Construction of a new artificial biomineralization system

Hydroxyapatite (HAP) was mineralized in poly(vinyl alcohol) (PVA)/poly(acrylic acid) (PAA) complex hydrogel immersed in a salt solution containing PAA. The transparent HAP/polymer composite swelled in water depending on the HAP content; high HAP content gave small swelling and vice versa. The HAP content reached about 80 wt % at most. Observation of the cross section of the composite by energy-dispersive analysis of X-ray (EDAX) revealed that the composite consisted of two phases, i.e., a hard HAP-rich phase and a soft polymer-rich phase. In the HAP-rich phase, the space inside the hydrogel was occupied by HAP, while HAP was not mineralized in the polymer-rich phase. The nucleation seemed to take place, at first, at the middle depth of the hydrogel where the HAP-rich phase was formed. The HAP-rich phase grew its size toward the surface of the hydrogel at the cost of the polymer-rich phase. The presence of phosphorus, P, in the polymer-rich phase indicated the adsorption of HPO(4)(2-) on the polymer chain of the hydrogel via hydrogen bonding, accompanied with Ca(2+) because of electrostatic constraints. This adsorption of ions in addition to Donnan distribution of ions leads to the formation of a hypercomplex that can be regarded as a precursor of the HAP-rich phase. The change of the hypercomplex into the HAP-rich phase is discontinuous and hence concluded as a phase transition. By comparison of our mineralization system with the biomineralization system of HAP and CaCO(3), the physicochemical mechanism of the mineralization process in the present system was found to be similar to that in biological systems. In this sense, we termed the present system an artificial biomineralization system.     

Characteristics of a new rapidly proliferating rabbit cell line

Summary A new tissue culture cell line (Calg-ARLC) has been established from an explant culture of adult rabbit lung. The Calg-ARLC line has been characterized with respect to morphology, chromosome constitution, tissue culture requirements, proliferative capacity, cell cycle, attainable synchrony, radioisotopically labeled precursor incorporation into nucleic acids and protein, and radioisotope sensitivity. The cells are fibroblast-like in appearance with a stabilized heteroploid chromosomal modal number of 35. They grow exponentially from high split ratios in several commercially available defined media with a generation time of 12 hr and are easily synchronized. Although sensitive to some isotopically labeled precursors, high specific activity nucleic acids have been isolated. The ARLC line is especially useful for the isolation of high specific activity nucleic acids and proteins of rabbit origin. The Calg-ARLC line should be invaluable in the fractionation of reiterated DNA sequences since no very rapidly reassociating DNA sequences such as those found in mouse are evident. This work was supported by operating grants from the National Cancer Institute of Canada and the National Research Council of Canada.     

Evaluation of a new anaerobic power testing system

The purpose of this study was to evaluate the reliability of a new anaerobic athletic performance system. This system is proposed to assess vertical jump height, anaerobic power through repetitive jumping, and reaction to both an auditory and visual stimulus. One hundred twenty-three subjects (92 men and 31 women; mean +/- SD: age, 20.5 +/- 2.1 years; body weight, 83.1 +/- 20.4 kg; height, 176.0 +/- 9.2 cm) volunteered to participate. To assess reliability of the new testing device, subjects were tested on 3 separate occasions (T1, T2, and T3). At least 72 hours but not more than 1 week separated each laboratory visit. During each testing session subjects performed a countermovement jump (CMJ), a 30 consecutive jumps anaerobic power test (30JT), and reaction to both an auditory and visual stimulus. Results showed no differences between T1, T2, and T3 in either CMJ height or 30JT assessments. However, reaction to an audible or visual stimulus significantly improved during each testing session. Intraclass reliability of the CMJ and the 30JT was greater than 0.96 across the 3 trials. Pearson correlation coefficients of r > 0.90 were seen for the CMJ and 30JT, indicating a high test-retest reliability. The test-retest reliability for the reaction tests were lower (r ranging from 0.72 to 0.83). A Bland-Altman plot showed limited agreement between methods of vertical jump height assessment. Results indicate that this new testing device shows high reliability to assess both CMJ height and anaerobic power. In addition, anaerobic power assessment in a jump test provides a specific measure of anaerobic power for many sports incorporating similar performance patterns.     

