Aid Of Trypsin Research Articles

Optimization of the biological processing of rice dregs into nutritional peptides with the aid of trypsin.

A protein hydrolysate was prepared from rice dregs (RD) using trypsin as a suitable protease. The hydrolysis conditions were optimized with response surface methodology, and then a mathematical model was developed to demonstrate the effect of each process parameter on the degree of hydrolysis (DH) and recovery of protein (RP). A hydrolysate with relatively high RP (75.81%) and low DH (6.95) was obtained from RD by hydrolyzing for 2.4h at pH7.6 52.8°C with a enzyme/RD ratio of 0.89:1000 (w/w) and RD/water level of 0.22g/mL. The dried hydrolysate was low molecular weight peptides (predominantly <1,000Da) and it possessed good solubility at various pH levels. Result of amino acid analysis revealed that the hydrolysate was considerably enriched in essential amino acids. Thus, the protein hydrolysate has a potential to be an excellent protein ingredient as a balanced milk replacer.

Development of a rapid and sensitive quantitative assay for rotavirus based on flow cytometry

A very sensitive and accurate flow cytometry (FC) based method have developed to quantitate rotavirus infection in MA104 cells. Confluent cell monolayers were infected with serial dilutions of rotavirus SA11. After infection, the cells were recovered with the aid of trypsin and then reacted with monoclonal antibody M60 (specific for the rotavirus outer capsid protein, VP7), followed by a second antibody (anti-mouse IgG-FITC). A FACScan FC was used to estimate the number of infected cells, as well as the level of infection. Viral infection was optimised by varying the concentration of trypsin used in the maintenance medium. The FC method enables many cells to be screened quickly for infectivity, and can detect low levels of virus. This method can be adapted to monitor the presence of other viruses in clinical and environmental samples without the need for prolonged periods of adaptation to growth in tissue culture.

Application of tryptic condensation strategy for the synthesis of α‐melanocyte stimulating hormone

The use of trypsin in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) paired with N,N-dimethylformamide (DMF) has been proposed for the enzymatic condensation of peptides. The tryptic condensation strategy was applied to the synthesis of the 13-peptide, alpha-melanocyte stimulating hormone (alpha-MSH), which contains two susceptible points to trypsin, in a low-water-containing solvent system, 4% H2O in HFIP/DMF (1/1, v/v). The N-terminal 8-peptide segment (S1) ending with Arg and C-terminal 5-peptide with the side-chain protections at Lys and Trp (S2) were prepared by the stepwise coupling on p-nitrobenzophenone oxime resin and by a conventional method, respectively. Both segments were condensed by the aid of trypsin. The acid component was converted into the partially protected alpha-MSH at as high as 95% conversion determined by reversed-phase HPLC. When the side-chain of Lys at the 11-position was not protected, alpha-MSH was obtained only in 35% yield, and was contaminated with products of secondary hydrolysis. Although the Lys11-Pro12 sequence was very poorly susceptible to trypsin, the side-chain of Lys had to be protected in order to be inert to trypsin under the synthetic conditions. HFIP is demonstrated as a good solvent with DMF to allow the efficient tryptic condensation of peptides. The strategy increased the value of the enzymatic condensation as practical method by avoidance of secondary hydrolysis, high dissolution of peptides and retention of activity of enzyme.

Proteolytic Enhancement of Human Rotavirus Infectivity

Rotaviruses are the most important etiologic agents of severe diarrhea worldwide. Despite great advances in vaccine development, little is known about host protection mechanisms other than immunity. This presentation focuses on the proteolytic enhancement of rotavirus infection, with emphasis on the functions of VP4, an outer capsid protein. The in vitro growth of human rotavirus is enhanced by trypsin, which selectively cleaves VP4. Treatment with trypsin increases the infectivity of human rotavirus while decreasing its hemagglutination activity. There are two modes of rotavirus internalization: direct penetration with the aid of trypsin and endocytosis without trypsin. Direct penetration via VP4 cleaved by trypsin is essential for the replication of the virus, whereas endocytotic internalization does not give rise to viral replication.

Cytopathic astrovirus isolated from porcine acute gastroenteritis in an established cell line derived from porcine embryonic kidney

A cytopathic astrovirus was isolated from pigs with acute diarrhea in an established cell line that was derived from porcine embryonic kidneys with the aid of trypsin. The virus showed a distinct cytopathic effect characterized by an enlargement of cells and the appearance of fine granules in the cytoplasm. Porcine astrovirus was shown to have an RNA genome, as determined by the effect of 5-iodo-2'-deoxyuridine on its replication, and five polypeptides with molecular masses of 13,000, 30,000, 31,000, 36,000, and 39,000 daltons; and it was shown to be stable to lipid solvents and heating at 50 degrees C for 30 min but somewhat labile to acid (pH 3.0). The buoyant density of the isolate determined in CsCl was 1.35 g/ml. Seroconversion to the virus was evident in the paired serum specimens obtained from pigs with diarrhea that were housed at the farm where the disease occurred. The neutralization test on serum specimens collected randomly from 128 adult pigs of eight herds revealed that 50 of the serum specimens were positive for antibody to porcine astrovirus, although there was considerable variation in the prevalence among herds, ranging from 0 to 83%. Hysterectomy-produced, colostrum-deprived, 4-day-old pigs developed mild diarrhea after oral exposure to porcine astrovirus propagated in the cell culture; and the virus was isolated again from diarrheal stool specimens.

