Bioequivalence comparison of a new freezing bag (CryoMACS(®)) with the Cryocyte(®) freezing bag for cryogenic storage of human hematopoietic progenitor cells

Background aimsWe investigated two different plastic freezing bags, namely the most recently U.S. Food and Drug Administration (FDA)-approved CryoMACS^® freezing bag (200-074-402) from Miltenyi Biotec and the familiar Cryocyte^® freezing bag (R4R9955) from (Baxter Healthcare, Deerfield, IL, United States) for the cryogenic storage of human hematopoietic progenitor cells (HPC).MethodsThe study material consisted of 12 frozen HPC pairs (= 24 transplant units) that were no longer needed for autologous treatment of patients. After thawing, one unit of a pair was transferred into the Miltenyi (M) bag; the other unit remained in the original Baxter (B) bag. After refreezing both units, all units were stored again under cryogenic conditions either partially immersed in liquid nitrogen (n = 22) or in the vapor phase over liquid nitrogen, n = 2, ResultsThe correlation coefficients (r) between the results obtained from the two bag types were high for white blood cells (WBC) content (r = 0.98), mononuclear cells (MNC) (r = 0.97), lymphocytes (r = 0.98), monocytes (r = 0.96), membrane integrity (r = 0.93), concentration of ‘free’ hemoglobin (r = 0.97) and hemolysis rate (r = 0.95). With regard to clonogenicity, there were no significant differences (Student's paired t-test) for the three parameters investigated [i.e. total number of colonies, including the numbers of burst-forming units–erythroid (BFU-E) and colony-forming units–granulocyte-macrophage (CFU-GM) colonies, respectively).ConclusionsThe CryoMACS freezing bag 200-074-402 is bioequivalent to the Cryocyte freezing container R4R9955. An advantageous feature of the CryoMACS is that its double-sterile wrapping provides additional safety regarding potential cross-contamination during cryogenic storage.     

A novel prenylated C6–C3 compound with estrogen-like activity from the fruits of Illicium arborescens

A novel C₆–C₃ prenylated compound, illicarborene A (1), together with illioliganfunone D (2), 1-allyl-3,5-dimethoxy-4-(3-methylbut-2-enyloxy)benzene (3), (?)-illicinone A (4), (?)-illicinone B (5) and (?)-illicinone A derivative (6) was isolated and characterized from the fruits of Illicium arborescens Hayata. Compound 1 possesses a new class of tricyclic 6/6/5 ring system. The structure of 1 was determined by spectroscopic analysis such as ¹H–¹H COSY, HMQC, HMBC, and NOESY, and confirmed by chemical reaction to yield 7. Compounds 1–5 were found to increase proliferative activity in primary cell culture of osteoblast cells.     

The occurrence of “mixed” nuclear bag intrafusal fibers in the cat muscle spindle

Summary A total of 147 muscle spindles was studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Nuclear bag₁, nuclear bag₂ and nuclear chain intrafusal muscle fibers were distinguished by the differential staining resulting from the reactions for myosin adenosine 5-triphosphatase and nicotinamide adenine dinucleotide tetrazolium reductase. The majority of intrafusal fibers were of the same histochemical type at both fiber poles. However, seven muscle spindles contained one nuclear bag fiber each that presented as a bag₁ in one pole and as a bag₂ in the other pole. These mixed nuclear bag fibers were found in spindles that also contained at least one bag₁ and one bag₂ fiber of equivalent histochemical presentation in both fiber poles. The mixed bag fibers displayed differences of apparent fiber diameter and relative polar length between the two fiber poles. The motor innervation pattern, as revealed by staining for cholinesterase, was also dissimilar between the two poles of mixed bag fibers. The study indicates that the spindle equatorial region may in some instances serve as a boundary between two morphologically and histochemically different poles of the same intrafusal fiber.     

Reclassification of “Flavobacterium arborescens” (Frankland and Frankland) Bergey et al. in the genusMicrobacterium (Orla-Jensen) Collins et al., asMicrobacterium arborescens comb. nov., nom. rev.

Flavobacterium arborescens (Frankland and Frankland) Bergey et al. IFO 3750 (ATCC 4358) is a Gram-positive, coryneform bacterium and the only available reference strain of the species. The cell wall peptidoglycan of the organism possesses alanine, glycine, lysine, glutamic acid plus 3-hydroxyglutamic acid, and homoserine at a ratio of 1:3:1:1:1, and a possible peptidoglycan structure is the B1 type described by Schleifer and Kandler. Cell wall sugars are galactose, mannose, and 6-deoxy-l-talose, but not rhamnose. Major menaquinones are unsaturated MK-11 and MK-12. These findings and other taxonomic properties suggest that F. arborescens should be reclassified in the genusMicrobacterium (Orla-Jensen) Collins et al., asMicrobacterium arborescens comb. nov., nom. rev.     

