Experimental measurement of enzyme activity during initial crustacean growth using the ornamental shrimp, <i>Neocaridina denticulata</i> (De Haan, 1844) as a model and changes in activity with growth

Abstract. Kusmintarsih ES, Mahmoud HHA, Sastranegara MH, Syakuri H,Nuryanto A, Ambarningrum TB. 2023. Barcoding of ornamental freshwater shrimp, Neocaridina denticulata (De Haan, 1844) from the aquatic ornamental market in Purbalingga, Central Java, Indonesia. Biodiversitas 24: 3601-3608. Indonesia is one of the world’s leading aquatic ornamental shrimps producers and exports. One of the aquatic ornament shrimp species is a Neocaridina denticulate (De Haan, 1844). There is a wide range of colors including red, bright red, yellow, orange, green, blue, violet, black, and color combinations. It is a landlocked Atydiae family species comprising31 species and subspecies. Due to taxonomic difficulties, this species is continually under revision, and the validation of a few species is currently questionable and uncertain for the relationships among the various colors of N. denticulata. The Cytochrome Oxidase gene I (CO1) gene sequences of mitochondrial DNA were used for species DNA barcoding studies. This study aimed to validate the taxonomic status of different colors of N. denticulata species using the mitochondrial DNA or mtDNA Cytochrome oxidase (CO1) gene sequences. Neighbour Joining trees were constructed based on 545 bp of CO1 gene from white, black, red, yellow, blue colors, and their combination. Several sequences were derived from GenBank for relevant species from different countries and the outgroup. The result showed that all samples are homolog, even to N. denticulata from China NC_023823.1.

Freshwater ornamental shrimp, particularly Neocaridina denticulata

Primary hyperoxaluria type 2 (PH2) is a rare monogenic genetic disorder with an autosomal recessive pattern of inheritance. The disease is caused by mutations in the GRHPR gene encoding the glyoxylate/hydroxypyruvate reductase enzyme [2,12]. The high urinary excretion of oxalate and L-glycerate is a characteristic biochemical feature of PH2. Pathologically, the increased plasma and urinary oxalate leads to calcium oxalate supersaturation in the collecting ducts, which causes progressive renal deposition of calcium oxalate in the kidney, in the form of urolithiasis and/or nephrocalcinosis. In severe cases, this occasionally leads to renal failure and/or systemic oxalosis. The diagnostic tools of PH2 include the measurement of urinary oxalate and L-glycerate, genotyping for mutations of the GRHPR gene, the measurement of GRHPR enzymatic activity in the blood cells [5,13] and measurement of enzymatic activity in liver tissue. None of these assays are ideal. Measurement of enzymatic activity in liver biopsy is complicated by comorbidity, measurement in blood cells requires sensitive assays not generally available. Measurement of urinary metabolites can occasionally lead to misdiagnosis. While genetic screening offers potential solutions to problems created by other methods, there are currently too few mutations described and evaluated for functional consequences to use this method for efficient diagnosis. The GRHPR gene is located in the pericentromeric region of chromosome 9 [2]. The gene has 9 exons and encodes a 328 amino acid, 36 kDa protein [2,8]. To date, 13 mutations have been identified [2-4,6,11,12]. All mutations reported lead to a loss of enzyme expression or function. The most common of all the mutations is 103delG in exon 2, which results in a frameshift and induces a premature stop at codon 45 [2]. The prevalence of this mutation in PH2 is around 40% [3,11] of all reported mutations and appears to have originated in a founder of Northern European origin [11]. However, it has not been found in any patient of Asian origin. The 103delG mutation is a candidate mutation for DNA screening especially in patients of Caucasian origin. The sensitivity of diagnosis using 103delG mutation in DNA samples from the individuals referred for PH2 was 33% [9]. We describe a Japanese patient with PH2 who suffers from recurrent urolithiasis without nephrocalcinosis. This 19 year old patient was diagnosed through biochemical urine analysis by gas chromatographymass spectrometry at 10 months of age [14]. In the current report, we provide the molecular basis of PH2 in this patient.

