Scalable diversification options delivers sustainable and nutritious food in Indo-Gangetic plains.

Indo-Gangetic plains (IGP) of South Asia have supported bulk of human and bovine population in the region since ages, and a spectacular progress has been made in food production. However, malnutrition, diminishing total factor productivity, and natural resource degradation continue to plague this cereal-dominated region, which is also vulnerable to climate change. Addressing these challenges would require a transition towards diversifying cereal rotations with agroecological cropping systems. A study was, therefore, conducted at the experimental farm of ICAR-CSSRI, Karnal on crop diversification and sustainable intensification options using agro-ecological approaches such as Conservation Agriculture (CA) and diversified cropping systems to ensure food and nutritional security while sustaining the natural resources. On 2 years mean basis, CA-based cropping system management scenarios (mean of Sc2-Sc7) using diversified crop rotations; increased the system yield by 15.4%, net return by 28.7%, protein yield by 29.7%, while using 53.0% less irrigation water compared to conventional tillage (CT)-based rice-wheat system (Sc1). Maize-mustard-mungbean on permanent beds (PBs) (Sc4) recorded the highest productivity (+ 40.7%), profitability (+ 60.1%), and saved 81.8% irrigation water compared to Sc1 (11.8 Mg ha-1; 2190 USD ha-1; 2514 mm ha-1). Similarly, Sc5 (maize-wheat-mungbean on PBs) improved productivity (+ 32.2%), profitability (+ 57.4%) and saved irrigation water (75.5%) compared to Sc1. In terms of nutritional value, Sc5 was more balanced than other scenarios, and produced 43.8, 27.5 and 259.8% higher protein, carbohydrate and fat yields, respectively, compared to Sc1 (0.93, 8.55 and 0.14 Mg ha-1). Scenario 5 was able to meet the nutrient demand of 19, 23 and 32 additional persons ha-1 year-1 with respect to protein, carbohydrate and fat, respectively, compared to Sc1. The highest protein water productivity (~ 0.31 kg protein m-3 water) was recorded with CA-based soybean-wheat-mungbean (Sc6) system followed by maize-mustard-mungbean on PBs (Sc4) system (~ 0.29 kg protein m-3) and lowest under Sc1. Integration of short duration legume (mungbean) improved the system productivity by 17.2% and profitability by 32.1%, while triple gains in irrigation water productivity compared to CT-based systems. In western IGP, maize-wheat-mungbean on PBs was found most productive, profitable and nutritionally rich and efficient system compared to other systems. Therefore, diversification of water intensive cereal rotations with inclusion of legumes and CA-based management optimization can be potential option to ensure nutritious food for the dwelling communities and sustainability of natural resources in the region.

Energy and economic efficiency of climate-smart agriculture practices in a rice-wheat cropping system of India.

Intensive tillage operations, indiscriminate use of irrigation water, chemical fertilizers, and pesticides and crop biomass burning have made the conventional rice-wheat (RW) system highly energy-intensive and inefficient. In the recent past, portfolios of climate-smart agricultural practices (CSAP) have been promoted as a potential alternative to improve the energy efficiency in conventional RW system. Therefore, to evaluate the energy input-output relation, energy flow and economic efficiency in various combinations of crop management options, a 3-year (2014-2017) on-farm study was conducted at Karnal, India. Various portfolio of management practices; Sc1-Business as usual (BAU) or Conventional tillage (CT) without residue, Sc2-CT with residue, Sc3-Reduce tillage (RT) with residue + recommended dose of fertilizer (RDF), Sc4-RT/Zero tillage (ZT) with residue + RDF, Sc5-ZT with residue + RDF + GreenSeeker + Tensiometer, Sc6-Sc5 + Nutrient expert were investigated. Present study results revealed that net energy, energy use efficiency and energy productivity were 11-18, 31-51 and 29-53% higher under CSAP (mean of Sc4, Sc5 and Sc6) in RW system than Sc1, respectively. However, renewable and non-renewable energy inputs were 14 and 33% higher in Sc1 compared to CSAP (4028 and 49,547 MJ ha-1), respectively, it showed that BAU practices mostly dependents on non-renewable energy sources whereas CSAP dependents on renewable energy sources. Similarly, the adoption of CSAP improved the biomass yield, net farm income and economic efficiency by 6-9, 18-23 and 42-58%, respectively compared to Sc1. Overall, the adoption of CSAP could be a viable alternative for improving energy use efficiency, farm profitability and eco-efficiency in the RW system.

