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Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Our previous studies revealed that the double variants of CYP105A1- R73A/R84A and R73V/R84A-show high levels of activity with respect to conversion of vitamin D3 to its biologically active form, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study, we found that both the double variants were also capable of converting vitamin D2 to its active form, that is, 1α,25-dihydroxyvitamin D2 (1α,25(OH)2D2), via 25(OH)D2, whereas its 1α-hydroxylation activity toward 25(OH)D2 was much lower than that toward 25(OH)D3. Comparison of the wild type and the double variants revealed that the amino acid substitutions remarkably enhanced both 25- and 26-hydroxylation activity toward vitamin D2. After 25-hydroxylation of vitamin D2, further hydroxylation at C26 may occur frequently without the release of 25(OH)D2 from the substrate-binding pocket. Thus, the double variants of CYP105A1 are quite useful to produce 25,26(OH)2D2 that is one of the metabolites of vitamin D2 detected in human serum.

The emergence of Magnaporthe oryzae strains with resistance to quinone outside inhibitor (QoI) fungicides necessitates the development of easy techniques for the rapid, sensitive monitoring of such strains. A QoI-resistant mutant and a wild-type strain showed a single nucleotide polymorphism (SNP). For simple, fast, low-cost, and reliable detection of this SNP, we developed two types of PCR-based DNA markers that enable easy detection of resistant strains by using DNA templates prepared from filter paper permeated with the fungus. The method enabled us to obtain reliable results by using a detection procedure that takes only a few hours without special kits.

Bioconversion processes, including specific hydroxylations, promise to be useful for practical applications because chemical syntheses often involve complex procedures. One of the successful applications of P450 reactions is the bioconversion of vitamin D₃ to 1α,25-dihydroxyvitamin D₃. Recently, a cytochrome P450 gene encoding a vitamin D hydroxylase from the CYP107 family was cloned from Pseudonocardia autotrophica and is now applied in the bioconversion process that produces 1α,25-dihydroxyvitamin D₃. In addition, the directed evolution study of CYP107 has significantly enhanced its activity. On the other hand, we found that Streptomyces griseolus CYP105A1 can convert vitamin D₃ to 1α,25-dihydroxyvitamin D₃. Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity. To date, multiple vitamin D hydroxylases have been found in bacteria, fungi, and mammals, suggesting that vitamin D is a popular substrate of the enzymes belonging to the P450 superfamily. A combination of these cytochrome P450s would produce a large number of compounds from vitamin D and its analogs. Therefore, we believe that the bioconversion of vitamin D and its analogs is one of the most promising P450 reactions in terms of practical application.

Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V/R84A, has a high ability to convert vitamin D(3) to its biologically active form, 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)], suggesting the possibility for R73V/R84A to produce 1α,25(OH)(2)D(3). Because Actinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance, we examined the expression of R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected, the metabolites 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1α,25(OH)(2)D(3) were detected in the cell culture of the recombinant S. lividans. A large amount of 1α,25(OH)(2)D(3), the second-step metabolite of vitamin D(3), was observed, although a considerable amount of vitamin D(3) still remained in the culture. In addition, novel polar metabolites 1α,25(R),26(OH)(3)D(3) and 1α,25(S),26(OH)(3)D(3), both of which are known to have high antiproliferative activity and low calcemic activity, were observed at a ratio of 5:1. The crystal structure of the double variant with 1α,25(OH)(2)D(3) and a docking model of 1α,25(OH)(2)D(3) in its active site strongly suggest a hydrogen-bond network including the 1α-hydroxyl group, and several water molecules play an important role in the substrate-binding for 26-hydroxylation. In conclusion, we have demonstrated that R73V/R84A can catalyze hydroxylations at C25, C1 and C26 (C27) positions of vitamin D(3) to produce biologically useful compounds.