Chondrocyte proliferation in a new culture system

Objective:  This study has aimed to study different culture systems that might stimulate an increase in cell proliferation of normal and osteoarthritis chondrocytes from articular cartilage in rat model.
Material and Methods:  Three culture systems using chondrocytes embedded in alginate beads were tested: chondrocytes cultured in Dulbecco's modified Eagle's medium (DMEM) as control, a co-culture system consisting of a monolayer of de-differentiated chondrocytes as a source of mitotic factors, and an enriched medium containing culture medium obtained from a monolayer of chondrocytes and DMEM. Normal and osteoarthritis chondrocytes were stained with 5-carboxyfluorescein diacetate succinimidyl ester and were cultured in each of the three systems. After 5 days of culture cell, proliferation was detected by flow cytometry. Chondrocyte phenotype was confirmed by collagen type II and MMP-3 expression. To determine possible molecules released into the medium by the cultured chondrocyte monolayer and which would probably be involved in cell proliferation, a study of mRNA and expression of transforming growth factor-β1 (TGF-β1), fibroblastic growth factor-2 (FGF-2), epidermal growth factor (EGF), platelet derived growth factor-A (PDGF-A) and insulin-like growth factor-1 (IGF-1) proteins was conducted.
Results and Conclusions:  Chondrocytes in the co-culture system or in enriched medium showed an increase in proliferation; only when osteoarthritis chondrocytes were cultured in enriched medium would they display a statistically significant increase in their proliferation rate and in their viability. When chondrocytes from the monolayer were analysed, differential mRNA expression of TGF-β1 and IGF-1 was found during all passages, which suggests that these two growth factors might be involved in chondrocyte proliferation.     

Characteristics of M-GTFI, a new inhibitor of Streptococcus mutans glucosyltransferase

M-GTFI, originally screened as an inhibitor of Streptococcus mutans glucosyltransferase, strongly inhibited alpha-glucosidase, in a non-competitive manner especially when the synthetic substrate p-nitrophenyl-alpha-D-glucopyranoside was used. It also inhibited beta-glucosidase, beta-amylase and, to a lesser extent, beta-glucuronidase. The inhibitor was stable in neutral and alkaline pH ranges and dependency of the inhibition on pH and temperature was not observed. Some proteinases and polysaccharides-hydrolyzing enzymes as well as human saliva did not inactivate the inhibitor. There was a correlation between the release of sulfate anions from the inhibitor molecule on incubation with HCl (0.2 N) at 100 degrees C and loss of inhibitory properties of the molecule. It is suggested that the presence of sulfate ester linkages in the inhibitor molecule play an important role in the inhibition process.     

Development of a new host vector system in mycobacteria

The hybrid plasmid pYT72/pYT92 constructed from an Escherichia coli plasmid pACYC177 and mycobacterial plasmid pMSC262 isolated from Mycobacterium scroflaceum strain W262 transformed both E. coli and BCG. Phage-sensitive mutants S-10 and S-20 isolated from BCG Tokyo strain showed higher frequency of transformation than the wild-type strain. Frequency of transformation was dependent on age of the culture and the electroporation condition used. Several deletion mutants were generated from pYT72/92 to determine the minimum region for the replication in the mycobacteria. A 2.3-kb fragment of pMSC262 was found to contain an essential region. Using this fragment and pACYC177, a small shuttle vector pYT937 containing two drug-resistance markers, kanamycin- and ampicillin-resistance, was constructed. pYT937 contains AatII, BamHI, BbvII, GsuI, HincII, PstI, ScaI and XbaI cloning sites.     

Bacteriophage exclusion,a new defense system

The ability to withstand viral predation is critical for survival of most microbes. Accordingly, a plethora of phage resistance systems has been identified in bacterial genomes (Labrie et al, 2010 ), including restriction‐modification systems (R‐M) (Tock & Dryden, 2005 ), abortive infection (Abi) (Chopin et al, 2005 ), Argonaute‐based interference (Swarts et al, 2014 ), as well as clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) adaptive immune system (CRISPR‐Cas) (Barrangou & Marraffini, 2014 ; Van der Oost et al, 2014 ). Predictably, the dark matter of bacterial genomes contains a wealth of genetic gold. A study published in this issue of The EMBO Journal by Goldfarb et al ( 2015 ) unveils bacteriophage exclusion (BREX) as a novel, widespread bacteriophage resistance system that provides innate immunity against virulent and temperate phage in bacteria.     