Assessing hunters’ ability to identify shot geese: implications for hunting bag accuracy

Reliable hunting bag statistics are a prerequisite for sustainable harvest management. Recently, Internet-based hunting bag reporting systems have been introduced in some European countries, e.g. Denmark, which may enable faster and more detailed reporting. However, reporting of waterfowl bags on a species-specific level may be biased from the individual hunters’ ability to correctly identify species, particularly because juvenile birds can only be identified from subtle differences. We assessed hunters’ ability to identify the five goose species huntable in Denmark. Identifications were made from a line-up of ten full-bodied geese including adults and juveniles. From a total of 2160 identifications made by active hunters, 85.5% were correct while 14.5% were assigned to a wrong species. Active hunters had on average an identification accuracy of 76.0%, highest for Canada goose (99.1%) and lowest for white-fronted goose (74.6%) and bean goose (73.7%). Identification accuracy was significantly lower for juvenile than for adult individuals of white-fronted and bean geese. Correcting the official Danish Bag Record (2013/2014) for identification accuracy, the bags of white-fronted and bean geese increase by 56.5 and 104.4%, respectively, while the bags of greylag and pink-footed geese decrease by 6.7 and 9.0%; the bag for Canada goose remains unchanged. Although identification accuracy is probably higher under field conditions, the study documents that inaccurate species identification is a source of bias in national bag statistics. Hence, improving identification skills by hunters is important to improve bag data accuracy when based on Internet reporting.     

Where is the pulse to have the finger on? A retrospective analysis of two decades of Alpine Galliforms (Aves: Galliformes) census and game bag data in Italy

Information on the abundance of the Italian populations of black grouse (Lyrurus tetrix), Alpine rock ptarmigan (Lagopus muta helvetica) and Alpine rock partridge (Alectoris graeca saxatilis) rely only on extrapolations of local data to the national scale, since there is no national standardized survey. Consequently, their status is virtually unknown. We performed a first-ever assessment of a medium-term (1996–2014) population trend of these species using and comparing post-breeding count and bag data at hunting district scale. These data were collected from various authorities in charge of wildlife management and allowed us to test the influence of hunting policies on the estimated trends. Rock partridge showed a stable trend with numbers fluctuating between years, while there was evidence of a severe decline for rock ptarmigan. No general conclusion could be drawn for the black grouse, as we detected lack of consistency of count and bag data. Counts were greatly overdispersed as a result of an uneven count effort among hunting districts. Adding the game management authority as model covariate resulted in more robust trend estimations, suggesting a significant effect of different policies that emerged also as similar hunting pressure across species within authorities. Hunting effort variation over the time was instead negligible. Species-specific game management bias is discussed. Our results highlight the need for a survey scheme or guidelines to be applied uniformly at a national scale.     

Sand Aggregation by Exopolysaccharide-Producing <Emphasis Type="Italic">Microbacterium arborescens––</Emphasis>AGSB

In the rhizosphere, exopolymers are also known to be useful to improve the moisture-holding capacity. The ability of the isolates from coastal sand dunes to produce exopolymers was determined. Among which the isolate, showing very high production of exopolysaccharide (EPS), Microbacterium arborescens––AGSB, a facultative alkalophile was further studied for exopolymer production. The isolate a gram-positive non-spore forming, slender rod, catalase positive, oxidase negative, showed growth in 12% sodium chloride. The culture was found to produce exopolymer which showed good aggregation of sand which has an important role in the stabilization of sand dunes. The exopolymer was further analysed. The cold isopropanol precipitation of dialysed supernatants grown in polypeptone yeast extract glucose broth produced partially soluble EPSs with glucose as the sole carbon source. Chemical analysis of the EPS revealed the presence of rhamnose, fucose, arabinose, mannose, galactose and glucose. On optimization of growth parameters (sucrose as carbon source and glycine as nitrogen source), the polymer was found to be a heteropolysaccharide containing mannose as the major component. It was interesting to note that the chemical composition of the exopolymers produced from both unoptimized and optimized culture conditions of Microbacterium arborescens––AGSB is different from those of other species from the same genera. This study shows that marine coastal environments such as coastal sand dunes, are a previously unexplored habitat for EPS-producing bacteria, and that these molecules might be involved in ecological roles protecting the cells against dessication especially in nutrient-limited environments such as the coastal sand dunes more so in the extreme conditions of pH. Such polysaccharides may help the bacteria to adhere to solid substrates and survive during the nutrient limitations.     

