Conversion of cellulose to glucose with cellulase of Trichoderma viride ITCC-1433

Trichoderma viride ITCC-1433 secretes a cellulase complex that is rich in β-glucosidase and therefore well suited for the saccharification of cellulosic materials. The cellulase was investigated with respect to optimum conditions of reaction and enzyme stability. Avicelase, CMCase, and β-glucosidase differed considerably in their physicochemical properties. At temperatures above 50°C, β-glucosidase is not very stable. Therefore, as a compromise the conditions of hydrolysis were chosen to be 50°C and pH 4.5. With the crude culture filtrate of T. viride ITCC-1433 a nearly pure glucose solution of 4% is reached from a 10% cellulose suspension. Wood pulp and newsprint are hydrolyzed to a much smaller extent. With an enzyme concentrate up to 8% glucose accumulated in the reaction fluid within 48 hr. At this time the glucose-cellobiose ratio was 75:1. Glucose was demonstrated to be the most potent inhibitor of total hydrolysis. The addition of glucose to the enzyme-substrate solution at zero time completely stopped its own formation and cellobiose and reducing groups (oligosaccharides) accumulated. By removing glucose through an ultrafilter device about 90% saccharification of cellulose to glucose was achieved in 48 hr without any accumulation of cellobiose.     

Paleobiogeographic relationships of angiosperms from the Cretaceous and early Tertiary of the North American area

The biogeographic affinities of the Cretaceous and early Tertiary angiosperm floras of the North American area (which includes Meso-America, and the Greater Antilles) have been the subject of considerable interest. Although recent treatments of isolated taxa have shown affinities between North American, European, east Asian and Neotropic floras, the relationships have not been quantified. This study compiles the records of fossils whose familial relationships seem secure. This provides a carefully culled, and uniformly presented review of the Cretaceous and Paleogene record from 1950 to 1989 and supplements LaMotte (1950). A subset of these records, which showed compelling evidence of subfamilial relationships, was analyzed to quantify the relationships of the Cretaceous, Paleocene, Eocene and Oligocene floras to other regions. The analysis suggests that for the entire period 24% of the fossil species had affinities with extant taxa from the Northern Hemisphere; 10% with taxa from the Northern Hemisphere that have a few species in South America; 17% with taxa from Eurasia; 3% with taxa with a disjunct Eurasian-South American pattern; 19% with taxa from South America and/or Africa; 8% with taxa from South America and/or Africa that have an important sister group in southeast Asia; 5% with taxa from the Old World; and 13% with taxa having other distribution patterns. Those fossils with affinities to Laurasian taxa are mostly found in the northern and western portions of the North American area. The fossils with affinities to South American and/or African taxa are found in the southern portions of North America, Meso-America, and the Greater Antilles. The taxa with disjunct distributions show both patterns. These patterns suggest that during this time there were wide-spread temperate elements, found throughout Laurasia; Boreotropical flora elements, distributed in North America, Europe and along the Tethys seaway to southeast Asia; and West Gondwana elements which show dispersion from South America across the proto-Caribbean. The paleobotanical data are compatible with current geological, paleontological and biogeographical studies.     

Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins.

Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and microsequencing of the intra-acrosomal protein SP-10. Evidence that SP-10 heterogeneity results from endoproteolytic processes.

The human sperm antigen SP-10 has been shown to be a testis-specific, intra-acrosomal protein that is associated with the membranes and matrix of the acrosomal vesicle. Sperm extracts, analyzed on Western blots with a monoclonal antibody to SP-10, have shown heterogeneity of SP-10 peptides ranging from 17.5-34 kDa. Although the entire SP-10 amino acid sequence of 265 amino acids (28.3 kDa) has been deduced from sequencing SP-10 cDNAs, the nature of multiple SP-10 peptide bands is incompletely understood. In this study, we developed a three-step purification method for SP-10 peptides using monoclonal antibody affinity chromatography, reverse-phase HPLC, and preparative gel electrophoresis. Eight SP-10 peptides separated by this protocol and sequenced using Edman degradation showed amino termini that corresponded to regions on the deduced SP-10 amino acid sequence. Peptides with progressively lower apparent mass aligned further toward the carboxy terminus. On the basis of putative cleavage sites on the SP-10 sequence, endoproteases that act at five different peptide bonds are predicted to cleave SP-10: these hydrolyze following arginine (a trypsin-like protease, possibly acrosin), and following serine, proline, glycine, and glutamic acid (previously undescribed intra-acrosomal protease specificities). The present studies 1) provide a purification method for SP-10 peptides; 2) confirm that the SP-10 cDNAs previously sequenced encode authentic SP-10; and 3) yield indirect evidence that endoproteases act to contribute to SP-10 heterogeneity.     

The effect of coupled cardiovascular and respiratory afferent signals on the central processes of human information processing

On the basis of contemporary knowledge on close functional connections between respiratory and cardiovascular afferents a computerized experimental device was developed by means of which central information processing can be studied in dependence on respiration phase and the level of baroreceptor activity. In all the examined parameters (time of motor reaction of two hands, latency of eye-lid reflex) the greatest changes were observed at maximum baroreceptor activity. These changes were clearly distinguished by direction during the phases of inhalation and exhalation. Such effect is probably based on the fact that the influence of cardiovascular afferents on the level of central activity is modulated in respiration rhythm through the mechanism of breath "gating" of postsynaptic STN structures.     

