Mosquito Iridescent Virus: New Records from Nature and Infections Using Strelkovimermis spiculatus (Mermithidae) as a Vector Under Laboratory Conditions.

Iridoviridae is a DNA virus family that affects both vertebrates and invertebrates. Immature aquatic stages of many dipteran species infected with iridovirus have been found in different places worldwide. The most represented genera of the Culicidae family are Aedes and Psorophora. To date, sixteen species of Aedes naturally infected with iridoviruses have been reported. Moreover, there are four records for the genus Psorophora, one for Culiseta, and two for Culex. In this paper, we report two new mosquito species as natural hosts of iridoviridae in Argentina: Aedes albifasciatus (Macquart) and Culex dolosus (Lynch Arribalzaga). We also analyzed the ability of a Cx. pipiens-Invertebrate Iridescent Virus to replicate in vivo in the larval stage of two mosquito species, Culex apicinus Philippi and Ae. aegypti (L.) using Strelkovimermis spiculatus as a vector, under laboratory conditions. Although Ae. aegypti is the most recognized mosquito vector of important arboviruses responsible for emergent diseases, Cx. apicinus and Ae. albifasciatus may also be implicated in enzootic or epizootic cycles of virus transmission, such as the St. Louis Encephalitis virus and the Western Equine Encephalomyelitis virus.

Identification of Multiple Replication Stages and Origins in the Nucleopolyhedrovirus of Anticarsia gemmatalis.

To understand the mechanism of replication used by baculoviruses, it is essential to describe all the factors involved, including virus and host proteins and the sequences where DNA synthesis starts. A lot of work on this topic has been done, but there is still confusion in defining what sequence/s act in such functions, and the mechanism of replication is not very well understood. In this work, we performed an AgMNPV replication kinetics into the susceptible UFL-Ag-286 cells to estimate viral genome synthesis rates. We found that the viral DNA exponentially increases in two different phases that are temporally separated by an interval of 5 h, probably suggesting the occurrence of two different mechanisms of replication. Then, we prepared a plasmid library containing virus fragments (0.5-2 kbp), which were transfected and infected with AgMNPV in UFL-Ag-286 cells. We identified 12 virus fragments which acted as origins of replication (ORI). Those fragments are in close proximity to core genes. This association to the core genome would ensure vertical transmission of ORIs. We also predict the presence of common structures on those fragments that probably recruit the replication machinery, a structure also present in previously reported ORIs in baculoviruses.

Bacillus wiedmannii biovar thuringiensis: A Specialized Mosquitocidal Pathogen with Plasmids from Diverse Origins.

Bacillus cereus sensu lato also known as B. cereus group is composed of an ecologically diverse bacterial group with an increasing number of related species, some of which are medically or agriculturally important. Numerous efforts have been undertaken to allow presumptive differentiation of B. cereus group species from one another. FCC41 is a Bacillus sp. strain toxic against mosquito species like Aedes aegypti, Aedes (Ochlerotatus) albifasciatus, Culex pipiens, Culex quinquefasciatus, and Culex apicinus, some of them responsible for the transmission of vector-borne diseases. Here, we report the complete genome sequence of FCC41 strain, which consists of one circular chromosome and eight circular plasmids ranging in size from 8 to 490 kb. This strain harbors six crystal protein genes, including cry24Ca, two cry4-like and two cry52-like, a cry41-like parasporin gene and multiple virulence factors. The phylogenetic analysis of the whole-genome sequence of this strain with molecular approaches places this strain into the Bacillus wiedmannii cluster. However, according with phenotypical characteristics such as the mosquitocidal activity due to the presence of Cry proteins found in the parasporal body and cry genes encoded in plasmids of different sizes, indicate that this strain could be renamed as B. wiedmannii biovar thuringiensis strain FCC41.

A comprehensive bioinformatic analysis of hepatitis D virus full-length genomes.