Erratum: Elastase and metalloproteinase 9 concentrations in saliva in patients with chronic periodontitis.

[This corrects the article DOI: 10.5114/ceji.2014.45948.].

Genetic engineering and molecular characterization of yeast strain expressing hybrid human-yeast squalene synthase as a tool for anti-cholesterol drug assessment.

AIMS: The main objective of the study is molecular and biological characterization of the human-yeast hybrid squalene synthase (SQS), as a promising target for treatment of hypercholesterolaemia. METHODS AND RESULTS: The human-yeast hybrid SQS, with 67% amino acids, including the catalytic site derived from human enzyme, was expressed in Saccharomyces cerevisiae strain deleted of its own SQS gene. The constructed strain has a decreased level of sterols compared to the control strain. The mevalonate pathway and sterol biosynthesis genes are induced and the level of triacylglycerols is increased. Treatment of the strain with rosuvastatin or zaragozic acid, two mevalonate pathway inhibitors, decreased the amounts of squalene, lanosterol and ergosterol, and up-regulated expression of several genes encoding enzymes responsible for biosynthesis of ergosterol precursors. Conversely, expression of the majority genes implicated in the biosynthesis of other mevalonate pathway end products, ubiquinone and dolichol, was down-regulated. CONCLUSIONS: The S. cerevisiae strain constructed in this study enables to investigate the physiological and molecular effects of inhibitors on cell functioning. SIGNIFICANCE AND IMPACT OF THE STUDY: The yeast strain expressing hybrid SQS with the catalytic core of human enzyme is a convenient tool for efficient screening for novel inhibitors of cholesterol-lowering properties.

Total and high molecular weight adiponectin levels in the rat model of post-myocardial infarction heart failure.

Adiponectin is a protein secreted primarily by adipose tissue. It has been suggested that adiponectin plays a protective role in the early phase following myocardial infarction. Our primary aim was to investigate the effects of post-myocardial infarction heart failure well-characterized by left ventricular hemodynamic parameters on the total and high molecular weight adiponectin concentrations in plasma, fat and cardiac tissue. Eight weeks after myocardial infarction or sham operation, total and high molecular weight adiponectin concentrations in plasma, fat, and cardiac tissues were assayed in rats. In addition, hemodynamic parameters and expression of the genes encoding atrial natriuretic peptide and brain natriuretic peptide in left ventricle were evaluated. Atrial natriuretic peptide and brain natriuretic peptide mRNA levels in left ventricle tissue were higher in rats with myocardial infarction-induced heart failure compared with the controls. Similarly, total adiponectin concentration was increased in left ventricle (but not in right ventricle) in rats with post-myocardial infarction heart failure. In contrast, adiponectin levels in plasma and cardiac adipose tissue in rats with post-myocardial infarction heart failure were lower than in sham-operated animals. Furthermore, there were no significant differences in levels of high molecular weight adiponectin in plasma, cardiac tissue or adipose tissue between these two groups. We conclude that in the rat model of post-myocardial infarction heart failure, adiponectin level is increased in left ventricle tissue. This is accompanied by decreased adiponectin levels in plasma and cardiac adipose tissue.

MicroRNA expression profiles in liver and colon of sexually immature gilts after exposure to Fusarium mycotoxins.

