mol% G+C. It had a restricted range of sulfur-containing growth substrates, could not grow methylotrophically or on autotrophic substrates other than CS₂, and is not yet classifiable These organisms extend the range of eubacteria known to be capable of CS₂ breakdown and demonstrate that several types of facultatively chemolithotrophic bacteria, able to grow exclusively on CS₂, are associated with a CS₂-producing plant.     

Genetic analysis of Karnal bunt (Neovossia indica) resistance in wheat

Embryos excised from seeds of six generations (P₁, P₂, F₁, BC₁, BC₂ and F₂) of a cross WH 283 x WH 533 were cultured on modified MS medium already inoculated with secondary sporidia ofNeovossia indica. Significant variations for callusing response (CR) (54.55–75.55%) were observed among generations but the presence or absence ofN. indicia did not affect callusing response. A clear inhibition zone (IZ) was formed around each embryo showing callusing. The diameter of IZ varied significantly among generations and was maximum in the resistant genotype, WH 283 (3.60 cm). Fresh weight and dry weight of calli, initiated from embryo cultured and inoculated withN. indica, varied significantly among generations. Coefficient of infection as well as percentage of infection reflected the overdominance of susceptibility. Generation mean analysis showed that the three parameter model was adequate for diameter of IZ only. Six-parameter model showed that additive (in presence ofN. indica), additive and additive x dominance (in absence ofN. indica) effects were also significant. Complementary type of epistasis for fresh weight of calli and dominance, and dominance x dominance effects for dry weight of calli were observed in the presence ofN. indica. Magnitude of additive effects was higher for diameter of IZ in three parameter model. Therefore, selection might assist in improving this trait and thus indirectly help in attaining the resistance towardsN. indica.     

A Selection Procedure for Selecting Good Exponential Populations

Consider k independent exponential populations with location parameters μ₁,…, μ_k and a common scale parameter or standard deviation θ. Let μ_(k) be the largest of the μ's and define a population to be good if its location parameter exceeds μ_(k) –Δ₁. A selection procedure is proposed to select a subset of the k populations which includes the good populations with probability at least P*, a pre-assigned value. Simultaneous confidence intervals, that can be derived with the proposed selection procedure, are discussed. Moreover, if populations with locations below μ_(k) –δ₂, (δ₂ > δ₁) are “bad”, a selection procedure is proposed and a sample size is determined so that the probability of omitting a “good” population or selecting a “bad” population is at most 1 – P*.     

Conformational Similarity Indices Between Different Residues in Proteins and α-Helix Propensities

Abstract

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS_XX′, between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS_AX′ indicates the extent of similarity in the main-chain and side-chain conformations (φ ψ and χ₁) of different residues (X) with Ala (A) and is found to have strong correlation with α-helix propensities. However, no subset of CS_XX′ provides any linear relationship with β-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an a-helix is different from the one favouring the β-sheet. Conformationally similar residues (close CS_AX values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS_AX values.     

Role of Thiobacillus thioparus in the biodegradation of carbon disulfide in a biofilter packed with a recycled organic pelletized material

This study reports the biodegradation of carbon disulfide (CS₂) in air biofilters packed with a pelletized mixture of composted manure and sawdust. Experiments were carried out in two lab-scale (1.2 L) biofiltration units. Biofilter B was seeded with activated sludge enriched previously on CS₂-degrading biomass under batch conditions, while biofilter A was left as a negative inoculation control. This inoculum was characterized by an acidic pH and sulfate accumulation, and contained Achromobacter xylosoxidans as the main putative CS₂ biodegrading bacterium. Biofilter operation start-up was unsuccessfully attempted under xerophilic conditions and significant CS₂ elimination was only achieved in biofilter A upon the implementation of an intermittent irrigation regime. Sustained removal efficiencies of 90–100 % at an inlet load of up to 12 g CS₂ m^?3 h^?1 were reached. The CS₂ removal in this biofilter was linked to the presence of the chemolithoautotrophic bacterium Thiobacillus thioparus, known among the relatively small number of species with a reported capacity of growing on CS₂ as the sole energy source. DGGE molecular profiles confirmed that this microbe had become dominant in biofilter A while it was not detected in samples from biofilter B. Conventional biofilters packed with inexpensive organic materials are suited for the treatment of low-strength CS₂ polluted gases (IL <12 g CS₂ m^?3 h^?1), provided that the development of the adequate microorganisms is favored, either upon enrichment or by inoculation. The importance of applying culture-independent techniques for microbial community analysis as a diagnostic tool in the biofiltration of recalcitrant compounds has been highlighted.     

