Isolation and Characterization of Two Novel Bacteria Afipia cberi and Mesorhizobium hominis from Blood of a Patient Afflicted with Fatal Pulmonary Illness

We recently isolated and discovered new Bradyrhizobiaceae microbes from the cryopreserved culture broth of blood samples from 3 patients with poorly defined illnesses using modified SP4 media and culture conditions coupled with genomic sequencing. Using a similar protocol, we studied a previously cryopreserved culture broth of blood sample from a patient who had succumbed to an acute onset of fulminant pulmonary illness. We report that two phases of microbial growth were observed in the re-initiated culture. Biochemical and genomic characterization revealed microbes isolated from the first phase of growth were new Afipia species of Bradyrhizobiaceae, tentatively named A. cberi with a ~ 5 MB chromosome that was different from those of all previously known Afipia microbes including the newly discovered A. septicemium. The microbes isolated from the second phase of growth were prominent sugar assimilators, novel Phyllobacteriaceae, phylogenetically most closely related to Mesorhizobium and tentatively named M. hominis with a ~ 5.5 MB chromosome. All A. cberi isolates carry a circular ~ 140 KB plasmid. Some M. hominis isolates possess a circular ~ 412 KB plasmid that can be lost in prolonged culture or passage. No antibiotics resistant genes could be identified in both of the A. cberi and M. hominis plasmids. Antibiotic susceptibility studies using broth culture systems revealed isolates of A. cberi could be sensitive to some antibiotics, but all isolates of M. hominis were resistant to essentially all tested antibiotics. However, the cell-free antibiotics susceptibility test results may not be applicable to clinical treatment against the microbes that are known to be capable of intracellular growth. It remains to be determined if the 2 previously unknown Rhizobiales were indeed pathogenic and played a role in the pulmonary disease process in this patient. Specific probes and methods will be developed to re-examine the diseased lungs from patient''s autopsy.     

Enhancing macrolide production in Streptomyces by coexpressing three heterologous genes

Antibiotic production in Streptomyces can often be increased by introducing heterologous genes into strains that contain an antibiotic biosynthesis gene cluster. A number of genes are known to be useful for this purpose. We chose three such genes and cloned them singly or in combination under the control of the strong constitutive ermE* promoter into a ?C31-derived integrating vector that can be transferred efficiently by conjugation from Escherichia coli to Streptomyces. The three genes are adpA, a global regulator from Streptomyces coelicolor, metK, encoding S-adenosylmethionine synthetase from S. coelicolor, and, VHbS, hemoglobin from Vitreoscilla. The substitutions with GC in VHbS was intended to convert codons from lower usage to higher, yet causing no change to the encoded amino acid. Plasmids containing either one of these genes or genes in various combinations were introduced into Streptomyces sp. FR-008, which produces the macrolide antibiotic FR-008-III (also known as candicidin D). The largest increase in FR-008-III production was achieved by the plasmid containing all three genes. This plasmid also increased avermectin production in Streptomyces avermitilis, and is likely to be generally useful for improving antibiotic production in Streptomyces.     

A sensitive radiochemical enzyme assay for S-adenosyl-l-homocysteine and l-homocysteine

The reversibility of the S-adenosylhomocysteine hydrolase reaction allows both nonradiolabeled S-adenosyl-l-homocysteine and l-homocysteine to serve as l-homocysteine donors for the synthesis of radiolabeled S-adenosyl-l-homocysteine from radiolabeled adenosine. Using high specific activity, radiolabeled adenosine, and high-performance liquid chromatography to separate products and reactants, as little as 0.1 pmol of l-homocysteine donor can be detected by its ability to be converted to radiolabeled S-adenosyl-l-homocysteine. Reduction of samples with dithiothreitol during the enzymatic reaction allows l-homocystine and mixed disulfides of l-homocysteine to be assayed as well. S-Adenosyl-l-homocysteine can be distinguished from other l-homocysteine donors by assaying samples before and after degradation of the former with the nonspecific adenosine deaminase from Aspergillus oryzae. A less sensitive version of the assay employs thin-layer chromatography in place of high-performance liquid chromatography, and is useful for the assay of l-homocysteine and its disulfides in quantities of 25 pmol or more.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

Sesquiterpenoid metabolites from Stereum complicatum

A new sesquiterpene antibiotic, complicatic acid, isolated from cultures of Stereum complicatum (Fr.)Fr. has been shown to be dehydrohirsutic acid C. Hirsutic acid C was also isolated from the same fungus. [2-¹⁴C]-MVA was incorporated into both metabolites and complicatic acid has been shown to be formed from hirsutic acid C both in vivo and in vitro.     

