[Cancer survival analysis in Tianjin, 2010 to 2016].

Objective: Survival analysis of cancers' incidence data in Tianjin from 2010 to 2016 was conducted to provide the basis for formulating and evaluating regional health policies on cancer prevention and treatment. Methods: Registration data in Tianjin were used between January 1, 2010 to December 31, 2016 and patients were followed-up till 31 December, 2021. Life-table method was used to calculate the observed survival rate and Edered Ⅱ was used to calculate the relative survival rate. The data were stratified by year, gender, age group and cancer sites. Difference in survival curves between group was analyzed by Kaplan-Meier method and Log rank test. Joinpoint regression model was used to analyze the trend change. Results: The 5-year relative survival rates of cancer were 41.92% to 53.65% from 2010 to 2016 for residents in Tianjin, with an increasing trend (t=4.81, P=0.005), and the average was 48.56%. The survival rate of females was higher than that of males (57.71%vs. 39.20%), and the survival rate of urban residents was higher than that of rural residents (49.38% vs. 47.24%). The 5-year relative survival rates were 63.14%, 78.39%, 58.25% and 32.67% in 0-14, 15-44, 45-64 and 65 and above age groups, respectively. The median relative survival times of all cancer were 2.34 to 6.00 years from 2010 to 2016 in Tianjin, with an increasing trend (t=3.86, P=0.012). The average of median relative survival times was 4.11 years. The median survival time of females was longer than that of males (11.99 years vs. 2.03 years), and the time of urban residents were longer than that of rural residents (4.60 years vs. 3.43 years). The median relative survival time were 12.07, 11.92 and 1.34 years in 15-44, 45-64 and 65 and above age groups, respectively. Conclusions: The cumulative survival rate of cancer increased significantly from 2010 to 2016 in Tianjin, indicating that the prevention and treatment effect of cancer is obvious. The focus should be on male, rural areas, higher age group, and targeted prevention and treatment measures should be taken to lung, esophagus, liver, gallbladder and pancreatic cancer.

Efficacy and safety of external application of Chinese herbal medicine for psoriasis vulgaris: a systematic review of randomized controlled trials.

OBJECTIVE: To evaluate the clinical efficacy and safety of external application of Chinese herbal medicine (ex-CHM) for psoriasis vulgaris (PV). METHODS: Different search portals, including the China National Knowledge Infrastructure Database, China Science and Technology Journal Database, Wanfang, SinoMed, clinicaltrials, PubMed, Cochrane Library, and Cochrane Central Register of Controlled Trials, were searched for randomized controlled trials (RCTs) that compared the ex-CHM intervention with other treatment protocols for PV, with available data as of November 25, 2020. The Review Manager 5.3 software was used for analysis. RESULTS: Nineteen RCTs involving 1988 participants were included, of which twelve RCTs qualified for the Meta-analysis. The results showed that the addition of CHM bath to narrow-band ultraviolet B (NB-UVB) had a higher response rate [RR = 1.29; 95% CI (1.19, 1.40); P < 0.000 01]; lower psoriasis area severity index (PASI) [MD = -3.15; 95% CI (-4.79, -1.52); P = 0.000 2)], adverse reactions rate [RR = 0.32; 95% CI (0.15, 0.66); P = 0.002], and recurrence rate [RR = 0.48; 95% CI (0.30, 0.79); P=0.004] than NB-UVB alone. The addition of CHM fumigation to NB-UVB also showed a higher response rate [RR = 1.29; 95% CI (1.11, 1.49); P = 0.000 7] and lower adverse reactions rate [RR = 0.44; 95% CI (0.24, 0.79); P=0.006]. In addition, CHM bath could reduce the adverse reactions induced by 308 nm excimer laser and improve patients' quality of life better than phototherapy. CHM fumigation could improve the efficacy of calcipotriol ointment and reduce the adverse reactions. CHM fumigation combined with external washing plus acitretin showed better results than using acitretin alone. No statistical difference was observed between CHM external washing and calcipotriol ointment or CHM ointment and retinoic acids. CONCLUSIONS: Current evidence showed that CHM bath and fumigation appeared to be efficient and safe for PV treatment. However, no definite conclusion could be drawn due to the low quality of included studies and thus more well-designed studies are needed for further assessment.

