Colorado potato beetle chymotrypsin genes are differentially regulated in larval midgut in response to the plant defense inducer hexanoic acid or the Bacillus thuringiensis Cry3Aa toxin.

When Colorado potato beetle larvae ingested potato plants treated with the plant defense inducer compound hexanoic acid, midgut chymotrypsin enzyme activity increased, and the corresponding chymotrypsin genes were differentially expressed, evidence of the larval digestive proteolytic system's plasticity. We previously reported increased susceptibility to Cry3Aa toxin in larvae fed hexanoic acid treated plants. Here we show that the most expressed chymotrypsin gene in larvae fed hexanoic acid treated plants, CTR6, was dramatically downregulated in Cry3Aa intoxicated larvae. lde-miR-965-5p and lde-miR-9a-5p microRNAs, predicted to target CTR6, might be involved in regulating the response to hexanoic acid but not to Cry3Aa toxin.

Treatment and mechanism of BMMSCs on deep II degree scald of hamster skin.

In this study, we examined the treatment and mechanism of BMMSC on a deep II degree scald of the hamster skin. A deep II degree scald model on the skin of 40 hamsters was duplicated and divided randomly into a stem cell plantation group (group A) and model control group (group B). Skin cells were cultured in vitro until the allogeneic BMMSCs of the 5th generation formed with a cell count of 1 x 10(7)/mL. Local injection plus liquid supernatant smearing was used to plant the cells into the position of the scald in the stem cell plantation group. The control group was given an equivalent amount of normal saline to observe the healing action, and 5 samples were taken in each group after 1, 3, 7, and 14 days for hematoxylin and eosin staining for physiological observation. Polymerase chain reaction was used to detect the amount of chymotrypsin in mast cells. The speed of healing in the stem cell transplantation group was greater than that in the control group; staining results showed that the quality of healing in the transplantation group was better than that in the control group. Chymotrypsin expression was detected in both groups, reaching a peak on day 3. BMMSCs can accelerate wound healing, and chymotrypsin in mast cells may participate in the wound healing process.

[The effect of carbon tetrachloride poisoning on the activity of digestive proteases in rats and correction of the disorders with vegetable oils].

The results of the study of activity of digestive proteases (pepsin, trypsin, chymotrypsin) in homogenates of stomach, pancreas and duodenum in experimental animals have been presented. Rats were exposed to intoxication with carbon tetrachloride (subcutaneous administration of a 50% oil solution of CCl4 in the dose of 0.5 ml per 100 g body weight) for three days and then they were given analysed oils (black nut, walnut and flax oil) intragastrically by gavage at a dose of 0.2 ml per day within 23 days. Pepsin level in gastric mucosa homogenates and chymotrypsin activity in pancreatic homogenates were determined by method of N.P. Pyatnitskiy based on on the ability of enzymes to coagulate dairy-acetate mixture, respectively, at 25 degrees C and 35 degrees C. Trypsin activity in homogenates of pancreatic was determined by method of Erlanger - Shaternikova colorimetrically. It has been established that intoxication with CCl4 decreased the synthesis of proteolytic enzymes of the stomach (by 51%) and pancreas (by 70-78%). Injections of analysed vegetable oils to animals contributed to the normalization of proteolytic enzymes synthesis. The conclusion that there are prospects of using the analysed vegetable oils containing large quantity of polyunsaturated fatty acids (omega-3 and omega-6) for the correction of detected biochemical abnormalities has been done.

Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells.

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.

Regulation of major acute-phase plasma proteins by hepatocyte-stimulating factors of human squamous carcinoma cells.