The effects of bisphenol A (BPA) on UDP-glucuronosyltransferase 1A6 (UGT1A6) activities in microsomes from human livers and yeast cells expressing human UGT1A6 (humUGT1A6) were investigated. Serotonin (5-HT) and 4-methylumbelliferone (4-MU) were used as the substrates for UGT1A6. BPA dose-dependently inhibited 5-HT and 4-MU glucuronidation activities in both enzyme sources. The IC(50) values of BPA for 5-HT and 4-MU glucuronidation activities were 156 and 163 microM for liver microsomes, and 84.6 and 80.3 microM for yeast cell microsomes expressing humUGT1A6, respectively. The inhibitory pattern of BPA for 5-HT and 4-MU glucuronidation activities in human liver microsomes exhibited a mixture of competitive and noncompetitive components, with K(i) values of 84.9 and 72.3 microM, respectively. In yeast cell microsomes expressing humUGT1A6, 5-HT glucuronidation activities were noncompetitively inhibited by BPA (K(i) value, 65.5 microM), whereas the inhibition of 4-MU glucuronidation activities by BPA exhibited the mixed type (K(i) value, 42.5 microM). These results suggest that BPA interacts with human UGT1A6 enzyme, and that the interaction may contribute to the toxicity, such as hormone disruption and reproductive effects, of BPA.

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D(3) [25(OH)-19-nor-D(3)] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)], suggesting that 25(OH)-19-nor-D(3) might be converted to 1 alpha,25-dihydroxy-19-nor-vitamin D(3) [1 alpha,25(OH)(2)-19-nor-D(3)] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) and 25-hydroxyvitamin D(3) [25(OH)D(3)] and CYP24A1-dependent hydroxylation of 1 alpha,25(OH)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3), we found that k(cat)/K(m) for 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) was less than 0.1% of that for 25(OH)D(3), and the k(cat)/K(m) value for 24-hydroxylation was not significantly different between 1 alpha,25(OH)(2)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3). The data suggest a much slower formation and a similar rate of degradation of 1 alpha,25(OH)(2)-19-nor-D(3) compared with 1 alpha,25(OH)(2)D(3). We then analyzed the metabolites of 25(OH)D(3) and 25(OH)-19-nor-D(3) in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1 alpha,25(OH)(2)D(3) was detected in cells incubated with 25(OH)D(3), whereas no 1 alpha,25(OH)(2)-19-nor-D(3) was detected in cells incubated with 25(OH)-19-nor-D(3). Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D(3) is converted to 1 alpha,25(OH)(2)-19-nor-D(3) by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D(3) by itself may have a novel mechanism to activate vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1 alpha-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D(3) has potential as an attractive agent for prostate cancer therapy.

Sepsis is characterized by various symptoms, signs and underlying pathophysiology. To investigate possible mechanisms underlying this diversity, we compared the cardiovascular effects of lipopolysaccharide (LPS) derived from Escherichia coli (E-LPS) with those of LPS from Pseudomonas aeruginosa (P-LPS) in rats. We also examined the possible roles of tumor necrosis factor-alpha (TNF-alpha) and oxidative stress in LPS-induced cardiovascular damage. E-LPS (10 mg/kg body weight) or P-LPS (2 mg/kg body weight) was administered intravenously to Wistar rats. Echocardiography was serially performed. E-LPS induced an increase in left ventricular fractional shortening that persisted for at least 6 h, whereas P-LPS elicited an initial increase and a subsequent decrease in this parameter. Histological analysis revealed that P-LPS induced interstitial edema, congestion, intramyocardial bleeding, myocardial necrosis, infiltration of inflammatory cells, and formation of fibrin thrombi in the heart, whereas no pathological changes were apparent in the hearts of rats treated with E-LPS. Furthermore, the plasma concentration of TNF-alpha in rats treated with P-LPS was greater than that in rats treated with E-LPS, but the glutathione redox ratio in the heart was not affected by either type of LPS. In conclusion, E-LPS and P-LPS induced distinct patterns of functional and structural responses in the cardiovascular systems of rats. These differential responses may be attributable in part to the difference in the associated increases in the plasma concentration of TNF-alpha. The cardiovascular effects of LPS thus depend on the causative organisms.

The hypersensitive response (HR) of plants is one of the earliest responses to prevent pathogen invasion. A brown dot lesion on a leaf is visual evidence of the HR against the blast fungus Magnaporthe oryzae in rice, but tracking the browning process has been difficult. In this study, we induced the HR in rice cultivars harboring the blast resistance gene Pit by inoculation of an incompatible M. oryzae strain, which generated a unique resistance lesion with a brown ring (halo) around the brown fungal penetration site. Inoculation analysis using a plant harboring Pit but lacking an enzyme that catalyzes tryptamine to serotonin showed that high accumulation of the oxidized form of serotonin was the cause of the browning at the halo and penetration site. Our analysis of the halo browning process in the rice leaf revealed that abscisic acid enhanced biosynthesis of serotonin under light conditions, and serotonin changed to the oxidized form via hydrogen peroxide produced by light. The dramatic increase in serotonin, which has a high antioxidant activity, suppressed leaf damage outside the halo, blocked expansion of the browning area and attenuated inhibition of plant growth. These results suggest that serotonin helps to reduce biotic stress in the plant by acting as a scavenger of oxygen radicals to protect uninfected tissues from oxidative damage caused by the HR. The deposition of its oxide at the HR lesion is observed as lesion browning.