Effects of drought and root injury on plant-generated CS2 emissions in soil

Conditions under which some plants emit carbon disulfide (CS₂) in the soil are unknown. A pot assembly was constructed to measure soil CS₂ emissions by Mimosa pudica under conditions of undisturbed growth, root injury, and drought stress. When M. pudica was grown without disturbance, soil CS₂ emissions were below the limit of detection (0.1 ng CS₂ mL^–1) for the entire 8-wk sampling period. However, when the roots of 6-wk-old M. pudica plants were cut to a depth of 10 cm, a maximum of 0.5 and 0.4 ng CS₂ mL^–1 was emitted within minutes at the 5- and 10-cm depths, respectively. These emissions declined slowly to undetectable levels after 50 min. No detectable CS₂ emissions were observed at the 0- and 15-cm depths. No CS₂ was emitted when 6-wk-old M. pudica plants were subjected to drought stress, however, when the same plants were watered, a maximum of 0.3, 0.4, and 0.5 ng CS₂ mL^–1 was emitted within minutes at the 5-, 10- and 15-cm depths, respectively. These emissions were detectable for at least 2 hr at the 10- and 15-cm depths. No detectable CS₂ emissions were observed at the 0-cm depth after watering. No detectable CS₂ emissions were observed at any depth under any conditions of undisturbed growth, root injury, or drought stress followed by watering for assemblies containing either no plants or Albizia julibrissin, a plant that is closely related to M. pudica but does not emit CS₂. Mimosa pudica emitted detectable CS₂ under conditions of root injury and rewetting of dry soil but not under conditions of undisturbed growth. Release of such a biocidal sulfide only during conditions of root injury or rewetting of dry soil would be advantageous to M. pudica.     

Degradation of carbondisulphide by a Thiobacillus isolate

During experiments investigating the purification of waste gas a bacterium capable of using carbon disulphide (CS₂) als sole energy source was isolated. It could be identified as a Thiobacillus sp.; however, the species remains unclear. Both the properties of T. thioparus and T. thiooxidans have been observed. Since the organism could be used for removing CS₂ in the environment, the degradation kinetics have been investigated by different methods. Substrate concentrations of up to 100 mg CS₂·l^–1 were oxidized at maximum rates of 2.5 mg CS₂·g^–1 protein·min^–1 at pH 7.0 and at 30°C. CS₂ levels above 150 mg CS₂·l^–1 caused termination of degradative activity. Correspondence to: Ch. Plas     

Gene stacking strategies with doubled haploids derived from biparental crosses: theory and simulations assuming a finite number of loci

