Analysis of Nuclear DNA content in plant cells by Flow cytometry

Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.     

Isolation of Bacteria of the Family <Emphasis Type="Italic">Enterobacteriaceae</Emphasis> from Plant Tissues

Bacteria of the family Enterobacteriaceae were isolated from the tissues of a number of wild and cultivated plants. All the cultures isolated had a broad spectrum of resistance to antibiotics and were highly adhesive to human erythrocytes. The studies conducted indicate the possibility of a concentration of microorganisms pathogenic for humans in plant tissues.     

The Isolation of DNA from Plant Materials

Total DNA was isolated from seedling and tissues of many plants. The nuclei and chloroplasts were prepared from plant tissues, and then the nuclear DNA and chloroplast DNA were isolated from them. According to the chemical analysis and the physical properties determined by ultraviolet absorbance, hyperchromicities, ultracentrifugation, gel electrophoresis and the electro-microscopical observations it is suggested that DNA obtained possessed a considerable purity and to a certain extent retained the natural status of the large molecule.     

Expression of FGF23 is correlated with serum phosphate level in isolated fibrous dysplasia

Fibrous dysplasia (FD) patients sometimes suffer from concomitant hypophosphatemic rickets/osteomalacia, resulting from renal phosphate wasting. It was recently reported that FD tissue in the patients with McCune-Albright syndrome (MAS) expressed fibroblast growth factor-23 (FGF-23), which is now known to be as a pathogenic phosphaturic factor in patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets. Since it remains controversial whether serum phosphate levels are influenced by FGF23 expressions in FD tissue, isolated FD patients without MAS syndrome were examined for the relationship between FGF23 expressions, circulating levels of FGF-23 and phosphate to negate the effects of MAS-associated endocrine abnormalities on serum phosphate. Eighteen paraffin embedded FD tissues and 2 frozen tissues were obtained for the study. Sixteen of 18 isolated FD tissues were successfully analyzed GNAS gene, which exhibited activated mutations observed in MAS. Eight of 16 FD tissues, which exhibited GNAS mutations, revealed positive staining for FGF-23. These evidence indicate that postzygotic activated mutations of GNAS is necessary for the FD tissue formation by mosaic distribution of mutated osteogenic cell lineage, but is not sufficient to elevate FGF23 expression causing generalized osteomalacia with severe renal phosphate wasting. The expression level of FGF23 in isolated FD tissue with hypophosphatemic osteomalacia determined by real-time PCR was abundant close to the levels in OOM tumors. Osteoblasts/osteocytes in woven bone were predominant source of circulating FGF-23 in FD tissues by immunohistochemistry. A negative correlation of the intensity of FGF-23 staining with serum inorganic phosphate levels indicated that the expression of FGF23 in focal FD tissues could be a prominent determinant of serum phosphate levels in isolated FD patient. These data provide novel insights into the regulatory mechanism of serum inorganic phosphate levels in isolated FD patients and extend the notion that FGF-23 originating from FD tissue may cause hypophosphatemia not only in isolated FD patients but also in the patients with MAS syndrome.     

Widespread distribution of a 210,000 mol wt microtubule-associated protein in cells and tissues of primates

Antisera prepared against a 210,000 mol wt microtubule-associated protein (210k MAP) isolated from the human cell line, HeLa, were used to survey a variety of cells and tissues for the presence of immunologically related proteins. The antisera were employed to test extracts of the cells and tissues, using a sensitive indirect immunofluorescence technique applied to polyacrylamide gels. Cross- reactive material of 210,000 mol wt was found in 10 kinds of cells and tissues derived from humans and four lines of cells from monkeys. Indirect immunofluorescent staining was also carried out on fixed cells and showed that the cross-reactive material was localized to interphase and mitotic microtubules as assayed in nine human and seven monkey cell lines. No protein that cross-reacted with 210k MAP antisera was detected in cells and tissues derived from two rodents, an ungulate, a marsupial, or a chicken. Therefore, the 210k MAP isolated from HeLa cells is present in a wide variety of cells and tissues of humans and other primates but is antigenically distinct from MAPs present in lower organisms.     

THE IN VITRO SECRETION OF GROWTH REGULATORS BY ISOLATED CALLUS TISSUES

Tissues from a wide variety of plants were surface sterilized, isolated, and grown on different media. These isolated tissues were bioassayed for growth regulatory activity. The secretions from four of the 20 callus tissues inhibited the growth of Bacillus subtilis. An aseptic method for measuring the growth of Lemna was developed and used to detect inhibitory materials in medium which had supported the growth of five isolated callus tissues. In the seed (Lycopersicon esculentum) germination test five callus tissues had an inhibitory influence while two callus tissues showed a stimulatory effect. The study also included expressed juices and extracts of callus tissues which secreted regulatory materials. The expressed juice of five callus tissues contained materials which inhibited the growth of Lemna. Two expressed juices inhibited the growth of Bacillus subtilis. The water extract of two callus tissues inhibited the growth of Lemna. Fifty percent of the plants which have been reported to produce growth regulatory materials in nature also produced callus tissue which was capable of regulating growth of assay organisms.     