This article describes the use of a microdensitometer for the measurement of formazan deposits in tissue sections. Some examples are given to illustrate the various applications of this technique in the assessment of glucose-6-phosphate dehydrogenase activity. These are (I) the separate measurement of the red half-formazan intermediate and purple diformazan of neotetrazolium, and the effect of incubation time on their production, (2) the measurement of activities in different regions of the liver lobule, and the selective effect of phenobarbitone, and (3) the measurement of enzyme activity in individual cartilage cells in normal and osteoarthrosis-prone animals. All activities can be expressed in absolute units as nmol hydrogen/mm3/hr, and thus compared with standard biochemical data. The activities obtained all fall within the range of published values for biochemical systems.

Mucolipidosis II α/β, mucolipidosis III α/β, and mucolipidosis III γ are autosomal recessive disorders belonging to the family of lysosomal storage disorders caused by deficiency of the UDP-N-acetylglucosamine, a lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) localized in the Golgi apparatus, which is essential for normal processing and packaging of soluble lysosomal enzymes with initiating the first step of tagging lysosomal enzymes with mannose-6-phosphate (M6P). Mucolipidosis II and III are caused by mutations in the GNPTAB and GNPTG genes, and patients with these diseases are characterized by short stature, skeletal abnormalities, and developmental delay. In this study we report 38 patients with mucolipidosis II and III enrolled in Eastern China during the past 8 years. The diagnosis was made based on clinical characteristics and measurement of plasma lysosomal enzyme activity. Sanger sequencing of GNPTAB and/or GNPTG for all patients and real-time quantitative PCR were performed to confirm the diagnosis. In addition, 11 cases of prenatal mucolipidosis II were diagnosed based on measurement of the enzyme activity in amniotic fluid supernatant and genetic testing of cultured amniotic cells. Based on molecular genetic tests, 30 patients were diagnosed with mucolipidosis II α/β, 6 were diagnosed with III α/β and 2 were diagnosed with III γ. Thirty-seven different GNPTAB gene mutations were identified in 29 patients with mucolipidosis II α/β and six patients with III α/β. These mutations included 22 new mutations (p.W44X, p.E279X, p.W416X, p.W463X, p.Q802X, p.Q882X, p.A34P, p.R334P, p.D408N, p.D534N, p.Y997C, p.D1018V, p.L1025S, p.L1033P, c.88_89delAC, c.890_891insT, c.1150_1151insTTA, c.1523delG, c.2473_2474insA, c.2980_2983delGCCT, c.3094delA, and deletion of exon 9). Four new GNPTG gene mutations were identified (c.13delC, p.Y81X, p.G126R and c.609+1delG) in two mucolipidosis III γ patients. Among the 11 cases of prenatal diagnosis, four were mucolipidosis II fetuses, three were heterozygous, and the remaining four were normal fetuses. This study expands the mutation spectrum of the GNPTAB and GNPTG genes and contributes to specific knowledge of mucolipidosis II/III in a population from Eastern China.

Operation control of anaerobic digesters on the basis of enzyme activity tests

Application of enzymatic activity measurements to biological phosphate removal from wastewater

The human kidney is a comparatively rich source of the enzyme acid phosphatase; the highest activity is found in the glomeruli. Since glomerular damage might be reflected by excretion of large quantities of this enzyme into the urine, the present study was undertaken to determine the biochemical characteristics of renal acid phosphatase as it appears in human urine and to establish optimum assay conditions for quantitative measurement of urinary enzyme activity. Enzyme kinetics, including substrate concentration requirement, Km values, and incubationtime characteristics, were determined for renal and urinary acid phosphatase. Stability of enzyme activity during storage and the effect of activators and inhibitors of the enzyme were examined. The influence of acid phosphatase activity derived from sources other than the kidney on the measurement of renal enzyme activity in the urine had to be considered; therefore, the selective inhibitory effects of various compounds on acid phosphatase derived from human kidney, prostate, and red and white blood cells have been studied. Renal and prostatic acid phosphatase share similar biochemical features, particularly with regard to selective inhibition. It was not possible to inhibit the urinary acid phosphatase contributed by prostate without simultaneously strongly affecting renal acid phosphatase. On the other hand, a selective inhibition of the urinary acid phosphatase contributed by erythrocytes and partially that contributed by leukocytes could be obtained. Bacteriuria was considered as a further source of error. The results indicate that it may be possible to utilize the measurement of urinary acid phosphatase activity as an index of glomerular damage in children and adult females but not in adult males, because of the contribution of prostatic acid phosphatase.