Human squamous carcinoma (COLO-16) cells release factors which specifically stimulate the synthesis of major acute-phase plasma proteins in human and rodent hepatic cells. Anion exchange, hydroxyapatite, lectin, and gel chromatography of conditioned medium of COLO-16 cells result in separation into three distinct forms of hepatocyte-stimulating factors (designated HSF-I, HSF-II, and HSF-III) with apparent molecular weights of 30,000, 50,000 and 70,000, respectively. None of the preparations contains detectable amounts of thymocyte-stimulating activity. Each of the three HSF forms stimulates the accumulation of mRNA for alpha 1-antichymotrypsin in the human hepatoma cell line, HepG2. When the same factors were added to primary cultures of adult rat hepatocytes, the expression of the same set of plasma proteins was modulated as by nonfractionated medium. The hormonally induced accumulation of mRNA for acute phase proteins is qualitatively comparable to that occurring in the liver of inflamed rats. Unlike in human cells, in rat liver cells dexamethasone acts additively and synergistically with HSFs. The only functional difference between the three HSF forms lies in the level of maximal stimulation. HSF-I represents the predominant form produced by normal human keratinocytes and closely resembles in molecular size and isoelectric point the activity produced by activated peripheral blood monocytes while the larger molecular weight forms are more prevalent in human as well as mouse squamous carcinoma cells. The observation that HSFs from different sources elicit essentially the same pleiotropic response in hepatic cells led to the hypothesis that the species-specific reaction of adult liver cells to inflammatory stimuli is pre-programmed and that the function of any HSF is to trigger and tune the execution of this fixed cellular process.

Isoleucine Regulates the Synthesis of Pancreatic Enzymes via the Activation of mRNA Expression and Phosphorylation in the Mammalian Target of Rapamycin Signalling Pathways in Pancreatic Tissues.

This study aimed to investigate the effects of isoleucine (Ile) on the synthesis and secretion of digestive enzymes and cellular signalling in the pancreatic tissue of dairy goats. The pancreatic tissues were incubated in buffer containing 0, 0.40, 0.80, and 1.60 mM Ile. High levels of Ile significantly increased the buffer release and total concentration of ɑ-amylase in the tissues (P < 0.001). The total trypsin and chymotrypsin concentrations in each of the Ile groups were significantly higher than those in the control group (P < 0.05); however, lipase was not affected. High levels of Ile significantly increased ɑ-amylase mRNA expression (P < 0.001) but had no effect on the mRNA expression of trypsin, chymotrypsin, or lipase. Ile did not affect S6K1 phosphorylation levels. High levels of Ile significantly increased the expression of the γ isoform of 4EBP1 (P < 0.001), which indicated that the phosphorylation of 4EBP1 was significantly increased. The phosphorylation level of eEF2 gradually decreased with the addition of Ile (P < 0.001). These results suggested that high doses of Ile can regulate the excretion of enzymes, especially ɑ-amylase, in the pancreatic tissues of dairy goats by modulating mTOR signalling, and this regulation is independent of the mTOR-S6K1 pathway.

Synthesis of alpha 1-antichymotrypsin and alpha 1-acid glycoprotein by human breast epithelial cells.

Malignant and uninvolved human breast tissues were maintained in organ culture for 3 to 6 days. Under these conditions, the three-dimensional glandular architecture is maintained with the least disruption of tissue integrity. The biosynthesis and release of glycoproteins were studied by using the incorporation of [14C]glucosamine and [14C]leucine by the breast surgical specimens. Five major families of labeled glycoproteins were identified from culture supernatants using two-dimensional gel electrophoresis. Quantitative immunoprecipitation established that 16 to 30% of the total of labeled glycoproteins were recognized as normal serum components. Two of these glycoproteins were antigenically related to normal human serum components as demonstrated with crossed immunoelectrophoresis. Evidence was obtained for the active synthesis of alpha 1-antichymotrypsin and alpha 1-acid glycoprotein by human breast epithelial cells. alpha 1-Antichymotrypsin accounted for 0.9 to 7.8% of the biosynthetically labeled glycoproteins from organ culture supernatants. This component was 11.9% of the glycoproteins released by a monolayer culture of the established breast carcinoma cell line, MCF-7. alpha 1-Acid glycoprotein made up 0.7 to 3.1% of the labeled glycoproteins. alpha 1-Antichymotrypsin is a known neutral serine proteinase inhibitor with a particularly strong affinity for cathepsin G. alpha 1-Acid glycoprotein may function primarily as a potent immunomodulator by suppressing lymphoblastogenesis. These glycoproteins may thus have regulatory roles in the proteolytic modification of breast tissue and represent the tissue's own protecting shield against invading leukocytes.

