Serum cytochrome c to indicate the extent of ongoing tumor cell death.

Despite the significant implication of apoptosis in tumorigenesis, there is no biomarker to assess the extent of ongoing apoptosis in vivo for hematological malignancies. We investigated the potential of serum cytochrome c (cyto-c) as a biomarker for apoptosis. Cyto-c and lactate dehydrogenase (LD) were released into the culture medium from apoptotic cells induced by tumor necrosis factor-related apoptosis-inducing ligand in a time-dependent manner in vitro, with different kinetic patterns. Only one-third of 153 patients with hematological malignancies showed high levels of serum cyto-c (>20 ng/ml). Although serum cyto-c level was roughly correlated to serum LD activity, their different kinetic patterns from serial measurements indicated that serum cyto-c rather than LD is a more sensitive indicator for tracking changes of tumor status. Furthermore, serum cyto-c level stratified patients with acute adult T-cell leukemia into favorable and unfavorable subgroups with 5-year survival rates of 67%vs. 11%. In conclusion, serum cyto-c may provide a fast real-time biomarker for tracking changes of tumor status involved in apoptotic cell death, but lacking disease or cell-type specificity.

Minilaparotomy may be independently associated with reduction in inflammatory responses after resection for colorectal cancer.

OBJECTIVES: A minilaparotomy approach (skin incision less than 7 cm) to resection of colon cancer is technically feasible, but objective data supporting its benefit are scarce. The aim of this study was to clarify whether minilaparotomy is independently associated with a reduction in the acute inflammatory response after resection of colorectal cancer. DESIGN: Thirty-one patients who underwent surgical resection of colorectal cancer using minilaparotomy or conventional laparotomy were included in this nonrandomized prospective study. Inflammatory responses were evaluated with serum interleukin-6 (IL-6) and C-reactive protein (CRP) levels. RESULTS: In both the minilaparotomy and conventional laparotomy groups, serum IL-6 and CRP levels significantly increased 24 h after the operation (1POD) compared to preoperative levels (p < 0.0001 and p < 0.0001, respectively). Median serum levels of IL-6 and CRP in the minilaparotomy group were significantly lower at 1POD versus the conventional group (p = 0.0066 and p = 0.0033, respectively). Multivariate analyses showed that a smaller increase in serum IL-6 or CRP levels at 1POD [less than 75th percentile (112.9 or 10.6 mg/ml, respectively)] was independently related to only minilaparotomy. CONCLUSIONS: These data in this nonrandomized trial suggest that minilaparotomy may be independently associated with reduced inflammatory responses in colorectal cancer resection.

Amino acid sequence and characterization of a rice bran ribonuclease.

A base-nonspecific and acid ribonuclease (RNase Os) belonging to the RNase T2 family was purified from rice bran to a homogeneous state by SDS-PAGE. The primary structure of RNase Os was determined by protein chemistry and molecular cloning. The RNase Os was a simple protein and consisted of 205 amino acid residues. Its molecular weight was 22578 and its amino acid sequence showed that it was most similar to barley RNase among the known RNase T2 family enzymes having 157 amino acid residues identical with barley RNase. However, its N-terminus was blocked by a gamma-pyroglutamyl residue. The optimal pH of RNase Os was around 5.5. The base preference at the B1 and B2 site of RNase Os was estimated from the rates of hydrolysis of 16 dinucleoside phosphates, to be guanine as the case of RNase LE from tomato. RNase Os was successfully expressed from yeast cells using the E. coli yeast expression vector pYE-RNAP.

Vitreous levels of insulin-like growth factor-I in patients with proliferative diabetic retinopathy.

