Understanding the mechanism(s) of mosaic trisomy 21 by using DNA polymorphism analysis.

In order to investigate the mechanism(s) underlying mosaicism for trisomy 21, we genotyped 17 families with mosaic trisomy 21 probands, using 28 PCR-detectable DNA polymorphic markers that map in the pericentromeric region and long arm of chromosome 21. The percentage of cells with trisomy 21 in the probands'' blood lymphocytes was 6%-94%. There were two classes of autoradiographic results: In class I, a "third allele" of lower intensity was detected in the proband''s DNA for at least two chromosome 21 markers. The interpretation of this result was that the proband had inherited three chromosomes 21 after meiotic nondisjunction (NDJ) (trisomy 21 zygote) and subsequently lost one because of mitotic (somatic) error, the lost chromosome 21 being that with the lowest-intensity polymorphic allele. The parental origin and the meiotic stage of NDJ could also be determined. In class II, a "third allele" was never detected. In these cases, the mosaicism probably occurred either by a postzygotic, mitotic error in a normal zygote that followed a normal meiosis (class IIA mechanism); by premeiotic, mitotic NDJ yielding an aneusomic zygote after meiosis, and subsequent mitotic loss (class IIB mechanism); or by a meiosis II error with lack of crossover in the preceding meiosis I, followed by mitotic loss after fertilization (class IIC mechanism). Among class II mechanisms, the most likely is mechanism IIA, while IIC is the least likely. There were 10 cases of class I and 7 cases of class II results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Theoretical modeling of the hydroxylation of methane as mediated by the particulate methane monooxygenase

We present here the results of density functional theory (DFT) calculations directed toward elucidation of the CH bond activation mechanism that might be adopted by the particulate methane monooxygenase (pMMO) in the hydroxylation of methane and related small alkanes. In these calculations, we considered three of the most probable models for the transition metal active site mediating the "oxo-transfer": (i) the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1, recently proposed by Chan et al. [S.I. Chan, K.H.-C. Chen, S.S.-F. Yu, C.-L. Chen, S.S.-J. Kuo, Biochemistry 43 (2004) 4421-4430.]; (ii) the most frequently used model complex, bis(mu-oxo)Cu(III)(2) complex 2; and (iii) the mixed-valence bis(mu-oxo)Cu(II)Cu(III) complex 3. The results obtained indicate that the methane hydroxylation chemistry mediated by the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1 offers the most facile pathway for methane hydroxylation, and this model yields KIE values that are in good agreement with experiment. In this mechanism, the reaction proceeds along a "singlet" potential surface and a "singlet oxene" is directly inserted across a CH bond in a concerted manner. Kinetic isotope effects (k(H)/k(D) or KIE) associated with the concerted oxene insertion process mediated by complex 1 are calculated to be 5.2 at 300K when tunneling effects are included. Overall rate constants for the methane hydroxylation by the three models have been calculated as a function of temperature, and the rates are at least 5-6 orders of magnitude more facile when the chemistry is mediated by complex 1 compared to complex 2 or complex 3.     

Optical studies on complexes between DNA and pseudoisocyanine.

Linear dichroism (LD) results when pseudoisocyanine=PIC (1,1-diethy1-2,2-cyannine iodide) binds to flow-oriented DNA. LD may be used to follow the complexation both stoichmetrically and structurally, since when specified to unti complex concentration LD provides a measure of the average orientation of the absorbing transition dipole. Two different types of complexes can be distinguished: I. One strong, ionic-strength insensitive complex with monomeric PIC with an orientation indication intercalation. II. Several weaker complexes of electrostatic nature (only observed at I less than 0.2M Na Cl) among which those with dimeric (IIa) and with polymeric (IIb) PIC are concluded both from LD and from circular dichroism (CD). The dimer is probably formed by employing one intercalated PIC as a second site. The polymer complex is characterized by a very sharp absorption band at 553 nm polarized parallel to the DNA-axis (with positive LD and positive CL). The structure, a right-handed helical array of PIC molecules, is discussed in terms of exciton theory in relation to that of polymeric free PIC ("Scheibe polymer") which is also shown to interact with DNA (IIc) yielding a large aggregate which is degraded at a distinct flow force field...     

Lipid II induces a transmembrane orientation of the pore-forming peptide lantibiotic nisin

Nisin is an antimicrobial peptide produced by Lactococcus lactis and used as a food preservative in dairy products. The peptide kills Gram-positive bacteria via the permeabilization of the membrane, most probably via pore formation using the cell wall precursor Lipid II as its docking molecule. In this study, site-directed tryptophan spectroscopy was used to determine the topology of nisin in the Lipid II containing membrane, as a start to elucidate the mechanism of targeted pore formation. Three single tryptophan mutants were used, which are viable representatives of the wild-type peptide. The emission spectra of tryptophans located at the N-terminus, the center, and the C-terminus as well as quenching by acrylamide and spin-labeled lipids were investigated using model membrane vesicles composed of DOPC containing 1 mol % Lipid II. Nisin was shown to adopt an orientation where the most probable position of the N-terminus was found to be near the Lipid II headgroup at the bilayer surface, the position of the center of nisin was in the middle of the phospholipid bilayer, and the C-terminus was located near the interface between the headgroups and acyl chain region. These results were used to propose a model for the orientation of nisin in Lipid II containing membranes. Our findings demonstrated that Lipid II changes the overall orientation of nisin in membranes from parallel to perpendicular with respect to the membrane surface. The stable transmembrane orientation of nisin in the presence of Lipid II might allow us to determine the structure of the nisin-Lipid II pores in the lipid bilayer.     