Production of human alpha 1-antichymotrypsin-like protein by a human malignant melanoma transplanted into nude mice.

A tissue culture line of a human malignant melanoma, SEKI, induced cachexia in nude mice (BALB/c-nu/nu) (Kondo et al., Cancer Res., 41: 2912-2916, 1981). During the investigation of the cause of the cachexia, the melanoma was found to produce a protein immunologically identical to human alpha 1-antichymotrypsin (alpha 1-Ach). Tissues of the SEKI melanoma contained the protein immunologically equivalent to 0.29 +/- 0.11 (S.D.) mg of human alpha 1-Ach per g of wet tissue, while the other six human malignant tumors transplanted into nude mice did not contain a detectable amount of it. In the serum of nude mice bearing the melanoma, this protein appeared soon after the tumor growth occurred and gradually increased up to the level equivalent to 5 mg of human alpha 1-Ach per dl. Removal of the tumor resulted in a rapid decrease of the protein in the serum to an undetectable level within 1 day. This problem was never detected in the serum of nude mice bearing the other 27 human malignant tumors or controls. Purification of this protein was carried out by the column chromatography using DE-52, Blue-Sepharose, and SP-Sephadex. The elution patterns were the same as those of alpha 1-Ach in human serum, and the molecular weight of the protein was estimated as 69,000 by Sephadex G-100 column chromatography and 65,000 by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. This purified protein, however, did not exhibit inhibitory activity against chymotrypsin. These results show that this melanoma produced a protein immunologically identical and physicochemically very similar to human alpha 1-Ach. This melanoma-nude mouse system may provide a useful model for investigating the synthesis of human alpha 1-Ach and analysis of its physiological roles.

Adaptive regulation of digestive serine proteases in the larval midgut of Helicoverpa armigera in response to a plant protease inhibitor.

Protease inhibitors (PIs) are direct defenses induced by plants in response to herbivory. PIs reduce herbivore digestive efficiency by inhibiting insects' digestive proteases; in turn insects can adapt to PIs by generally increasing protease levels and/or by inducing the expression of PI-insensitive proteases. Helicoverpa armigera, a highly polyphagous lepidopteran insect pest, is known for its ability to adapt to PIs. To advance our molecular and functional understanding of the regulation of digestive proteases, we performed a comprehensive gene expression experiment of H. armigera exposed to soybean Kunitz trypsin inhibitor (SKTI) using a custom-designed microarray. We observed poor larval growth on the SKTI diet until 24 h, however after 48 h larvae attained comparable weight to that of control diet. Although initially the expression of several trypsins and chymotrypsins increased, eventually the expression of some trypsins decreased, while the number of chymotrypsins and their expression increased in response to SKTI. Some of the diverged serine proteases were also differentially expressed. The expression of serine proteases observed using microarrays were further validated by qRT-PCR at different time points (12, 24, 48, 72 and 96 h) after the start of SKTI ingestion. There were also large changes in transcriptional patterns over time in the control diet. Carbohydrate metabolism and immune defense genes were affected in response to SKTI ingestion. Enzyme assays revealed reduced trypsin-specific activity and increased chymotrypsin-specific activity in response to SKTI. The differential regulation of trypsins and chymotrypsins at the transcript and protein levels accompanying a rebound in growth rate indicates that induction of SKTI-insensitive proteases is an effective strategy of H. armigera in coping with this protease inhibitor in its diet.

Geotrichum candidum P-5 produces an intracellular serine protease resembling chymotrypsin.

