Ca lectin gene insertion has the structural features of a transposable element

The single gene Le1, coding for soybean seed lectin, was compared to le1, a naturally occurring mutant allele containing a 3.4 kb insertion within its coding region. Le1 is devoid of introns and produces a 1.0 kb mRNA. It codes for a signal sequence of 32 amino acids and a mature protein of 253 amino acids. With the exception of six single-base substitutions, the coding and flanking sequences in le1 are identical with those in the uninterrupted gene. The insertion termini are imperfect inverted repeats flanked by a 3 bp duplication of lectin target DNA. Inverted repeats within the lectin gene are located symmetrically with respect to the insertion site and are homologous to a region of the insertion termini. These molecular traits conform with the structural aspects of transposable elements in other organisms and imply some degree of site specificity.     

Differential patterns of incorporation and remodeling among various types of bone grafts

The early (3 months) and later (6 months) patterns of incorporation and bone formation have been evaluated histomorphometrically for different types of bone grafts; that is, vascularized and nonvascularized autografts with and without ciclosporin, and vascularized and nonvascularized dog leukocyte antigen (DLA)-mismatched allografts with and without ciclosporin. The vascularized bones were superior to the nonvascularized ones in healing and remodeling their grafted segments. In the autograft bones, ciclosporin did not alter the incorporation process 3 months after transplantation but delayed and increased the remodeling activities in the long run (6 months). Nonvascularized allografts underwent vigorous resorption, and were markedly porotic. Ciclosporin administration significantly reduced resorption and enhanced remodeling in nonvascularized allografts. The remodeling of allografts was similar to that of autografts in the presence of ciclosporin, but stopped soon after the administration of ciclosporin ceased.     

1H-NMR conformational analysis of a high-affinity antigenic 11-residue peptide from the tryptophan synthase beta 2 subunit.

Two synthetic peptides from the beta 2 subunit of tryptophan synthase have been studied by 1H-NMR spectroscopy at 300 MHz. One peptide, His-Gly-Arg-Val-Gly-Ile-Tyr-Phe-Gly-Met-Lys (peptide 11; Ile, isoleucine) is antigenic and binds with a high affinity to a monoclonal antibody that recognizes the native beta 2 subunit. The second peptide, His-Gly-Arg-Val-Gly-Ile-Tyr-Phe (peptide 8) reacts very weakly with the antibody. The 1H-NMR spectra of the two peptides have been assigned from two-dimensional techniques in H2O, 2H2O and (2H6) dimethyl sulfoxide [(2H6)Me2SO]. The structure has been evaluated through analysis of nuclear Overhauser effects, coupling constants, amide-proton exchange rates and their temperature coefficients, and chemical shifts. In aqueous solvent, the C-terminal part of peptide 11 presents some structure centered around residues Phe-Gly-Met. The relationship between the structure found in peptide 11 and its antigenic nature is discussed.     

ATP dependent histone phosphorylation and nucleosome assembly in a human cell free extract.

Physiologically spaced nucleosome formation in HeLa cell extracts is ATP dependent. ATP hydrolysis is required for chromatin assembly on both linear and covalently closed circular DNA. The link between the phosphorylation state of histones and nucleosome formation has been examined and we demonstrate that in the absence of histone phosphorylation no stable and regularly spaced nucleosomes are formed. Phosphorylated H3 stabilizes the nucleosome core; while phosphorylation of histone H2a is necessary to increase the linker length between nucleosomes from 0 to approximately 45 bp. Histone H1 alone, whether phosphorylated or unphosphorylated, does not increase the nucleosome repeat length in the absence of core histone phosphorylation. Phosphorylations of H1 and H3 correlate with condensation of chromatin. Maximum ATP hydrolysis which is necessary to increase the periodicity of nucleosomes from approximately 150 to approximately 185 bp, not only inhibits H1 and H3 phosphorylation but facilitates their dephosphorylation.     

Specific interchain cross-linking of antibodies using bismaleimides. Repression of ligand leakage in immunoaffinity chromatography.

The extensive use of antibody-containing affinity columns in the purification of biologically active compounds (e.g., genetically engineered proteins) is severely hampered by the leaching of antibody (or portions thereof) from the immunoaffinity resin during elution of the target antigen. One of the major problems in this context is the combined use of reducing (i.e., thiols) and chaotropic (e.g., detergents and denaturants) agents in the elution step, which causes the disassociation of heavy and/or light chains from the immobilized antibody, thereby contaminating the resultant product. In order to overcome this problem, we have cross-linked the four antibody chains at their sites of disulfide interlinkage, thus producing a single antibody chain. To accomplish this, interchain disulfide bonds were reduced, and the resultant thiol groups were cross-linked by using bifunctional SH-specific reagents (particularly bismaleimides). Cross-linking of up to 95% of the available SH groups produced was achieved with concomitant retention of antigen-binding activity. The cross-linked antibody was immobilized onto CNBr-activated Sepharose, and the resultant column was found to be substantially more stable to harsh elution conditions than similar columns which contain the un-cross-linked antibody.     

