Complete Genome Sequence of Macrococcus caseolyticus Strain JSCS5402, Reflecting the Ancestral Genome of the Human-Pathogenic Staphylococci

We isolated the methicillin-resistant Macrococcus caseolyticus strain JCSC5402 from animal meat in a supermarket and determined its whole-genome nucleotide sequence. This is the first report on the genome analysis of a macrococcal species that is evolutionarily closely related to the human pathogens Staphylococcus aureus and Bacillus anthracis. The essential biological pathways of M. caseolyticus are similar to those of staphylococci. However, the species has a small chromosome (2.1 MB) and lacks many sugar and amino acid metabolism pathways and a plethora of virulence genes that are present in S. aureus. On the other hand, M. caseolyticus possesses a series of oxidative phosphorylation machineries that are closely related to those in the family Bacillaceae. We also discovered a probable primordial form of a Macrococcus methicillin resistance gene complex, mecIRA_m, on one of the eight plasmids harbored by the M. caseolyticus strain. This is the first finding of a plasmid-encoding methicillin resistance gene. Macrococcus is considered to reflect the genome of ancestral bacteria before the speciation of staphylococcal species and may be closely associated with the origin of the methicillin resistance gene complex of the notorious human pathogen methicillin-resistant S. aureus.Among various bacterial genera, Macrococcus is the most closely related to the genus Staphylococcus. Historically, it had been included in the staphylococcal family until it was reassigned to an independent genus because of its distinctively smaller genome size than that of staphylococci (19). Currently, seven species are included in genus Macrococcus: Macrococcus bovicus, M. carouselicus, M. caseolyticus, M. equipercicus, M. brunensis, M. hajekii, and M. lamae (26). Unlike staphylococcal species, macrococci do not cause human or animal diseases and are typically isolated from animal skin and food such as milk and meat. The physiological features of this organism are, however, largely unknown, and only a small number of reports on macrococci have been published.M. caseolyticus, previously classified as Staphylococcus caseolyticus (34), was reportedly isolated from cow''s milk, bovine organs and food-processing factories. Phylogenetic relationship analysis based on 16S rRNA sequences revealed that, in addition to Staphylococcus, Bacillus species are also closely related to M. caseolyticus. The morphology of this organism is globular; however, the cell size is larger than for staphylococci.Since the genomes of macrococci are much smaller than those of staphylococci (34), we considered that knowledge of the macrococcal genome would be important in elucidating the evolution of staphylococci along with its acquisition of pathogenic potential to humans. We describe here a unique metabolic feature of M. caseolyticus as inscribed in its genome and the discovery of a primordial form of a mecA gene complex, the causative genetic determinant of the notorious hospital pathogen methicillin-resistant S. aureus (MRSA).     

The dynamic expression of tenascin-C and tenascin-X during early heart development in the mouse

One of a family of extracellular matrix proteins, tenascin-C (TNC) is expressed in a spatiotemporally restricted pattern associated with tissue remodeling during embryonic development, wound healing, cancer invasion and tissue regeneration. Another form, tenascin-X (TNX), is found in most tissues but most predominantly in heart and muscle, often complementarily to TNC. The present analysis demonstrated their expression during early heart development, using mouse lines containing the lacZ gene targeted to the TNC locus, by RT-PCR, immunohistochemistry, and in situ hybridization. TNC was transiently expressed at important steps during heart development: (1) precardiac mesodermal cells differentiating to cardiomyocytes and endocardial cells at E 7.5 - 8.5; (2) cardiomyocytes in the outflow tract at E 8.5 - 12; (3) endocardial cells forming cushion tissue at E 9.5 - 13; and (4) mesenchymal cells in the proepicardial organ (PEO), the precursors of coronary vessels, at E 9.5. When PEO cells were transferred onto the heart surface, the expression of TNC was downregulated, while TNX was upregulated at E 11. Initially, epicardial cells around the AV groove and atrium started to express TNX. TNX-positive cells then gradually spread all over the entire surface of the heart and invaded and formed primitive vascular channels in the myocardium. Despite restricted expression at important sites and steps during cardiogenesis, the hearts of TNC deficient mice developed normally. No difference in the expression pattern of TNX were observed in TNC knockout and wild mice. These results suggest; (1) TNC could play important roles in the differentiation of cardiomyocytes and the early morphogenesis of the heart; (2) TNX could be involved in coronary vasculogenesis; (3) TNX does not compensate for the loss of TNC.     