A wide range of intra- and extracellular microbial proteases has been studied and characterized. These enzymes are mostly extracellular and in some cases they may resemble 'classical' serine proteases. As part of a programme in which the lipase and protease activities of the fungus Geotrichum candidum are being studied, an intracellular protease with an apparent chymotrypsin-like specificity was detected. The serine protease was isolated from biomass using ion-exchange and exclusion chromatography. Kinetic characterization was done using a series of synthetic substrates and inhibitors. Aprotinin-sepharose affinity chromatography was used to isolate a fraction for molecular size determination on SDS-PAGE. The purified protease, which could hydrolyse haemoglobin as protein substrate, was obtained with a 30-fold purification and a yield of 44%, but it was very unstable and rapidly lost activity. The enzyme which bound to the affinity column had a single subunit mass of 278 kDa. Kinetic analysis showed a similarity with trypsin and chymotrypsin, but tending more towards chymotrypsin in that a bulky aromatic group, e.g. phenylalanine in the P1 position, was preferred. The optimum pH was in the region of 7-8.25. Inhibition patterns indicated that the enzyme was a serine protease with no metal dependence, although it was stabilized by magnesium ions. The enzyme seems to share some properties with other intra- and extracellular microbial serine proteases. The exact function of the enzymatic activity is still unclear, but it is suggested that it may be involved with intracellular protein turnover.

Biosynthesis of abnormally glycosylated hepatoma secretory proteins in cell cultures.

We studied, by electrophoretic techniques, the physiochemical properties of 4 glycoproteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein and transferrin synthesized by three different human hepatoma cell lines. A common feature was the export of glycoproteins with retarded electrophoretic mobility, indicating incomplete sialylation, and a predominance of atypical, highly branched carbohydrate chains. The abnormal glycosylation pattern may be specific for malignant transformation of hepatocytes and possibly related to the intracellular accumulation of some of these proteins in malignant cells.

Limited proteolyses in pancreatic chymotrypsinogens and trypsinogens.

In the 1950's, the specific cleavages of the peptide bonds occurring in bovine cationic chymotrypsinogen and trypsinogen were among the first examples of limited proteolyses. According to the split bond(s), the precursor is transformed into enzyme or different forms of zymogen or again into inert protein. The conversion of trypsinogen into trypsin triggers the activations of all the other enzyme precursors of pancreatic juice. In the pancreas, several factors oppose trypsinogen autoactivation, whereas, in the duodenum, all the conditions are favorable for trypsinogen activation by enteropeptidase.

Estradiol increases the production of alpha 1-antichymotrypsin in MCF7 and T47D human breast cancer cell lines.

alpha 1-Antichymotrypsin (Achy) is an antiprotease of the acute inflammation phase, which is also released by MCF7 human breast cancer cells in culture. Using a fluorimetric assay with the synthetic substrate L-Seryl-L-Tyrosyl-2-N-naphthylamide, we have shown that a medium conditioned by MCF7 cells treated by estradiol inhibits the activity of alpha-chymotrypsin. This inhibition increased when physiological concentrations of estradiol were added to the cells for 2 days. It was due to an increased production of Achy and not to a direct effect of estradiol on alpha-chymotrypsin activity as shown by double immunoprecipitation with an antiserum against human alpha 1-antichymotrypsin. An increased accumulation by estradiol of an antigen located in the cytoplasm of MCF7 cells, which was revealed by immunoperoxidase staining with antibodies to Achy, also indicated that estradiol increased the production of Achy in these cells. Similar immunostaining was observed in a breast cancer tissue. Most of the estrogen regulated 60-68 kDa protein secreted by T47D cells (Chalbos et al. (1982) J. Clin. Endocrinol. Metab. 55, 276-283) was also specifically immunoprecipitated by the antibodies to Achy. Thus, alpha 1-antichymotrypsin is the first protein to be identified which is induced by estradiol and secreted by breast cancer cells.

Mass spectrometry and characterization of Aedes aegypti trypsin modulating oostatic factor (TMOF) and its analogs.