Purpose: To understand better the relationship between the macular ganglion cell complex (mGCC) thickness and visual field sensitivity assessed by frequency-doubling technology (FDT) perimetry in the standard automated perimetry (SAP) normal hemifields of glaucomatous eyes, a model of preperimetric stage of glaucoma. Patients and Methods: Thirty-four eyes of 34 patients with glaucomatous visual field defects restricted to the superior or inferior hemifield were included. Patients underwent the mGCC and circumpapillary retinal nerve fiber layer (cpRNFL) thickness measurements using spectral domain optical coherence tomography and the FDT testing with N-30 full-threshold protocol. SAP and FDT sensitivity values were averaged in the area corresponding to thickness measurements and the thickness sensitivity relationships were assessed in the SAP normal and SAP abnormal halves (Spearman’s rank correlation coefficient). Results: FDT sensitivity was significantly correlated with both the cpRNFL and mGCC thicknesses either in the SAP normal (ρ=0.384 and 0.462, respectively) or in the SAP abnormal (ρ=0.402 and 0.717, respectively) halves. Correlation between the FDT sensitivity and the mGCC thickness was significantly (P=0.016) stronger than that with the cpRNFL thickness in the SAP abnormal half. SAP sensitivity was correlated significantly (ρ=0.570) only with the mGCC thickness in the SAP abnormal half. Conclusions: Similarly strong correlations of the mGCC thickness with the FDT sensitivity in the SAP normal and SAP abnormal halves, but not with the SAP sensitivity, indicates that the mGCC thickness and the FDT sensitivity may be more optimal structure-function indicator in preperimetric stage of glaucoma.

We treated rapidly growing Jurkat cells with 40 nmol/l of doxorubicin for 72 h. After 36 h, the G2-arrested cells became larger and some of them started endoreplication. Nuclear staining with Hoechst 33342 combined with propidium iodide (PI) exclusion revealed that about 90% of the cells were necrotic at 72 h, although apoptotic cells accounted for only 8%. Incubation with 40 nmol/l of aclarubicin or cytosine beta-d-arabinofuranoside for 60 h induced necrosis both in Jurkat and ml-1 cells. Pre-necrotic Jurkat cells incubated with 40 nmol/l of doxorubicin had much higher intracellular reactive oxygen species (ROS) levels than pre-apoptotic ones. Addition of Tempol or Desferal accelerated doxorubicin-induced necrosis and partially converted it into apoptosis. Both antioxidants reduced surviving colony numbers of prenecrotic Jurkat cells. n-acetyl-l-cysteine had little effect on the apoptotic conversion but profoundly accelerated necrosis. Because an apoptosis-resistant Jurkat subclone was also refractory to doxorubicin-induced necrosis, apoptosis and necrosis might share some common pathways. Low-dose doxorubicin increased micronuclei-positive cell percentages and also suppressed high-dose doxorubicin-induced apoptosis in Jurkat and ml-1 cells. Some of the prenecrotic cells, therefore, might survive and obtain genomic instability. Antioxidants may be useful to suppress, at least to some extent, this vicious consequence.