In association with hepatitis B virus (HBV), hepatitis delta virus (HDV) is a subviral agent that may promote severe acute and chronic forms of liver disease. Based on the percentage of nucleotide identity of the genome, HDV was initially classified into three genotypes. However, since 2006, the original classification has been further expanded into eight clades/genotypes. The intergenotype divergence may be as high as 35%-40% over the entire RNA genome, whereas sequence heterogeneity among the isolates of a given genotype is <20%; furthermore, HDV recombinants have been clearly demonstrated. The genetic diversity of HDV is related to the geographic origin of the isolates. This study shows the first comprehensive bioinformatic analysis of the complete available set of HDV sequences, using both nucleotide and protein phylogenies (based on an evolutionary model selection, gamma distribution estimation, tree inference and phylogenetic distance estimation), protein composition analysis and comparison (based on the presence of invariant residues, molecular signatures, amino acid frequencies and mono- and di-amino acid compositional distances), as well as amino acid changes in sequence evolution. Taking into account the congruent and consistent results of both nucleotide and amino acid analyses of GenBank available sequences (recorded as of January, 2017), we propose that the eight hepatitis D virus genotypes may be grouped into three large genogroups fully supported by their shared characteristics.

Variability study of entomopathogenic nematode populations (Heterorhabditidae) from Argentina / Estudo da variabilidade entomopatogênicos nematóides populações (Heterorhabditidae) da Argentina

Abstract Entomopathogenic nematodes (EPN) belonging to the Heterorhabditidae family are lethal parasites of soil-dwelling insects. Two species were reported in Argentina: Heterorhabditis argentinensis and Heterorhabditis bacteriophora characterized mainly by morphometric features. In this work a comparative and phylogenetic study between five Heterorhabditis populations from Argentina was conducted to analyze the variability between strains and to evaluate the taxonomic position of Heterorhabditis argentinensis. The PCA analyses of morphometric characters separated the larger juvenile, female and male H. argentinensis from H. bacteriophora populations. The juvenile (IJs) stage provided the clearest separation of Heterorhabditis populations presenting the least variability between strains. The variable L and MBW were highly related to H. argentinensis IJs. Three groups were separated by this stage considering PC1 and PC2: one formed by H. bacteriophora OLI, RIV and RN strains, (isolates from Córdoba and Río Negro province), one for H. bacteriophora VELI strain (Buenos Aires province) and one for H. argentinensis (Santa Fe province). Heterorhabditis bacteriophora VELI and H. argentinensis isolated from regions with more rainfalls and humidity presented larger values for morphometric features. Molecular analyses showed the Argentinian populations (H. bacteriophora VELI strain and H. argentinensis), forming a same clade, with six other H. bacteriophora populations (not from Argentina) with a genetic similarity between them of 99%. Heterorhabditis argentinensis presented one unique nucleotide that was not present in any of the other species of the clade. Considering the results of this study H. argentinensis would be conspecific to H. bacteriophora, constituting a strain with a great morphometric variation where the host and climatic conditions could have influenced on the measurements.

Resumo Nematóides entomopatogênicos (EPN) pertencentes à família Heterorhabditidae são parasitas letais de insetos que vivem no solo. Duas espécies foram relatados na Argentina: Heterorhabditis argentinensis e Heterorhabditis bacteriophora, caracterizada principalmente por características morfométricas. Neste trabalho um estudo comparativo e filogenética entre cinco populações do Heterorhabditis da Argentina foi conduzido para analisar a variabilidade entre as linhagens e avaliar a posição taxonômica das Heterorhabditis argentinensis. Características morfométricas de Heterorhabditis bacteriophora VELI e H. argentinensis isoladas de regiões com mais chuvas e umidade apresentaram dimensões maiores. Analisa o PCA de personagens morfométricas separou a maior juvenil, feminino e masculino H. argentinensis de H. bacteriophora populações. A fase juvenil (JIs) fornece a mais clara separação de populações Heterorhabditis apresentando a menor variação entre as cepas. A L variável e MBW foram altamente relacionada com H. argentinensis JIs. Três grupos foram separados por esta fase considerando PC1 e PC2: um formado por H. bacteriophora OLI, RIV e estirpes RN, (isolados de Córdoba e província de Rio Negro), um para a estirpe H. bacteriophora VELI (província de Buenos Aires) e um para H. argentinensis (província de Santa Fe). Heterorhabditis bacteriophora VELI e H. argentinensis isolado a partir de regiões com mais chuvas e umidade apresentaram maiores valores para as características morfométricas. A análise molecular mostrou as populações da Argentina (estirpe H. bacteriophora VELI e H. argentinensis), formando um mesmo subtipo, com seis outras populações H. bacteriophora (não da Argentina), com uma similaridade genética entre eles de 99%. Heterorhabditis argentinensis apresentado um único nucleótido que não estava presente em nenhum dos outros espécies do clado. Considerando os resultados deste estudo H. argentinensis seria conspecific a H. bacteriophora, constituindo uma estirpe com uma grande variação morfométrica onde o anfitrião e as condições climáticas podem ter influenciado nas medições.

Variability study of entomopathogenic nematode populations (Heterorhabditidae) from Argentina.

Entomopathogenic nematodes (EPN) belonging to the Heterorhabditidae family are lethal parasites of soil-dwelling insects. Two species were reported in Argentina: Heterorhabditis argentinensis and Heterorhabditis bacteriophora characterized mainly by morphometric features. In this work a comparative and phylogenetic study between five Heterorhabditis populations from Argentina was conducted to analyze the variability between strains and to evaluate the taxonomic position of Heterorhabditis argentinensis. The PCA analyses of morphometric characters separated the larger juvenile, female and male H. argentinensis from H. bacteriophora populations. The juvenile (IJs) stage provided the clearest separation of Heterorhabditis populations presenting the least variability between strains. The variable L and MBW were highly related to H. argentinensis IJs. Three groups were separated by this stage considering PC1 and PC2: one formed by H. bacteriophora OLI, RIV and RN strains, (isolates from Córdoba and Río Negro province), one for H. bacteriophora VELI strain (Buenos Aires province) and one for H. argentinensis (Santa Fe province). Heterorhabditis bacteriophora VELI and H. argentinensis isolated from regions with more rainfalls and humidity presented larger values for morphometric features. Molecular analyses showed the Argentinian populations (H. bacteriophora VELI strain and H. argentinensis), forming a same clade, with six other H. bacteriophora populations (not from Argentina) with a genetic similarity between them of 99%. Heterorhabditis argentinensis presented one unique nucleotide that was not present in any of the other species of the clade. Considering the results of this study H. argentinensis would be conspecific to H. bacteriophora, constituting a strain with a great morphometric variation where the host and climatic conditions could have influenced on the measurements.

Telomerase as a Cancer Target. Development of New Molecules.

Telomeres are the terminal part of the chromosome containing a long repetitive and noncodifying sequence that has as function protecting the chromosomes. In normal cells, telomeres lost part of such repetitive sequence in each mitosis, until telomeres reach a critical point, triggering at that time senescence and cell death. However, in most of tumor cells in each cell division a part of the telomere is lost, however the appearance of an enzyme called telomerase synthetize the segment that just has been lost, therefore conferring to tumor cells the immortality hallmark. Telomerase is significantly overexpressed in 80-95% of all malignant tumors, being present at low levels in few normal cells, mostly stem cells. Due to these characteristics, telomerase has become an attractive target for new and more effective anticancer agents. The capability of inhibiting telomerase in tumor cells should lead to telomere shortening, senescence and apoptosis. In this work, we analyze the different strategies for telomerase inhibition, either in development, preclinical or clinical stages taking into account their strong points and their caveats. We covered strategies such as nucleosides analogs, oligonucleotides, small molecule inhibitors, G-quadruplex stabilizers, immunotherapy, gene therapy, molecules that affect the telomere/ telomerase associated proteins, agents from microbial sources, among others, providing a balanced evaluation of the status of the inhibitors of this powerful target together with an analysis of the challenges ahead.

Telomere structure and telomerase in health and disease (review).

Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells, proliferation potential is strictly limited and senescence follows approximately 50-70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. Undoubtedly, DNA polymerase is not capable of completely copying DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis and cell death. However, in tumor cells the system of telomere length maintenance is activated. Much work has been done regarding the complex telomere/telomerase as a unique target, highly specific in cancer cells. Telomeres have additional proteins that regulate the binding of telomerase. Telomerase, also associates with a number of proteins forming the sheltering complex having a central role in telomerase activity. This review focuses on the structure and function of the telomere/telomerase complex and its altered behavior leading to disease, mainly cancer. Although telomerase therapeutics are not approved yet for clinical use, we can assume that based on the promising in vitro and in vivo results and successful clinical trials, it can be predicted that telomerase therapeutics will be utilized soon in the combat against malignancies and degenerative diseases. The active search for modulators is justified, because the telomere/telomerase system is an extremely promising target offering possibilities to decrease or increase the viability of the cell for therapeutic purposes.

Production of heterologous polygalacturonase I from Aspergillus kawachii in Saccharomyces cerevisiae in batch and fed-batch cultures.

The pg1 gene from the filamentous fungus Aspergillus kawachii, which codifies for an acid polygalacturonase, was cloned into the pYES2 expression vector, giving rise to the pYES2:pg1ΔI construct. Engineered Saccharomyces cerevisiae, transformed with pYES2:pg1ΔI construct, both expressed and exported an active polygalacturonase with a MW of ~60 kDa and an isoelectric point of 3.7, similar to those reported for the wild-type enzyme. The recombinant enzyme has the ability to hydrolyze polygalacturonic acid at pH 2.5. Heterologous PG1 production was studied under controlled conditions in batch and fed-batch systems. A simultaneous addition of glucose and galactose was found to be the most suitable feeding strategy assayed, resulting in a final PG1 production of 50 U/ml. The production process proposed in this study could be applied for the industrial production of a novel and useful polygalacturonase.

Expression and purification of Z protein from Junín virus.

Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z). The arenavirus Junín (JUNV) is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one). One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.

Argentine hemorrhagic fever diagnostic test based on recombinant Junín virus N protein.

Junín arenavirus is the etiologic agent of Argentine hemorrhagic fever. Due to its morbidity and high mortality rate in untreated cases, this endemic disease is of mandatory report in Argentina. Secure and accurate diagnostic methods are needed for the epidemiological surveillance of the disease. Current assays rely on antigens prepared from lysates of virus infected mammalian cells. The bio-safety issue related to the manipulation of large quantities of virus restricts such antigen production to laboratories with the appropriate containment facilities. In this report, we describe the development of an enzyme linked immunosorbent assay for the etiologic confirmation of the disease, based on recombinant antigens expressed in insect cells. Eight different variables of the assay were optimized with the Taguchi approach for experimental design (L18 design, seven three-level factors and one two-level factor). The area under the receiver operating characteristics (ROC) curve was 0.966, showing the high accuracy of the test discriminating positive from negative samples. Taking into account the biosafety benefits, the high yields of antigen in cell culture, and the general performance of the assay, it is expected that it will be a useful alternative to the current ELISA for the detection of antibodies in sera from convalescent patients.

Two simultaneous hepatitis B virus epidemics among injecting drug users and men who have sex with men in Buenos Aires, Argentina: characterization of the first D/A recombinant from the American continent.