The levels of IGF-I in the vitreous body and the intraocular fluid of patients with proliferative diabetic retinopathy (PDR) were determined using radioimmunoassay (RIA). Eleven vitreous specimens were obtained from the intraocular fluid of eyes of patients with PDR who underwent surgery during the operation. Eleven intraocular fluids from the same patients during reoperations were compared with controls. The expression of IGF-I mRNA in cultured human Muller glial cells was evaluated using reverse transcription-polymerase chain reaction (RT-PCR). The mean IGF-I level in the vitreous samples during initial PDR surgery and reoperation was significantly higher than that found in the vitreous of the control (p < 0.05). The level of IGF-I increased in 6 of the 11 cases. Cultured human Muller cells expressed IGF-I mRNA. The results indicate increased levels of IGF-I both in the initial vitreous and ocular fluid at post-operative re-proliferation. Muller cell is suggested as an origin of local IGF-I production.

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.

Relationship of two ribonucleases with molecular masses of 45 kDa and 37 kDa from the culture medium of Lentinus edodes.

Lentinus edodes (shiitake) produces three base non- specific and acid ribonucleases, RNase Le2, RNase Le37 and RNase Le45. The primary structures of the former two RNases, having molecular masses about 24 and 37 kDa, respectively, have been elucidated to be members of the RNase T2 family. The latter two are excreted from mycelia into the medium. In this report, we estimated the primary structure of RNase Le45 using the following experimental evidence. (i) The partial amino acid sequence of RNase Le45 determined that up to about 60% of total protein was identical with that of RNase Le37. (ii) The amino acid composition of RNase Le45 was identical to that of RNase Le37. (iii) The elution profiles on HPLC of lysylendopeptidase and Staphylococcus aureus V8 protease digests of RCM-RNase Le45 (reduced and S-carboxymethylated RNase Le45) were very similar to those of RNase Le37, except for the absence of C-terminus peptide which contained O-glycosylated peptides. However, RNase Le45 contained about 70 residues of mannose and 4 residues of hexosamine. These values were more than twice those of RNase Le37. (iv) RNase Le45 was immunologically indistinguishable from RNase Le37. (v) After treatment with both glycosidase EndoH and alpha-mannosidase, RNases Le37 and Le45 gave complex bands by slab-gel electrophoresis. However, one of the major bands with the highest mobility from RNase Le45 and Le37 showed the molecular mass of 29 kDa in common, which is slightly larger than that of RNase Le2 containing no carbohydrate. These results indicated that RNase Le45 is an enzyme which is a heavily glycosylated species of RNase Le37.

Amino acid sequence of a nuclease (nuclease Le1) from Lentinus edodes.

The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5'-GMP. To obtain the basic information on the mechanism of production of 5'-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide. Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.

Crystal structure of a plant ribonuclease, RNase LE.

Ribonuclease LE (RNase LE) from cultured tomato (Lycopersicon esculentum) cells is a member of the RNase T(2) family showing broad base specificity. The crystal structure of RNase LE has been determined at 1.65 A resolution. The structure consists of seven alpha-helices and seven beta-strands, belonging to an alpha+beta type structure. Comparison of the structure of RNase LE with that of RNase Rh, a microbial RNase belonging to the RNase T(2) family, reveals that while the overall folding topologies are similar to each other, major insertions and deletions are found at the N-terminal regions. The structural comparison, an amino acid sequence alignment of the RNase T(2) enzymes, and comparison of the disulfide-bonding pattern of these enzymes show that the structure of RNase LE shown here is the basic framework of the animal/plant subfamily of RNase T(2) enzymes (including a self-incompatibility protein called S-RNase), and the structure of RNase Rh is that of the fungal subfamily of RNase T(2) enzymes (including RNase T(2)). Subsequently, we superposed the active-site of the RNase LE with that of RNase Rh and found that (1) His39, Trp42, His92, Glu93, Lys96, and His97 of RNase LE coincided exactly with His46, Trp49, His104, Glu105, Lys108, and His109, respectively, of RNase Rh, and (2) two conserved water molecules were found at the putative P(1) sites of both enzymes. These facts suggest that plant RNase LE has a very similar hydrolysis mechanism to that of fungal RNase Rh, and almost all the RNase T(2) enzymes widely distributed in various species share a common catalytic mechanism. A cluster of hydrophobic residues was found on the active-site face of the RNase LE molecule and two large hydrophobic pockets exist. These hydrophobic pockets appear to be base binding sites mainly by hydrophobic interactions and are responsible for the base non-specificity of RNase LE.