Recent progress in genotyping and doubled haploid (DH) techniques has created new opportunities for development of improved selection methods in numerous crops. Assuming a finite number of unlinked loci (ℓ) and a given total number (n) of individuals to be genotyped, we compared, by theory and simulations, three methods of marker-assisted selection (MAS) for gene stacking in DH lines derived from biparental crosses: (1) MAS for high values of the marker score (T, corresponding to the total number of target alleles) in the F₂ generation and subsequently among DH lines derived from the selected F₂ individual (Method 1), (2) MAS for augmented F₂ enrichment and subsequently for T among DH lines from the best carrier F₂ individual (Method 2), and (3) MAS for T among DH lines derived from the F₁ generation (Method 3). Our objectives were to (a) determine the optimum allocation of resources to the F₂ ( n₁^* \, n_{1}^{*} ) and DH generations (n - n₁^* ) (n - n_{1}^{*} ) for Methods 1 and 2 by simulations, (b) compare the efficiency of all three methods for gene stacking by simulations, and (c) develop theory to explain the general effect of selection on the segregation variance and interpret our simulation results. By theory, we proved that for smaller values of ℓ, the segregation variance of T among DH lines derived from F₂ individuals, selected for high values of T, can be much smaller than expected in the absence of selection. This explained our simulation results, showing that for Method 1, it is best to genotype more F₂ individuals than DH lines ($ n_{1}^{*} :n > 0.5 $ n_{1}^{*} :n > 0.5 ), whereas under Method 2, the optimal ratio n₁^* :n n_{1}^{*} :n was close to 0.5. However, for ratios deviating moderately from the optimum, the mean [`(X)] \overline{X} of T in the finally selected DH line ( T<sub>\textDH</sub><sup>*</sup> T_{\text{DH}}^{*} ) was hardly reduced. Method 3 had always the lowest mean [`(X)] \overline{X} of T_\textDH^* T_{\text{DH}}^{*} except for small numbers of loci (ℓ = 4) and is favorable only if a small number of loci are to be stacked in one genotype and/or saving one generation is of crucial importance in cultivar development. Method 2 is under most circumstances the superior method, because it generally showed the highest mean [`(X)] \overline{X} and lowest SD of T_\textDH^* T_{\text{DH}}^{*} for the finally selected DH.     

Genetic Predisposition to Alcoholic Toxic Cirrhosis

Comprehensive analysis of the contribution of genetic factors into predisposition to alcoholic toxic cirrhosis (TC) was performed. The AB0, RH, HP, TF, GC, PI, ACP1, PGM1, ESD, GLO1, and GST1 genetic polymorphisms were compared in 34- to 59-year-old male TC patients and control donors of the same sex and age. The phenotypic frequencies in the TC group deviated from the theoretically expected values; the main difference was the excess of rare homozygotes for the lociGC, ACP1, ESD, and GLO1.In the TC patients, the observed heterozygosity (H _o) was considerably lower than the theoretically expected value (H _e). Wright's fixation index (F) in the TC patients was 30 times higher than in the control group (0.0888 and 0.0027, respectively). A considerable decrease in the ABO*0allele frequency at the expense of an increase in the ABO*Aallele frequencywas observed in the TC patients as compared to the control sample. The TF*C2allele frequencywas two times higher in the patients than in the control group (0.2571 and 0.1308, respectively). The frequencies ofPI*Zand PI*S, the PIalleles that are responsible for lower concentrations of proteinase inhibitor, were 12 and 6 times higher in the TC than in the control group. The TC patients exhibited a significantly higher frequency of the liver glutathione-S-transferase GST1*0allele, whereas the GST1*2frequency was two times higher in the control subjects than in the TC patients (0.2522 and 0.0953, respectively). The TC and control groups showed statistically significant differences in the frequencies of the following alleles of six independent loci: ABO*0, TF*C1, TF*C2, PI*M1, PI*Z, ACP1*C, PGM1*1+, PGM1*1–, PGM1*2–, GST1*0, andGST1*2. The haptoglobin level was significantly higher and the serum transferrin level was drastically lower in all phenotypic groups of TC patients than in control subjects. The concentrations of IgM and IgG depended on the HP, GC, and PI phenotypes. The total and direct reacting bilirubin concentrations depended on the red cell-enzyme phenotypes (ACP1, PGM1, and GLO1) in both TC and control groups.     