A Highlights from MBoC Selection: Calcineurin-dependent cofilin activation and increased retrograde actin flow drive 5-HT–dependent neurite outgrowth in Aplysia bag cell neurons

Neurite outgrowth in response to soluble growth factors often involves changes in intracellular Ca²⁺; however, mechanistic roles for Ca²⁺ in controlling the underlying dynamic cytoskeletal processes have remained enigmatic. Bag cell neurons exposed to serotonin (5-hydroxytryptamine [5-HT]) respond with a threefold increase in neurite outgrowth rates. Outgrowth depends on phospholipase C (PLC) → inositol trisphosphate → Ca²⁺ → calcineurin signaling and is accompanied by increased rates of retrograde actin network flow in the growth cone P domain. Calcineurin inhibitors had no effect on Ca²⁺ release or basal levels of retrograde actin flow; however, they completely suppressed 5-HT–dependent outgrowth and F-actin flow acceleration. 5-HT treatments were accompanied by calcineurin-dependent increases in cofilin activity in the growth cone P domain. 5-HT effects were mimicked by direct activation of PLC, suggesting that increased actin network treadmilling may be a widespread mechanism for promoting neurite outgrowth in response to neurotrophic factors.     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Selection of optimal variants of Gō-like models of proteins through studies of stretching

The Gō-like models of proteins are constructed based on the knowledge of the native conformation. However, there are many possible choices of a Hamiltonian for which the ground state coincides with the native state. Here, we propose to use experimental data on protein stretching to determine what choices are most adequate physically. This criterion is motivated by the fact that stretching processes usually start with the native structure, in the vicinity of which the Gō-like models should work the best. Our selection procedure is applied to 62 different versions of the Gō model and is based on 28 proteins. We consider different potentials, contact maps, local stiffness energies, and energy scales—uniform and nonuniform. In the latter case, the strength of the nonuniformity was governed either by specificity or by properties related to positioning of the side groups. Among them is the simplest variant: uniform couplings with no i, i + 2 contacts. This choice also leads to good folding properties in most cases. We elucidate relationship between the local stiffness described by a potential which involves local chirality and the one which involves dihedral and bond angles. The latter stiffness improves folding but there is little difference between them when it comes to stretching.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.     

Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

The kinetics of renaturation of the β₂-subunit of Escherichia coli tryptophan-synthetase (l-serine hydrolyase (adding indole) E.C. 4.2.1.20) and those of its two proteolytic fragments F₁ and F₂ are studied and compared. Steps corresponding to the refolding of F₁, to the association of the folded F₁ and F₂ fragments, and to an isomerization of the associated protein are identified. These steps are ordered on the pathway of renaturation and some of their kinetic parameters are determined. This leads to a tentative kinetic model for the renaturation of nicked-β₂ starting from the denatured F₁ and F₂ fragments.The step corresponding to the refolding of the F₁ domain, as well as that corresponding to the last rate-limiting isomerization leading to the native protein, is shown to be the same in the refolding of the entire, uncleaved β₂-protein. It is concluded that the refolded F₁ fragment corresponds to a folding intermediate on the pathway of renaturation of the β₂-subunit.     

Determination of the number of degrees of freedom of the trapeziometacarpal joint–An in vitro study

ObjectiveMost of the studies about trapeziometacarpal joint assume that it exhibits only two independent degrees of freedom, but the experimental or theoretical support for considering a two-degrees of freedom model is not always clear.Materials and methodsTherefore, an in vitro kinematic study has been designed to demonstrate, from experimental data, that only two of the trapeziometacarpal degrees of freedom (i.e., flexion/extension and adduction/abduction) are non-null and independent. Several movements of maximal amplitude in flexion, abduction and circumduction have been realized and the relative position and orientation of the segment coordinate system embedded on the first metacarpal with respect to that embedded on the trapezium have been collected using electromagnetic sensors. The trapeziometacarpal rotations have been described using a joint coordinate system and the joint displacements have been evaluated on the axes of this coordinate system.ResultsThe root mean square (RMS) values of the joint displacement components have been found small enough to assume that the trapeziometacarpal joint has no translation degrees of freedom. A paraboloid coupling equation has been found between the internal/external rotation angle and the two other, flexion/extension and adduction/abduction, angles.ConclusionThus, this study demonstrates that the trapeziometacarpal joint has only two independent rotational degrees of freedom, and further, the described methodology could also be used to determine the coupling laws between degrees of freedom of various joints.