Germ-Tube Formation is Independent of DNA Synthesis during Spore Germination in the Fission Yeast, Schizosaccharomyces pombe

Germ-tube emergence took place at almost the same time as DNAsynthesis during spore germination in the fission yeast, Schizosaccharomycespombe. Neither the emergence nor the elongation of the germ-tubeswas inhibited by hydroxyurea. Spores harboring a temperature-sensitivecdc 10 mutation produced germ-tubes even at a restrictive temperature,which indicates that germ-tube formation is not dependent onDNA synthesis. ¹ Present address: Department of Microbial Genetics, ResearchInstitute for Microbial Diseases, Osaka University, Yamada-Oka,Suita, Osaka 565, Japan. (Received June 3, 1982; Accepted July 5, 1982)     

Intraarterial combined immunochemotherapy for unresectable hepatocellular carcinoma: preliminary results

An important objective for patients with unresectable hepatocellular carcinoma (HCC) is the development of effective chemotherapy. We administered a combination of biological response modifiers and anticancer agents to 24 patients with unresectable HCC. Each case had an implanted infuser port which was connected to a catheter placed in the hepatic artery for the intraarterial (i.a.) administration of chemotherapy. The following agents were administered to each patient: recombinant interleukin-2 (800 000 JRU/day infused i. a. continuously for 6 days/week); OK-432 (5 KE injected i. a. twice in 4 weeks and i. m. three times per week); Adriamycin (10 mg injected i.a. twice in 4 weeks); cyclophosphamide (300 mg injected i. a. twice in 4 weeks), and famotidine (40 mg/day administered orally). Objective response was assessed according to tumor size measured by computed tomography and angiography before and after treatment. We observed a complete response (CR) in 4, partial response (PR) in 3, minor response (MR) in 7, no change (NC) in 7, and progressive disease (PD) in 3. The response rate (CR+PR+MR) was 58.3%. The overall 2-year survival rate was 52%. The 2-year survival rate of the responders (CR+PR+MR) was 80%, while that of the non-responders (NC+PD) was 0%. There was a significant difference between the responders and non-responders in respect to survival rate (P<0.05). The percentages of CD25+ cells, CD56+ cells, and Leu7-CD16+ cells and NK activity in the peripheral blood showed a significant increase following the regimen. Serum levels of tumor necrosis factor TNF rose after the initiation of OK-432. TNF levels were higher in the responders than in the non-responders. Adverse effects included high fever (all patients) and severe transient hypotension (15 patient) that was controlled by convervative therapy. Combined immunochemotherapy administered intraarterially may be a new strategy for treating unresectable HCC.     

Protease II from Escherichia coli: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.

Protease II gene of Escherichia coli HB101 was cloned and expressed in E. coli JM83. The transformant harboring a hybrid plasmid, pPROII-12, with a 2.4 kbp fragment showed 90-fold higher enzyme activity than the host. The whole nucleotide sequence of the inserted fragment of plasmid pPROII-12 was clarified by the dideoxy chain-terminating method. The sequence that encoded the mature enzyme protein was found to start at an ATG codon, as judged by comparison with amino terminal protein sequencing. The molecular weight of the enzyme was estimated to be 81,858 from the nucleotide sequence. The reactive serine residue of protease II was identified as Ser-532 with tritium DFP. The sequence around the serine residue is coincident with the common sequence of Gly-X-Ser-X-Gly, which has been found in the active site of serine proteases. Except for this region, protease II showed no significant sequence homology with E. coli serine proteases, protease IV and protease La (lon gene), or other known families of serine proteases. However, 25.3% homology was observed between protease II and prolyl endopeptidase from porcine brain. Although the substrate specificities of these two enzymes are quite different, it seems possible to classify protease II as a member of the prolyl endopeptidase family from the structural point of view.     

Effectivein vivo induction of lymphokine-activated killer (LAK) cells by pretreatment with a streptococcal preparation,OK-432

Effects of a streptococcal preparation, OK-432, on precursors of lymphokine-activated killer (LAK) cells were observedin vivo. Total number of splenocytes and the ratio of asGM ₁ ⁺ cells increased gradually after i.v. administration of OK-432, reaching their peaks at 3 to 4 days. It was found that as GM ₁ ⁺ cells were nonadherent and large in size. There were little differences in the ratios of Thy-1⁺, Lyt-2⁺, and L3T4⁺ cells before and after OK-432 treatment. Mice were injected i.p. with recombinant interleukin 2 (rIL-2) at a dose of 5 × 10⁴ U per mouse 4 days after OK-432 administration and LAK activity in their splenocytes was examined using natural killer (NK) resistant EL-4 target cells. Splenocytes in mice treated with both OK-432 and rIL-2 showed higher LAK activity than those in mice treated with rIL-2 alone.In vivo treatment with anti asGM, antibody prior to rIL-2 injection abolished completely such augmentation of LAK activity in OK-432 treated mice. These results demonstrated that asGM ₁ ⁺ LAK precursor cells induced by OK-432 were effectively differentiated into LAK cells by rIL-2.     

