[To enhance students' sense of purpose in learning].

The increase in the number of universities in Japan in spite of a decrease in the number of enrollees is causing a decline in the academic ability of undergraduates. The diversification of selection methods also contributes to the deterioration of the situation. Some students and teachers in high schools still hold the prejudice that only chemistry is important in the entrance examination for schools of pharmacy. To study pharmaceutical sciences, biology is as important as chemistry, and the number of students who have difficulty in obtaining biology course credits is increasing. Logical thinking based on the established knowledge in basic sciences is necessary for a successful clinical clerkship. However, students are inexperienced in logical thinking using the knowledge learned in their classes. This is why practice is needed during the basic pharmaceutical course. We made it compulsory for all second- and third-year students to take practical courses in physics, chemistry, and biology. In addition, a program in which a tutor conducted individual practice for students was carried out. A change in students' sense of purpose in learning was achieved by changing the method and environment of learning.

The unusual high molecular mass of Bothrops protease A, a trypsin-like serine peptidase from the venom of Bothrops jararaca, is due to its high carbohydrate content.

Bothrops protease A (BPA) is a serine peptidase isolated from the venom of Bothrops jararaca. Unlike many venom enzymes, it is stable at pHs between 3 and 9 and resists heating at 86 degrees C for 10 min. Mature snake venom serine peptidases of the chymotrypsin family are in general glycoproteins composed of around 232 amino acids and their molecular masses vary between 25 and 40 kDa. BPA is a glycosylated protein that migrates on SDS-polyacrylamide gel electrophoresis (PAGE) as a single band of 67 kDa. In order to find out whether BPA has the typical serine peptidase primary structure or if it is composed of a longer amino acid sequence, we cloned a cDNA encoding BPA. Its deduced amino acid sequence showed that BPA is composed of 234 residues with a calculated molecular mass of 25,409 Da implying that approximately 62% of its molecular mass assessed by SDS-PAGE is due to carbohydrate moieties. Eight putative N-glycosylation and two putative O-glycosylation sites were found in BPA amino acid sequence. Deglycosylation experiments indicated that all 10 potential glycosylation sites in BPA are utilized. Complete N- and O-deglycosylation was only achieved under denaturing conditions and generated main products of 25 and 55 kDa, respectively, which were enzymatically inactive. N-deglycosylation under non-denaturing conditions was only partial and gave a main product of 50 kDa and fragments ranging from 25 to approximately 10 kDa. Kinetic parameters K(m) and V(max) of partially N-deglycosylated BPA upon substrate Bz-Arg-pNA were similar to the native form. However, when partially N-deglycosylated BPA was submitted to pH 3 and pH 10, it appeared to be unstable as it underwent hydrolysis, as shown by the presence of two main products of 30 and 12 kDa while the 50 kDa protein band disappeared. These changes also had effects on V(max) upon Bz-Arg-pNA which dropped to approximately 45%, while K(m) values remained unchanged. Fluorescence emission spectroscopy indicated that in partially N-deglycosylated BPA, tryptophan residues are more exposed to a polar environment than in the fully glycosylated protein. Taken together, these studies indicate that glycosylation has a stabilizing effect on BPA.

Molecular cloning of serine proteinases from Bothrops jararaca venom gland.

Snake venom is known to contain an abundance of enzyme isoforms, and various disorders associated with envenomation have been ascribed partially to their diversified functions. Crude venom of Bothrops jararaca was subjected to conventional two-dimensional SDS-PAGE, followed by immunoblot analysis using an antiserum raised against KN-BJ 2, a serine proteinase previously isolated from this venom. A number of immunoreactive proteins with comparable molecular masses and different pIs emerged, implying the venom contains yet-unknown serine proteinases. A B. jararaca venom gland cDNA library was subsequently screened with a labeled KN-BJ 2 cDNA as a probe. Among a number of positive cDNA clones, three--HS112, HS114, and HS120--were selected and sequenced. These clones each had an open reading frame of 759-774 bp, and their deduced amino acid sequences illustrated considerable similarities to that of KN-BJ 2 as well as to those of serine proteinases of different origins. However, no apparent match to any of the deposited sequences was found in the current GenBank/EMBL databases, indicating that each of these cDNA clones encodes a serine proteinase distinct from the known enzymes. Analyses of the nucleotide and amino acid sequences of these cDNA clones support the accelerated evolution hypothesis proposed for snake venom enzymes.

Identification of multiple cytochrome P450 genes belonging to the CYP4 family in Xenopus laevis: cDNA cloning of CYP4F42 and CYP4V4.

In order to obtain cDNA clones coding for CYP4 proteins in frog Xenopus laevis, degenerate primers were designed utilizing the conserved sequences of known CYP4s and were used to amplify partial cDNA fragments from liver mRNA. Five new CYP genes were identified. Three of these genes, XL-1, -2 and -3, were assigned to the CYP4T subfamily found previously in fish and amphibians. The other two genes, XL-4 and XL-5, were quite similar to CYP4F and CYP4V subfamilies, respectively. Subsequently, two full-length cDNA clones corresponding to XL-4 and XL-5 were isolated and characterized. The resultant cDNAs, designated as CYP4F42 and CYP4V4, had open reading frames encoding proteins of 528 and 520 residues, respectively. RT-PCR analysis indicated that the expression of CYP4F42 was limited to the liver, kidney, intestine and brain. In contrast, CYP4V4 mRNA was expressed ubiquitously.

A novel peptide from the ACEI/BPP-CNP precursor in the venom of Crotalus durissus collilineatus.

In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of a gene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii, ACEI/BPP sequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are located together at the N-terminus, but without repeats. An additional sequence for CNP was unexpectedly found at the C-terminus. Homologous genes for the ACEI/BPP-CNP precursor suggest that most crotaline venoms contain both ACEIs/BPPs and CNP. The sequence of ACEIs/BPPs is separated from the CNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study, analysis of the cDNA clone from C. d. collilineatus revealed a nearly identical sequence in the ACEI/BPP-CNP spacer. Fractionation of the crude venom by reverse phase HPLC (C(18)), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acid sequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired proline residues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activity when injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidence suggests that these novel toxins probably activate anaphylatoxin C3a receptors.

Molecular cloning and expression analysis of the aryl hydrocarbon receptor of Xenopus laevis.

The aryl hydrocarbon receptor (AHR) is a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors. Although this receptor has been known to mediate the toxic effects of environmental pollutants, its physiological functions remain elusive. Here, we describe the isolation and expression pattern of the Xenopus AHR gene. The predicted amino acid sequence contained regions characteristic of other vertebrate AHRs. However, in line with previously described fish AHR genes, no distinct Q-rich domain was found. Phylogenetic analysis demonstrated that Xenopus AHR was clustered within the AHR1 clade. As in the case of mammalian AHR genes, the Xenopus AHR gene was expressed in all the adult tissues tested. Xenopus AHR was also expressed during early development, in parallel with expression of the CYP1A7 gene, which is thought to be regulated by AHR. These results suggest that while frogs are relatively tolerant to TCDD toxicity, the AHR of frogs has characteristics similar to those of other vertebrate AHRs.