Development of sertoli cell junctions in vitro—A freeze-fracture study

Summary Seminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo. This study was supported by Grant 801D185 (Dr. Posalaky) from the Medical Education and Research Foundation, St. Paul-Ramsey Hospital.     

Improved method of in vitro regeneration in Leucaena leucocephala — a leguminous pulpwood tree species

Leucaena leucocephala is a fast growing multipurpose legume tree used for forage, leaf manure, paper and pulp. Lignin in Leucaena pulp adversely influences the quality of paper produced. Developing transgenic Leucaena with altered lignin by genetic engineering demands an optimized regeneration system. The present study deals with optimization of regeneration system for L. leucocephala cv. K636. Multiple shoot induction from the cotyledonary nodes of L. leucocephala was studied in response to cytokinins, thidiazuron (TDZ) and N⁶-benzyladenine (BA) supplemented in half strength MS (½-MS) medium and also their effect on in vitro rooting of the regenerated shoots. Multiple shoots were induced from cotyledonary nodes at varied frequencies depending on the type and concentration of cytokinin used in the medium. TDZ was found to induce more number of shoots per explant than BA, with a maximum of 7 shoots at an optimum concentration of 0.23 µM. Further increase in TDZ concentration resulted in reduced shoot length and fasciation of the shoots. Liquid pulse treatment of the explants with TDZ did not improve the shoot production further but improved the subsequent rooting of the shoots that regenerated. Regenerated shoots successfully rooted on ½-MS medium supplemented with 0.54 µM α-naphthaleneacetic acid (NAA). Rooted shoots of Leucaena were transferred to coco-peat and hardened plantlets showed ≥ 90 % establishment in the green house.Key words: Cotyledonary nodes, Multiple shoot induction, Pulse treatment, TDZ     

In vitro regeneration of plantlets in Fagraea fragrans Roxb. — a tropical tree

Different vegetative parts of Fagraea fragrans Roxb., a valuable timber tree of South East Asia, were used as explants in in vitro studies.Nodal segments showed the best growth response as numerous adventitious shoots were regenerated in MS medium supplemented with Benzyl adenine (BA, 8.8 um) and 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 um). Shoot buds also developed from the leaf and root segments that were subcultured. The detailed process of callus growth and differentiation leading to the formation of whole plantlets is described. Uniform plantlets obtained could be transplanted successfully in soil.     

Direct regeneration in vitro and transient GUS expression in Mentha×piperita

Genetic transformation of peppermint is known to be very difficult essentially because of low efficiency regeneration. A regeneration protocol allowing 51% shooting frequency is proposed. Transient -glucuronidase expression and adjustment of selection pressure with kanamycin are also reported. The final retained method to attempt peppermint transformation is:Agrobacterium inoculation or biolistic treatment of the first apical leaves ofin vitro clones, regeneration in the dark with kanamycin (1 mg l^–1) and 6-benzylaminopurine (2 mg l^–1), followed by selection of regenerated shoots with 200 mg 1^–1 kanamycin.Abbreviations BA 6-benzylaminopurine - GUS -glucuronidase - MS Murashige and Skoog (1962) - NAA -naphthalenacetic acid - PIG particle inflow gun - SEM scanning electron microscope     

Can prematurity risk in twin pregnancies after in vitro fertilization be predicted? A retrospective study

Background  

Assisted reproduction (ART) contributes to world-wide increases of twin pregnancies, in turn raising prematurity risks. Whether characteristics of ART cycles, resulting in twin gestations, can predict prematurity risks was the subject of this study.     

Multiple sites in αB-crystallin modulate its interactions with desmin filaments assembled in vitro

The β3- and β8-strands and C-terminal residues 155-165 of αB-crystallin were identified by pin arrays as interaction sites for various client proteins including the intermediate filament protein desmin. Here we present data using 5 well-characterised αB-crystallin protein constructs with substituted β3- and β8-strands and with the C-terminal residues 155-165 deleted to demonstrate the importance of these sequences to the interaction of αB-crystallin with desmin filaments. We used electron microscopy of negatively stained samples to visualize increased interactions followed by sedimentation assays to quantify our observations. A low-speed sedimentation assay measured the ability of αB-crystallin to prevent the self-association of desmin filaments. A high-speed sedimentation assay measured αB-crystallin cosedimentation with desmin filaments. Swapping the β8-strand of αB-crystallin or deleting residues 155-165 increased the cosedimentation of αB-crystallin with desmin filaments, but this coincided with increased filament-filament interactions. In contrast, substitution of the β3-strand with the equivalent αA-crystallin sequences improved the ability of αB-crystallin to prevent desmin filament-filament interactions with no significant change in its cosedimentation properties. These data suggest that all three sequences (β3-strand, β8-strand and C-terminal residues 155-165) contribute to the interaction of αB-crystallin with desmin filaments. The data also suggest that the cosedimentation of αB-crystallin with desmin filaments does not necessarily correlate with preventing desmin filament-filament interactions. This important observation is relevant not only to the formation of the protein aggregates that contain both desmin and αB-crystallin and typify desmin related myopathies, but also to the interaction of αB-crystallin with other filamentous protein polymers.     

Multiple glucose 6-phosphate pools or channelling of flux in diverse pathways?

Glucose 6-phosphate is an intermediate of pathways of glucose utilization and production as well as a regulator of enzyme activity and gene expression. Studies on the latter functions are in part based on measurement of the glucose 6-phosphate content in a whole-cell extract. Several studies have suggested that there are multiple subcellular pools of glucose 6-phosphate. It is proposed that this data can be interpreted in terms of channelling of metabolic intermediates through multiple pathways of glucose metabolism with leakage of glucose 6-phosphate from the channels into a single free pool. It is also proposed that measurement of total tissue content of glucose 6-phosphate approximates the free pool.     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

Loss of viability in hybridoma cell culture—A kinetic study

In the cultivation of hybridoma cells HB8178 in suspension, exponential growth was followed by cell death after reaching a maximum cell concentration. We examined the cause of such transition from growth to death. Exogenously added lactate and ammonium did not cause cell death at the concentrations observed at the end of exponential growth. The rate of cell death decreases by diluting the conditioned medium with phosphate buffer saline, suggesting the presence of inhibitory factor(s) in the conditioned medium. This inhibitory factor(s) is dialyzable. Furthermore, the conditioned medium obtained from HB8178 culture also causes transition to death phase for another hybridoma cell line AFP.     

Trends in breast cancer mortality in Trinidad and Tobago—A 35-year study

Background: Breast cancer is the most frequently diagnosed cancer among women worldwide. This study examines the breast cancer mortality patterns and trends in the Caribbean island state, Trinidad and Tobago for the 35-year period, 1970–2004. Methods: A retrospective analysis of the trends in breast cancer mortality from 1970 to 2004 was conducted. Crude mortality per 100,000 women, age-standardized mortality using World Standard population and age-stratified mortality were calculated and comparison was made between age groups above and below 50 years. Results: A general pattern of increase was observed in both crude and age-standardized mortality. The overall average crude mortality was 15.6 per 100,000 women (95% confidence interval (CI) 13.9–17.1) and the average age-standardized mortality was 18.0 per 100,000 women (95% CI 16.7–19.2). There was a pattern of increase in mortality with increasing age. The mortality rate was considerably higher for the age groups above 50 years than those less than 50 years of age both showing an upward trend over the 35-year period. Conclusions: Breast cancer mortality continued to increase over the 35-year period in Trinidad and Tobago. This study did not identify the exact reasons for this increasing trend. However, it is known that Trinidad and Tobago is becoming much more industrialized. It may be speculated that decrease in fertility rates, increase in the incidence of obesity and hormone utilization could have influenced this trend.     

The use of charcoal in in vitro culture – A review

Activated charcoal is commonly used in tissue culture media. Its addition to culture medium may promote or inhibit in vitro growth, depending on species and tissues used. The effects of activated charcoal may be attributed to establishing a darkened environment; adsorption of undesirable/inhibitory substances; adsorption of growth regulators and other organic compounds, or the release of growth promoting substances present in or adsorbed by activated charcoal.     

7,7′-Diazaindirubin—A small molecule inhibitor of casein kinase 2 in vitro and in cells

Aza- and diaza-bisindoles were synthesized by coupling of 7-azaisatin, 7-azaoxindol, 7-azaindoxyl acetate, and their non-aza counterparts, respectively. Whereas 7,7′-diazaindigo (10) and 7,7′-diazaisoindigo (11) did not show antiproliferative activity in several human tumor cell lines up to 100 μM, 7-azaindirubin (12) and 7′-azaindirubin (13) were more active than the parent molecule, indirubin, in LXFL529L cells (human large cell lung tumor xenograft), and 7,7′-diazaindirubin (14) was exhibiting substantially enhanced growth inhibitory activity in these cells. In the NCI 60 cell line panel, 14 displayed antiproliferative activity preferentially in certain melanoma and non-small cell lung cancer cells. In contrast to the potent serine/threonine/tyrosine kinase inhibition observed for indirubins, kinase inhibition profiling of 14 in 220 kinases revealed largely a loss of kinase inhibitory activity towards most kinases, with retained inhibitory activity for just a few kinases. At 1 μM concentration, especially casein kinases CK1γ3, CK2α, CK2α2, and SIK were inhibited by more than 50%. In cell-based assays, 14 markedly affected CK2-mediated signaling in various human tumor cells. In MCF7 cells, 14 induced cell cycle arrest at G1 and G2/M and apoptosis, whereas CK2-deficient MCF7 cells were resistant. These findings reveal a novel key mechanism of action for 14, suggesting primarily CK2 inhibition to be causally related to growth inhibition of human tumor cells.     