To improve our knowledge of the role of microRNAs (miRs) in responses of the porcine digestive system to two Fusarium mycotoxins, zearalenone (ZEN) and deoxynivalenol (DON), we examined the expression of 7 miRs (miR-9, miR-15a, miR-21, miR-34a, miR-122, miR-125b, and miR-192), previously found to be deregulated in diseased liver and colon cells. In this study, immature gilts were exposed to NOEL doses of ZEN (40 µg/kg/d), DON (12 µg/kg/d), ZEN + DON (40 + 12 µg/kg/d), andplacebo (negative control group) for 7, 14, 21, 28, 35, and 42 days. Before the treatment, expression levels of the selected miRs were measured in the liver, the duodenum, the jejunum, and the ascending and the descending colon of the gilts. Hierarchical clustering of the tissues by their miR expression profiles was consistent with what would be expected based on the anatomical locations and the physiological functions of the organs, suggesting that functions of the miRs are related to the specificities of the tissues in which they are expressed. A subset of 2 pairs of miRs (miR-21+miR-192 and miR-15a+miR-34a), which were assigned to two distinct clusters based on their tissue abundance, was then evaluated in the liver and the ascending and the descending colon during the treatment. The most meaningful results were obtained from the ascending colon, where a significant effect of the treatment was observed, suggesting that during the exposure to mycotoxins, the pathways involved in cell proliferation and survival were disordered. Changes in miR expression in the liver and the descending colon of the treated gilts were smaller, and were associated more with treatment duration than the exposure to ZEN, DON, or ZEN + DON. Further research should focus on identification of genes whose expression is regulated by these aberrantly expressed miRs. This should facili- tate understanding of the miRNA-regulated biological effects of mycotoxins.

Assessment of temporomandibular joint disease.

The diagnosis of temporomandibular joint (TMJ) disorders consists of clinical (Reaserch Diagnostic Criteria for Temporomandibular Disorders, RDC/TMD) and additional (computer tomography, CT or magnetic resonance imaging, and MRI) examinations. Due to the growing knowledge of pathologic changes within the TMJ, the researches become more aware of the difficulty in detection the early symptoms of disorders using conventional examination. Therefore, it is now expected that the collected samples of synovial fluid, serum, or urine samples could enable easier identification of inflammatory process course, and degenerative cartilage changes state.

The evaluation of the efficacy of sodium carbonate as zearalenone destructor in feeding stuffs.

Contamination of feed with zearalenone (ZEA) is still a serious problem in farm animals feeding, especially in gilts, sensitive to this compound. The relative failure of current methods of decontamination and quality control lead us to look for new techniques. The commonly accepted method for breaking down ZEA was performed in controlled temperature and time conditions. Various sodium carbonate doses (0.5 - 4%) were added to feed naturally contaminated with ZEA (ZEA biosynthesis by F. graminearum isolates). These doses were found to be effective in in vitro studies. The addition of 2% sodium carbonate gave the best results in reducing the phytoestrogen in the feed.

Real-time PCR analysis of p53 mRNA levels in tissues of whitefish (Coregonus lavaretus) exposed to benzo[a]pyrene.

We developed a real-time PCR assay for measuring relative quantities (RQ) of p53 tumor suppressor mRNA in the whitefish (Coregonus lavaretus, Salmonidae, Teleostei). Real-time PCR primers for the p53 gene were designed from a region that was found to be conserved among salmonid p53 genes. To test for the usefulness of the assay we performed a treatment study, using benzo[a]pyrene (B[a]P) a putative p53-inducer. Two groups of hatchery raised whitefish, with an average body mass of 15 g and total length of 12 cm were either given an intraperitoneal injection (10 mg x kg(-1)) of B[a]P in corn oil (2 mg B[a]P ml(-1) corn oil) or corn oil alone (Control). After treatment (48 h, 7 degrees C), two random fish from each group were anesthetized and the liver, head kidney and brain were collected for mRNA isolation and analysis. In the control fish, relative quantification analysis based on the p53 mRNA levels in liver (RQ=1.00) showed higher basal levels of p53 mRNA in the head kidney (RQ= 1.69), and lower in the brain (RQ=0.41). In all three tissues sampled, p53 mRNA was affected by treatment with B[a]P. Liver tissue showed the greatest induction (RQ=1.53) from base levels (RQ=1.00), followed by brain (RQ=1.36), and head kidney (RQ=1.23). These results confirm that p53 mRNA is generally present at lower levels in differentiated tissues (liver and brain) than in those tissues with cell lines (head kidney), and demonstrate that p53 is moderately inducible by B[a]P in the whitefish. The approach presented here has the advantage of providing rapid and accurate measures of p53 induction in various tissues of fish responding to PAH contaminant exposure.