Allozyme variability within and divergence among populations of the liverwortConocephalum conicum (Marchantiales: Hepaticae) in Japan

Genetic variation within, and divergence among, populations of the liverwortConocephalum conicum were estimated from the study of 17 populations and 23 putative gene loci. Two additional multilocus genotypes (“T” and “FS”) were detected in Japan, along with the previously reported “J” type. These three multilocus genotypes differed both morphologically and ecologically. All eight populations from western Japan included only the J-type and exhibited low genetic variation within populations: Nei's (1973) average gene diversity (Ĥ)=0.080±0.029. In contrast, co-occurrence of several multilocus genotypes in each population from the Kanto District resulted in much higher levels of genetic variation (Ĥ=0.218±0.037). If the three genotypes are distinguished,Ĥ values are 0.113±0.030 for T-type, 0.107±0.033 for FS-type, and 0.083±0.018 for J-type. UsingC. japonicum, which showed low genetic variation (0.014±0.010) as an outgroup, each genotype formed a monophyletic clade, and the J- and FS-types were more closely related to each other than to the T-type. Populations of western Japan and the Kanto District also differed in the degree of gene diversity among populations, but the reasons for these differences are obscure.     

Dual origin of mesenchymal tissues participating in mouse mammary gland embryogenesis

By the 14th day of gestation, two different mesenchymes can be identified which affect mouse mammary gland embryogenesis: the fibroblastic mammary mesenchyme (MM) closely surrounding the epithelial rudiment, and a condensed mesenchymal tissue (FP) appearing separately, posterior to the mammary rudiment, the precursor tissue of the fat pad. Late on the 16th day, the mammary epithelium (ME), surrounded by MM, starts to elongate, puts out branches, and penetrates the FP. A fatty substance appears in the FP at this stage. Interaction between ME and FP is necessary for typical mammary morphogenesis. When 17-day ME is combined with 14- or 17-day FP, the resulting mammary gland has the normal mammary pattern, but when 17-day ME is combined with 12- to 17-day MM, a ductal hyperplasia is formed by frequent branching, without the “stretching out” of these ducts. All the glands formed by combining ME with either FP or MM will lactate, if the mice carrying the grafts are allowed to mate and give birth. Adult ME also shows a different response to MM and FP.     

Experience of reviewing the follow-on biologics including Somatropin and erythropoietin in Japan

To share the experience of reviewing clinical data required for the licensing of follow-on biologic products (biosimilar products and similar biotherapeutical products as EU and WHO terminology, respectively) in Japan, the data packages of two follow-on biologics, "Somatropin BS s.c. [Sandoz] (Omnitrope?)" and "Epoetin alfa BS [JCR]", which have been recently approved in Japan according to the "Guidelines for the Quality, Safety and Efficacy Assurance of Follow-on Biologics" published on March 4th 2009, are described. The clinical data package and indication of Somatropin BS/Omnitrope(?) were different in each country. In case of Epoetin alfa BS [JCR], non-clinical and clinical data-package was different from those of erythropoietin biosimilar products approved in EU. Submission of post-marketing surveillance plans for both products was required. Even though there seem to be differences in data requirements by each national regulatory authority, the accumulation of experience will provide the rationale and consensus on how to design the clinical trials for follow-on biologics.     

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4–7 N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix.Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.     

Quality, safety and efficacy of follow-on biologics in Japan

Recently, WHO, EU, Japan and Canada have published guidelines on biosimilar/follow-on biologics. While there seems to be no significant difference in the general concept in these guidelines, the data to be submitted for product approval are partially different. Differences have been noted in the requirements for comparability studies on stability, prerequisites for reference product, or for the need of comparability exercise for determination of process-related impurities. In Japan, there have been many discussions about the amount and extent of data for approval of follow-on biologics. We try to clarify the scientific background and rational for regulatory pathway of biosimilar/follow-on biologics in Japan in comparison with the guidelines available from WHO, EU and Canada. In this article, we address and discuss the scientific background underlying these differences to facilitate the harmonization of follow-on biologic principles in the guidelines in future.     

In vitro chemotaxis of Brugia pahangi infective larvae to the sera and hemolymph of mammals and lower animals

The jird (Mongolian gerbil) is a highly susceptible experimental host for the lymphatic filarial nematode, Brugia pahangi. The chemotactic activity of serum from this host for B. pahangi infective larvae was compared in vitro to that of sera or hemolymph of a wide variety of other organisms including mammals, reptiles, fishes and invertebrates. The range of the Chemotactic Index (CI) was from 96.0 for the jird to 56.2 for a snail. An average of CI of saline control was 4.5. Significant chemotactic activity was present in many organisms, especially mammals, but was not closely related to either the phylogenetic position of the organism and to its known susceptibility as definitive host for B. pahangi. Migratory response was diminished in a consistent way by serial dilution of sera of humans, jirds and fetal bovine serum. Pre-incubation of larvae in fetal bovine serum inhibited migration, especially towards the sera of humans. Inhibition could be reversed by rinsing larvae in saline, longer rinse periods resulting in greater recovery of CI. These results are the first to suggest the activity of the specific amphid chemoreceptors in the chemotaxis of the infective larvae of B. pahangi.     

A Novel Gene,fudoh, in the SCCmec Region Suppresses the Colony Spreading Ability and Virulence of Staphylococcus aureus

Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis.