Anin vitro model for chick embryonic notochords

A two step method to obtain mesenchymal free 3.5 day old chick embryonic notochordsin vitro is presented. 1.) Notochords are isolated by mechanical microdissection from the embryos below the head and above the leg-buds. 2.) The dissected notochords are trypsinized to eliminate contaminating mesenchymal cells, while the perinotochordal sheath (PNS) is retained. After isolation and trypsinization, notochords are cut in standard 8mm lengths, explantedin vitro and incubated at 37°C. Immediately before incubation and after 3 and 6 daysin vitro, notochords are fixed and stained to follow the morphological changes. The total DNA content of notochords is measured before and during maintenancein vitro to evaluate their metabolic activities. Results show that during thein vitro period, the isolated mesenchymal free notochordal fragments can conserve their characteristic architecture. The total DNA content measurements indicate proliferative activity and a high viability of the notochords in ourin vitro system. In the present study, an isolation andin vitro method is offered which might be an effective tool to study the metabolic activities of chick embryonic notochordsin vitro in comparison toin vivo behaviour, in order to study the underlying mechanism of notochord regression.     

The Escherichia coli recA gene increases resistance of the yeast Saccharomyces cerevisiae to ionizing and ultraviolet radiation

Summary The Escherichia coli recA protein coding region was ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase promoter region to produce plasmid pADHrecA. Transformation of the wild-type yeast strains YNN-27 and 7799-4B, as well as the recombination-deficient rad52-t C5-6 mutant, with this shuttle plasmid resulted in the expression of the bacterial 38 kDa RecA protein in exponential phase cells. The wild-type YNN27 and 7799-4B transformants expressing the bacterial recA gene showed increased resistance to the toxic effects of both ionizing and ultraviolet radiation. RecA moderately stimulated the UV-induced mutagenic response of 7799-4B cells. Transformation of the rad52-t mutant with plasmid pADHrecA did not result in the complementation of sensitivity to ionizing radiation. Thus, the RecA protein endows the yeast cells with additional activities, which were shown to be error-prone and dependent on the RAD52 gene.     

In vitro synthesis of the Bowman-Birk and related soybean protease inhibitors

Poly (A)⁺ RNAs from immature soybean seeds were size fractionated in denaturing sucrose gradients to identify mRNA that directs the cell-free synthesis of the Bowman-Birk protease inhibitor and the related inhibitors PI I–IV. Polypeptides synthesized in vitro were labeled with (³⁵S)-cysteine and (³H)-serine and detected by immunoprecipitation with anti Bowman-Birk and anti PI I–IV sera. Immunoprecipitates of the translation products comigrated on SDS-polyacrylamide gels with the dimeric or trimeric aggregates of the authentic inhibitor proteins, which self-associate under certain conditions. Further evidence that these immunoprecipitates contained authentic polypeptides corresponding to the Bowman-Birk or PI IV inhibitor was shown by sequential amino acid analyses of peptides generated by cleavage with cyanogen bromide.     

In vitro study of gas effects on alveolar macrophages

Summary To evaluate the biological effects of gas pollutants on alveolar macrophages several in vitro systems ave been developed. We described here an original method of cell culture in aerobiosis, which permitted direct contact between the atmosphere and the target cells. We studied the long term (24 h) and short term (30 min) effects of NO₂ on alveolar macrophages. Our results demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either cell injury or cell activation associated with the release of various bioactive mediators (superoxide anion, neutrophil chemotactic activity). Cell culture in aerobiosis opens new ways for the research on the biological effects of gas pollutants.Abbreviations AM alveolar macrophages - CL Chemiluminescence     