Identification and characterization of adipokinetic hormone (Locusta migratoria)-like immunoreactivity in the human cerebrospinal fluid

Using an antiserum raised against locust adipokinetic hormone I, considerable quantity of adipokinetic hormone-like immunoreactivity was demonstrated in the human cerebrospinal fluid. The immunoreactivity was characterized by gel permeation and high performance liquid chromatography. The main immunoreactive component in the cerebrospinal fluid coeluted with adipokinetic hormone I. These results suggest that adipokinetic hormone may contribute to the neuronal function in the human central nervous system.     

Local antibody response in Peyer's patches to the orally administered dietary protein antigen

To understand local antibody production to dietary protein antigens in the gut, the reactivity of the monoclonal antibodies (mAbs) from Peyer's patches of BALB/c mice raised against orally administered hen egg lysozyme (HEL) was studied. These mAbs were of IgG1 (7 clones), IgA (5 clones) and IgM (13 clones) isotypes. Some of the HEL-binding mAbs preferentially reacted with reduced, carboxy-methylated HEL, rather than with native HEL. MAbs of the IgA and IgM isotypes had cross-reactivity with other unrelated environmental antigens such as E. coli, single-strand DNA, and soluble components of mouse food. In contrast, the IgG1 mAbs did not cross-react with these antigens. The average of the Kd values for HEL of these mAbs was in the order of 10(-6) M, which is moderately higher than those of mAbs from the preimmune repertoire. These results suggest that, under normal physiological conditions, orally administered dietary proteins predominantly induce the local production of polyreactive IgA/IgM antibodies cross-reacting with environmental luminal antigens.     

Identification of an Arylphorin-type Storage Protein in the Sweet Potato Hornworm, Agrius convolvuli

A major hemolymph protein (M_r 480,000) in the larvae of the sweet potato hornworm, Agrius convolvuli, was purified and characterized. This protein was isolated with a high yield from the hemolymph of day 3 fifth final instar larvae by ammonium sulfate precipitation and Phenyl-Sepharose and Q-Sepharose column chromatographies. The protein has two subunits, an M_r 84,000 subunit (α) and an M_r 80,000 subunit (β), and the native protein was composed of a heterohexamer (α₃β₃). The two subunits have similar amino acid compositions, with high contents of aromatic amino acids (about 15% phenylalanine plus tyrosine) and low levels of methionine. The N-terminal amino acid sequences of both subunits showed high homologies with insect arylphorin-type storage proteins. The protein concentration in the hemolymph increased steeply from day 3 final instar larva and reached a maximum level of 42 mg/ml in females and 41 mg/ml in males among wandering larvae. The concentration in the hemolymph declined once during the larval–pupal transformation but remained high during the early–mid pupal period and almost disappeared after adult emergence. These quantitative changes were the same for males and females. Based on these characteristics, we identified the hemolymph protein as an arylphorin-type storage protein.     

Germination and outgrowth of Schizosaccharomyces pombe ascospores isolated by Urografin density gradient centrifugation.

A simple method for the isolation of single ascospores of the fission yeast Schizosaccharomyces pombe was examined. Single spores in the 7-day-old sporulating culture of a homothallic strain were separated from remaining vegetative cells by isopycnic centrifugation in the linear gradient from 10 to 60% of Urografin solution at 700 X g for 20 min. Protein content of isolated spores was very low as compared with that of vegetative cells. The isolated spores germinated through the following steps when cultured in a liquid medium at 25--35 degrees C; loss of refractility (darkening) under a phase-contrast microscope, spherical growth (swelling), emergence of germ tubes, elongation of germ tubes, cell plate formation, and cell separation. The absorbance at 650 nm of the spore suspension initially decreased, accompanied by darkening of spores, and then increased with spherical growth. The germination rate of isolated spores reached almost 100%.