Sterols have important functions in membranes and signaling. Plant sterols are synthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to cycloartenol. Plants also convert 2,3-oxidosqualene to other sterol-like cyclization products, including the simple triterpene β-amyrin. The function of β-amyrin per se is unknown, but this molecule can serve as an intermediate in the synthesis of more complex triterpene glycosides associated with plant defense. β-Amyrin is present at low levels in the roots of diploid oat (Avena strigosa). Oat roots also synthesize the β-amyrin–derived triterpene glycoside avenacin A-1, which provides protection against soil-borne diseases. The genes for the early steps in avenacin A-1 synthesis [saponin-deficient 1 and 2 (Sad1 and Sad2)] have been recruited from the sterol pathway by gene duplication and neofunctionalization. Here we show that Sad1 and Sad2 are regulated by an ancient root developmental process that is conserved across diverse species. Sad1 promoter activity is dependent on an L1 box motif, implicating sterol/lipid-binding class IV homeodomain leucine zipper transcription factors as potential regulators. The metabolism of β-amyrin is blocked in sad2 mutants, which therefore accumulate abnormally high levels of this triterpene. The accumulation of elevated levels of β-amyrin in these mutants triggers a “superhairy” root phenotype. Importantly, this effect is manifested very early in the establishment of the root epidermis, causing a greater proportion of epidermal cells to be specified as root hair cells rather than nonhair cells. Together these findings suggest that simple triterpenes may have widespread and as yet largely unrecognized functions in plant growth and development.

A fusion between the plastid psbA promoter and the green fluorescent protein gene (gfp) was introduced into the tobacco chloroplast genome by stable plastid transformation. GFP was synthesized actively and exclusively in the chloroplasts. Tubular projections filled with GFP but containing no chlorophyll were visualized for the first time in chloroplasts of these transplastomic plants. Occasionally, the tubules connect chloroplasts with each other, suggesting the possibility of the exchange of endogenous proteins. However, the fusion of protoplasts between the transplastomic and wild-type plants showed that such chloroplast connections might be rare in mesophyll protoplasts.

Purpose To report a case of optic neuropathy associated with prolonged use of a new antibiotic, linezolid. Design Interventional case report. Methods Optic and peripheral neuropathy occurred in a 27-year-old woman who was undergoing prolonged linezolid therapy for osteomyelitis. The use of corticosteroid exacerbated visual loss. Discontinuation of the linezolid therapy resulted in marked improvement of optic neuropathy. Results Linezolid-induced optic neuropathy was diagnosed in this case, because prolonged linezolid therapy was performed at a higher dose than usual and because the patient recovered after linezolid therapy was discontinued. Conclusions With prolonged linezolid therapy, ophthalmologists should be aware that bilateral optic neuropathy can occur and that corticosteroids are not indicated for this neuropathy.

PURPOSE: To evaluate the cross-sectional configurations of peripapillary atrophy (PPA)-alpha and -beta in ophthalmologically normal subjects using spectral domain-optical coherence tomography (SD-OCT). METHODS: One hundred twenty normal subjects had a complete ophthalmic examination including axial length measurement, standard automated perimetry, fundus imaging with photography, and SD-OCT (3D OCT-1000; Topcon Inc., Tokyo, Japan). PPA-alpha and -beta were identified in color photographs of the optic disc. Cross-sectional B-mode images of the peripapillary retina and sclera, including PPA-alpha and -beta obtained with SD-OCT, were analyzed. RESULTS: Of 120 normal eyes, 120 (100%) had PPA-alpha and 90 (75%) had PPA-beta. In OCT images of the peripapillary retina, the ganglion cell layer and the inner and outer plexiform layers were observed to end in a tapering fashion at the edge of the optic disc, whereas the retinal nerve fiber layer continued into the optic cup. The external limiting membrane (ELM), inner-outer segments (IS-OS), and retinal pigment epithelium(RPE)/Bruch's membrane complex were significantly more commonly absent before the optic disc edge within the PPA-beta compared with areas outside the PPA-beta (P < 0.0001). Specific findings in the peripapillary area including slope and step configurations of the scleral bed and hump- and wedge-shaped appearances of the RPE-Bruch's membrane complex were identified in 63 (52.5%), 6 (5.0%), 19 (15.8%), and 6 (5.0%) of 120 eyes, respectively. The presence of the step configuration was associated with myopia and longer axial length (P = 0.0014 and 0.0105, respectively). CONCLUSIONS: The cross-sectional anatomic configurations of the peripapillary atrophy were evaluated by using SD-OCT. The termination of the retinal layers and configurations of the scleral bed in the peripapillary area varied among normal subjects.

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.

Background Oxidative stress is implicated in cardiac remodeling and failure. We tested whether xanthine oxidase (XO) inhibition could decrease myocardial oxidative stress and attenuate left ventricular (LV) remodeling and dysfunction in the TO-2 hamster model of dilated cardiomyopathy. Methods and Results TO-2 hamsters were randomized to treatment with the XO inhibitor, allopurinol, or vehicle from 6 to 12 weeks of age. F1B hamsters served as controls. TO-2 hamsters treated with vehicle progressively developed severe LV systolic dysfunction and dilation between 6 and 12 weeks. Marked cardiac fibrosis was apparent in these hamsters at 12 weeks in comparison with F1B controls. The ratio of reduced to oxidized glutathione (GSH/GSSG) was decreased and malondialdehyde levels were increased in the hearts of vehicle-treated TO-2 hamsters. Treatment with allopurinol from 6 to 12 weeks attenuated LV dysfunction and dilation as well as myocardial fibrosis and the upregulation of a fetal-type cardiac gene. Allopurinol also inhibited both the decrease in GSH/GSSG ratio and the increase in malondialdehyde levels in the heart. Conclusions These results indicate that chronic XO inhibition with allopurinol attenuates LV remodeling and dysfunction as well as myocardial oxidative stress in this model of heart failure. Allopurinol may prove beneficial for the treatment of heart failure.

The biosynthetic gene cluster of goadsporin, a polypeptide antibiotic containing thiazole and oxazole rings, was cloned from Streptomyces sp. TP-A0584. The cluster contains a structural gene, godA, and nine god (goadsporin) genes involved in post-translational modification, immunity and transcriptional regulation. Although the gene organization is similar to typical bacteriocin biosynthetic gene clusters, each goadsporin biosynthetic gene shows low homology to these genes. Goadsporin biosynthesis is initiated by the translation of godA, and the subsequent cyclization, dehydration and acetylation are probably catalysed by godD, godE, godF, godG and godH gene products. godI shows high similarity to the 54 kDa subunit of the signal recognition particle and plays an important role in goadsporin immunity. Furthermore, four goadsporin analogues were produced by site-directed mutagenesis of godA, suggesting that this biosynthesis machinery is used for the heterocyclization of peptides.

Most plastid promoters recognized by bacteria-like plastid RNA polymerase (PEP) are similar to E. coli sigma(70)-type promoters comprising "-35" and "-10" elements. Among them, psbA promoter is unique in bearing additional elements between the conserved -35 and -10 elements. The psbA promoter activity is differentially maintained in the mature chloroplasts where the activity of most PEP promoters declines. Previously, we identified two types of PEP activities in wheat seedlings [Satoh et al. (1999) Plant J. 18: 407]; PEP present in the mature chloroplasts of the leaf tip (tip-type PEP) can initiate transcription from the -35-destructed psbA promoter, but the -35 element is essential for transcription by PEP present in immature chloroplasts of the leaf base (base-type PEP). To reveal which type of PEP functions in various types of plastids in tobacco, we analyzed the tobacco psbA promoter by means of a transplastomic approach. The promoter core context (-42 to +9) was sufficient for developmental regulation of the psbA promoter activity. The -35 promoter element was important for transcription initiation at the psbA promoter in all types of plastids, including chloroplasts in mature leaves, leucoplasts in roots, etioplasts in etiolated cotyledons. The conclusion is that the PEP bearing a promoter preference, similar to the wheat base-type PEP, functions dominantly in tobacco chloroplasts.

The proliferation activity of component cells and its regional differences in the regenerating rat pancreas after 90% pancreatectomy were examined by bromodeoxyuridine (BrdU) immunohistochemistry. Cells of the ductal system and the centroacinar cells showed a rapid increase in labeling indices at day 2 after pancreatectomy, followed by a second peak of a mild increase at days 5 to 7. No regional difference in the labeling index was recognized in the ductal elements. In contrast, the labeling index of acinar cells started to increase at day 3, reaching a definite peak at day 5. Furthermore, acinar cells in the region close to the duodenum had labeling indices more than 2 times higher than those in the portions further away from the duodenum. Acinar cells increased in number as early as from day 3 after surgery. These result suggested that the parental cells of regeneration were located in the ductal epithelium. It is highly probable that the proliferation of acinar cells is controlled by some unknown trophic factor(s) which is released locally from the duodenum, but does not involve a neural or a circulatory route. The phenomenon may be closely linked to the known fact that the incidence of pancreatic cancer is highest in the head region.

Thermal degradation and solid-state polymerization of poly-trans-4-γ-propoxycyclohexyl amide (PPCA) were investigated. From the results of thermal gravimetric analysis with the multiple heating rates method, the activation energy for thermal degradation reaction of PPCA was found to be 53.7kcal/mole, and this value was essentially identical with that obtained from the investigation at the constant temperatures between 340 and 380°C (51.7kcal/mole; in vacuo, 48.1kcal/mole, in N2). The small value of activation energy (37.7kcal/mole) for a first stage of degradation suggests that PPCA is thermally unstable.The activation energy for solid-state polymerization of PPCA at the temperature from 170 to 230°C was found to be a small value 10.7kcal/mole, this indicates a high mobility of polymer chain at this temperature range. The results are in good agreement with the consideration being described in the previous paper for relationship between the high value of Tg/Tm (0.73, °K/°K) and the chemical structure of PPCA.

Luteinized rat ovaries contain a high concentration of particulate luteinizing hormone (lutropin, LH) receptors and a small quantity of lipid-associated receptors that float in the 360,000 X g supernatant fraction of ovarian homogenates. During fractionation of Lubrol-solubilized LH receptors and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from the ovary and testis, LH receptors and adenylate cyclase were coincident on gel filtration, but could be resolved during ion-exchange chromatography of soluble ovarian preparations and were completely separated by lectin-affinity chromatography on Sepharose-concanavalin A. For further analysis of receptor-adenylate cyclase coupling, the lipid-rich fraction of ovarian luteal cells was used to transfer gonadal LH receptors to isolated adrenal fasciculata cells. The lipid vesicles obtained from ovarian homogenates by flotation at 360,000 X g contained 5--10% of the ovarian LH receptors and were devoid of adenylate cyclase activity. During incubation of lipid-associated receptors with dispersed rat fasciculata cells at 16 degrees C, progressive incorporation of LH binding sites into the adrenal cells was observed. When adrenal cells bearing heterotopic LH receptors were incubated with 1 nM human choriogonadotropin, cyclic AMP production was consistently stimulated, with an accompanying increase in corticosterone production. These results indicate that LH receptors exist as separate entities from adenylate cyclase in the gonadal cell membrane and can become functionally coupled to adenylate cyclase to evoke cyclic AMP production and steroidogenesis in the host adrenal cells to which they are transferred.

The effects of various additives to protect Soya lecithin granules from moisture were investigated. Additives used were Adsolider-101®, Aerosil R-972® Neusilin UFL-2® and carnauba wax (mean diameter: 3μm). Weight increase, angle of repose, aggregation rate and disintegration time of the lecithin granules were measured to evaluate the effect of additives on moisture protection and fluidity.The hygroscopicity of the lecithin granules decreased with an increase in the amount of additives. Aerosil R-972® was not adhered uniformly to the lecithin granules. The addition of 2-3% of Adsolider-101® and Neusilin UFL-2® prevented the hygroscopicity and enhanced the fluidity of lecithin granules, though the appearance changed to white. It was considered. that carnauba wax in the proportion of 5% was very useful for the prevention of hygroscopicity and the enhancement of fluidity of the lecithin granules.

Peritoneal macrophages elicited by Lactobacillus casei YIT9018 (LCEPM) were incubated in culture for 18 h with L. casei; the culture supernatant (LCM) was then harvested and tested for its ability to increase the cytostatic activity of resident peritoneal macrophages (RPM) and LCEPM. Treatment of RPM with LCM induced activation of macrophages to a cytostatic state against L929, Colon 26, P815, P388D1 and L1210 cells. A combination of recombinant human tumor necrosis factor (rhTNF), recombinant mouse TNF (rmTNF), recombinant human interleukin-1 (rhIL-1) or bacterial lipopolysaccharide with recombinant mouse interferon gamma (rmIFN-gamma) resulted in the synergistic induction of cytostatic activity in RPM. Recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) plus rhTNF increased the cytostatic activity of RPM a little but rmGM-CSF or rhTNF combined with rhIL-1 or alone had no effect. The effect of LCM on RPM was not inhibited by polymyxin B, anti-mTNF antiserum or below 20 U/ml monoclonal anti-rmIFN-gamma antibody (anti-rmIFN-gamma) but was inhibited by more than 40 U/ml anti-rmIFN-gamma, and LCM did not have any interferon antiviral activity. These results suggest that the cytostatic activity of RPM was augmented by the LCM, and that the effect of the LCM may be not due to IFN-gamma, TNF, GM-CSF, IL-1 or a small amount of contaminating lipopolysaccharide.