Amino acid sequence of an unique ribonuclease with a C-terminus rich in O-glycosylated serine and threonine from culture medium of Lentinus edodes.

The mushroom Lentinus edodes produces three base-non-specific and acid ribonucleases, RNases Le2, Le37, and Le45. The latter two are excreted from mycelia into the medium. The primary structure of RNase Le37, which had a molecular mass of 37 kDa, was sequenced. It was a member of the RNase T2 family, as is RNase Le2. RNase Le37 was some 30 amino acid residues longer at the C-terminal end than RNase Le2. The C-terminal region of RNase LE37 was rich in O-glycosylated serine and threonine. In fungal glucoamylases and chitinases, which hydrolyze raw-starch and chitin, respectively, have structures resembling the structure of the C-terminal of RNase Le37.

Organization, structure, and evolution of the nonadult rat beta-globin gene cluster.

The beta-globin gene cluster of Wistar rat was extensively cloned and the embryonic genes were mapped and sequenced. Four overlapping lambda Dash recombinant clones cover about 31 kb and contain four nonadult beta-globin genes, 5'-epsilon1-gamma1-gamma2-psigamma3-3'. The epsilon1 and gamma2 are active genes, since their protein products were detected in the fetal stage of the rat (Iwahara et al., J Biochem 119:360-366, 1996). The gamma1 locus might be a pseudogene, since the ATA box in the promoter region is mutated to GTA; however, no other defect is observed. The psigamma3 locus is a truncated pseudogene because a 19-base deletion, which causes a shift of the reading frame, is observed between the second nucleotide of the putative codon 68 and codon 76. A sequence comparison suggests that the psigamma3 might be produced by a gene conversion event of the proto-gamma-globin gene set. Possible histories of the evolution of rat nonadult beta-globin genes are discussed.

A case of an intraocular foreign body due to graphite pencil lead complicated by endophthalmitis.

We report a case of an 8-year-old boy who presented with an intraocular foreign body composed of graphite pencil lead. The patient had been accidentally poked in the right eye with a graphite pencil. Primary care consisted of corneal suturing and lens extraction. Two pieces of the pencil lead remained in the vitreous cavity following surgery, and 2 days later the patient developed endophthalmitis. Pars plana vitrectomy was performed immediately and the intraocular foreign bodies were removed through the scleral wound. Cultures of the vitreous fluid revealed no bacterial organisms. X-ray fluoroscopic analysis of the vitreous detected 1 ppm of aluminum (a constituent of the pencil lead). Although the clinical presentation indicated probable bacterial endophthalmitis, the detection of elemental aluminum within the vitreous cavity also suggested the possibility of further retinal toxicity due to some dissolving of the pencil lead.

Molecular cloning and characterization of two sets of alpha-theta genes in the rat alpha-like globin gene cluster.

The highly heterogeneous rat hemoglobin system was investigated at the gene level. Two regions of the alpha-like globin gene cluster from a Wistar rat were isolated. Four lambda Dash recombinant clones carrying rat alpha-like globin genes were localized on two distinct gene regions. A region of approximately 16kb was found to contain the 5'-IIalpha1-psi theta 1-3' loci, and another of approximately 24kb the 5'-IIalpha2-psi theta2-psiI alpha3-3' loci. Both IIalpha1 and IIalpha2 are considered to be active, coding the IIalpha-globin chain. The nt sequences of IIalpha1 and IIalpha2 are identical except for six nt in the non-coding region. The psiI alpha3 locus is a truncated pseudogene. The putative promoter region of an alpha-like globin gene is joined directly to the third exon, homologous to that of Ialpha-globin cDNA. psi theta1 and psi theta2 are also pseudogenes, as evidenced by several deletions located in the protein-coding regions of these loci. The psi theta1 and psi theta2 loci exhibit extensive homology, but the restriction maps of these genes and their flanking regions differ considerably. Genomic Southern blot analyses of the total liver DNA from six rats showed the existence of three theta-globin-related genes, including psi theta1 and psi theta2. These results indicate that the two gene regions investigated are not allelic variants, but may be generated by block duplication. This is the first report of the existence of rodent theta-globin genes.