[Terpenoids from Toona sinensis].

Eight terpenoids(1-8) were isolated from the ethyl acetate soluble fraction of 80% ethanol extract of leaf of Toona sinensis through various column chromatographies on silica gel, Sephadex LH-20, MCI and ODS. Their structures were elucidated as 8ß-hydroxypimar-15-en-19-oic acid methyl ester(1), cedrodorol B(2), 11ß-acetoxyobacunol(3), toonayunnanin D(4), toonaciliatone D(5), toonaciliatone A(6), cedrelone(7), and 11ß-hydroxygedunin(8) based on their chemical and physicochemical methods and spectroscopic data. Compound 1 was a new pimaradienediterpenoid and terpenoid 2-7 were isolated and identified from this plant for the first time. Compound 1 was tested in vitro for cytotoxic potential by employing MTT method and radical scavenging potential using DPPH test. As a result, 1 exhibited weak cytotoxic activity against three tested tumor cell lines(SMMC-7721, A549 and MCF-7) with IC_(50) values less than 40 µmol·L~(-1) and moderate radical scavenging activities with IC_(50) values of 74.3 µmol·L~(-1).

Influence of surgical resection of hepatocellular carcinoma(HCC) for hematogenous dissemination of HCC cells and its effect on recurrence and metastasis: 3 years prospective study.

Aims was to study whether hepatal surgery leads to hematogenous dissemination of hepatocellular carcinoma (HCC) cells and determine period of its persistence by nested reverse transcription-polymerase chain reaction (RT-PCR). Peripheral vein blood (5ml) samples were obtained from 54 HCC patients of T1N0M0 and 6 HCC patients of T2N0M0 stage and 20 patients with liver cavernous hemangioma(LCH) before operation and 48h,72h and 1 week after surgical resection of hepatoma between Janurary 1st, 2007 and December 31th, 2010. We detected alpha-fetoprotein (AFP) messenger RNA(mRNA) by nested RT-PCR perioperatively.FPmRNA of all 60 cases of HCC patients and 20 cases of LCH were negative before hepatal surgery, 28 of 60 HCC cases became positve at 48h after surgery (46.7%), 16 of 60 HCC cases remained positive at 72h postoperatively(26.7%), none of 60 HCC patients AFPmRNA was detected at 1 week after hepatal surgery(0.0%). None of LCH patients were detected AFPmRNA after hepatal surgery(0.0%). For 28 HCC patients with AFPmRNA positive after hepatal surgery, 4 HCC patients developed intrahepatic tumor recurrences in 1st year (4/28, 14.3%),6 HCC patients relapsed in the second year(6/28,21.4%),10 HCC patients relapsed in the third year(10/28,35.7%). For 32 HCC patients with AFPmRNA negative group postoperation, 5 cases relapsed in 1st year (5/32, 15.6%),7 cases developed intrahepatic tumor recurrences in the second year (7/32, 21.9%),11 cases relasped in the third year (11/32, 34.4%,P>0.05),none of HCC patients occured distal metastasis after surgical resection of hepatoma. None of LCH patients relapsed postoperation within three years.Hepatal surgery may cause HCC cells spreading into peripheral blood shortly, but it may be not concerning with recurrence or metastasis of HCC.

Effect of lifestyle exposures on sperm aneuploidy.

Lifestyle exposures including cigarette smoke, alcohol, and caffeine have all been studied in relationship to male reproductive health. Over the years the focus has primarily been on semen quality and/or fertility. More recently, literature evaluating direct adverse effects of lifestyle exposures on sperm chromosomes and chromatin has grown due to concern that induced damage could be transmitted to offspring causing transgenerational health effects. In this paper we present a new analysis that summarizes published studies of smoking effects on sperm chromosome number and demonstrates a statistically significant increase in sperm disomy among smokers compared to nonsmokers (P < 0.001). In addition, new data on the effect of alcohol intake on sperm chromosome number are presented showing a rate ratio of 1.38 (95% CI 1.2, 1.6) for XY frequency in sperm of alcohol drinkers compared to nondrinkers.