AMPK phosphorylates NAMPT to regulate NAD+ homeostasis under ionizing radiation

Radiation-induced oral mucositis is the most common complication for patients who receive head/neck radiotherapy. Nicotinamide adenine dinucleotide (NAD⁺) is vital for DNA damage repair under ionizing radiation, through functioning as either the substrate for protein poly(ADP-ribosyl)ation at DNA break sites or the cofactor for multiple DNA repair-related enzymes, which therefore can result in a significant consumption of cellular NAD⁺ during DNA repair. Mammalian cells produce NAD⁺ mainly by recycling nicotinamide via the salvage pathway, in which the rate-limiting step is governed by nicotinamide phosphoribosyltransferase (NAMPT). However, whether NAMPT is co-opted under ionizing radiation to timely fine-tune NAD⁺ homeostasis remains elusive. Here we show that ionizing radiation evokes NAMPT activation within 30 min without apparent changes in its protein expression. AMPK rapidly phosphorylates NAMPT at S314 under ionizing radiation, which reinforces the enzymatic activity of NAMPT by increasing NAMPT binding with its substrate phosphoribosyl pyrophosphate (PRPP). AMPK-mediated NAMPT S314 phosphorylation substantially restores NAD⁺ level in the irradiated cells and facilitates DNA repair and cell viability. Our findings demonstrate a new post-translational modification-based signalling route, by which cells can rapidly orchestrate NAD⁺ metabolism to support DNA repair, thereby highlighting NAMPT as a potential target for the prevention of ionizing radiation-induced injuries.     

Effects of ionizing radiation on the growth and allyl isothiocyanate accumulation of Wasabia japonica in vitro and ex vitro

Mutations were induced in tissue-cultured wasabi (Wasabia japonica Matsumura) by treating in vitro-derived shoot tips with either γ-rays or X-rays at 0, 10, 20, 40 or 80 Gy. Doses of up to 40 Gy of either γ- or X-ray treatments resulted in a survival rate of more than 60% in culture after 3 mo. The use of γ- or X-rays at doses between 10 Gy and 40 Gy to induce mutation in W. japonica resulted in an alteration of the growth and allyl isothiocyanate (AITC) content of multiple shoots after 3 mo. in culture on Murashige and Skoog medium containing 5 μM N⁶-benzyladenine (BA). Putative mutants from the 40 Gy treatments of either γ- or X-rays exhibited a reduction in shoot weight, number, and height, whereas treatments of either γ-rays or X-rays at 10 Gy and 20 Gy doses showed no significant differences in shoot growth. All shoots treated with 80 Gy were either necrotic or irregenerable, while those treated with 40 Gy produced deformed leaves, from both types of ionizing radiation. Concentrations of AITC were measured by the use of gas chromatography-mass spectrometry (GC-MS). The accumulation of AITC was shown to decrease when doses increased in both γ- and X-ray treatments, compared with the controls. Positive responses were solely occurred at 18 mo. after transfer of in vitro rooted shoots to the shade house. The survival rate, rhizome weight and AITC content of plants derived from shoots treated with 20 Gy or 40 Gy of either γ-rays or X-rays were significantly greater than those of the controls.     

Restorative Morphogenesis in vitro in the Annual Medic in the Presence of Benzylaminopurine

Four annual medic species (Medicago orbicularis (L.) All., M. rigidula (L.) Desr., M. scutellata (L.) Miller, and M. rugosa Desr.) were used as model objects for studying the spectrum of morphogenetic reactions in vitro. The seeds were incubated on nutrient media with benzylaminopurine at different concentrations until germination and, thereafter, the explants of seedlings were cultivated in order to obtain primary calluses and morphogenetic structures. Normal and abnormal (with reduced root and/or apex) seedlings were cultivated in the presence of benzylaminopurine. Further cultivation of explants from the seedlings of both types showed a considerable intra- and interspecific polymorphism by the capacity for callusogenesis, frequency of primary restorative reactions, and pattern of microreproduction in vitro. In the control (the seeds were incubated on a hormone-free medium), no cases of microreproduction by way of organogenesis or somatic embryogenesis were observed. In all experimental variants, the restorative reactions preceded microreproduction in vitro.     

The effect of bacterial contamination on the growth and gas evolution ofIn vitro cultured apricot shoots

Summary Shoots of “San Castrese” and “Portici” apricots (Prunus armeniaca L.) free of cultivable bacteria, shoots of the same origin exhibiting bacterial contamination after repeated subcultures, and contaminated shoots treated with cefotaxime were compared for gas exchange, proliferation rate, and fresh and dry weight. Cultures of San Castrese contaminated byBacillus circulans andSphingomonas paucimobilis, and of Portici contaminated withStaphylococcus hominis andMicrococcus kristinae, including those treated with cefotaxime, showed comparable shoot weights and lower proliferation rates than healthy cultures. Bacteria, even if not visible until the end of subculture, markedly influenced the gaseous composition of the jar headspace. Healthy cultures clearly showed photosynthetic activity at 60 μM·m⁻²·s⁻¹ photosynthetically active radiation; in contrast, oxygen quickly decreased and carbon dioxide increased in contaminated cultures, including those treated with cefotaxime, in which bacteria became visible in the culture medium only after repeated subcultures.     

A study of the effects of near UV radiation on the pigmentation of the blue-green alga Gloeocapsa alpicola

The UV survival characteristics of a wildtype and a UV resistant strain of Gloeocapsa alpicola were compared. Except for a higher carotenoid content the resistant strain was similar to the wild-type.However, on exposure to UV radiation the level of carotenoids in the wild-type fell sharply whereas no such decrease occurred in the resistant strain.Growth of cells in diphenylamine and under red and blue light reduced the survival rate. This phenomenon is linked with a reduced carotenoid level in the cell, indicating that they perform an important protective function.     

Organ culture of human articular cartilage: Studies on sulphated glycosaminoglycan synthesis

Summary An organ culture system is described for adult human articular cartilage obtained from joints afterfemoral head replacement operations. Cartilage slices maintain maximal viability for 2 days in culture as assessed by uptake of [³H]uridine and [³H]leucine into whole tissue, and³⁵SO₄ into sulphated glycosaminoglycans (GAGs). Since GAGs are the components of cartilage matrix, the depletion of which is associated with osteoarthrosis, a method for measuring sulphated GAG synthesis in culture has been investigated.     

Hormonal stimulation of avian embryonic cartilage growth in vitro: Histologic and ultrastructural features

Summary We studied the histologic and ultrastructural features of embryonic chick cartilage after the cartilage had been incubated in serum-free medium that contained hormones and growth factors known to stimulate in vitro cartilage growth. Pelvic cartilages from 9 d chick embryos were incubated in BGJ_b (Fitton-Jackson modification) medium alone (control) or medium containing one of the following:N ⁶ monobutyryl cyclic AMP 0.5 mM, forskolin 100 μM, triiodothyronine (T₃) 10 nM, insulin 45 nM, or somatomedin C 0.67 nM. At the end of 3 d of incubation the cartilages were fixed in buffered formalin. Significant growth (increases in size, wet and dry weight) was seen with each treatment group. N⁶-Monobutyryl cAMP treated cartilage had an increased number of flattened immature chondrocytes with large nuclei and prominent nucleoli. The histologic and ultrastructural features of forskolin treated cartilage were indistinguishable from N⁶-monobutyryl cAMP treatment. The T₃ treated cartilage contained large hypertrophic chondrocytes with prominent lacunar typical of mature cartilage. T₃ Treated cartilage had considerable vacuole formation and dilated endoplasmic reticulum. Insulin and somatomedin treated cartilage had histologic appearance similar to control cartilage. Thus, the effects of various hormones on embryonic cartilage growth in vitro can be separated as to whether growth is the result of chondrocytic hyperplasia (cyclic AMP mediated), chondrocytic hypertrophy with maturation (T₃), or a combination of both hyperplasia and hypertrophy (insulin and somatomedin-C). This study was supported by a grant (K08 AM 01021-01) from the National Institute of Arthritis, Metabolism, Diabetes, and Kidney Diseases, Bethesda, MD, and a Basil O'Connor Starter Grant (0-400) from the March of Dimes.     

The influence of growth regulators on proliferation and rooting of in vitro propagated myrtle

The influence of various growth regulators, in different quantities, and the physiological and biological stage of the plant, on the in vitro propagation of myrtle (Myrtus communis L.) were evaluated. It was found that the proliferation was dependent on both the medium and the period in which the sampling was performed. The highest rate of in vitro shoot proliferation was obtained by using 6-benzyladenine and α-naphthyleneacetic acid as growth regulators, starting from nodes sampled at the beginning of May. Rooting was achieved by either transplanting the shoots directly into soil or by culturing on a medium containing 1 mg 1⁻¹ of indole-3-acetic acid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Protective effect of hypothermia on brain neurons in rats exposed to ionizing radiation

The protein-synthesizing system of hippocampal (CA1, CA3) and sensorimotor cortex neurons is damaged less and recovers much quicker in rats exposed to 8 Gy of γ-radiation under hypoxia/hypercapnia (body temperature 16–18°C) than under usual conditions, as evidenced by microfluorimetry and electron microscopy. The radioprotective effect does not cover the membrane structures (endoplasmic reticulum and Golgi complex), and their restoration is not so prompt.     

The in Vitro Growth of Human Chondrocytes

Autologous chondrocyte implantation (ACI) for the treatment of articular cartilage defects has been described by other workers, however, relatively few details of the in vitro growth of the cells have been published. Here we describe the release of cells from adult human articular cartilage and their growth characteristics in vitro.Cultures were successfully established from 29 of 30 biopsies taken from patients aged 20–72 year. No significant relationship was found between donor age and initial cell yield following cartilage digest, however, the time to primary confluence increased in direct proportion to age. Thereafter the kinetics of cell proliferation was independent of donor age.The proportion of apoptotic or necrotic cells in the cartilage digest was low and increased with time in culture only in those cells which remained non-adherent. Conversely, entry into cell cycle was restricted to those cells which had become adherent.These results illustrate that previously reported techniques for isolating and culturing chondrocytes are reproducible, that adherent chondrocytes have considerable proliferative potential, and that concern about cell growth and viability need not, in itself, limit the clinical application of ACI to younger patients.     

The effects of ultraviolet-B radiation on loblolly pine

Summary Depletion of stratospheric ozone and the resulting increase in ultraviolet-B (UV-B) radiation may negatively impact the productivity of terrestrial ecosystems. This concern has led to a number of studies that report the influence of supplementing UV-B radiation on plant growth and development. However, only two of these field studies have included tree species and both were singleseason experiments. In this study, loblolly pine (Pinus taeda L.) from seven seed sources was grown under natural and supplemental levels of UV-B radiation. Irradiation treatments were continued for three seasons on plants from four of the seven groups and for 1 year only for three groups. The supplemental irradiances simulated those that would be anticipated with stratospheric ozone reductions of 16% and 25% over Beltsville, Md. The effects of UV-B radiation during the 1st year on plant growth varied among the seed sources. The growth of plants from two of the seven seed sources tested showed significant reductions following a single irradiation season and plants from one group tended to be larger under increased UV-B radiation. However, after 3 years of supplemental irradiation, plant biomass was reduced in all four groups by 12–20% at the highest simulated ozone depletion. These results suggest that the effects of UV-B radiation may accumulate in trees and that increased UV-B radiation could significantly reduce the growth of loblolly pine over its lifetime. However, they also point to a need for multiple season research in any analysis of potential consequences of global change on the long-term growth of trees.     

Virulence of Paracoccidioides brasiliensis: The influence of in vitro passage and storage

Stability of virulence in P. brasiliensis isolates was studied with respect to the in vitro culture history and methods used for storage. Virulence in yeast-form P. brasiliensis isolates was tested in a chronic pulmonary murine model of paracoccidioidomycosis where progression of disease was quantitated in terms of colony forming units recoverable from lungs. Four isolates of P. brasiliensis, including recently isolated from patients or experimental animals, caused chronic progressive disease. Two isolates with a history of subculturing showed attenuation by causing resolving but chronic disease. An attenuated isolate became avirulent subsequent to 15 more years of subculturing. These findings suggest that virulence of P. brasiliensis can be attenuated or lost subsequent to cycles of subculturing over long periods. Our data suggest that the use of fresh P. brasiliensis isolates may be needed to provide reproducible virulence for experimental systems.Abbreviations ATCC American Type Culture Collection - CFU colony-forming units - LD Lethal dose - MMv-M modified McVeigh Morton - PMN polymorphonuclear neutrophil