Trypsin modulating oostatic factor (TMOF), a decapeptide that directly inhibits the biosynthesis of trypsin- and chymotrypsin-like enzymes in epithelial cells of mosquito midgut and indirectly inhibits vitellogenesis in anautogenous females, has been sequenced by Fourier transform mass spectrometry analysis. The peptide has a primary amino acid sequence of NH2-Tyr-Asp-Pro-Ala-(Pro)6-COOH and probably exhibits left-handed helical conformation as was shown by computer stereoview simulation. The factor is metabolized very rapidly (half-life of 1.6 h) in intact mosquitoes when injected after the blood meal. Inhibition of trypsin biosynthesis was followed in ligated abdomens, which synthesize trypsin but do not metabolise TMOF. At concentrations of 3 x 10(-9) M and 6.8 x 10(-6) M, TMOF inhibited 50 and 90% of trypsin-like enzyme biosynthesis, respectively. Several analogs of varying chain lengths were synthesized and evaluated for biological activity using dose-response curves. Switching the positions of Tyr and Asp at the N-terminus reduced the activity of the hormone, indicating that the N-terminus is important for biological activity. Removal of two to five prolines at the C-terminus also reduced activity, indicating that both the N- and C-termini are important. Synthesis of trypsin-like isozyme was followed in several insect species using [1,3-3H]diisopropyl-fluorophosphate (DFP) in the presence of tosylamide-2-phenylethyl chloromethyl ketone. Marked reduction of [1,3-3H]diisopropyl-phosphoryl-trypsin-like derivatives was noted after TMOF treatment, as assessed by polyacrylamide gel electrophoresis. These results indicate that the biosynthesis of trypsin-like enzyme in mosquitoes and other insects may be regulated by sequence-related TMOFs.

Characterization of major midgut proteinase cDNAs from Helicoverpa armigera larvae and changes in gene expression in response to four proteinase inhibitors in the diet.

A Helicoverpa armigera larval midgut cDNA library from larvae raised on an artificial, protein-rich, inhibitor-free diet contained very large numbers of serine proteinase positive clones. DNA sequencing of six random positive cDNAs and 12 PCR derived products identified trypsin genes classifiable into three families, and chymotrypsin and elastase genes classifiable into a single family each. Genomic blots established that the most highly expressed of the trypsin families contained about 18 genes, and that the chymotrypsin and elastase families contained about 14 and 2 genes respectively. The levels of mRNA corresponding to the highly expressed trypsin and chymotrypsin families were determined following chronic ingestion of four proteinase inhibitors. Compared to insects on an inhibitor-free diet, chymotrypsin mRNA was increased by all inhibitors, and trypsin mRNA levels decreased. This occurred independent of whether the inhibitor was solely a trypsin inhibitor (aprotinin), an inhibitor of both trypsin and chymotrypsin (proteinase inhibitor II, soybean trypsin inhibitor) or predominantly a chymotrypsin inhibitor (proteinase inhibitor I). Changing the protein level of the diet did not affect trypsin mRNA levels, but chymotrypsin mRNA levels decreased with increasing dietary protein.

Effects of chronic administration of somatostatin on rat exocrine pancreas.

We studied the effects of somatostatin on synthesis of pancreatic DNA, RNA and protein and on pancreatic weight and contents of DNA, protein, amylase and chymotrypsinogen in rats. In short term synthesis studies, rats were injected with 100 micrograms . kg-1 somatostatin or 0.15 M NaCl (control) at times 0, 8 and 16 h. Eight rats from each treatment group were killed 2, 4, 8, 12, 16, 20 and 24 h after beginning treatment. Incorporation rates in vivo of [3H]thymidine into DNA, [3H]uridine into RNA and [14C]phenylalanine into total protein were significantly depressed by somatostatin. In long term studies, four groups of 12 rats were injected every 8 h for 5 days with 0.15 M NaCl or 11, 33 or 100 micrograms . kg-1 somatostatin. Body weight was unaffected but pancreatic contents of DNA, protein and enzymes were significantly decreased by somatostatin. Administration of somatostatin inhibits DNA, RNA and protein synthesis in exocrine pancreas with resulting decreases in DNA and enzyme contents.

