Mutational screening of SF1 and WNT4 in Tunisian women with premature ovarian failure

Background

WNT4 and SF1 genes play an important role in ovarian development. They constitute coherent candidate genes associated with premature ovarian failure (POF) pathogenesis.

Methods

We sequenced the coding region of WNT4 and SF1 in 55 Tunisian women with POF and 100 healthy controls.

Results

We identified a synonymous variation in WNT4 (c.99G>A, p.Ser33Ser) and a substitution (c.G437C) in SF1 gene inducing G146 to Ala (GGG–GCG) missense mutation. WNT4 (c.99G>A, p.Ser33Ser) was not associated with POF pathology. However, a positive association of SF1 Gly146Ala polymorphism was noted. Gly146Ala minor allele frequency was significantly higher (p = 0.029) in POF patients versus controls and Ala allele containing genotypes (p = 0.005) were positively associated with POF pathology. The carriage of 146Ala allele was also associated with a significant reduction in estradiol plasma levels.

Conclusions

SF1 Gly146Ala polymorphism seems to be associated with POF pathology in the Tunisian population likely by reducing estradiol levels.     

46,XX, der(15),t(Y;15)(q12;p11) karyotype in an azoospermic male

We report on a Yq/15p translocation in a 23-year-old infertile male referred for Klinefelter Syndrome testing, who had azoospermia and bilateral small testes. Hormonal studies revealed hypergonadotropic hypogonadism. Conventional cytogenetic procedures giemsa trypsin giemsa (GTG) and high resolution banding (HRB) and molecular cytogenetic techniques Fluorescence In Situ Hybridization (FISH) performed on high-resolution lymphocyte chromosomes revealed the karyotype 46,XX, t(Y;15)(q12;p11). SRY-gene was confirmed to be present by classical Polymerase Chain Reaction (PCR) methods. His father carried de novo derivative chromosome 15 [45,X, t(Y;15)(q12;p11)] and was fertile; the karyotype of the father using G-band technique confirmed a reciprocal balanced translocation between chromosome Y and 15. In the proband, the der (15) has been inherited from the father because the mother had a normal karyotype (46,XX). In the proband, the der (15) could have produced genetic imbalance leading to unbalanced robertson translocation between chromosome Y and 15, which might have resulted in azoospermia and infertility in the proband. The paternal translocation might have lead to formation of imbalanced ova, which might be resulted infertility in the proband. Sister''s karyotypes was normal (46,XX) while his brother was not analyzed.     

Chromosomal abnormalities and hormonal disorders of primary amenorrhea patients in Egypt

BACKGROUND:

Primary amenorrhea is defined as the absence of menstruation and secondary sexual characteristics in phenotypic women aged 14 years or older. Hormonal disorders are main causes of primary amenorrhea. Common hormonal cause of primary amenorrhea includes pituitary dysfunction and absent ovarian function. The aim of this study was to estimate the incidence and types of chromosomal abnormalities in patients with primary amenorrhea in Egypt.

MATERIALS AND METHODS:

Chromosomal analysis and hormonal assay were carried out on 223 patients with primary amenorrhea that were referred from different parts of Egypt to Cytogenetic laboratory of Genetic Unit, Children Hospital Mansoura University, from July 2008 to December 2010. FISH technique was carried out in some of cases to more evaluation.

RESULTS:

The frequency of chromosomal abnormalities was 46 (20.63%) in primary amenorrhea patients. The chromosomal abnormalities can be classified into four main types. (1) The numerical abnormalities of the X chromosome were detected in 23 (50 %). (2) Structural abnormalities of the X chromosome were detected in 11 (23.91%). (3) Mosaicism of X chromosome was found in 10 (21.74%). (4) Male karyotype 46, XY was presented in 2 (4.35%).

CONCLUSION:

The present study showed that karyotype and FISH are necessary to detect the causes of primary amenorrhea. This study also revealed the incidence of chromosomal abnormalities in women with primary amenorrhea in Egypt is similar to that reported in previous literatures.     

Enzyme dimension of the ribosomal protein S4 across plant and animal kingdoms

Background

The protein S4 of the smaller ribosomal subunit is centrally important for its anchorage role in ribosome assembly and rRNA binding. Eubacterial S4 also facilitates synthesis of rRNA, and restrains translation of ribosomal proteins of the same polycistronic mRNA. Eukaryotic S4 has no homolog in eubacterial kingdom, nor are such extraribosomal functions of S4 known in plants and animals even as genetic evidence suggests that deficiency of S4X isoform in 46,XX human females may produce Turner syndrome (45,XO).