Modification of zearalenone structure in model and natural conditions.

Zearalenone is a mycotoxin widely occurring in cereals and animal feed, and it is associated with hyperestrogenism and other reprodutive disorders in animals. A new method of detoxication of feedstuffs involves alkaline hydrolysis of toxic macrolactone (1) (as well as model compounds (2a, 2b)). The method caused modification of zearalenone structure under mild conditions and the toxin underwent irreversible hydrolysis with high efficiency.

Metabolic profile of pigs fed feed containing zearalenone destructor.

Zearalenone (ZEA) is a member of macrocyclic lactons family. It is a toxin--phytosteride produced by fungi of Fusarium ssp. genus. Zearalenone contaminates food and animal feeding stuffs and its destruction is difficult. It requires application of particular compounds that would bind zearalenone in the feed or feeding stuff or in the gastrointestinal tract and decrease its bio-accessibility. It should also fulfil all the safety requirements regarding the plant supplements and animals that are fed with this feed. The aim of the study was to estimate if the feed supplemented with different doses of zearalenone and zearalenone destructor causes changes of the metabolic profile in gilts. The results obtained show that applied destructor did not cause negative haematological and biochemical changes in the blood of the gilts examined. It can be suggested that it is a safe feed supplement pigs in prevention of zearalenone micotoxicosis.

Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice.

Murine experimental autoimmune thyroiditis (EAT), characterized by thyroid destruction after immunization with thyroglobulin (Tg), has long been a useful model of organ-specific autoimmune disease. More recently, porcine thyroid peroxidase (pTPO) has also been shown to induce thyroiditis, but these results have not been confirmed. When (C57BL/6 x CBA)F(1) mice, recently shown to be susceptible to mouse TPO-induced EAT, were immunized with plasmid DNA to human TPO (hTPO) and cytokines IL-12 or GM-CSF, significant antibody (Ab) titres were generated, but minimal thyroiditis was detected in one mouse only from the TPO + GM-CSF immunized group. However, after TPO DNA immunization of HLA-DR3 transgenic class II-deficient NOD mice, thyroiditis was present in 23% of mice injected with TPO + IL-12 or GM-CSF. We also used another marker for assessing the closeness of the model to human thyroid autoimmunity by examining the epitope profile of the anti-TPO Abs to immunodominant determinants on TPO. Remarkably, the majority of the anti-TPO Abs was directed to immunodominant regions A and B, demonstrating the close replication of the model to human autoimmunity. TPO protein immunizations of HLA-DR3 transgenic mice with recombinant hTPO did not result in thyroiditis, nor did immunization of other mice expressing HLA class II transgenes HLA-DR4 or HLA-DQ8, with differential susceptibility to Tg-induced EAT. Moreover, our efforts to duplicate exactly the experimental procedures used with pTPO also failed to induce thyroiditis. The success of hTPO plasmid DNA immunization of DR3(+) mice, similar to our reports on Tg-induced thyroiditis and thyrotropin receptor DNA-induced Graves' hyperthyroidism, underscores the importance of DR3 genes for all three major thyroid antigens, and provides another humanized model to study autoimmune thyroid disease.

Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis.

Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549-563), P14 (aa 599-617) and P18 (aa 210-225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75%. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81.5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model.

Graves' hyperthyroidism and thyroiditis in HLA-DRB1*0301 (DR3) transgenic mice after immunization with thyrotropin receptor DNA.