Comparison of reaction rates in trypsin-catalysed transamidation of porcine insulin and its B29-arginine analogue.

[B29-Arginine]porcine insulin was prepared from des-(B23-30)-insulin and synthetic octapeptide with the aid of trypsin. Comparison of reaction rates in trypsin-catalysed transamidation of this compound and porcine insulin with threonine ether ester showed that this reaction is determined only by conformational effects and structural features of amino acids leaving from and entering into B30, not by the structure and the pKa value of the basic amino acid in B29.

Extraction of Carotenoprotein from Shrimp Process Wastes with the Aid of Trypsin from Atlantic Cod

ABSTRACTAtlantic cod trypsin or bovine trypsin were used to aid the extraction of carotenoprotein from shrimp wastes at 4°C. When 25 mg% cod trypsin was added to extraction medium containing 0.5N ethylene diaminetetraacetic acid (EDTA) 64% of the astaxanthin and 81% of the protein of shrimp waste was recovered as carotenoprotein in 24 hr. With 25 mg% bovine trypsin, under otherwise identical conditions, the carotenoprotein recovered represented 49% of the astaxanthin and 65% of the protein of the waste. Semi‐purified cod trypsin was not as effective as pure trypsin in facilitating recovery of carotenoprotein from shrimp waste. The recovery of carotenoprotein from shrimp waste, during extraction at 4°C with or without trypsin, was facilitated by EDTA.

Serial propagation of astrovirus in tissue culture with the aid of trypsin.

Astrovirus could be serially passed at least 13 times in primary human embryo kidney (HEK) cells when 10 micrograms/ml of crystalline trypsin was incorporated in a serum-free maintenance medium. In the presence of trypsin the virus was also passed and adapted to a continuous line of rhesus monkey kidney cells (LLCMK2) and primary baboon kidney (PBK) cells in which it was passed 25 and 16 times respectively, without evidence of diminishing infectivity. Attempts to adapt the virus to other cell lines (Vero, Hep II, MRC-5, BHK and HRT-18) were unsuccessful. After 11 passages in HEK cells, a titration of virus grown in different concentrations of trypsin showed that virus propagation was still trypsin-dependent.

A comparative study of chromosome G-banding using trypsin, papain, and pretreatment with emulphogene.

G-banding of chromosome metaphase preparations derived from haemic cells of healthy individuals and from patients with acute myeloid leukaemia was performed with the aid of trypsin, papain, and pretreatment of the chromosome spreads with emulphogene before proteolytic digestion. Papain digestion revealed more distinguishable bands than did trypsin digestion. Pretreatment of the chromosome spreads with emulphogene greatly enhanced the number of distinguishable bands for both enzymes. The combination of pretreatment with emulphogene and digestion with papain revealed optimal numbers of bands for individual chromosomes essentially identical with those agreed at the Paris Conference 1971. The use of the emulphogene-papain technique appears also to offer an advantage in the banding of chromosomes from leukaemic cells.

Isolation and comparative study of the nucleocapsids of measles and canine distemper viruses from infected cells

Nucleocapsids were isolated and purified from cells infected with measles and canine distemper virus (CDV). Electron microscopy of negatively stained nucleocapsids revealed the mean outside diameter was approximately 17–18 nm in each case and was not significantly different from similar measurements obtained with SV5 nucleocapsids and tobacco mosaic virus (TMV). All nucleocapsids also revealed central hollow cores which ranged from 4.3 to 5.2 nm. Direct chemical determinations showed that both CDV and measles virus nucleocapsids contained about 5% RNA. UV absorption spectra of measles and CDV nucleocapsids were typical for nucleoproteins and 280 260 nm ratios gave estimates of 5% RNA in each case. Buoyant density determinations in CsCl with both unlabeled and 32P-labeled nucleocapsids gave figures of 1.29–1.30 g/cc. Sedimentation analysis by analytical and sucrose gradient centrifugation revealed considerable heterogeneity, with nucleocapsids ranging from about 200–300 S. This heterogeneity was shown by electron microscopy to be due to the highly fragmented nature of the nucleocapsids. Analysis of CDV and measles nucleocapsids by SDS-polyacrylamide gel electrophoresis revealed a single major polypeptide when infected cells were harvested in the absence of proteolytic enzymes. These had estimated molecular weight of 54,000 (CDV) and 61,000 (measles). However, nucleocapsids prepared from cells harvested with the aid of trypsin or pronase contained two polypeptides (38,000 and 24,000 for measles; 27,000 and 22,500 for CDV), while similarly prepared SV5 nucleocapsid contained only a single polypeptide (44,500). These differences in polypeptide composition were correlated with differences in morphologic appearance of the nucleocapsids. Nucleocapsids harvested from cells in the absence of proteolytic enzymes had a loosely coiled, flexible appearance, while those harvested from cells treated with trypsin or pronase had a tightly coiled, rigid appearance. RNA was extracted from 32P-labeled measles and CDV nucleocapsids. Treatment with RNase A converted most of the 32P to a TCA-soluble form and DNase had no effect. Base compositions were determined on the 32P-labeled RNA and both viruses were rich in uracil, the molar ratios for the four bases were similar for each virus and were not significantly different from values reported in the literature for the better-characterized members of the Paramyxovirus group (NDV, Sendai and SV5).