Methods

Recombinant human S4X and rice S4 were used to determine their enzymatic action in the cleavage of synthetic peptide substrates and natural proteins. We also studied autoproteolysis of the recombinant S4 proteins, and examined the growth and proliferation of S4-transfected human embryonic kidney cells.

Results

Extraribosomal enzyme nature of eukaryotic S4 is described. Both human S4X and rice S4 are cysteine proteases capable of hydrolyzing a wide spectrum of peptides and natural proteins of diverse origin. Whereas rice S4 also cleaves the -XXXD↓- consensus sequence assumed to be specific for caspase-9 and granzyme B, human S4 does not. Curiously, both human and rice S4 show multiple-site autoproteolysis leading to self-annihilation. Overexpression of human S4 blocks the growth and proliferation of transfected embryonic kidney cells, presumably due to the extraribosomal enzyme trait reported.

Conclusions

The S4 proteins of humans and rice, prototypes of eukaryota, are non-specific cysteine proteases in the extraribosomal milieu.

General significance

The enzyme nature of S4 is relevant toward understanding not only the origin of ribosomal proteins, but also processes in cell biology and diseases.     

Deoxyribonucleic acid damage study in primary amenorrhea by comet assay and karyotyping

AIM:

This study aims at evaluating the chromosomal abnormalities and deoxyribonucleic acid (DNA) damage in cases with primary amenorrhea by karyotyping and comet assay.

STUDY DESIGN:

A total of 30 cases of primary amenorrhea were recruited. Secondary sexual characters were assessed by Tanner staging. Chromosomal analysis was performed by conventional phytohemagglutinin stimulated lymphocyte cell culture technique. Alkaline version of comet assay was used to evaluate DNA damage.

RESULTS:

The chromosomal pattern of 20 subjects (66.7%) was found to be normal (46,XX). Two subjects had 46,XY pattern and eight subjects had Turner syndrome (45,X or 45,X/46,XX). The comet parameters were found to be increased among subjects with 45,X monosomy, when compared to the rest of the study group and also in subjects with Tanner stage 1 when compared to stage 2.

CONCLUSION:

Comet assay revealed increased DNA damage in cases with 45,X monosomy, compared with subjects with 46,XX and 46,XY karyotype, which correlated with clinical features.     

Mosaic triple X syndrome in a female with primary amenorrhea

BACKGROUND:

Turner''s syndrome is the most common chromosomal abnormality in females, affecting 1 in 2,500 live female births. It is a result of absence of an X chromosome or the presence of a structurally abnormal X chromosome. Its most consistent clinical features are short stature and ovarian failure.

AIM:

The aim of the study was to report a rare case of mosaic triple X syndrome in a female with primary amenorrhea.

MATERIALS AND METHODS:

The chromosomal analysis using GTG banding was carried out, which revealed a mosaicism with 45,XO/47,XXX chromosomal constitution. Fluorescent in situ hybridization was also carried out to further confirm the observation made in the study.

CONCLUSION:

The physical features presented by the female could be due to the 45,XO/47,XXX mosaicism and the karyotype analysis was consistent with the diagnosis and clinical symptoms. Triple X mosaicism was confirmed with conventional and molecular cytogenetic analysis.     

22q11.2 Microduplication in a patient with 19p13.12–13.13 deletion

Background

The chromosome 22q11.2 region microduplication has been described in patients with variable phenotypes. Here we present a 3-month-old girl with both 22q11.2 microduplication and 19p13.12–13.13 deletion. The presence of both genomic imbalances in one patient has not been previously reported in literature.

Methods

A routine G-banding karyotype analysis was performed using peripheral lymphocytes. Chromosome microarray analysis (CMA) was done using Affymetrix CytoScan™ HD array.

Results

The result of karyotyping showed that the patient is 46,XX,t(12;19)(q24.3;p13.1), but CMA detected a 2.8 Mb microduplication within the region 22q11.2 (chr22: 18,648,866–21,465,659) and a 1.2 Mb deletion on the chromosome 19at band p13.12–p13.13 (chr19: 13,107,938–14,337,347) in her genome, while no abnormalities were identified on 12q24.3. The 3-month-old girl presented with microcephaly, cleft palate, low set and retroverted ears, and facial dysmorphism which consisted of the following: a long narrow face, widely spaced eyes, downslanting palpebral fissures, broad nasal base, short philtrum, thin upper lip, and micro/retrognathia. She also had a congenital right pulmonary artery sling and tracheal stenosis and suffered from significant hypotonia and partial bilateral mixed hearing loss.