Previous studies have revealed that hepatitis B virus (HBV)/D and HBV/F predominate among blood donors from Buenos Aires, Argentina. In the present study, blood samples from two high-risk groups were analysed: 160 corresponding to street- and hospital-recruited injecting drug users [81.2% showing the 'anti-hepatitis B core antigen (anti-HBc) only' serological pattern] and 20 to hepatitis B surface antigen (HBsAg)(+)/anti-HBc(+) men who have sex with men. HBV genotypes were assigned by polymerase chain reaction amplification followed by restriction fragment length polymorphism and confirmed by nucleotide sequencing of two different coding regions. HBV DNA was detected in 27 injecting drug users (16.9%, occult infection prevalence: 7.7%), and 14 men who have sex with men (70%). HBV/A prevailed among injecting drug users (81.8%) while HBV/F was predominant among men who have sex with men (57.1%). The high predominance of HBV/A among injecting drug users is in sharp contrast to its low prevalence among blood donors (P = 0.0006) and men who have sex with men (P = 0.0137). Interestingly, all HBV/A S gene sequences obtained from street-recruited injecting drug users encoded the rare serotype ayw1 and failed to cluster within any of the known A subgenotypes. Moreover, one of the HBV strains from a hospital-recruited injecting drug user was fully sequenced and found to be the first completely characterized D/A recombinant genome from the American continent. Data suggest that two simultaneous and independent HBV epidemics took place in Buenos Aires: one spreading among injecting drug users and another one sexually transmitted among the homosexual and heterosexual population.

Patient re-training in peritoneal dialysis: why and when it is needed.

The aim of this multicenter, quantitative, observational study was to analyze compliance and re-training needs of patients on peritoneal dialysis (PD) through the assessment of patient knowledge (with a Patient Questionnaire; phase 1) and patient behavior (home visit with a Score Card; phase 2). A total of 353 patients from 11 Italian centers participated in the first phase and 191 patients from nine centers in the second phase. Overall, 66% of questions on the Patient Questionnaire were answered correctly. Correct answers were more frequent in females than males, in patients under 55 years of age, and in those with higher education. The lowest rate of correct answers involved questions related to diet and physical activity (67% and 51%, respectively). Data collected during the home visit showed that 25% of patients were partially compliant with their drug therapy. Twenty-three percent of patients were non-compliant with the exchange protocol procedures, with a significant association between compliance and the incidence of peritonitis, and 11% were non-compliant with the exit-site protocol procedures without a statistically significant correlation to peritonitis. By combining the two evaluations, we found that approximately one-third (29%) of patients needed reinforcement of knowledge and ability to correctly perform PD as related to infection control and 27% for the correct use of drugs. Looking at the combined evaluation of infection control and drug use, results showed that 47% of patients needed re-training. This need for re-training was greater for younger patients (less than 55 years old), patients with lower education degree and patients in the early or late phase of PD therapy (less than 18 months or more than 36 months). Gender and degree of autonomy had no effect on the need for re-training.

[Temporomandibular joint involvement in juvenile idiopathic arthritis: treatment with an orthodontic appliance]. / Trattamento con un dispositivo ortodontico per l'impegno temporomandibolare nell'artrite idiopatica giovanile. Osservazioni su 72 casi.

OBJECTIVE: About 65% of children suffering from juvenile idiopathic arthritis (JIA) shows a more or less marked involvement of temporo-mandibular joint (TMJ) with altered mandibular growth, resorption of the condyles, occlusary instability, reduced chewing ability and facial dysmorphia. The purpose of our study is to prevent and to treat the progressive evolution of JIA on craniofacial growth and morphology with a functional appliance; surgery should be considered only in so far as the adequacy of TMJ movement is concerned. METHODS: From 1992 until now 72 children with proved JIA and TMJ involvement have been treated (50 females, 22 males, aged 6 to 16 years old). TMJ involvement was bilateral in 61% and unilateral in 39% of patients. A diagnostic workup was carried out involving tomograms of TMJ and cephalometric radiograph and analysis. The authors used a bimaxillary activator in the attempt to modify the unfavourable growth pattern and provide a gradual ante-rotation of the jaw. RESULTS: Almost all JIA patients showed satisfactory long term results, easing of pain, reduced skeletal discrepancy, increased function and good facial profile. CONCLUSIONS: The long term results of this study indicate that orthopaedic therapy might control the vicious circle of the malocclusion in children with JIA, preventing exacerbation of mandibular clockwise rotation. Surgical intervention for the improvement of TMJ function should be considered only if a severe restricted state is imminent.

Physical and genetic map of Epinotia aporema granulovirus genome.

