Differential nuclease sensitivity of the ovalbumin and beta-globin chromatin regions in erythrocytes and oviduct cells of laying hen.

We have monitored the differential nuclease sensitivity of defined regions of the chicken genome in different cells using a method which combines restriction enzyme digestion and blotting to diazobenzyloxymethyl (DBM)-paper (see Ref. 11). By using different specific probes and by scanning the bands on the autoradiograms, it is possible to compare on the same blot the digestion patterns of similar-sized fragments from different regions of the genome corresponding to "active" and reference "inactive" genes. We have demonstrated the preferential sensitivity to DNaseI and micrococcal nuclease digestion of the ovalbumin gene region in hen oviduct chromatin. The beta-globin gene region (containing both an adult and an embryonic gene) is also preferentially digested by DNaseI in hen mature erythrocyte nuclei, but at a lower rate than the ovalbumin gene region in oviduct. These observations raise the possibility that there may be several types of preferential nuclease sensitivities, all characterized by increased rates of digestion but to different levels, the highest corresponding to the very actively transcribing genes.     

Studies in heterochromatin DNA: accessibility of late replicating heterochromatin DNA in chromatin to micrococcal nuclease digestion

Heterochromatin DNA in cactus mouse (Peromyscus eremicus) replicates in the late S phase of cell cycle. A method of obtaining cells which contain DNA preferentially labeled at heterochromatic areas by a pulse-labeling of late replicating DNA is described. When the nuclei of P. eremicus cells containing radioactively labeled DNA in heterochromatin were digested with micrococcal nuclease and the resultant nucleosomal DNA was separated by gel electrophoresis, it was found that the repeat length of nucleosomal DNA in the heterochromatin DNA is not different from that of the bulk of the genomic DNA. Furthermore, there was no significant difference in the accessibility to digestion by micrococcal nuclease between the late replicating heterochromatin DNA and the total DNA under our digestion conditions. Two dimensional gel electrophoresis patterns of nucleosomal DNAs isolated from micrococcal nuclease digested nuclei from P. eremicus, P. collatus, and P. crinitus cells in culture were very similar. Cytogenetic data showed that these three species are different in heterochromatin but similar in euchromatin.     

Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis.

The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.     

Decreased activity of cytidine 3':5'-monophosphate (cyclic CMP) phosphodiesterase in the fast-growing Morris hepatoma 3924A, but not in the slow-growing Morris hepatoma 9618A.

The activity level of the newly-identified cyclic CMP phosphodiesterase in the fast-growing Morris hepatoma 3924A was found to be much lower than the control (normal or host) liver. Its level in the slow-growing Morris hepatoma 9618A (a minimal deviation tumor), on the other hand, was the same as the host liver. The level of cyclic AMP phosphodiesterase was higher, whereas that of cyclic GMP phosphodiesterase was lower, in hepatoma 3924A than the control liver. In comparison, the levels of the two enzymes were both depressed in hepatoma 9618A. These findings suggest that depression of cyclic CMP phosphodiesterase may be related to the process and the rate of malignant growth, and that metabolism of cyclic CMP may be more crucial than that of cyclic AMP or cyclic GMP in the neoplastic cell proliferation.     

Differential accumulation of proteinase inhibitor I in normal and crown gall tissue of tobacco, tomato, and potato

A proteinase inhibitor (inhibitor I) is induced in crown gall tumors of tobacco (Nicotiana tabacum) initiated through infection with the tumorinducing bacterium, Agrobacterium tumefaciens, strains B6 or CG-14. Uninfected tissues do not contain immunologically detectable quantities of inhibitor I. Inhibitor I synthesis in tobacco crown gall tumors paralleled tumor growth at the average rate of about 4.5 μg of inhibitor I per 200 mg of fresh tissue per day. Infection of variegated tobacco mutant Dp-I with A. tumefaciens strain CG-14 produced tumors with 25% more inhibitor than tumors induced with strain B6. Unlike tobacco, tumors induced by either bacterial strain on potato (Solanum tuberosum) and on tomato (Lycopersicum esculentum) did not accumulate inhibitor I. Consequently, inhibitor I accumulation is modulated by the type of plant host used in spite of familial relatedness (Solanaceae) and the strain of A. tumefaciens used for infection.     