Apoptosis occurs in implantation site of the rhesus monkey during early stage of pregnancy.

The exact role of apoptosis that occurs in human placenta during the early stage of pregnancy remains unknown because of the difficulty in obtaining the intact implantation site. In this study, we used rhesus monkey as an animal model to examine apoptosis occurring in the implantation site at various stages of early pregnancy. It was shown that Fas and FasL mRNA and protein were localized in both the chorionic villi and glandular epithelium from day 15 to day 30 of pregnancy. Fas and FasL protein were also expressed in the epithelial plaque on day 15 of pregnancy. In situ 3'-end-labeling results showed that glandular epithelial cells underwent extensive apoptosis with obvious morphological degradation during the early stage of pregnancy. It was found that the cells that were 3'-end-labeled in the chorionic villi and anchoring villi were mainly localized in cytotrophoblast and cytotrophoblast column. It is therefore suggested that in primates apoptosis, which may be involved in the regulation of proliferation of trophoblast villi and degradation of epithelial plaque, as well as remodeling of the glands in the maternal decidua, may play an important role during the early stage of implantation and placentation.

Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1.

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35 degrees C and 8, respectively. The apparent Km for IDA was 4.0 mM with a kcat of 5.3 s(-1). When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.

Cloning, sequencing, and characterization of a gene cluster involved in EDTA degradation from the bacterium BNC1.

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.

Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase.

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH(2))-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH(2) in vitro, HpaB was able to use FADH(2) and O(2) for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH(2) for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH(2)-utilizing monooxygenase. FADH(2) generated by Fre was rapidly oxidized by O(2) to form H(2)O(2) in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH(2) and transitorily protected it from rapid autoxidation by O(2). When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O(2) for FADH(2) utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH(2) produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH(2) was autoxidized by O(2), causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH(2)-utilizing monooxygenase that uses FADH(2) as a substrate rather than as a cofactor.

Expression of titin in skeletal muscle varies with hind-limb unloading.

The effects of prolonged hind-limb unloading on titin antibody localization and expression of titin isozymes of single fibers from the synergistic slow-twitch soleus (SOL) and fast-twitch plantaris (PLN) of adult rats were studied after 14 and 28 days of hind-limb unloading (HU). Titin antibody localization and expression was not altered at 14 days of HU. However, there was a 4% loss in antibody to Z-band distance (Ab-Z) in the SOL and an increase of 8% in PLN Ab-Z after 28 days of HU. The titin and myosin heavy chain composition of single fibers and small bundles of fibers from control and unloaded muscles were examined using 2% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked loss of relative amounts of titin in both SOL and PLN following 28 days of HU. As the protein loads for these measures were identical, the authors conclude that these findings represent an actual loss of titin density rather than a decreased value due to a loss of total muscle mass. Laser scanning densitometry of the titin bands show a marked decrease in density and molecular weight in unloaded SOL. In the PLN, marked losses of titin density were accompanied by decreased electrophoretic motility. The results demonstrate that the titin isoform composition and titin antibody localization of skeletal muscle is altered during hind-limb unloading. Furthermore, as titin is responsible for positional stability of the sarcomere and the fiber during contraction, change in isoforms during HU may predispose atrophied muscle to injury during reuse and recovery.

Characterization of 2,6-dichloro-p-hydroquinone 1,2-dioxygenase (PcpA) of Sphingomonas chlorophenolica ATCC 39723.

Pentachlorophenol (PCP) is a general biocide and a major environmental pollutant. The initial steps of PCP degradation by Sphingomonas chlorophenolica ATCC 39723 have been studied and characterized. Two enzymes are responsible for converting PCP to 2, 6-dichloro-p-hydroquinone (2,6-DiCH) which is a common metabolic intermediate of the biodegradation of polychlorinated phenols. 2, 6-DiCH is degraded by PcpA from strain ATCC 39723, but the reaction end product has been misidentified as 6-chlorohydroxyquinol and has been elusive to detection. We report here the overproduction of PcpA in Escherichia coli and the demonstration of quantitative conversion of 2,6-DiCH to 2-chloromaleylacetate with the coconsumption of one equivalent O(2) and release of one equivalent Cl(-) by purified PcpA. On the basis of the reaction stoichiometry, the enzyme is proposed to be 2,6-DiCH 1,2-dioxygenase.