Aromatic l-amino acid decarboxylase enzyme activity in deficient patients and heterozygotes

Characterization of drug-neutralizing antibodies in patients with Fabry disease during infusion

Alignment of sequences of vertebrate beta-carotene 15,15'-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His172, His237, His308, His514, Asp52, Glu140, Glu314, Glu405, and Glu457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate non-conserved in the overall family with alanines by site-directed mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V(max). The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase.

Effect of the cosolvent type on the extraction of α-amylase with reversed micelles: Circular dichroism study

ectoenzymes and the latter enzymes. Substrates for hydrolysis are generally proteins, carbohydrates, fats and organic P- or S- compounds. Mechanisms of decomposition of individual compounds within these groups may be studied by in vitro experiments. However, aquatic microbial ecologists require, in many cases, a more general measurement of the in situ hydrolytic capacity of the prevailing bacterial community. This has led to the adaptation of biochemical methods for determination of overall bacterial extracellular enzyme activities (peptidases, a- and P-glucosidases, chi- tinases, etc.) in natural waters (Table 1). These methods enable us to study the impact of extracellular enzyme activity (EEA) on bacterial substrate uptake, bacterial growth, and water chemistry. The quantitative estimates of total bacterial extracellular enzyme activity are completed by rapid and sensitive tests for the detection of enzymatic properties of bacterial isolates. The methods used for these purposes are based on the application of fluorogenic model substrates. These substrates have some characteristics in common: (1) they contain an artificial fluorescent molecule and one or more natural molecules (e.g., glucose, amino acids), linked by a specific binding (e.g., peptide binding, ester binding); fluorescence is observed after enzymatic splitting of the complex molecule (Figure 1); (2) the hydrolysis of model substrates is competitively inhibited by a variety of natural compounds with the same structural characteristics; (3) hydrolysis of model substrates follows first order enzyme kinetics; and (4) application of those model substrates allows enzyme activity measurements under natural (in situ) conditions within short incubation periods. The latter is highly im- portant for microbial ecologists, because the process of enzymatic hydrolysis is fully

All worldwide newborn screening (NBS) for lysosomal storage diseases (LSDs) is performed as a first-tier test by measurement of lysosomal enzymatic activities in dried blood spots (DBS). The currently two available methodologies used for measurement of enzymatic activities are tandem mass spectrometry (MS/MS) and digital microfluidics fluorimetry (DMF-F). In this chapter we summarize the workflows for the two platforms. Neither platform is fully automated, but the relative ease of workflow will be dependent upon the specific operation of each newborn screening laboratory on a case-by-case basis. We provide the screen positive rate (the number of below cutoff newborns per 100,000 newborns) from all NBS laboratories worldwide carrying out MS/MS-based NBS of one or more LSDs. The analytical precision of the MS/MS method is higher than that for DMF-F as shown by analysis of a common set of quality control DBS by the Centers for Disease Control and Prevention (CDC). Both the MS/MS and DMF-F platforms enable multiplexing of the LSD enzymes. An advantage of MS/MS over DMF-F is the ability to include assays of enzymatic activities and biomarkers for which no fluorimetric methods exist. Advantages of DMF-F over MS/MS are: (1) simple to use technology with same-day turn-around time for the lysosomal enzymes with the fastest rates compared to MS/MS requiring overnight analytical runs.; (2) the DMF-F instrumentation, because of its simplicity, requires less maintenance than the MS/MS platform.

Immunocapture-based fluorometric assay for the measurement of insulin-degrading enzyme activity in brain tissue homogenates