Conclusions

We report a case of 22q11.2 duplication syndrome with 19p13.12–13.13 deletion. Synergistic effect from the two genomic imbalances is likely responsible for the complicated clinical features observed in this patient.     

Triple X Egyptian woman and a Down's syndrome offspring

The 47, XXX karyotype (triple X) has a frequency of 1 in 1000 female newborns. However, this karyotype is not usually suspected at birth or childhood. Female patients with a sex chromosome abnormality may be fertile. In patients with a 47, XXX cell line there appears to be an increased risk of a cytogenetically abnormal child but the extent of this risk cannot yet be determined; it is probably lower in the non-mosaic 47, XXX patient than the mosaic 46, XX/47, XXX one. We describe a new rare case of triple X woman and a Down''s syndrome offspring. The patient is 26 years of age. She is a housewife, her height is 160 cm and weight is 68 kg and her physical features and mentality are normal. She has had one pregnancy at the age of 25 years resulted in a girl with Down''s syndrome. The child had 47 chromosomes with trisomy 21 (47, XX, +21) Figure 1. The patient also has 47 chromosomes with a triple X karyotype (47, XX, +X) Figure 2. The patient''s husband (27 years old) is physically and mentally normal. He has 46 chromosomes with a normal XY karyotype (46, XY). There are neither Consanguinity between her parent''s nor she and her husband.Open in a separate windowFigure 1Karyotype 47, XX + 21 of the daughter of Triple X syndromeOpen in a separate windowFigure 2Karyptype 47, XX + X of the Down syndrome''s mother     

Sulfation pattern of the fucose branch is important for the anticoagulant and antithrombotic activities of fucosylated chondroitin sulfates

Background

The aim is to compare the structures, anticoagulant and antithrombotic activities of two fucosylated chondroitin sulfates isolated from sea cucumbers Isostichopus badionotus (fCS-Ib) and Pearsonothuria graeffei (fCS-Pg), which were known to have different sulfation patterns on the fucose branches.

Methods

The structures of fCSs were identified using 2D NMR. Anticoagulant activities were measured by activated partial thromboplastin time (APTT) and thrombin time (TT), and inhibition of factors IIa, Xa and XIIa was assessed in vitro. Antithrombotic activity was determined ex vivo by measuring the length and weight of the thrombus generated.

Results

The two fCSs had identical chondroitin sulfate E backbones and similar fucose branches, but different sulfation patterns of the fucose branches. The fucose branch in fCS-Ib was mainly 2,4-O-sulfated whereas that in fCS-Pg was mainly 3,4-O-sulfated. In vitro assay indicated that fCS-Pg possessed much lower potency than fCS-Ib in prolonging APTT/TT and in inhibition of thrombin. However, they both exhibited similar inhibitory effects on factor X activation by intrinsic tenase complex, and on thrombus generation. Furthermore, both fCSs significantly activated factor XII, which has been proved to be associated with adverse clinical events associated with heparin contaminated by oversulfated chondroitin sulfate.

Conclusion

The 2,4-O-sulfated fucose branch is the key structural factor of fCSs for prolonged APTT/TT and inhibition of thrombin, whereas the inhibitory effect of fCSs on factor X, XII activation and thrombus generation was attributed to the overall structure of fCS polysaccharide.

General importance

Both fCSs have well defined structures and can be readily quality-controlled, and therefore may be potential alternatives for heparin as anticoagulant and antithrombotic drugs.     

Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects

BACKGROUND AND OBJECTIVE:

Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario.

MATERIALS AND METHODS:

A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup.

RESULTS:

Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals.

CONCLUSION:

Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India.     

Novel homozygous p.Y395X mutation in the CYP11B1 gene found in a Vietnamese patient with 11β-hydroxylase deficiency

Context

The deficiency of steroid 11β-hydroxylase is caused by mutations in the CYP11B1 gene and is the second major form of congenital adrenal hyperplasia associated with hypertension.

Objective

The objective of this study was to screen the CYP11B1 gene for mutations in one Vietnamese male suffering from congenital adrenal hyperplasia.

Patient

The patient (46,XY) had congenital adrenal hyperplasia. The clinical manifestations presented precocious puberty, hyper-pigmentation and high blood pressure at 4 years.

Results

The patient was a homozygous carrier of a novel mutation located in exon 7 containing a premature stop codon instead of tyrosine at 395 (p.Y395X).

Conclusion

We have identified a novel mutant of the CYP11B1 gene in one Vietnamese family associated with phenotypes of congenital adrenal hyperplasia. The mutant gene p.Y395X produces a truncated form of the polypeptide and abolishes the enzyme activities, leading to a severe phenotype of congenital adrenal hyperplasia.     