Chemical compositions of elastins isolated from aortas and pulmonary tissues of humans of different ages

1. Elastins were isolated from the visceral pleuras and parenchymas of lungs of humans of different ages. 2. The elastin content of pleuras increased whereas that of parenchymas remained constant with increasing age. 3. The amino acid compositions and carbohydrate contents of elastins isolated from both pulmonary tissues changed in the same way with increasing age of the subjects. These changes were similar to those observed in elastins isolated from the aorta. 4. Similar glycoproteins were isolated from pleuras and aortas, and were more difficult to extract from the elastins of older subjects. Contamination with these glycoproteins was responsible for the changes in composition of elastin, as the age of the tissue from which it was extracted increased. 5. The amount of the cross-linking amino acids desmosine and isodesmosine was lower in elastins isolated from both aorta and pulmonary tissues of senile subjects than those from younger subjects.     

Nucleotide sequence of a cDNA clone for human aldolase: a messenger RNA in the liver

Nearly complete cDNA clones for human aldolase A mRNA were isolated from human liver cDNA library and the nucleotide sequence determined. Using the cDNA clone as a probe the length of human aldolase A mRNAs, isolated from the skeletal muscle, liver and placenta tissues, was measured by RNA blotting and estimated to be 1,600 nucleotides for skeletal muscle mRNA and 1,700 nucleotides for both the liver and placenta mRNAs, indicating that different species of mRNA coding for human aldolase A were expressed in the different tissues.     

Occurrence of ungulic acid in some epidermal tissues

The lipids isolated from different animal tissues have been studied qualitatively, by TLC, for the occurrence of the ungulic acid fraction. This fraction was found in considerable amounts only in epidermal tissues and its keratinized derivatives. In the present study it was isolated from human keratinous epidermis, hair, and nails, pig bristles, wool, and feathers. The analytical results indicated that a lipid fraction from all of these sources contained ceramide, galactose, galactosamine, sulfate, and sialic acid in equimolar amounts, and that the fractions were similar to the ungulic acid isolated earlier from a horse's hoof.     

几种植物组织及其原生质体过氧化物酶同工酶的比较研究

对马尾松幼苗子叶和胚轴、甘蔗、小麦、烟草、黄花菜等幼叶及其原生质体的过氧化物酶同工酶(POI)分别作比较研究。观察到凡经纤维素酶处理的各植物组织POI酶带数均多于未经纤维素酶处理的组织的酶带数;除个别例外,后者一般又比无壁原生质体的酶带数多。此种差异随植株生长年龄而增大,表明植物组织内大部分POI主要存于质外体中。     

The presence of arsenobetaine in marine animals

Methanol soluble arsenic compounds were isolated from the muscle tissues of a crayfish, Jasus novaehollandiae, a prawn, Penaeus latisulcatus, a scallop, Pecten alba, a squid, Sepioteuthis australis and a fish, Sillaginodes punctatus. Arsenobetaine was found to be the major arsenic compound present in all muscle tissues. The prawn and scallop tissues also contained another organo arsenic compound that could be partially degraded to characterized methylated arsenic species.     

Isolation of rat mammary epithelial cells for polyamine analysis.

Mammary epithelial cells were isolated from either abdominal-inguinal glands or mammary tumours of rats, after enzymic digestion of the tissues, and were analysed for polyamine content. Optimum conditions were developed for the isolation of cells in sufficient yield for the analysis of polyamines from 1 g of mammary gland or 0.5 g of tumour tissue. Complete recoveries of the polyamines in the tissues were achieved in the isolated epithelial cells.     

Natural occurrence of gliotoxin in turkeys infected with Aspergillus fumigatus,Fresenius

Thirteen samples of infected turkey lung tissue from cases of airsacculitis were collected either at the processing plant or from a local turkey farm and subjected to cultural and gliotoxin analysis. Aspergillus fumigatus was isolated from 6 of the 13 samples; all isolates were determined to be gliotoxin producers when grown in laboratory culture and assayed by HPLC procedures. Gliotoxin was isolated from 5 of the 13 tissues but was not isolated from all tissues that were infected with A. fumigatus. Gliotoxin was isolated from two tissues from which no A. fumigatus was isolated and it was not detected in three tissues from which gliotoxin-producing isolates of A. fumigatus were obtained. The ability of this pathogenic fungus to produce this immunomodulating compound in naturally infected turkeys provides further evidence that gliotoxin may be involved in the pathogenesis of the disease, aspergillosis of turkeys. Disclaimer: Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the products, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