Comparison of base specificity and other enzymatic properties of two protozoan ribonucleases from Physarum polycephalum and Dictyostelium discoideum.

Base specificity and other enzymatic properties of two protozoan RNases, RNase Phyb from a true slime mold (Physarum polycephalum) and RNase DdI from a cellular slime mold (Dictyostelium discoideum), were compared. These two RNases have high amino acid sequence similarity (83 amino acid residues, 46%). The base specificities of two base recognition sites, The B1 site (base recognition site for the base at 5'-side of scissile phosphodiester bond) and the B2 site (base recognition site for the base at 3'-side of the scissile bond) of the both enzymes were estimated by the rates of hydrolysis of 16 dinucleoside phosphates. The base specificities estimated of B1 and B2 sites of RNase Phyb and RNase DdI were A, G, U > C and A > or = G > C > U, and A > or = G, U > C and G > U > A, C, respectively. The base specificities estimated from the depolymerization of homopolynucleotides and those from the releases of four mononucleotides upon digestion of RNA coincided well with those of the B2 sites of both enzymes. Thus, in these enzymes, the contribution of the B2 site to base specificity seems to be larger than that of the B1 site. pH-stability, optimum temperature, and temperature stability, of both enzymes are discussed considering that RNase Phyb has one disulfide bridge deleted, compared to the RNase DdI with four disulfide bridges.

Characterization of ribonucleases from culture medium of Lentinus edodes.

Lentinus edodes (shiitake) cultivated in potato dextrose medium produced five RNases in the culture filtrate. The two major RNases (RNase Le37 and RNase Le45) were highly purified and their molecular masses, base specificities, N-terminal amino acid sequences, and amino acid compositions were analyzed and compared to RNase Le2 isolated from the fruit bodies of the same mushroom. RNase Le37 and RNase Le45 are base non-specific and adenylic acid preferential RNases like RNase Le2 and their N-terminal sequences are very similar to RNase Le2, but they are glycoproteins and their amino acid compositions are significantly different from that of RNase Le2. In addition to these enzymes, a guanylic acid-specific RNase with a molecular mass 13 kDa was partially purified. Since RNase Le2, which has very similar N-terminal sequence to RNase Le37 and RNase Le45, was not excreted from the mycelia, the analysis of the structures of these two excreted RNase may shade a light on the mechanism of excretion of RNases in this organism.

Characterization and primary structure of a base non-specific and acid ribonuclease from Dictyostelium discoideum.

A base non-specific and acid RNase was isolated from cellular slime mold (Dictyostelium discoideum) cells in a homogeneous state (about 2.4 kDa) by SDS-polyacrylamide gel electrophoresis. The RNase (RNase DdI) has a pH optimum of 5.0. The amino acid sequence of RNase DdI was determined by a combination of protein chemistry, a search of Data base, Dicty cDB and further sequence analysis of cDNA from the same bank. RNase DdI consists of 198 amino acid residues, and about 13.3, 0.9, 1.2, 3.3, and 1.0 residues of mannose, xylose, glucose, GlcNAc, and GalNAc, respectively. RNase DdI has two characteristic conserved segments of the RNase T2 family, and thus belongs to the RNase T2 family. Considering the fact that most of the RNase activity of D. discoideum is present in the lysosomal fraction [Wiener and Ashworth (1970) Biochem. J. 118, 505-512], it was concluded that the lysosomal RNase in D. discoideum is a member of the RNase T2 family. The amino acid sequence of RNase DdI is highly homologous with that of Physarum polycephalum RNase (RNase Phyb), and its amino acid sequence seems to be similar to those of plant/animal type RNases, rather than fungal RNases. The location of RNase DdI in the phylogenetic tree of the RNase T2 family was estimated.

Prevalence of hepatitis B and C infection markers among adult women in urban and rural areas in Shaanxi Province, China.