Rapid detection of Down's syndrome using quantitative real-time PCR (qPCR) targeting segmental duplications on chromosomes 21 and 11

Objective

Development of a qPCR test for the detection of trisomy 21 using segmental duplications.

Methods

Segmental duplications in the TTC3 gene on chromosome 21 and the KDM2A gene on chromosome 11 were selected as molecular markers for the diagnostic qPCR assay. A set of consensus primers selected from the conserved regions of these segmental duplications were used to amplify internal diverse sequences that were detected and quantified with different probes labeled with distinct fluorescence. The copy numbers of these two fragments were determined based on the ΔCq values of qPCR. The results of qPCR for prenatal and neonatal screening of Down's syndrome were compared with the conventional karyotype analysis by testing 82 normal individuals and 50 subjects with Down's syndrome.

Results

The ΔCq values of segmental duplications on chr21 and 11 ranged between 0.33 and 0.75 in normal individuals, and between 0.91 and 1.18 in subjects with Down's syndrome. The ΔCq values of these two segmental duplications clearly discriminated Down's syndrome from normal individuals (P < 0.001). Furthermore, the qPCR results were consistent with karyotype analysis.

Conclusion

Our qPCR can be used for rapid prenatal and neonatal screening of Down's syndrome.     

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP reductase

Background

Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans.

Methods

A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectrophotometry and ¹H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners.

Results

A plastidic type, high efficiently ferredoxin-NADP⁺ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis.

Conclusions

Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP⁺ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans.

General significance

Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.     

Effects of sex chromosome dosage on corpus callosum morphology in supernumerary sex chromosome aneuploidies

Background

Supernumerary sex chromosome aneuploidies (sSCA) are characterized by the presence of one or more additional sex chromosomes in an individual’s karyotype; they affect around 1 in 400 individuals. Although there is high variability, each sSCA subtype has a characteristic set of cognitive and physical phenotypes. Here, we investigated the differences in the morphometry of the human corpus callosum (CC) between sex-matched controls 46,XY (N =99), 46,XX (N =93), and six unique sSCA karyotypes: 47,XYY (N =29), 47,XXY (N =58), 48,XXYY (N =20), 47,XXX (N =30), 48,XXXY (N =5), and 49,XXXXY (N =6).

Methods

We investigated CC morphometry using local and global area, local curvature of the CC boundary, and between-landmark distance analysis (BLDA). We hypothesized that CC morphometry would vary differentially along a proposed spectrum of Y:X chromosome ratio with supernumerary Y karyotypes having the largest CC areas and supernumerary X karyotypes having significantly smaller CC areas. To investigate this, we defined an sSCA spectrum based on a descending Y:X karyotype ratio: 47,XYY, 46,XY, 48,XXYY, 47,XXY, 48,XXXY, 49,XXXXY, 46,XX, 47,XXX. We similarly explored the effects of both X and Y chromosome numbers within sex. Results of shape-based metrics were analyzed using permutation tests consisting of 5,000 iterations.

Results

Several subregional areas, local curvature, and BLDs differed between groups.Moderate associations were found between area and curvature in relation to the spectrum and X and Y chromosome counts. BLD was strongly associated with X chromosome count in both male and female groups.

Conclusions

Our results suggest that X- and Y-linked genes have differential effects on CC morphometry. To our knowledge, this is the first study to compare CC morphometry across these extremely rare groups.

     

Karyotyp-Phenotyp-Lorrelation bei einem 46,Xdel(X)(p22)-Befund

Summary In a patient with a height of 1.46 m, short neck and cubitus valgus the unbalanced karyotype 46,Xdel(X)(p22) was found. The mother of the proband has the balanced karyotype 46,Xt(X;15)(p22;p1). The mother is 1.56 m tall and has a short neck and cubitus valgus too. For this reason the deletion of the Xp22 band seems to cause the short stature of Turner patients. Our proband has had 2 pregnancies which ended as abortions in the 2nd and 4th month.As Giraud et al. (1974) have shown the deletion of the dark middle bande of the short X chromosome arm induces a slight dysgenesie of the gonade.     

Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background

An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.

Methods

We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.

Results

Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.

Conclusions

Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.

General significance

Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.     

Biosynthesis and function of chondroitin sulfate

Background

Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions.

Scope of review

Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo.

Major conclusions

Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes.

General significance

Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders.     

Residual Factor VIII-like cofactor activity of thioredoxin and related oxidoreductases

Background

Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood.

Methods

We screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay.

Results

We set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol–disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme.

Conclusion

Our findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases.

General significance

The possible involvement of thiol–disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.