Solenoid: a new aquiferous system to Porifera

The aquiferous system is an essential character of poriferans and supports their monophyly. Within the Calcarea, this system displays its greatest variety and traditionally is classified as: asconoid, syconoid, sylleibid, and leuconoid. Species of Leucascus, however, present a different type of aquiferous system composed of anastomosed (interconnected) choanocyte tubes and have an atrium lacking choanoderm. There is such confusion about the classification of the aquiferous system of Leucascus that, depending on the author, it has been classified as asconoid, syconoid, or leuconoid. Therefore, in the present work, we describe a new type of aquiferous system for Leucascus: the solenoid aquiferous system. This new aquiferous system is defined by the presence of anastomosed tubes internally lined by choanocytes and atrium without choanoderm. Although no deep phylogenetic significance has been attributed to the aquiferous system, the solenoid system raises important evolutionary questions about the variety of systems found among the poriferans.     

Characteristics of a new aromatic heptaenic antibiotic, flavumycin A]

The study of the products of alkaline and acid hydrolysis showed that flavumycin A included mycosamine, an aminosugar and n-aminoacetophen, an aromatic fragment. The main functional groups of the antibiotics aglicone were determined.     

Characteristics of a new hairless mutation (bald) in rats

Hereditarily hairless rats have been maintained by sibmatings since 1973. These animals were termed "bald rats", and examined for their characteristics on genetics, physiological, hematological and clinical chemistry values and histology. The bald rats began to lose their hair at approximately three weeks of age. At five weeks of age, the skin was devoid of all general body hair except for the vibrissae, and the skin became wrinkled at two months of age. Histologically, follicular cysts were observed in a large number, from three months of age onward. The bald condition was a simple recessive character (ba); homozygous animals lost hair and heterozygous ones did not. The bald females attained sexual maturity around eight weeks of age and littered normally, but failed to nurse their young. The incapability of supplying sufficient milk supposedly resulted from the mammary glands that involuted after delivery. The bald animals consumed more food but showed lower values for body weights, plasma triglyceride, and epididymal adipose tissue amount than the haired animals. In addition, the bald males had brown adipose tissue in the interscapulum which was histologically activated as in newborn animals. Aged bald animals showed spontaneous tumors of the thymus, pituitary, testis, uterus, mammary gland, and skin. The incidence of skin tumors was much higher than those of tumors of other origins.     

Characterization of AloI, a restriction-modification system of a new type.

We report the properties of the new AloI restriction and modification enzyme from Acinetobacter lwoffi Ks 4-8 that recognizes the DNA target 5' GGA(N)6GTTC3' (complementary strand 5' GAAC(N)6TCC3'), and the nucleotide sequence of the gene encoding this enzyme. AloI is a bifunctional large polypeptide (deduced M(r) 143 kDa) revealing both DNA endonuclease and methyltransferase activities. Depending on reaction cofactors, AloI cleaves double-stranded DNA on both strands, seven bases on the 5' side, and 12-13 bases on the 3' side of its recognition sequence, and modifies adenine residues in both DNA strands in the target sequence yielding N6-methyladenine. For cleavage activity AloI maintains an absolute requirement for Mg(2+) and does not depend on or is stimulated by either ATP or S-adenosyl-L-methionine. Modification function requires the presence of S-adenosyl-L-methionine and is stimulated by metal ions (Ca(2+)). The C-terminal and central parts of the protein were found to be homologous to certain specificity (HsdS) and modification (HsdM) subunits of type I R-M systems, respectively. The N-terminal part of the protein possesses the putative endonucleolytic motif DXnEXK of restriction endonucleases. The deduced amino acid sequence of AloI shares significant homology with polypeptides encoding HaeIV and CjeI restriction-modification proteins at the N-terminal and central, but not at the C-terminal domains. The organization of AloI implies that its evolution involved fusion of an endonuclease and the two subunits, HsdM and HsdS, of type I restriction enzymes. According to the structure and function properties AloI may be regarded as one more representative of a newly emerging group of HaeIV-like restriction endonucleases. Discovery of these enzymes opens new opportunities for constructing restriction endonucleases with a new specificity.