Characterization of α-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

α-mannosidase from Erythrina indica seeds is a Zn²⁺ dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 °C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol^− 1. N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3–8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of α-helical structure in the enzyme. α-Mannosidase from E indica exhibits immunological identity with α-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with α-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of α-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.     

cpcBA-IGS as an effective marker to characterize Microcystis wesenbergii (Komárek) Komárek in Kondrateva (cyanobacteria)

Microcystis wesenbergii (Komárek) Komárek in Kondrateva, a major bloom forming cyanobacterial species, possesses unique colonial characteristics which can be easily distinguished from other Microcystis species. However, there is still no genetic marker to effectively characterize M. wesenbergii. In this research, thirteen strains of M. wesenbergii, collected from eight locations in Chinese water bodies were examined for molecular characterization of both cpcBA-IGS sequences (phycocyanin intergenic spacer and flanking regions) and ITS sequences (internal transcribed spacer region between 16S and 23S rDNA). The phylogenetic analysis based on cpcBA-IGS sequences showed that the M. wesenbergii strains formed a distinct cluster with high support values, indicating the cpcBA-IGS region could be used to characterize and distinguish M. wesenbergii from other species of Microcystis. These developed primers were verified to be effective in distinguishing M. wesenbergii from other species of Microcystis and from other species in different genera of cyanobacteria.     

Purification and Characterization of Chorismate Synthase from Euglena gracilis: Comparison with Chorismate Synthases of Plant and Microbial Origin

Chorismate synthase was purified 1200-fold from Euglena gracilis. The molecular mass of the native enzyme is in the range of 110 to 138 kilodaltons as judged by gel filtration. The molecular mass of the subunit was determined to be 41.7 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified chorismate synthase is associated with an NADPH-dependent flavin mononucleotide reductase that provides in vivo the reduced flavin necessary for catalytic activity. In vitro, flavin reduction can be mediated by either dithionite or light. The enzyme obtained from E. gracilis was compared with chorismate synthases purified from a higher plant (Corydalis sempervirens), a bacterium (Escherichia coli), and a fungus (Neurospora crassa). These four chorismate synthases were found to be very similar in terms of cofactor specificity, kinetic properties, isoelectric points, and pH optima. All four enzymes react with polyclonal antisera directed against chorismate synthases from C. sempervirens and E. coli. The closely associated flavin mononucleotide reductase that is present in chorismate synthase preparations from E. gracilis and N. crassa is the main difference between those synthases and the monofunctional enzymes from C. sempervirens and E. coli.     

Afrochoerodon nov. gen. kisumuensis (MacInnes) (Proboscidea,Mammalia) from Cheparawa,Middle Miocene,Kenya

A proboscidean skull from Cheparawa, (Muruyur Formation, Kenya), differs markedly from those of Eurasian Choerolophodon (C. pentelici, C. dhokpathanensis). It is morphologically and metrically close to the holotype of Choerolophodon kisumuensis (MacInnes) a partial skull from Maboko, much of which has been reconstructed in plaster of Paris. The more complete remains of this species now available indicate that it should be placed in a genus separate from Choerolophodon. The new genus Afrochoerodon is erected for it. Choerolophodon ngorora from Ngorora and Fort Ternan (Kenya), Choerolophodon zaltaniensis from Gebel Zelten (Libya) and Choerolophodon chioticus from Chios, Greece, should be transferred to the genus Afrochoerodon. Late Miocene specimens from Nakali, Kenya are probably referrable to the genus Choerolophodon. Fossils from Burji-Soyama (Ethiopia) hitherto assigned to Choerolophodon sp. are excluded from the subfamily Choerolophodontinae.     

Water loss from aerially exposed mussels

The valve activity and water loss from aerially exposed Mytilus edulis L. and Modiolus modiolus (L.) have been investigated in a series of laboratory experiments. Mytilus is capable of maintaining long periods of complete, or almost complete, valve closure when exposed in air, and this allows the retention of water in the mantle cavity and protects the tissues from evaporative water loss. Over periods of three days or more the amount of water lost from emersed Mytilus was found to be less than that retained in the mantle cavity at the beginning of exposure, suggesting that during normal periods of exposure the tissues are never directly subject to water loss. In contrast, Modiolus shows periods of gaping during which water loss is rapid due to drainage from the mantle cavity and evaporation from the tissues. The exposure of the tissues to air that results from gaping, makes the water loss susceptible to environmental influences of which wind was found to be the factor which caused the greatest increase in water loss and, as a consequence, an important factor in causing death through dehydration. The different abilities of Mytilus edulis and Modiolus modiolus to control water loss may be related to their intertidal distributions.     

Stomatal Ultrastructure, Molecular Phylogeny, and Description of Parasitodiplogaster laevigata n. sp. (Nematoda: Diplogastridae), a Parasite of Fig Wasps

Parasitodiplogaster comprises a potentially large radiation of nematode species that appear to be parasitically bound to their Agaonid fig wasp hosts, which are mutualistically associated in the syconia (figs) of the diverse plant genus Ficus. Parasitodiplogaster laevigata n. sp. is described and illustrated as an associate of the fig wasp, Pegoscapus sp. from Ficus laevigata from southern Florida. It is the first species of Parasitodiplogaster reported from North America and is closest to P. trigonema from F. trigonata from Panama. Parasitodiplogaster laevigata n. sp. can be differentiated from all described species of Parasitodiplogaster based on stomatal morphology (presence of a large dorsal and a right subventral tooth) in the adults of both sexes, molecular comparisons of two expansion segments (D2,D3) of the large subunit (LSU) rRNAgene, and fig-fig wasp host affinities. The ultrastructure of P. laevigata n. sp. was elucidated using TEM and SEM for comparisons with other species of Parasitodiplogaster. The stoma of P. laevigata n. sp. possesses a nonsegmented cheilostomal ring that connects to the longitudinal body musculature per- and interradially, a claw-like dorsal tooth, a right subventral tooth, and telostegostomatal apodemes arising from the dorsal side of each subventral sector. The unification of the pro-, meso-, and metastegostom with the gymnostom in P. laevigata n. sp. and further simplification in other described species may be due to derived adaptations associated with the internal parasitism of fig wasps.     

The membrane-bound high-molecular-mass cytochromes c from Desulfovibrio gigas and Desulfovibrio vulgaris Hildenborough; EPR and Mössbauer studies

The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280?mV, and can be roughly divided into three groups: four to five hemes have E ⁰s of –30?mV to –100?mV, three to four hemes have E ⁰s around –170?mV, and seven to eight hemes have a lower E ⁰ of –250 to –280?mV. Several of these redox potentials are strongly pH dependent. Mössbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow.     

Pseudomonas putida and Pseudomonas fluorescens Species Group Recovery from Human Homes Varies Seasonally and by Environment

By shedding light on variation in time as well as in space, long-term biogeographic studies can help us define organisms’ distribution patterns and understand their underlying drivers. Here we examine distributions of Pseudomonas in and around 15 human homes, focusing on the P. putida and P. fluorescens species groups. We describe recovery from 10,941 samples collected during up to 8 visits per home, occurring on average 2.6 times per year. We collected a mean of 141 samples per visit, from sites in most rooms of the house, from the surrounding yards, and from human and pet occupants. We recovered Pseudomonas in 9.7% of samples, with the majority of isolates being from the P. putida and P. fluorescens species groups (approximately 62% and 23% of Pseudomonas samples recovered respectively). Although representatives of both groups were recovered from every season, every house, and every type of environment sampled, recovery was highly variable across houses and samplings. Whereas recovery of P. putida group was higher in summer and fall than in winter and spring, P. fluorescens group isolates were most often recovered in spring. P. putida group recovery from soils was substantially higher than its recovery from all other environment types, while higher P. fluorescens group recovery from soils than from other sites was much less pronounced. Both species groups were recovered from skin and upper respiratory tract samples from healthy humans and pets, although this occurred infrequently. This study indicates that even species that are able to survive under a broad range of conditions can be rare and variable in their distributions in space and in time. For such groups, determining patterns and causes of stochastic and seasonal variability may be more important for understanding the processes driving their biogeography than the identity of the types of environments in which they can be found.     

New materials of Dinocrocuta (Percrocutidae, Carnivora) from Lantian, Shaanxi Province, China, and remarks on Chinese Late Miocene biochronology

New materials from the middle part of the Bahe formation are described as Dinocrocuta gigantea. Review of the species reveals that it is derived in the evolutionary lineage of Dinocrocuta, and biochronologically later than Vallesian records from Turkey. The only possibly related Vallesian species from China is Crocuta gigantea xizangensis from Biru, Tibet, which may prove to be conspecific with D. senyureki. Based on the mammalian faunal sequence from Lantian, and with reference to Red Clay paleomagnetic data, the duration of D. gigantea in China should be later late Miocene, rather than the previously postulated early late Miocene (Vallesian equivalent) age.     

Trichoderma songyi sp. nov., a new species associated with the pine mushroom (Tricholoma matsutake)

A new species, Trichoderma songyi, was found to be associated with the pine mushroom (Tricholoma matsutake) in Korea. This species was isolated from three different substrates: Tricholoma matsutake basidiomata, as well as roots of Pinus densiflora and soil in the fairy ring. Based on its molecular and phenotypic characteristics, we demonstrate that Trichoderma songyi is unique and distinguishable from closely related species. We performed phylogenetic analyses based on two molecular markers, the genes for both translation elongation factor 1-alpha and the second largest subunit of RNA polymerase II. Phylogenetic analyses showed that Trichoderma songyi is closely related to Trichoderma koningii aggregate and Trichoderma caerulescens. Morphologically, Trichoderma songyi can be distinguished from these closely related taxa by its growth rates, colony morphology on PDA in darkness, and coconut-like odour. Due to the economic importance of the pine mushroom, the relationship between Trichoderma songyi and Tricholoma matsutake should be studied further.     

Nuclear Ribosomal DNA Variation and Pathogenic Specialization in Alternaria Fungi Known To Produce Host-Specific Toxins

A total of 99 strains of 11 Alternaria species, including 68 strains of seven fungi known to produce host-specific toxins, were subjected to analysis of restriction fragment length polymorphism (RFLP) in nuclear ribosomal DNA (rDNA). Total DNA was digested with XbaI, and the Southern blots were probed with a nuclear rDNA clone of Alternaria kikuchiana. The hybridization gave 17 different RFLPs from the 99 strains. On the basis of these RFLPs, populations of host-specific toxin-producing fungi could not be differentiated from one another nor from nonpathogenic A. alternata. Each population of the toxin-producing fungi carried rDNA variants. Nine different types, named A1 to A6 and B1 to B3, were detected among the toxin-producing fungi and nonpathogenic A. alternata. All of the populations contained the type A4 variant, and the other rDNA types were also shared by different toxin-producing fungi and A. alternata. In contrast, Alternaria species that are morphologically distinguishable from A. alternata could be differentiated from A. alternata on the basis of the rDNA RFLPs. Polymorphisms in rDNA digested with HaeIII and MspI were also evaluated in 61 Alternaria strains. These restriction enzymes produced 31 variations among all of the samples. The seven toxin-producing fungi and nonpathogenic A. alternata could not be resolved by phylogenetic analysis based on the RFLPs, although they could be differentiated from the other Alternaria species studied. These results provide support for the hypothesis that Alternaria fungi known to produce host-specific toxins are intraspecific variants of A. alternata specialized in pathogenicity.     

Forever in the dark: the cave-dwelling?azooxanthellate reef coral Leptoseris troglodyta sp. n. (Scleractinia,?Agariciidae)

The coral species Leptoseris troglodyta sp. n. (Scleractinia, Agariciidae) is described as new to science. It is the first known azooxanthellate shallow-water agariciid and is recorded from the ceilings of caves at 5-35 m depth in West Pacific coral reefs. The corals have monocentric cup-shaped calices. They may become colonial through extramural budding from the basal coenosteum, which may cause adjacent calices to fuse. The size, shape and habitat of Leptoseris troglodyta are unique compared to other Leptoseris species, many of which have been recorded from mesophotic depths. The absence of zooxanthellae indicates that it may survive well in darkness, but endolithic algae in some corals indicate that they may be able to get some light. The presence of menianes on the septal sides, which may help to absorb light at greater depths in zooxanthellate corals, have no obvious adaptive relevance in the new species and could have been inherited from ancestral species that perhaps were zooxanthellate. The new species may be azooxanthellate as derived through the loss of zooxanthellae, which would be a reversal in Leptoseris phylogeny.     

THE EFFECT OF ADVANCE IN LACTATION AND GESTATION ON MAMMARY ACTIVITY

The rate of milk secretion in farrow cows may be expressed as See PDF for Equation, in which y = yield and t = time from calving. Pregnancy causes a decrease in yield which may be expressed as See PDF for Equation, in which i = inhibition or decrease in yield and p = time from conception. The constant K appears to be the same for various groups but b is roughly proportional to a. The decrease in yield associated with pregnancy is interpreted as due to a hormone. The hormone hypothesis also affords an interpretation of the increasing rate of milk secretion which occurs for a short time following parturition.