Familial and twin studies in Caucasians have established that the MHC class II allele HLA-DRB1*0301 (DR3) is a strong susceptibility gene in Graves' hyperthyroid disease (GD). To determine if a DR3 transgene could help establish an animal model for GD, we expressed DR3 molecules in class II-knockout NOD mice (H2Ag7-). DR3+g7- mice were given cardiotoxin prior to immunization on weeks 0, 3 and 6 with plasmid DNA encoding human thyrotropin receptor (TSHR). Two groups of mice were also coimmunized with plasmid DNA for IL-4 or GM-CSF. Serial bleeds on weeks 8, 11 and 14 showed that approximately 20% of mice produced thyroid-stimulating antibodies (Abs), and approximately 25% had elevated T4 levels. In particular, a subset displayed both signs of hyperthyroidism, resulting in approximately 30% with some aspect of GD syndrome. Additional mice had thyroid-stimulating blocking Abs and/or TSH-binding inhibitory immunoglobulins, while most mice showed strong labelling of TSHR+ cells by flow cytometry. Interestingly, lymphocytic infiltration with thyroid damage and Abs to mouse thyroglobulin were also noted. Vector controls were uniformly negative. Thus, DR3 transgenic mice can serve as a model for GD, similar to our earlier reports that this allele is permissive for the Hashimoto's thyroiditis model induced with human thyroglobulin.

Suppressors of translation initiation defect in hem12 locus of Saccharomyces cerevisiae.

A system for the positive selection of transational initiation suppressors in S. cerevisiae has been developed. A mutant with an ATA initiation codon in the HEM12 gene, encoding uroporphyrinogen decarboxylase, was used to select cis- and trans-acting suppressors. These suppressors partially restore growth on nonfermentable carbon sources, such as glycerol, but still allow the accumulation of porphyrins. All extragenic suppressors are mapped to the SUI1 locus, encoding initiation factor eIF1. The effect of the hem12 mutation is also partially reversed by the known SUI3 suppressor encoding the beta subunit of eIF2. In contrast, the sui2 suppressor encoding the a subunit of eIF2 does not affect the hem12 phenotype. The intragenic suppressors are able to restore the translation of hem12 due to the generation of additional, in frame AUG codons upstream of the hem12-14 mutation. Mutational analysis of the HEM12 leader sequence was also performed to determine the role of small open reading frames (uORFs) present upstream of the HEM12 ORF. Studies on the expression of integrated hem12-1/4-lacZ fusion, devoid of all upstream ATGs, indicate a lack of regulatory effect of uORFs on HEM12 translation.

Activity and cellular location in Saccharomyces cerevisiae of chimeric mouse/yeast and Bacillus subtilis/yeast ferrochelatases.

We have constructed a series of chimeric yeast/mouse and yeast/Bacillus subtilis ferrochelatase genes in order to investigate domains of the ferrochelatase that are important for activity and/or association with the membrane. These genes were expressed in a Saccharomyces cerevisiae mutant in which the endogenous ferrochelatase gene (HEM15) had been deleted, and the phenotypes of the transformants were characterized. Exchanging the approximately 40-amino-acid C-terminus between the yeast and mouse ferrochelatases caused a total loss of activity and the hybrid proteins were unstable when overproduced in Escherichia coli. The water-soluble ferrochelatase of B. subtilis did not complement the yeast mutant, although a large amount of active protein accumulated in the cytosol. Addition of the N-terminal leader sequence of yeast ferrochelatase to the B. subtilis enzyme targeted the fusion protein to mitochondria, but both the precursor and the mature forms of the enzyme were inactive in vivo and had residual activity when measured in vitro. An internal approximately 45-amino-acid segment located at the N-terminus of yeast ferrochelatase was identified, which, when replaced with the corresponding 30-amino-acid segment of the B. subtilis enzyme, caused the yeast enzyme to be located in the mitochondrial matrix as a soluble protein. The fusion protein was inactive in vivo and had residual activity in vitro. We speculate that this segment, which shows the greatest variability between species, is responsible for the association of the enzyme with the membrane.

Cerebral blood flow assessed by brain SPECT with 99mTc-HMPAO utilising the acetazolamide test in systemic lupus erythematosus.