Single-molecule enzymatic dynamics.

Enzymatic turnovers of single cholesterol oxidase molecules were observed in real time by monitoring the emission from the enzyme's fluorescent active site, flavin adenine dinucleotide (FAD). Statistical analyses of single-molecule trajectories revealed a significant and slow fluctuation in the rate of cholesterol oxidation by FAD. The static disorder and dynamic disorder of reaction rates, which are essentially indistinguishable in ensemble-averaged experiments, were determined separately by the real-time single-molecule approach. A molecular memory phenomenon, in which an enzymatic turnover was not independent of its previous turnovers because of a slow fluctuation of protein conformation, was evidenced by spontaneous spectral fluctuation of FAD.

Novel pathway for conversion of chlorohydroxyquinol to maleylacetate in Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and beta-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.

Purification and characterization of EDTA monooxygenase from the EDTA-degrading bacterium BNC1.

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O2 were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH2 and O2. The FMN reductase provided EDTA monooxygenase with FMNH2 by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35 degreesC. Kms were 34.1 microM for uncomplexed EDTA and 8.5 microM for MgEDTA2-; this difference in Km indicates that the enzyme has greater affinity for MgEDTA2-. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH2-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.

Genes for 2,4,5-trichlorophenoxyacetic acid metabolism in Burkholderia cepacia AC1100: characterization of the tftC and tftD genes and locations of the tft operons on multiple replicons.

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2, 4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftD genes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences of tftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH of Klebsiella pneumonia. Expression of the tftC and tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes of B. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of the tftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, the tftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGH and one copy of tftCD.

Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp. strain ATCC 39723.

The biochemistry of pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723 has been studied, and two enzymes responsible for the conversion of PCP to 2,6-dichloro-p-hydroquinone (2,6-DiCH) have previously been purified and characterized. In this study, enzymatic activities consuming 2,6-DiCH were identified from the cell extracts of strain ATCC 39723. The enzyme was purified to apparent homogeneity by a purification scheme consisting of seven steps. Gel filtration chromatography showed a native molecular weight of about 40,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 42,500 Da. The purified enzyme converted 2,6-DiCH to 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene), which was easily oxidized by molecular oxygen and hard to detect. The end product, 6-chlorohydroxyquinol, was detected only in the presence of a reductase and NADH in the reaction mixture. The enzyme dechlorinated 2,6-DiCH but not 2,5-DiCH. The enzyme required Fe2+ for activity and was severely inhibited by metal chelating agents. The optimal conditions for activity were pH 7.0 and 40 degrees C. The Kcat for 2,6-DiCH was 35 microM, and the kcat was 0.011 s-1.

Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation.

Purification, characterization and gene sequence analysis of a novel cytochrome c co-induced with reductive dechlorination activity in Desulfomonile tiedjei DCB-1.

The sulfate-reducing bacterium, Desulfomonile tiedjei DCB-1, conserves energy for growth from reductive dechlorination of 3-chlorobenzoate via halorespiration. To understand this respiratory process better, we examined electron carriers from different cellular compartments of D. tiedjei. A 50-kDa cytochrome from the membrane fraction was found to be co-induced with dechlorination activity. This inducible cytochrome was extracted from the membrane fractions by Tris-HCl buffer containing ammonium sulfate at 35% saturation and was purified to electrophoretic homogeneity by phenyl superose, Mono Q, and hydroxyapatite chromatography. The purified cytochrome had a high-spin absorption spectrum. In a pH titration experiment, the absorption spectrum of the inducible cytochrome shifted to low spin at pH 13.2. The midpoint potential of the inducible cytochrome at pH 7.0 was -342 mV. The NH2-terminal amino acid sequence of the inducible cytochrome was determined and was used to obtain inverse PCR products containing the sequence of the gene encoding the inducible cytochrome. The ORF was 1398 bp and coded for a protein of 52.6 kDa. Two c-type heme-binding domains were identified in the COOH-terminal half of the protein. A putative signal peptide of 26 residues was found at the NH2-terminal end. The protein sequence was not found to have substantial sequence similarity to any other sequence in GenBank. We conclude that this is a c-type cytochrome substantially different from previously characterized c-type cytochromes.