Callus culture and an unconventional pattern of sporophyte regeneration in Drynaria quercifolia—A medicinal fern

Summary Callus induction and regeneration studies were carried out on a medicinal fern, Drynaria quercifolia native to Asian countries. It is a seasonal fern that regenerates only during the monsoons. Callus was induced on Knop’s (1865) medium supplemented with 20 gl⁻¹ sucrose, 8gl⁻¹ agar, and either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), or indole-3-butyric acid at different concentrations. Morphogenetic callus obtained on 5 mgl⁻¹ 2,4,5-T was subcultured onto solid and liquid media (shaken flask and discontinuously stirred bioreactor cultures) for callus proliferation and regeneration studies. A significant amount of sporophyte regeneration was observed on solid medium containing 10 mgl⁻¹ 6-(δ, δ-dimethylallylamino) purine (2iP). Sporophyte regeneration from callus followed an atypical pattern of development. Leafy structures of single-cell thickness with a microrhizome were formed as sporophyte initials. Prolonged cultures of these structures resulted in the formation of juvenile sporophytes in vitro. The use of liquid media resulted in increased biomass in culture. The present study is the first report of a successful system for callus production and regeneration of sporophytes from leafy structures in ferns. The method can be successfully applied for generation of biomass of D. quercifolia, throughout the year.     

Specificity of membrane specializations in mechanoreceptors of birds—A freeze-etching study

The detailed knowledge of the molecular process of mechanotransduction is still an unsolved question. The investigation of the intramembranous structure of the cutaneous mechanoreceptors may play an important role in elucidating this problem. In this relation, Herbst sensory corpuscles in ducks were studied for the first time using the freeze-etching and thin sectioning techniques. Herbst corpuscles have the basic structural components valid for most of the encapsulated mechanoreceptors in mammals: a capsule made of perineural cells, a lamellar complex of modified Schwann cells, surrounding the non-myelinated part of the receptor nerve fiber and its ending. Freeze-etching replicas reveal that the plasmalemmae of the capsule cells, modified Schwann cells and axolemmae of parts of the nerve fiber differ in both density and pattern of distribution of intramembranous particles (IMPs) as well as IMP size. On all the plasmalemmae the IMP density is higher on the P-face (2000–3300?µm^?2) than the respective E-face (800–1500?µm^?2). The axolemma of the ending of the receptor nerve fiber expresses higher density of IMPs than its shaft. The mean IMP size for all the plasmalemmae varies between 5.5 and 7.5?nm. Many tight junctions occur between the capsule cells. These results indicate that the non-myelinated axolemma as well as the plasmalemmae of other components of Herbst corpuscles are specialized in terms of content and distribution of IMPs. The IMPs may represent various kinds of mechanosensitive channel proteins or related membrane-bound proteins participating in the process of mechanotransduction.     

Dynamical correlation of non-stationary signals in time domain—A comparative study

In a thorough study, the multitaper (MTM) and the extended continuous wavelet-transform (CWT) coherence-analysis methods were compared in terms of there application in determining the dynamics from the electroencephalogram (EEG) and electromyogram (EMG) signals of patients with Parkinsonian tremor. The main aim of the study in a biological point of view is to analyze whether the basic tremor frequency and its “first harmonic” frequency of Parkinsonian tremor are really harmonically related or are in fact distinct processes.The extension of the CWT is achieved by using a Morlet wavelet as the analysis window with an adjustable relative bandwidth which gives the flexibility in setting a desired frequency resolution. In order to obtain a perspective view of the two methods, they were applied to two different model signals to determine their actual threshold in detecting short-lived changes in the analysis of non-stationary signals and to determine their noise thresholds by adding external noise to the signals to test the reduction in coherence to be not merely due to the random fluctuations in stochastic signals. Beyond applying an autoregressive 2nd-order and a coupled van der Pol model system, however, also true EEG and EMG data from five Parkinson patients were used. The results were compared in terms of the time and frequency resolutions of these two methods, and it was determined that the multitaper method was able to detect reduction in power and coherence as short as 1 s. The extended CWT analysis only revealed gaps that were longer than 3 s.The time gaps in the coherence indicate the loss of connection between the cortex and muscle during the respective time intervals. This more accurate analysis of the MTM was also seen in the dynamical EEG–EMG coherence at the tremor frequency and its “first harmonic” of Parkinsonian patients.In terms of our “biological” aim, this shows distinct prevalence of the corticomuscular coupling at those frequencies over time. Applying this method to biological data reveals important aspects about their dynamics, e.g., the distinct dynamics between basic frequency and “first harmonic” frequency over time in Parkinsonian tremor.     

Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. & C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5–8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30–35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea—an endangered medicinal plant.     

Seasonal dynamic of plant phenophases in Beijing—A case study in Beijing Botanical Garden

季节性是北京植物景观的典型特征, 而个体物候是植物景观季节动态的基础。该研究基于北京植物园内120种落叶树种的周年物候数据, 对北京植物景观的物候季节动态进行分析。物候观测以中国物候观测法为标准, 采用a、b、c三级物候代码进行物候记录; 数据分析以二十四节气中的候(5日)为基本时间变量, 基于频率分布型法探究北京物候季节划分及其物候构成动态, 基于SPSS 20.0频数分布统计等探究各类物候现象发生期及持续期的时间分布特征等。物候季节划分及物候构成特征结果为: 6-19候为春, 物候期发生频数占全年总量的54.03%, 以发芽、展叶、开花为主要物候特征, 后期有少数树种结果; 20-45候为夏, 物候量占全年的12.95%, 此期全部观测树种完成展叶, 春花树种进入结果期, 并有较少夏花开放及秋色叶出现; 46-59候为秋, 物候量占全年的27.19%, 以秋色叶及落叶物候为主并伴有较少结果和开花物候发生; 60候至次年春季起始前为冬, 其中60-72候物候量仅占全年的0.6%, 全为落叶物候。各类物候期的时间分布特征如下: 展叶物候期分布于3-23候, 华北珍珠梅(Sorbaria kirilowii)、旱柳(Salix matsudana)等展叶最早, 展叶期平均持续3.27候。秋色物候期分布于40-63候, 49-56候为最佳观赏期, 蒙椴(Tilia mongolica)、山杏(Armeniaca sibirica)等最早显秋色; 秋色期平均持续8.2候, 卫矛(Euonymus alatus)、接骨木(Sambucus williamsii)等秋色期较长。叶幕期平均持续44.2候, 糯米条(Abelia chinensis)、旱柳、棣棠(Kerria japonica)等叶幕期最长。花物候期分布于1-53候, 始花期为1-41候, 盛花期平均发生于始花后1.81候, 8-23候为集中观赏期, 蜡梅(Chimonanthus praecox)、迎春(Jasminum nudiflorum)、榆(Ulmus pumila)、毛白杨(Populus tomentosa)等开花最早, 木香薷(Elsholtzia stauntoni)开花最晚; 花期平均持续 6.7候, 华北珍珠梅、木槿(Hibiscus syriacus)、紫薇(Lagerstroemia indica)等夏秋开花树种花期最长。果物候期分布于8-59候, 榆、郁香忍冬(Lonicera fragrantissima)等果实成熟最早; 持果期平均持续29.0候, 果实宿存树种及黑果荚蒾(Viburnum melanocarpum)、‘金叶’风箱果(Physocarpus opulifolius ‘Luteus’)等具有较长的果实观赏期。与20世纪80年代同类研究结果对比分析, 北京2017年的物候季节与30年前相比, 入春提早1候, 夏季延长4候, 入秋延后3候, 秋季缩短2候, 且不同季节持续期长短的差距加大。     

Multiple Symmetrical Lipomatosis — A mitochondrial disorder of brown fat

Multiple Symmetrical Lipomatosis (MSL) is an unusual disorder characterized by the development of axial lipomas in adulthood. The pathoetiology of lipoma tissue in MSL remains unresolved. Seven patients with MSL were followed for a mean period of 12 years (8–20 years). All patients had cervical lipomas ranging from subtle lesions to disfiguring masses; six patients had peripheral neuropathy and five had proximal myopathy. Myoclonus, cerebellar ataxia and additional lipomas were variably present. All patients showed clinical progression. Muscle histopathology was consistent with mitochondrial disease. Five patients were positive for mtDNA point mutation m.8344A > G, three of whom underwent lipoma resection — all samples were positive for uncoupling protein-1 mRNA (unique to brown fat). Lipoma from one case stained positive for adipocyte fatty-acid protein-2 (unique to brown fat and immature adipocytes). This long-term study hallmarks the phenotypic heterogeneity of MSL's associated clinical features. The clinical, genetic and molecular findings substantiate the hypothesis that lipomas in MSL are due to a mitochondrial disorder of brown fat.