BACKGROUND: Cerebrovascular diseases are one of the most important complications of systemic lupus erythematosus (SLE). The diagnostic imaging of neuropsychiatric SLE complications presents many problems. This study was undertaken to investigate cerebral blood flow char s and its reactivity to hypercapnia by means of acetazolamide test in SLE patients. METHODS: Brain SPELT studies using 99mTc-HMPAO were performed in 50 patients with SLE. Acetazolamide test was performed in 35 patients 3 days after the baseline study by means of repetitive scanning 20 min after i.v. injection of 1.0 g of acetazolamide. RESULTS: Significant interhemispheric hypoperfusion areas were shown in 76.3% of all patients, 83.8% symptomatic and 63.1 % asymptomatic. Patients with antiphospholipid syndrome showed multifocal perfusion deficits. The reaction of cerebral perfusion to acetazolamide was heterogenous and showed increase, decrease, no change or mixed reaction of baseline-study-found focal hypoperfusion. Acetazolamide test revealed hypoperfusion in two patients with normal baseline study. MRI scanning revealed cerebral lesions in 41 % of patients. CONCLUSIONS: CBF asymmetries in symptomatic and asymptomatic patients with SLE are frequent. Regional CBF alterations seem to be different in patients with and without antiphospholipid syndrome. The part of the patients with SLE shows no or paradoxically inversed reaction to acetazolamide.

Subtelomeric duplications in Saccharomyces cerevisiae chromosomes III and XI: topology, arrangements, corrections of sequence and strain-specific polymorphism.

We have determined the sequence of a 3.42 kb segment from the left arm of chromosome III (coordinates 5394-8815 of Oliver et al., 1992). Instead of four open reading frames (ORFs) listed previously, the verified sequence reveals the presence of only one ORF, renamed YCL070/73c, encoding a protein of 615 amino acids. The putative product of ORF YCL070/73c shows 98.5% identity and 99% similarity with the protein of the same length encoded by ORF YKR106w from the right arm of chromosome XI and displays a topology characteristic for the Major Facilitators Superfamily of membrane proteins. These corrections will be deposited in the EMBL data library under the Accession Number X59720. In strain S288C the subtelomeric sequence 4319-11 215 of chromosome III is 98.3% identical with the subtelomeric sequence of 658 204-665 061 from the right arm of chromosome XI. Using various subtelomeric probes from chromosome III (coordinates 2097-3646 of S288C) we have analysed eight different Saccharomyces cerevisiae strains and the closely related species S. douglasii: some S. cerevisiae strains have additional duplications and longer chromosomes XI; in all strains chromosome III contains the 1200-11 000 segment (strain FL100 is disomic) while S. douglasii does not show any hybridization in this region.

Probing the active-site residues in Saccharomyces cerevisiae ferrochelatase by directed mutagenesis. In vivo and in vitro analyses.

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the insertion of ferrous iron into protoporphyrin, the terminal step in protoheme biosynthesis. The functional/structural roles of 10 invariant amino acid residues were investigated by site-directed mutagenesis in the yeast Saccharomyces cerevisiae ferrochelatase. The mutant enzymes were expressed in a yeast strain lacking the ferrochelatase gene HEM15 and in Escherichia coli. The kinetic parameters of the mutant enzymes were determined for the enzymes associated with the yeast membranes and the enzymes in the bacterial soluble fraction. They were compared with the in vivo functioning of the mutant enzymes. The main conclusions are the following. Glu-314 is critical for catalysis, and we suggest that it is the base responsible for abstracting the N-pyrrole proton(s). His-235 is essential for metal binding. Asp-246 and Tyr-248 are also involved in metal binding in a synergistic manner. The Km for protoporphyrin was also increased in the H235L, D246A, and Y248L mutants, suggesting that the binding sites of the two substrates are not independent of each other. The R87A, Y95L, Q111E, Q273E, W282L, and F308A mutants had 1.2-2-fold increased Vm and 4-10-fold increased Km values for protoporphyrin, but the amount of heme made in vivo was 10-100% of the normal value. These mutations probably affected the geometry of the active center, resulting in improper positioning of protoporphyrin.