Cyclic AMP-like effects of polyamines on phosphatidylcholine synthesis and protein phosphorylation in human promyelocytic leukemia HL60 cells. Comparison with the effects of phorbol ester

Spermine or putrescine increased cAMP levels through a catalase-sensitive mechanism, resulting in, most notably, a dephosphorylation of protein A (Mr 45,000, pI 5.15) and protein B (Mr 45,000, pI 4.9) and slightly increased phosphatidylcholine (PC) synthesis in HL60 cells. Exogenous dibutyryl cAMP mimicked the polyamine effects. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) also promoted the protein dephosphorylation and PC synthesis, the effects augmented by R59022 and mimicked by exogenous 1-oleoyl-2-acetylglycerol. The effects of spermine (or dibutyryl cAMP) and TPA on PC synthesis were synergistic. It was suggested that cAMP-dependent protein kinase and protein kinase C might mediate, in an independent but inter-related manner, the effects of polyamines and TPA.     

Using Optics to Measure Biological Forces and Mechanics

Spanning all size levels, regulating biological forces and transport are fundamental life processes. Used by various investigators over the last dozen years, optical techniques offer unique advantages for studying biological forces. The most mature of these techniques, optical tweezers, or the single-beam optical trap, is commercially available and is used by numerous investigators. Although technical innovations have improved the versatility of optical tweezers, simple optical tweezers continue to provide insights into cell biology. Two new, promising optical technologies, laser-tracking microrheology and the optical stretcher, allow mechanical measurements that are not possible with optical tweezers. Here, I review these various optical technologies and their roles in understanding mechanical forces in cell biology.     

Temperature-dependent development and population growth of Tetraneura nigriabdominalis (Homoptera: Pemphigidae) on three host plants

The root aphid Tetraneura nigriabdominalis (Sasaki) (Homoptera: Pemphigidae) is a pest of many Gramineae species; however, little is known about its biology and relationships with host plants. The objectives of this study were to quantify the effects of temperature on development, longevity, fecundity, and population growth of T. nigriabdominalis and to assess the effects of host plant on development of T. nigriabdominalis. The effects of temperature on performance of this root aphid were determined at 10, 15, 20, 25, 30, and 35 +/- 1 degrees C on rice roots, Oryza sativa L. Nymphal stages from birth to adult decreased from 46.3 d at 10 degrees C to 8.5 d at 30 degrees C. Aphid survival and development were lowest at 35 degrees C, and no aphid produced progeny at this temperature. Average adult longevity decreased from 23.3 d at 15 degrees C to 8.2 d at temperatures up to 35 degrees C. Average number of nymphs produced per female was highest at 25 degrees C; averaging near 30 nymphs per female, but it dropped to near zero at both 10 and 35 degrees C. The highest intrinsic rate of increase (r(m)) was 0.241 at 30 degrees C. Net reproductive rate (R(0)) ranged from 29.8 at 25 degrees C to 0.2 at 10 degrees C. The generation time (GT) decreased with increasing temperatures from 60.3 d at 10 degrees C to 13.8 d at 30 degrees C. In addition, root aphids reared at 30 degrees C on three host plants [O. sativa, Zea mays L. and Sorghum bicolor (L.) Moench] revealed that the developmental time of the nymphal stages averaged 6.9 d when reared on O. sativa and 10.7 d when reared on Z. mays. Comparison of the nitrogen content of the three host plants indicated that the root nitrogen content was highest in O. sativa. The effect of nitrogen content on aphid performance, however, is still not clear. Other factors, such as plant secondary chemistry, may play a role in affecting aphid performance.     

A novel in vitro retinal differentiation model by co-culturing adult human bone marrow stem cells with retinal pigmented epithelium cells

Human retinal pigment epithelium (HRPE) cells are important in maintaining the normal physiology within the neurosensory retina and photoreceptors. Recently, transplantation of HRPE has become a possible therapeutic approach for retinal degeneration. By negative immunoselection (CD45 and glycophorin A), in this study, we have isolated and cultivated adult human bone marrow stem cells (BMSCs) with multilineage differentiation potential. After a 2- to 4-week culture under chondrogenic, osteogenic, adipogenic, and hepatogenic induction medium, these BMSCs were found to differentiate into cartilage, bone, adipocyte, and hepatocyte-like cells, respectively. We also showed that these BMSCs could differentiate into neural precursor cells (nestin-positive) and mature neurons (MAP-2 and Tuj1-positive) following treatment of neural selection and induction medium for 1 month. Furthermore, the plasticity of BMSCs was confirmed by initiating their differentiation into retinal cells and photoreceptor lineages by co-culturing with HRPE cells. The latter system provides an ex vivo expansion model of culturing photoreceptors for the treatment of retinal degeneration diseases.