The bean shoot borer, Epinotia aporema (Lep. Tortricidae), is an economically important pest of legume crops in South America. Recently, a granulovirus (EpapGV) was isolated from E. aporema larvae, and evaluated as a potential biological control agent. In order to generate a restriction map and to investigate the gene organisation of EpapGV genome, DNA isolated from occlusion bodies as well as a set of cloned genomic fragments were analysed using combinations of restriction endonucleases and Southern blot analyses that lead to a first version of the physical map. It was subsequently confirmed and refined by sequencing the termini of the cloned fragments and assessing their contiguity by comparing the sequences with databases to identify putative ORFs spanning neighbour fragments. This was also aided by PCR amplifications with primers that pointed outwards of the cloned viral DNA. The granulin gene was positioned on the physical map, cloned and sequenced. Its 747-nucleotide-long ORF encodes a predicted protein of 29 kDa and the core of the baculovirus very late promoter ATAAG was found 29 nucleotides upstream the initiation codon. In addition, 27 putative ORFs were located on the map and used to explore the genome organisation by GeneParityPlot against the fully sequenced granulovirus genomes. These data, taken together with the phylogenetic tree generated by alignment of the major occlusion proteins, indicate that EpapGV is closely related to CpGV, but has a distinct gene organisation.

Characterization of a granulovirus isolated from Epinotia aporema Wals. (Lepidoptera: Tortricidae) larvae.

A granulovirus (GV) isolated from Epinotia aporema (Lepidoptera: Tortricidae)-a major soybean pest-was studied in terms of its main morphological, biochemical, and biological properties. The ovoidal occlusion bodies were 466 by 296 nm in size, and their most prominent protein had an apparent molecular mass of 29 kDa. Its amino-terminal sequence was remarkably homologous to that of the granulins of other GVs. The DNA genome size was estimated to be 120 kbp. The high specificity and pathogenicity of this newly described granulovirus (EpapGV) indicate that it is indeed a good candidate for the biological control of this pest.

Generation of a recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus expressing a foreign gene under the control of a very late promoter.

A recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) expressing beta-galactosidase under the control of the polyhedrin promoter was generated in our laboratory. To this end, we cloned the AgMNPV-2D genomic DNA fragment containing the polh gene and subcloned and sequenced the polyhedrin gene and its flanking regions. Based on this sequence information, sets of primers were designed to amplify the flanking regions by PCR, including appropriate restriction sites. The transfer vector (pAgPHZ) was constructed by the consecutive cloning of these PCR fragments flanking the Escherichia coli LacZ gene, in place of the polh gene. pAgPHZ was used for cotransfection of UFL-AG-286 insect cells with AgMNPV-2D DNA and the required recombinant, generated by homologous recombination with the polh locus, was identified by its polh(-)/LacZ+ plaque phenotype. Its genome structure was confirmed by PCR, restriction digestion and Southern blot analyses. The kinetics and levels of expression of beta-galactosidase in UFL-AG-286 cells infected with the recombinant were tested by SDS-PAGE and enzymatic activity assays.

Arenavirus nucleocapsid protein displays a transcriptional antitermination activity in vivo.

RNA polymerase pausing and transcriptional antitermination regulates gene activity in several systems. In arenavirus infected cells the switch from transcription to replication is subjected to a hairpin-dependent termination and requires protein synthesis to bypass this signal. The transcriptional antitermination control by Junín virus nucleocapsid protein N, has been demonstrated in vivo by infecting BHK-21 cells expressing this viral protein in the presence of translation inhibitors. This is the first demonstration in vivo of a transcriptional antitermination control in arenavirus-infected cells.

Zinc-binding properties of Junín virus nucleocapsid protein.

The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle. First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid. We have expressed the N protein of Junín virus in E. coli and shown that it binds zinc in vitro. This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX(2)HX(23)CX(4)C sequence, which resembles a classical zinc-finger motif. The specificity for zinc binding was demonstrated by competition with other divalent metal ions. The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions. In addition, alternative zinc-binding sites were found.