Ovarian development and modes of apoptosis during oogenesis in various castes of the termite Reticulitermes aculabialis

Reproductive division in termites is the most significant biological process that leads to the formation of caste‐specific differences in tasks and status. However, little is known about the mechanism of reproductive division that underlies caste differentiation. In the present study, ovarian development and stage‐specific apoptotic patterns are investigated during oogenesis in reproductive, worker and soldier termites Reticulitermes aculabialis Tsai & Hwang. The results show that the mean lengths of the ovaries of reproductives are two‐fold longer compared with those of workers and six‐fold longer compared with soldiers. By contrast to the reproductives, the process of oogenesis in the workers includes only the oogonium differentiation stage (stage I) and oocyte growth stage (stage II), and oogenesis in the soldiers stops at stage I. Vitelogenic oocytes (stage III) are absent from workers and soldiers. During stage II in the reproductives and workers, the layer of follicle cells has a thickness of 7.56 ± 0.52 and 2.81 ± 0.34 µm, respectively. In addition, there are significant differences in the number and size of the germ cells at the same stage in the various castes. The existence of two apoptotic patterns during oogenesis is demonstrated by the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assay. First, the majority of the cells showing apoptosis occur at stage I of oogenesis in reproductives, workers and soldiers. Second, DNA fragmentation is demonstrated by TUNEL staining of the follicle cell layers and oocytes at stage II in reproductives. Finally, the proliferation activity of follicle cells in the reproductives is observed by 5‐bromo‐2′‐deoxy‐uridine labelling. The level of oogenesis may explain the significant discrepancies in the reproductive capacity among the reproductives, soldiers and workers. These large discrepancies are controlled by apoptosis during early oogenesis.     

Some Properties of Neutral Proteolytic System in Rabbit Skeletal Muscle

The properties of the neutral proteolytic activity concentrated in a fraction (F–1) separated from rabbit muscle homogenate were examined by measuring the effects of various reagents and metal ions, the time course of the proteolysis and Ca-stability. The obtained results have indicated that F–1 contains two types of neutral protease active on proteins, tentatively named Protease I and II, The former, which is activated by Ca²⁺ and Ca-labile, shows an explosive production of Cu-Folin phenol reagent positive materials at the early stage of incubation. The latter, which is Ca-stable, shows a large production of ninhydrin positive materials throughout the incubation time. The proteolysis by F–1 was similar to the autolysis of muscle homogenate in all the properties examined. Therefore, Proteases I and II were assumed to be main enzymes responsible for the muscle proteolysis at the neutral pH region. As there has been no factor denying their functioning in living muscle, it is probable that Proteases I and II take important parts in the muscle catabolism.     

Characterization of Two Isozymes of Coniferyl Alcohol Dehydrogenase from Streptomyces sp. NL15-2K

We purified two isozymes of coniferyl alcohol dehydrogenase (CADH I and II) to homogeneity from cell-free extracts of Streptomyces sp. NL15-2K. The apparent molecular masses of CADH I and II were determined to be 143 kDa and 151 kDa respectively by gel filtration, whereas their subunit molecular masses were determined to be 35,782.2 Da and 37,597.7 Da respectively by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Thus, it is probable that both isozymes are tetramers. The optimum pH and temperature for coniferyl alcohol dehydrogenase activity were pH 9.5 and 45 °C for CADH I and pH 8.5 and 40 °C for CADH II. CADH I oxidized various aromatic alcohols and allyl alcohol, and was most efficient on cinnamyl alcohol, whereas CADH II exhibited high substrate specificity for coniferyl alcohol, and showed no activity as to the other alcohols, except for cinnamyl alcohol and 3-(4-hydroxy-3-methoxyphenyl)-1-propanol. In the presence of NADH, CADH I and II reduced cinnamaldehyde and coniferyl aldehyde respectively to the corresponding alcohols.     

Studies on Feeding and Digestion in Protozoa. III. Acid Phosphatase Activity in Food Vacuoles of Paramecium multimicronucleatum

SYNOPSIS. Acid phosphatase activity was studied in total mounts and sections of agnotobiotic Paramecium multimicronucleatum by the alpha-naphthyl phosphate-hexazotized rosanilin method. Timing was achieved by India ink marking of food vacuoles. Enzyme activity is present in small endoplasmic granules and in the greatest part of food vacuoles. Following an inactive stage (stage I) of an average length of 5 min the activity appears at the periphery of the vacuole, in most cases in the form of granules (stage II). A high activity level (stage III) is attained within 1 1/2 min and maintained for the most part of the vacuolar cycle. The activity disappears only in the latest vacuoles before egestion (stage IV). The appearance of activity is not concurrent with but succeeding to the maximum of vacuolar acidity as ascertained by feeding Congo red stained killed yeast cells. On the basis of these results the food vacuoles may be looked upon as belonging to the lysosomes sensu lato.     