烟草内生真菌多样性和种群结构

从云南“世界烟草品种园”10种烟草的根、茎、叶中分离得到199株内生真菌,根据rDNA-ITS系统发育分析鉴定为17属25种,其中格孢腔菌目Pleosporales内生真菌的种类和数量最多;茎点霉属Phoma、链格孢属Alternaria和镰孢菌属Fusarium为主要优势属,相对频率分别为25.1%、24.6%和11.6%,优势度Y值分别为0.251、0.172和0.104。烟草不同组织内生真菌的种群结构存在显著差异,分离自根的内生真菌的主要优势属为Fusarium和Phoma,Y值分别为0.235和0.123;分离自茎的内生真菌的主要优势属为Phoma和Alternaria,Y值分别为0.186和0.155;分离自叶片的内生真菌的主要优势属为Alternaria,Y值为0.286。Phoma从烟草根茎叶中均可分离得到,而Alternaria只分布在地上部茎叶中,Fusarium只分布在根茎中,表明这3个优势属真菌对根茎叶组织的专化性不同。     

Plasminogen activators and plasminogen activator inhibitors in connective tissues and connective tissue cells: influence of the neuropeptide substance P on expression

Tissue segments isolated from ligament, epiligament, and synovial tissues from mature female New Zealand White Rabbits were demonstrated to consitutively secrete a plasminogen activator. Several tissues were also observed to constitutively secrete a plasminogen activator inhibitor which was detected in the form of a PA-PAI complex. Heterogeneity was observed in PA and PAI activity between the different connective tissues. Heterogeneity also existed between and within the medial collateral (MCL), lateral collateral (LCL), and the anterior cruciate (ACL) ligaments. In addition to the differences in constitutive expression of PA and PAI activity, differences in the responsiveness to the neuropeptide substance P (10^?5?10^?9 M) were also detected. This responsiveness to substance P was displayed by an increase in PA and PAI activity in the conditioned medium. The pattern of responsiveness reflected the degree of innervation of these tissues. That is, synovium and epiligament tissue were the most responsive tissues to substance P while the MCL, LCL and ACL were less responsive to the neuropeptide. Parallel results were obtained using cell culture with fibroblasts isolated from the above mentioned tissues. That is, the pattern of responsiveness was similar between cells and tissue segments. More specifically, cells isolated from both synovium and epiligament increased their both their PA (slightly) and PAI activity following exposure to substance P. This was demonstrated at both the protein and RNA level. Thus, cells within a tissue maintain their phenotype when removed from their three-dimensional matrix. These results are unique in demonstrating that normal ligament and synovial cells and tissue respond to substance P by altering the expression of PA and PAI activity. This investigation further supports the concept that innervation may be important in normal connective tissue function.     

Target tissue specificity of retinoic acid-induced stress proteins and malformations in mice

Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.     

Isolation of vegetable wasps and plant worms, Cordyceps nutans, from fruit-body tissue

We isolated Cordyceps nutans from the stipe and abdominal tissues of fruit bodies using a surface sterilization method. Hyphal growth was observed in inocula from both the stipe and abdominal tissue. Some strains from discharged ascospores were obtained and colony characteristics were compared to the strains isolated from the tissues. Colonies of isolates from ascospores grew quite slowly. Isolates of 43 from the 52 examined fruit bodies formed colonies similar to those from ascospores. To confirm the success of isolation, we analyzed by PCR-RFLP of the ITS regions of rDNA samples from fruit bodies, isolates from fruit bodies, and isolates from ascospores. All the isolates obtained from stipe and abdominal tissues presented identical patterns. In this study, we report the first successful isolation of C. nutans from fruit-body tissue using a surface sterilization method.     

Isolation of Pathogenic Bacillus circulans from Callus Cultures and Healthy Offshoots of Date Palm (Phoenix dactylifera L.)

Pure cultures of a gram-negative, rod-shaped bacterium, which produced endospores after 3 to 5 days on solid medium, were isolated exclusively from tissue cultures of the date palm Phoenix dactylifera L. Electron microscopic examination of thin sections of the bacteria revealed the bilayer membrane typical of gramnegative bacteria and confirmed the nature of the spores as true endospores. Biochemical and physiological tests indicated that the bacteria were Bacillus circulans. B. circulans was consistently isolated from the internal tissues, including the meristem, of apparently healthy offshoots of date palm. When meristem and embryo callus tissue culture samples were injected with B. circulans isolated from similar tissue culture samples and from offshoots, the majority of the isolates produced a rapid, destructive soft rot of the tissues.     

Evaluation of a water concentration method for the detection of infectious pancreatic necrosis virus in a fish hatchery

A virus-adsorption-elution (viradel) method using electropositive microporous filters (Virosorb 1-MDS) was used for the concentration and détéction of infectious pancreatic necrosis virus (IPNV) at a hatchery with a past history of IPNV infection. Samples of fish tissue and unconcentrated water were also examined for the presence of IPNV. Virus was isolated from three of the nine concentrated water samples taken from various locations at the hatchery. Virus was also isolated from pooled fish tissues, but not from unconcentrated water samples. Representative numbers of viruses isolated from water and fish tissues were examined for virus neutralization in the presence of anti-IPNV amiserum; resistance to acid, ether, and heat; and replication in the presence of 5-bromo-2'-deoxyuridine (BUDR). When tested by dot-immunobinding assay using a panel of monoclonal antibodies (MABs), the viral isolates were found to belong to the West Buxton serotype of IPNV.