Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.     

Mice with Disrupted TGFβ Signaling Have Normal Cerebella Development,but Exhibit Facial Dysmorphogenesis and Strain-Dependent Deficits in Their Body Wall

The transforming growth factor betas (TGFβs) are context-dependent regulators of neurons in vitro, but their physiological functions in the brain are unclear. Haploinsufficiency of either Tgfβ1 or Tgfβ2 leads to age-related deterioration of neurons, but the development of the brain is normal in the full absence of either of these genes. However, some individuals with mis-sense mutations of TGFβ receptors are mentally retarded, suggesting that the TGFβ isoforms can compensate for each other during brain development. This possibility was tested by generating mice (NSE × PTR) with neuron-specific expression of a dominant-negative inhibitor of TGFβ signaling. The NSE × PTR mice with a FVBxC57Bl/6 genetic background were viable and developed normally despite strong neuronal expression of the inhibitor of TGFβ signaling. Their cerebella were of normal size and contained normal numbers of neurons. When the genetic background of the mice was changed to C57BL/6, the phenotype of the mice became neonatal lethal, with the neonates exhibiting various malformations. The malformations correlated with sites of non-neuronal expression of the transgenes and included facial dysmorphogenesis, incomplete closure of the ventral body wall and absence of intestinal motility. The C57BL/6 Tgfbm1–3 alleles, which modulate the phenotype of Tgfβ1^−/− mice, were not major determinants of the NSE × PTR phenotype. The data suggest that the development of the cerebellum is insensitive to the level of TGFβ signaling, although this may be dependent on the genetic background.     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Specific enzyme complex of beta-1,4-galactosyltransferase-II and glucuronyltransferase-P facilitates biosynthesis of N-linked human natural killer-1 (HNK-1) carbohydrate

Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.     

Nitrile hydratase involved in aldoxime metabolism from Rhodococcus sp. strain YH3-3 purification and characterization.

Nitrile hydratase responsible for aldoxime metabolism from the E-pyridine-3-aldoxime degrading bacterium, Rhodococcus sp. strain YH3-3 was purified and characterized. Addition of cobalt ion was necessary for the formation of enzyme. The enzyme activity was highly induced not only by nitriles and amides but also by several aldoxime compounds. The enzyme was purified approximately 108-fold with a 16% yield from the cell-free extract of the strain. The native enzyme had a Mr of approximately 130 000 and consisted of two subunits (alpha-subunit, 27 100; beta-subunit, 34 500). The enzyme contained approximately 2 mol cobalt per mol enzyme; it showed a maximum activity at 60 degrees C and at 40 degrees C under the rate assay and end-point assay conditions, respectively, and was stable over a wide range of pH (pH 2.5-11.0). The enzyme had a wide substrate specificity: it acted on aliphatic saturated and unsaturated as well as aromatic nitriles. The N-terminus of the beta-subunit showed good sequence similarities with those of other nitrile hydratases. Nitrile hydratase is part of the metabolic pathway for aldoximes in microorganisms.     

Micropropagation of horseradish hairy root by means of adventitious shoot primordia

Adventitious shoot primordia were formed on horseradish hairy root cultured in dark. Plantlet formation frequency from the primordia was higher than that from root fragments. Culture for 26 days provided the adventitious shoot primordia, which had the highest potential for plantlet formation (53% explants at 40 days). Benzyladenine supplementation in the dark caused primordium enlargement, but did not increase the number of primordia formed. After adventitious shoot primordia were encapsulated with calcium alginate, kinetin supplementation (2.0–4.0 M) increased the shoot formation frequency (65–80% explants at 20 days) in the light, but also promoted the undesirable formattion of multiple shoots. Supplementation with naphthaleneacetic acid (0.27–5.4 M) in the calcium alginate beads in light enhanced the root emergence from primordia without inhibition of plantlet formation when the encapsulated beads were put on the agar-medium without naphthaleneacetic acid.     

Effects of winter flooding on phosphorus dynamics in rice fields

Limnology - Controlling phosphorous (P) loads from rice fields is important for the conservation of aquatic ecosystems, in part because P is relatively concentrated at its sources. Recently, winter...     

Biochemical studies of rat liver Golgi apparatus. I. Isolation and preliminary characterization.

A method is described for the isolation of morphologically well-preserved Golgi apparatus from rat liver. The method is essentially the same as that of Morré et al. (Morré, D.J., Hamilton, R.L., Mollenhauser, H.H., Mahley, R.W., Cunningham, W.P., Cheetham, R.D., & Lequire, V.S. (1970) J. Cell Biol. 44, 484-491) except that mild cell disruption is achieved by means of a stainless-steel sieve. The average recoveries of protein and galactosyltransferase in the isolated fraction are about 6 mg from 10 g of perfused liver and about 35% from the homogenate, respectively. The preparation is virtually free from succinate-cytochrome c reductase, glucose-6-phosphatase, acid phosphatase, and 5'-nucleotidase. The Golgi fraction as well as its vesicular fragments is homogeneous upon isopycnic centrifugation in both sucrose and dextran density gradients. Their buoyant densities in sucrose are significantly higher than those in dextran, indicating that both forms of the organelle are closed systems which are impermeable to macromolecules. The galactosyltransferase activity of a freshly prepared Golgi fraction, measured with ovalbumin as galactosyl acceptor, is activated 26-fold by the addition of Triton X-100, whereas those of homogenized, sonicated, and aged preparations are only activated 2- to 4-fold.     

Biological activity of human granulocyte colony stimulating factor with a modified C-terminus

Granulocyte colony-stimulating factor (G-CSF) undergoes receptor-mediated internalization into target cells which are normally restricted to neutrophilic granulocytes and their committed progenitor cells, suggesting that it may be applicable as a myeloid cell-targeting vehicle. To test this notion, we constructed a cDNA encoding a human G-CSF/murine stem cell factor (mSCF) chimeric molecule in a mammalian expression vector and transfected NIH3T3 cells with this plasmid. The resulting chimeric cytokine consisted of the entire G-CSF sequences fused to Lys148 of mSCF. It can be released from the surface membrane of NIH3T3 transformants through proteolytic cleavage at Ala164 of mSCF. The culture media conditioned by a number of stable transformants, which were confirmed by an enzyme-linked immunosorbent assay (ELISA) to secrete an hG-CSF derivative, were examined for their ability to stimulate CFU-G-derived colony formation as well as the proliferation of G-CSF-dependent NFS-60 cells. The results indicated that this C-terminus modified version of hG-CSF is as potent as recombinant hG-CSF in both assays.