A case of leukemia in the desert spiny lizard (Sceloporus magister)

A leukemia characterized by the proliferation of undifferentiated hematopoietic stem cells is described from a desert spiny lizard (Sceloporus magister). This is the first neoplasm reported from this species and the first leukemia from the family Iguanidae. A massive invasion of primitive hematopoietic cells was noted in the vascular system and heart muscle. Spleen, bone marrow, lungs, adrenal gland, pancreas, brain, skin, testes, liver, lamina propria and submucosa of intestines were extensively involved. Tissue-fixed mast cells were noted in the kidney and intestinal submucosa but were not participating in the neoplastic proliferation.     

Kinetics and compartmentation of energy metabolism in intact skeletal muscle determined from 18O labeling of metabolite phosphoryls

Analyses of isolated intact diaphragm muscle show that at rest only about 30% of the total cellular Pi is metabolically reactive as indicated by 18O incorporation from [18O]water, whereas up to 90% becomes metabolically active incrementally with contractile frequency. Kinetics of [gamma-18O]ATP appearance show that about 90% of the cellular ATP is metabolically active and suggest slowly and rapidly metabolizing compartments of ATP in resting muscle and only rapidly metabolizing compartments in contracting muscle. Rates of [18O]creatine phosphate [( 18O]CrP) appearance are consistent with creatine kinase-catalyzed phosphoryl exchange functioning in an obligatory phosphoryl shuttle system. In noncontracting muscle, ATP turnover rate was 83 nmol.mg protein-1.min-1, and the P/O ratio was determined to be 3.2. ATP utilization increases in direct proportion to contractile frequency with each contracture consuming the equivalent of 0.96 nmol of ATP.mg protein-1 or 2.5-3.5 molecules of ATP/myosin active site. Basal concentrations of nucleotide polyphosphates are not altered when ATP utilization rates increase during contraction. At high contractile frequencies, decreases in CrP concentration occur, but this accounts for less than 4% of total high energy phosphoryls consumed. If metabolic intermediates are free in the aqueous cellular cytosol, each twitch contracture would result in a decrease in ATP concentration of no more than 2% and increases in ADP and AMP concentrations of less than 20 and 7%, respectively. Thus, changes in metabolite concentration must be highly localized or metabolic regulation can be accomplished by a nonallosteric mechanism.     

SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages

We have reported that SV40-transformed human lung fibroblasts secrete a 92-kDa metalloprotease which is not detectable in the parental cell line IMR-90. We now present the complete structure of this enzyme along with the evidence that it is identical to the 92-kDa metalloprotease secreted by normal human alveolar macrophages, phorbol ester-differentiated monocytic leukemia U937 cells, fibrosarcoma HT1080 cells, and cultured human keratinocytes. A similar, perhaps identical, enzyme can be released by polymorphonuclear cells. The preproenzyme is synthesized as a polypeptide of predicted Mr 78,426 containing a 19 amino-acid-long signal peptide and secreted as a single 92,000 glycosylated proenzyme. The purified proenzyme complexes noncovalently with the tissue inhibitor of metalloproteases (TIMP) and can be activated by organomercurials. Activation with phenylmercuric chloride results in removal of 73 amino acids from the NH2 terminus of the proenzyme, yielding an active form capable of digesting native types IV and V collagen. The in vitro substrate specificity of the enzyme using these substrates was indistinguishable from that of the 72-kDa type IV collagenase. The 92-kDa type IV collagenase consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-1, and a unique 54-amino-acid-long proline-rich domain homologous to the alpha 2 chain of type V collagen.     

Thermodynamics of the hydrolysis of sucrose

A thermodynamic investigation of the hydrolysis of sucrose to fructose and glucose has been performed using microcalorimetry and high-pressure liquid chromatography. The calorimetric measurements were carried out over the temperature range 298-316 K and in sodium acetate buffer (0.1 M, pH 5.65). Enthalpy and heat capacity changes were obtained for the hydrolysis of aqueous sucrose (process A): sucrose(aq) + H2O(liq) = glucose(aq) + fructose (aq). The determination of the equilibrium constant required the use of a thermochemical cycle calculation involving the following processes: (B) glucose 1-phosphate2-(aq) = glucose 6-phosphate2-(aq); (C) sucrose(aq) + HPO4(2-)(aq) = glucose 1-phosphate2-(aq) + fructose(aq); and (D) glucose 6-phosphate2-(aq) + H2O(liq) = glucose(aq) + HPO4(2-)(aq). The equilibrium constants determined at 298.15 K for processes B and C are 17.1 +/- 1.0 and 32.4 +/- 3.0, respectively. Equilibrium data for process D was obtained from the literature, and in conjunction with the data for processes B and C, used to calculate a value of the equilibrium constant for the hydrolysis of aqueous sucrose. Thus, for process A, delta G0 = -26.53 +/- 0.30 kJ mol-1, K0 = (4.44 +/- 0.54) x 10(4), delta H0 = -14.93 +/- 0.16 kJ mol-1, delta So = 38.9 +/- 1.2 J mol-1 K-1, and delta CoP = 57 +/- 14 J mol-1 K-1 at 298.15 K. Additional thermochemical cycles that bear upon the accuracy of these results are examined.