Cytoplasm-to-myonucleus ratios following microgravity.

The cytoplasmic volume-to-myonucleus ratio in the tibialis anterior and gastrocnemius muscles of juvenile rats after 5.4 days of microgravity was studied. Three groups of rats (n = 8 each) were used. The experimental group (space rats) was flown aboard the space shuttle Discovery (NASA, STS-48), while two ground-based groups, one hindlimb suspended (suspended rats), one non-suspended (control), served as controls. Single fibre analysis revealed a significant decrease in cross-sectional area (microns2) in the gastrocnemius for both the space and the suspended rats; in the tibialis anterior only the suspended rats showed a significant decrease. Myonuclei counts (myonuclei per mm) in both the tibialis anterior and gastrocnemius were significantly increased in the space rats but not in the suspended rats. The mean myonuclear volume (individual nuclei: microns3) in tibialis anterior fibres from the space rats, and in gastrocnemius fibres from both the space and the suspended rats, was significantly lower than that in the respective control group. Estimation of the total myonuclear volume (microns3 per.mm), however, revealed no significant differences between the three groups in either the tibialis anterior or gastrocnemius. The described changes in the cross-sectional area and myonuclei numbers resulted in significant decreases in the cytoplasmic volume-to-myonucleus ratio (microns3 x 10(3)) in both muscles and for both space and suspended rats (tibialis anterior; 15.6 +/- 0.6 (space), 17.2 +/- 1.0 (suspended), 20.8 +/- 0.9 (control): gastrocnemius; 13.4 +/- 0.4 (space) and 14.9 +/- 1.1 (suspended) versus 18.1 +/- 1.1 (control)). These results indicate that even short periods of unweighting due to microgravity or limb suspension result in changes in skeletal muscle fibres which lead to significant decreases in the cytoplasmic volume-to-myonucleus ratio.

Cytoplasm-to-myonucleus ratios in plantaris and soleus muscle fibres following hindlimb suspension.

In multinucleated skeletal muscle fibres the size of the cytoplasmic volume-to-myonucleus ratio is related to the myosin heavy chain phenotype, with the ratio being larger in those fibres expressing the fast myosin heavy chain phenotype. It is unknown, however, whether this ratio is modulated during muscle fibre adaptation, such as that which occurs following muscle unloading. In this study the relationship between cross sectional area, myonuclear number and myosin type, in single fibres from the plantaris and soleus muscles of adult rats following 28 days of hindlimb suspension was examined. Each fibre was cut transversely into two segments; one segment was used for immunohistochemical identification of myosin type, the other for determination of cross sectional area and myonuclei number. Single fibre analysis revealed significant atrophy of both plantaris fast and soleus slow fibres; the mean cross sectional area (microns2) of these fibres, 3104 +/- 183 and 2082 +/- 107 (mean +/- SE), being 70 and 45%, respectively, of control means. The decreases in cross sectional area were not accompanied by corresponding decreases in the number of myonuclei (myonuclei/mm); in plantaris fast fibres the mean myonuclei counts were within the control range (88 +/- 8 (hindlimb suspension), 76 +/- 7 (control), in soleus slow fibres the counts were significantly increased (185 +/- 12 (hindlimb suspension), 154 +/- 11 (control)). The changes resulted in a significant decrease in the cytoplasmic volume-to-myonucleus ratio (microns3 x 10(3) for both fibre types; the mean ratios of 39 +/- 3 and 12 +/- 1, were 60% and 36% of control means for the plantaris fast and soleus slow fibres, respectively. These results indicate that following hindlimb suspension atrophy of muscle fibres the myonuclei numbers remain constant or increase and, hence, the effective cytoplasmic-to-myonucleus ratio is decreased. Further, the decreased changes are significantly greater in soleus slow than plantaris fast fibres.