Effect of trypsin on the infectivity of Sendai virus grown in several host cells.

As described in a previous study, Sendai virus formed plaques in chick embryo (CE) cell monolayers with the aid of trypsin incorporated into the agar overlay medium. The virus also formed plaqůes in monkey kidney (MR) cell monolayers without the aid of trypsin. Sendai virus grown in CE cells, as well as that grown in either of other stable cell lines tested, showed much lower plaque-forming unit (PFU) titers in MK monolayers than in CE monolayers. However, a mild trypsin treatment of the virus grown in these cells enhanced its PFU titers in MK monolayers to the level obtained in CE monolayers. In spite of this marked infectivity enhancement, the enzyme treatment little affected the hemagglutinin titers of the virus. On the other hand, Sendai virus grown in embryonated eggs or MK cells showed similar PFU titers in both CE and MK monolayers and also showed no evidence of infectivity enhancement due to the enzyme treatment.

Studies on the conservation of epidermal specificies of skin and certain mucosas in adult mammals.

To determine whether the factor(s) responsible for the conservation of epidermal specificities in adult guinea pigs and hamsters resides in the germinal layer of the epidermis or in the dermis, thin grafts of skin, possessing qualitatively distinct regional characteristics, were separated into their superficial epidermal and dermal components with the aid of trypsin. Dermis of one type was combined with epidermis of another to produce "recombinant" grafts which were then transplanted to small, full thickness cutaneous sites on the thorax of geneticaily compatible hosts. A variant of this procedure involved transplanting sheets of superficial epidermis of various types to shallow split thickness recipient areas in the skin of the thorax. All grafts were maintained for 100 days before they were excised and examined histologically. The results indicate that, whereas the dermis determines the kind of epidermis produced in recombinant grafts involving the ear, the sole of the foot, and the trunk, this is not the case in recombinants which include tongue, esophageal, or cheek pouch epithelia. The one exception to this occurred when tongue or esophagus epithelia were transplanted to split thickness beds in trunk skin. Here they appeared to produce an epidermis characteristic of their new location. It is believed that this exception is probably due to the fact that the native follicular epidermis present in trunk dermis made such a substantial contribution to the new superficial epidermis that it behaved overtly as body skin epidermis. Taken together, these results suggest that basal layer cells of the superficial epidermis of sole of foot skin, ear skin, and the hair-bearing skin of the general integument behave as if they are equipotential, and that in adult life maintenance of these particular epidermal specificities is the outcome of persistent specific inductive stimuli from the underlying dermis. The results of subsidiary experiments are reported which indicate that the epithelial component of mammary gland tissue is also pluripotential, being capable of producing, under appropriate conditions, a normal-looking, fully stratified superficial epidermis.

Purification and properties of hemoprotein 559 in bovine heart microsomes

From the bovine heart microsomes, a CO-binding hemoprotein has been solubilized with the aid of trypsin and extensively purified by fractionation with ammonium sulfate, chromatography on Sephadex, and electrophoresis on starch. The purified sample that migrates as a single band in electrophoresis at pH 9.5 and 12 possesses an extremely low peroxidase activity and contains a small amount of phosphorus. The pigment, when reduced, absorbs light at 423, 530, and 559 mμ at pH 9.5. The reduced pigment which combines with carbon monoxide is rapidly autoxidizable. Based on the α-absorption band of the ferrous spectrum at pH 9.5, the pigment was named hemoprotein 559. At pH 12, the α-band shifts to 557 mμ with a concomitant increase in absorbancy. The pH-dependent hyperchromism has been studied and interpreted as arising from the increased electronic transition probability in the porphyrin π-system.

In vitro cultivation of Eimeria acervulina (Coccidia)

Eimeria acervulina oocysts were sterilized with 50 ppm chlorine and viable sporozoites were released mechanically with the aid of trypsin and bile. These sporozoites, when suspended in the appropriate cell culture nutrient, were used as inoculum for chick embryo kidney, chick embryo fibroblast, mouse fibroblast, human amnion, and HeLa cell cultures. Infection of the host cell by sporozoites occurred in cultures of each cell type. Apparent cell infection occurred as early as one hour post inoculation, and forms recognized as trophozoites were observed at 18 to 24 hours. Further development failed to occur.