Nucleotide sequence of the 3′ terminus of E. coli 16S ribosomal RNA

The 3′-terminal T1 oligonucleotide of E. coli 16S ribosomal RNA has been sequenced, using U2 and silkworm nucleases, and was found to be A-U-C-A-C-C-U-C-C-U-U-A_OH. This result is discussed in view of previously reported conflicting sequences and with respect to suggested functional roles for this region of 16S RNA.     

Nitrogen fixation and respiration by root nodules ofAlnus rubra Bong.: Effects of temperature and oxygen concentration

Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO₂=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1     

Decidual cells in the human ovary at term: II. Morphometric analysis of cytoplasmic processes and organelles

Morphometric analysis of human ovarian decidual cells was performed with a Videoplan computer, and mean values were established for the area and perimeter of cellular processes and organelles. Two-hundred forty electron micrographs representing 160 cells were analyzed. The mean decidual cell area was 218.7 microns2, of which 34.5 microns2 was occupied by the nucleus (15.8% of the cytoplasmic area); the nucleus contained 1.74 micron2 of nucleolar material (0.8%). The endoplasmic reticulum occupied 13.63 microns2 (6.2%). Mitochondria occupied 7.3 microns2 (3.3%) and the Golgi network 5.49 microns2 (2.5%). Decidual secretory bodies occupied 0.91 micron2 (0.42%) and cytoplasmic processes 1.89 micron2 (0.94%). The remainder of the cytoplasm, containing inclusions and cytoskeleton, represented 71% of the cell area. Perimeter measurements indicated an average decidual cell was surrounded by 87.8 microns of plasma membrane. The mean nuclear membrane measured 28.3 microns (representing 32.3% of the plasma membrane, pm, or 4.1% of total cellular membranes, cm). Outer mitochondrial membranes measured 156.6 microns (178% pm, 23.5% cm); endoplasmic reticulum membranes measured 350.3 microns (400% pm, 52.6% cm); Golgi membrane measured 30.77 microns (35% pm; 4.5% cm) and membrane surrounding secretory bodies measured 9.8 microns (11.2% pm; 1.4% cm). A mean of 280 secretory bodies per ovarian decidual cell was calculated. The plasma membranes of evaginated cytoplasmic processes represented 22.3% of the total pm (19.6 microns or 2.9% cm). A mean of seven such processes was observed per 87.8 microns of plasma membrane (160/cell). These morphometric data provide a baseline for comparisons of human ovarian decidual cells with uterine decidua, in vivo and in vitro, as well as with decidual cells of other species.     

The relation between antisperm antibodies and testicular alterations after vasectomy and vasovasostomy in Lewis rats

The relationship between antisperm antibodies as determined by enzyme-linked immunosorbent assay (ELISA) and the occurrence of alterations in testicular weight and histology was studied following vasectomy in Lewis rats. The effects of vasovasostomy on antisperm antibody levels were also examined. At 1, 3, and 4 months after vasectomy, the mean absorbance values in an ELISA for sera from animals with altered testes was significantly greater than that from animals lacking testicular alterations. However, animals showing positive antisperm antibody responses were represented both in the group with testicular alterations and among those that lacked testicular damage. Levels of antisperm antibody in both vasectomy and vasovasostomy groups significantly exceeded that for sham-operated animals, but the level of antisperm antibodies in vasovasostomized animals with positive responses was similar to vasectomized animals one and four months after reanastomosis. It is suggested that persistence of antisperm antibodies or testicular alterations, or both, may play roles in limiting the restoration of fertility after vasovasostomy.     

Mechanism of ribosomal subunit association: discrimination of specific sites in 16 S RNA essential for association activity.

Modification of 30 S ribosomal subunits with kethoxal causes loss of their ability to associate with 50 S subunits under tight couple conditions. To identify those 16 S RNA sequences important for the association. ³²P-labeled 30 S subunits were partially inactivated by reaction with kethoxal. The remaining association-competent 30 S subunits were selected from the modified population by their ability to form 70 S ribosomes. Comparison of kethoxal diagonal maps of the association-competent subunits with those of the total population of modified subunits reveals nine sites in 16 S RNA whose modification leads to loss of association activity. Eight of these sites were previously found to be protected from kethoxal attack and one was shown to have enhanced reactivity in 70 S ribosomes (Chapman &; Noller, 1977). As before, these sites are not distributed thoughout the molecule, but are found to be clustered in two regions, at the middle and at the 3′ terminus of the 16 S RNA chain.We interpret these findings in terms of a simple preliminary model for the functional organization of 16 S RNA, supported by the observations of other investigators, in which we divide the molecule into four domains. (1) Residues 1 to 600 are involved mainly in structural organization and assembly. (2) Residues 600 to 850 include sites which make contact with the 50 S subunit and are essential for subunit association. (3) Sites from the domain comprising residues 850 to 1350 line a pocket at the interface between the two ribosomal subunits. and contribute to the binding site(s) for transfer RNA. (4) Residues 1350 to 1541 also contain sequences which bind the 50 S subunit, but some sites in this domain alternatively participate in the initiation of protein synthesis.