In an attempt to investigate possible urban-rural difference in prevalence of hepatitis B and C virus (HBV and HCV, respectively) infection in continental China, triplet surveys on HBV and HCV infection markers (ie, HBsAg, anti-HBs, anti-HBc, and anti-HCV) and serum enzyme levels (AST, ALT and gamma-GTP) were conducted in 1997 on groups of apparently healthy adult women (49 to 50 subjects per group); one group (the City group) was in Xian, the provincial capital of Shaanxi Province, and two others (the Village A group and the Village B group) were in farming villages in the Province some 200 and 25 km away from Xian, respectively. Comparison among the three groups showed that there was no urban-rural difference in prevalence of HBV and HCV infection positive (HBV+ and HCV+) cases and that the overall prevalence of HBV+ and HCV+ cases was 70% and 3%, respectively. HBsAg+ prevalence was however higher in the villages (8% when the two villages were combined) than in the city (2%). HBV infection was not associated in general with apparent increase in emission enzyme levels in the serum, whereas HCV infection might be associated with an increase in ALT, AST and gamma-GTP. The present observation in combination with other previously published results suggests that urban-rural difference will not be remarkable in HBV and HCV infection prevalence in Continental China and that the public health problem is more serious with HBV infection and quite less so with HCV infection.

Primary structure of base non-specific and acid ribonuclease from bullfrog (Rana catesbeiana).

A base non-specific acid ribonuclease (RNase RCL2) was purified from bullfrog liver [H. Yagi et al. Biol. Pharm. Bull., 18, 219-222 (1992)]. The sequence study and comparison of the amino acid sequence of the enzyme with homologous RNases from oyster, drosophila and chicken liver suggested that the RNase RCL2 consisted of two components, large protein fraction (182 amino acid residues) and peptide 2 (20 amino acid residues) or peptide 1 (18 amino acid residues), and that both components bind with disulfide bridge. The RNase preparation was probably formed from a single polypeptide protein by processing with some proteases. The amino acid sequence of RNase RCL2 showed that the RNase belongs to the RNase of RNase T2 family and its sequence most resembles chicken liver acid RNase. In RNase RCL2, the amino acid residues which consist of the active site and major base recognition site of RNase Rh, a typical RNase of RNase T2 family, are very well conserved except for Tyr57 (RNase Rh numbering), and part of the amino acid residues of the minor base recognition site (Phe101 and Pro92) are also conserved.

[Optic neuritis and measles infection].

Optic neuritis has been reported as a complication of measles encephalitis. This resembles to parainfectious optic neuritis following other virus infections. We mentioned pathology, therapy of this disease, and summarized our previous cases. Intravenous methylpredonisolone treatment is effective from our experience.

The base specificities of tomato ribonuclease (RNase LE) and its Asp44 mutant enzyme expressed from yeast cells.

RNase LE from cultured tomato cells is a member of the RNase T2 family. It is, however, distinguishable from RNase Rh from Rhizopus niveus, a typical RNase of the RNase T2 family, by its CD spectrum in the 200-250 nm region. In order to reinvestigate the base specificity of RNase LE and to study the role of Asn44 in RNase LE, which is considered to correspond to the base recognition site Asp51 of RNase Rh, RNase LE, and its Asp mutant at the 44th position were expressed from yeast cells with the same expression system as RNase Rh [K. Ohgi, et al., J. Biochem., 109, 776-785 (1991)]. RNase LE with four extra amino acid residues at the 2nd amino acid residue of mature RNase LE and its Asp44 mutant were secreted from yeast cells to give a yield of 10 mg/liter and 0.5 mg/liter culture broth, respectively. The expressed RNase LE (RNase RNAP LE) had the same characteristics as native RNase LE in the CD spectrum and specific activity. This is the first example of the expression of plant RNase from microbes and in sufficient amount to perform further enzymological research. The base specificity of RNase LE was guanylic acid preferential and that of N44D was changed to a more adenylic acid preference as compared to that of RNase LE. These experiments showed that Asn44 of RNase LE is crucial for base recognition as the case of Asp51 in RNASE Rh, and also suggested that the base recognition mechanism of RNase LE is very similar to that of RNase Rh.