Thermal Stability Investigation and the Kinetic Study of Folnak<Superscript>®</Superscript> Degradation Process Under Nonisothermal Conditions

The nonisothermal degradation process of Folnak^® drug samples was investigated by simultaneous thermogravimetric and differential thermal analysis in the temperature range from an ambient one up to 810°C. It was established that the degradation proceeds through the five degradation stages (designated as I, II, III, IV, and V), which include: the dehydration (I), the melting process of excipients (II), as well as the decomposition of folic acid (III), corn starch (IV), and saccharose (V), respectively. It was established that the presented excipients show a different behavior from that of the pure materials. During degradation, all excipients increase their thermal stability, and some kind of solid–solid and/or solid–gas interaction occurs. The kinetic parameters and reaction mechanism for the folic acid decomposition were established using different calculation procedures. It was concluded that the folic acid decomposition mechanism cannot be explained by the simple reaction order (ROn) model (n?=?1) but with the complex reaction mechanism which includes the higher reaction orders (RO, n?>?1), with average value of <n?>?=?1.91. The isothermal predictions of the third (III) degradation stage of Folnak^® sample, at four different temperatures (T _iso?=?180°C, 200°C, 220°C, and 260°C), were established. It was concluded that the shapes of the isothermal conversion curves at lower temperatures (180–200°C) were similar, whereas became more complex with further temperature increase due to the pterin and p-amino benzoic acid decomposition behavior, which brings the additional complexity in the overall folic acid decomposition process.     

Red shift in the spectrum of a chlorophyll species is essential for the drought-induced dissipation of excess light energy in a poikilohydric moss, <Emphasis Type="Italic">Bryum argenteum</Emphasis>

Some mosses are extremely tolerant of drought stress. Their high drought tolerance relies on their ability to effectively dissipate absorbed light energy to heat under dry conditions. The energy dissipation mechanism in a drought-tolerant moss, Bryum argenteum, has been investigated using low-temperature picosecond time-resolved fluorescence spectroscopy. The results are compared between moss thalli samples harvested in Antarctica and in Japan. Both samples show almost the same quenching properties, suggesting an identical drought tolerance mechanism for the same species with two completely different habitats. A global target analysis was applied to a large set of data on the fluorescence-quenching dynamics for the 430-nm (chlorophyll-a selective) and 460-nm (chlorophyll-b and carotenoid selective) excitations in the temperature region from 5 to 77 K. This analysis strongly suggested that the quencher is formed in the major peripheral antenna of photosystem II, whose emission spectrum is significantly broadened and red-shifted in its quenched form. Two emission components at around 717 and 725 nm were assigned to photosystem I (PS I). The former component at around 717 nm is mildly quenched and probably bound to the PS I core complex, while the latter at around 725 nm is probably bound to the light-harvesting complex. The dehydration treatment caused a blue shift of the PS I emission peak via reduction of the exciton energy flow to the pigment responsible for the 725 nm band.     

Arm regeneration in two species of cuttlefish Sepia officinalis and Sepia pharaonis

To provide quantitative information on arm regeneration in cuttlefish, the regenerating arms of two cuttlefish species, Sepia officinalis and Sepia pharaonis, were observed at regular intervals after surgical amputation. The third right arm of each individual was amputated to ~10–20 % starting length. Arm length, suction cup number, presence of chromatophores, and behavioral measures were collected every 2–3 days over a 39-day period and compared to the contralateral control arm. By day 39, the regenerating arm reached a mean 95.5 ± 0.3 % of the control for S. officinalis and 94.9 ± 1.3 % for S. pharaonis. The process of regeneration was divided into five separate stages based on macroscopic morphological events: Stage I (days 0–3 was marked by a frayed leading edge; Stage II (days 4–15) by a smooth hemispherical leading edge; Stage III (days 16–20) by the appearance of a growth bud; Stage IV (days 21–24) by the emergence of an elongated tip; and Stage V (days 25–39) by a tapering of the elongated tip matching the other intact arms. Behavioral deficiencies in swimming, body postures during social communication, and food manipulation were observed immediately after arm amputation and throughout Stages I and II, returning to normal by Stage III. New chromatophores and suction cups in the regenerating arm were observed as early as Stage II and by Stage IV suction cup number equaled that of control arms. New chromatophores were used in the generation of complex body patterns by Stage V. These results show that both species of cuttlefish are capable of fully regenerating lost arms, that the regeneration process is predictable and consistent within and across species, and provide the first quantified data on the rate of arm lengthening and suction cup addition during regeneration.