Factors affecting production of COS and CS2 in Leucaena and Mimosa species

Carbon disulfide (CS₂) and carbonyl sulfide (COS) are colorless, foul-smelling, volatile sulfur compounds with biocidal properties. Some plants produce CS₂ or COS or both. When used as an intercrop or forecrop, these plants may have agronomic potential in protecting other plants. Most of the factors which affect production of these plant-generated organic sulfides are unknown. We determined the effects of sulfate concentration, plant age, nitrogen fixation, drought stress, root injury (through cutting), and undisturbed growth on COS production in Leucaena retusa or Leucaena leucocephala and the effect of some of these factors on CS₂ production in Mimosa pudica. In addition, we determined if organic sulfides were produced in all Leucaena species. When L. retusa and M. pudica seedlings were grown in a plant nutrient medium with different sulfate concentrations (50 to 450 mg SL^-1), COS or CS₂ from crushed roots generally increased with increasing sulfate concentration. COS production was highest (74 ng mg^-1 dry root) for young L. retusa seedlings and declined to low amounts (<5 ng mg^-1 dry root) for older seedlings. Nitrogen fixation reduced the amounts of COS or CS₂ produced in L. leucocephala and M. pudica. Under conditions of undisturbed growth, root cutting, or drought stress, no COS production was detected in 4-to 8-weeks-old L. retusa plants. COS or CS₂ or both was obtained from crushed roots or shoots of all 13 known Leucaena species.     

Effects of acute CS2 intoxication on liver protein and drug metabolism

Rats pretreated with phenobarbitone and their controls were exposed to 0.15% atmospheric CS₂ for 2 h. Liver protein metabolism and microsomal drug metabolizing enzyme activities were analyzed 1, 4 and 46 h later. In the pretreated rats the [¹⁴C]leucine uptake was at first inhibited, then liver RNA tended to increase. This increase was followed by a decline in the [¹⁴C]leucine uptake, while RNA content diminished to the control level. In the control rats (not pretreated with phenobarbital) the effects of CS₂ on liver protein metabolism were less; only at 4 h after the exposure was liver RNA increased and [¹⁴C]leucine uptake slightly stimulated. In the pretreated rats CS₂ had decreased microsomal P-450 by about 50% at 1 and 4 h, and the activity of 7-O-dealkylase of ethoxycoumarin had decreased even more. The measurable UDPglucuronosyltransferase of the liver microsomes of the pretreated rats had increased by 26% at 1 h and by 80% at 4 h after the CS₂ exposure. The in vivo activation of microsomal UDPglucuronosyltransferase may result from a stimulated lipid peroxidation of reticuloendothelial membranes by CS₂ metabolites in vivo, as suggested by the diene conjugation spectra. In control rats CS₂ depressed only the ethoxycoumarin deethylase activity. All the microsomal changes caused by CS₂ exposure were restored within 46 h.It is suggested that the different action of CS₂ on the liver protein metabolism of rats pretreated with phenobarbitone and controls results from the different stage of protein turnover in the two groups, i.e. in the barbituratetreated animals there is a phase of increased protein synthesis and accordingly the protein turnover is more sensitive to the action of CS₂.     

Characterization of the protein Z-dependent protease inhibitor interactive-sites of protein Z

Background

Protein Z (PZ) has been reported to promote the inactivation of factor Xa (FXa) by PZ-dependent protease inhibitor (ZPI) by about three orders of magnitude. Previously, we prepared a chimeric PZ in which its C-terminal pseudo-catalytic domain was grafted on FX light-chain (Gla and EGF-like domains) (PZ/FX-LC). Characterization of PZ/FX-LC revealed that the ZPI interactive-site is primarily located within PZ pseudo-catalytic domain. Nevertheless, the cofactor function and apparent K_d of PZ/FX-LC for interaction with ZPI remained impaired ~ 6–7-fold, suggesting that PZ contains a ZPI interactive-site outside pseudo-catalytic domain. X-ray structural data indicates that Tyr-240 of ZPI interacts with EGF2-domain of PZ. Structural data further suggests that 3 other ZPI surface loops make salt-bridge interactions with PZ pseudo-catalytic domain. To identify ZPI interactive-sites on PZ, we grafted the N-terminal EGF2 subdomain of PZ onto PZ/FX-LC chimera (PZ-EGF2/FX-LC) and also generated two compensatory charge reversal mutants of PZ pseudo-catalytic domain (Glu-244 and Arg-212) and ZPI surface loops (Lys-239 and Asp-293).

Methods

PZ chimeras were expressed in mammalian cells and ZPI derivatives were expressed in Escherichia coli.

Results

The PZ EGF2 subdomain fusion restored the defective cofactor function of PZ/FX-LC. The activities of PZ and ZPI mutants were all impaired if assayed individually, but partially restored if the compensatory charge reversal mutants were used in the assay.

Conclusions

PZ EGF2 subdomain constitutes an interactive-site for ZPI. Data with compensatory charge reversal mutants validates structural data that the identified residues are part of interactive-sites.

General significance

Insight is provided into mechanisms through which specificity of ZPI–PZ–FXa complex formation is determined.     

Bacterial CS2 Hydrolases from Acidithiobacillus thiooxidans Strains Are Homologous to the Archaeal Catenane CS2 Hydrolase

Carbon disulfide (CS₂) and carbonyl sulfide (COS) are important in the global sulfur cycle, and CS₂ is used as a solvent in the viscose industry. These compounds can be converted by sulfur-oxidizing bacteria, such as Acidithiobacillus thiooxidans species, to carbon dioxide (CO₂) and hydrogen sulfide (H₂S), a property used in industrial biofiltration of CS₂-polluted airstreams. We report on the mechanism of bacterial CS₂ conversion in the extremely acidophilic A. thiooxidans strains S1p and G8. The bacterial CS₂ hydrolases were highly abundant. They were purified and found to be homologous to the only other described (archaeal) CS₂ hydrolase from Acidianus strain A1-3, which forms a catenane of two interlocked rings. The enzymes cluster in a group of β-carbonic anhydrase (β-CA) homologues that may comprise a subclass of CS₂ hydrolases within the β-CA family. Unlike CAs, the CS₂ hydrolases did not hydrate CO₂ but converted CS₂ and COS with H₂O to H₂S and CO₂. The CS₂ hydrolases of A. thiooxidans strains G8, 2Bp, Sts 4-3, and BBW1, like the CS₂ hydrolase of Acidianus strain A1-3, exist as both octamers and hexadecamers in solution. The CS₂ hydrolase of A. thiooxidans strain S1p forms only octamers. Structure models of the A. thiooxidans CS₂ hydrolases based on the structure of Acidianus strain A1-3 CS₂ hydrolase suggest that the A. thiooxidans strain G8 CS₂ hydrolase may also form a catenane. In the A. thiooxidans strain S1p enzyme, two insertions (positions 26 and 27 [PD] and positions 56 to 61 [TPAGGG]) and a nine-amino-acid-longer C-terminal tail may prevent catenane formation.     

Transgenic indica rice cv. ADT 43 expressing a Δ<Superscript>1</Superscript>-pyrroline-5-carboxylate synthetase (<Emphasis Type="Italic">P5CS</Emphasis>) gene from <Emphasis Type="Italic">Vigna aconitifolia</Emphasis> demonstrates salt tolerance

To develop salt tolerant rice, the P5CS gene of Vigna aconitifolia, encoding for proline synthesis, was introduced into the popular indica rice cultivar ADT 43. Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1301/P5CS, carrying the proline synthesis encoding gene P5CS, was co-cultivated with embryogenic callus of rice. Adding 100 μM acetosyringone to the Linsmaier and Skoog (LS) liquid and solid co-culture medium, along with 30 mg/l hygromycin and 250 mg/l timentin, contributed to significantly higher efficiency of transformation. Southern blot analysis of T₁ independent transformants revealed that the copy number of transgene varied between one and three. When transgenic plants were subjected to salt stress, these plants grew well in the presence of up to 200 mM NaCl, while control plants died within 10 days under these treatment conditions. These transgenic plants grew under salt stress for a period of 4 weeks, and were capable of flowering and set seed. T₁ plants segregated into 3:1 ratio suggesting Mendelian segregation pattern of inheritance of the P5CS transgene.