Extracellular fibrinogenolytic enzyme of Aspergillus fumigatus: substrate-dependent variations in the proteinase synthesis and characterization of the enzyme.

To get a better understanding of the role of the previously reported fibrinogenolytic enzyme of Aspergillus fumigatus, we investigated the in vitro conditions of enzyme synthesis and attempted to characterize it. Modification of the nitrogen source did not influence the extracellular serine-proteinase profile, but resulted in important quantitative differences in the yields in batch cultures. The enzyme synthesis appeared to be an inducible phenomenon in A. fumigatus since it was initiated exclusively in the presence of proteins or protein hydrolysate. Free amino acids or inorganic nitrogen compounds could not promote significant enzyme production. Moreover, peptone at a concentration of 0.1% appeared to be the best inducer of enzyme synthesis. Conversely, modification of the carbon source did not affect fungal growth or enzyme synthesis. However, the production of chymotrypsin was highly sensitive to the carbohydrate level in the culture medium and, with peptone as nitrogen source, highest yields were obtained in the presence of 0.3 or 0.5% glucose. Culture filtrates of A. fumigatus CBS 113.26 grown with peptone or nitrate as nitrogen source were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the protein patterns suggested for the proteinase a molecular mass of 33 kDa which was confirmed by chromatographic purification of the enzyme through (N alpha-CBZ)-D-phenylalanine agarose.

Enzyme production by recombinant Trichoderma reesei strains.

The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.

Immunohistochemistry in the differential diagnosis of acinar and endocrine pancreatic neoplasms.

Histologic differential diagnosis of acinar cell carcinoma (ACC), mixed acinar-endocrine cell carcinoma (MAEC), and pancreatic endocrine tumors (PET) can be difficult but is important because of differences in their clinical behavior. This study investigates the utility of immunohistochemistry (IHC) in this differential diagnosis using immunohistochemical stains that are available in most laboratories. IHC was performed on paraffin-embedded tissue in ACC (n = 6), MAEC (n = 2), and PET (n = 13), using synaptophysin (SYN), chromogranin (CHR), chymotrypsin (CHY), and alpha-1-antitrypsin (AAT). Electron microscopy (EM) was performed in all cases to confirm the diagnosis. Long-term follow-up and death of disease (DOD) was known in all patients. The ACCs stained as follows: CHY (4/6), AAT (3/6), SYN (4/6); CHR was negative in all cases. Both cases of MAEC stained with CHY, AAT, and SYN (2/2); CHR was negative. PET stained as follows: SYN (13/13), CHR (8/13), CHY (4/13), AAT (5/13). In the ACC/ MAEC group, six of eight patients were DOD at mean follow-up of 11 months. Among the PET, two of 16 patients were DOD at mean follow-up of 37 months. Considerable immunophenotypic overlap exists between ACC, MAEC, and PET. Consequently, one can neither confirm nor rule out a diagnosis of ACC or MAEC using generally available immunohistochemical stains alone. These findings support a role for EM in the evaluation of exocrine and endocrine pancreatic neoplasms.

Biosynthesis of serine proteases in Lutzomyia anthophora (Diptera: Psychodidae).

Changes in the biosynthesis of serine proteases in adult Lutzomyia anthophora Addis were followed and compared with the larval and pupal stages. More chymotrypsinlike than trypsinlike enzyme was synthesized by 2-d-old and 3-d-old sugar-fed females and females that were fed blood 72 h earlier. A small increase in the amount of chymotrypsinlike enzyme occurred within the first 48 h after blood feeding, whereas trypsinlike enzyme activity increased rapidly after the blood meal and peaked at 72 h. [1,3-3H]DIP trypsinlike and chymotrypsinlike derivatives of sugar-fed and blood-fed females were compared using polyacrylamide gel electrophoresis.