Pulmonary hemodynamic reaction to foreign blood in goats and rabbits.

We have found that the goat is extraordinarily sensitive to very small quantities of rabbit or rat blood. As little as 0.004 ml/kg induces transient pulmonary hypertension [maximal rise in pulmonary arterial pressure 32 +/- 10 (SD) cmH2O] in goats. We hypothesized that this reaction may be related to the presence of the resident population of intravascular macrophages that reside in the pulmonary capillaries of goats. If that is so, then rabbits or rats, which have few or no intravascular macrophages, should not be reactive to foreign blood. We compared pulmonary hemodynamics and changes in blood thromboxane B2 concentrations among goats, rabbits, and rats in response to graded doses of foreign blood. The pulmonary reaction to foreign blood was much greater in goats than in rabbits or rats, even though we injected up to 10- or 60-fold larger amounts into the latter species. In goats the pulmonary vascular pressure response to rabbit blood was dose dependent in goats and correlated well with changes in systemic arterial thromboxane B2 concentrations [change in pulmonary arterial pressure = 0.07 (thromboxane B2) + 8.3, r = 0.79]. We also tested the prostaglandin H2 endoperoxide analogue (U-46619) and found that the goats are somewhat more reactive than rabbits. We conclude that the pulmonary hemodynamic reaction to foreign blood is consistent with the concept that the foreign erythrocytes are reacting with the pulmonary intravascular macrophages in goats. The lower reactivity of the rabbit pulmonary circulation to thromboxane may also have a role.     

Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site. The use of modular DNA

To obtain crystals of the Escherichia coli catabolite gene activator protein (CAP) complexed with its DNA-binding site, we have searched for crystallization conditions with 26 different DNA segments greater than or equal to 28 base-pairs in length that explore a variety of nucleotide sequences, lengths, and extended 5' or 3' termini. In addition to utilizing uninterrupted asymmetric lac site sequences, we devised a novel approach of synthesizing half-sites that allowed us to efficiently generate symmetric DNA segments with a wide variety of extended termini and lengths in the large size range (greater than or equal to 28 bp) required by this protein. We report three crystal forms that are suitable for X-ray analysis, one of which (crystal form III) gives measurable diffraction amplitudes to 3 A resolution. Additives such as calcium, n-octyl-beta-D-glucopyranoside and spermine produce modest improvements in the quality of diffraction from crystal form III. Adequate stabilization of crystal form III is unexpectedly complex, requiring a greater than tenfold reduction in the salt concentration followed by addition of 2-methyl-2,4-pentanediol and then an increase in the concentration of polyethylene glycol.     

On the ultrastructure and the supposed function of the mineralizing matrix coat of sea urchins (Echinodermata,Echinoida)

Summary Echinoderm ossicles are part of the mesenchyme. Their formation and growth, with respect to the underlying tissues, is studied using echinoid spines and teeth and applying different methods of fixation. The calcification process in echinoderms is strictly intracellular and needs (1) syncytial sclerocytes which completely enclose (2) a vacuolar cavity which in turn contains (3) an organic matrix coat. Strictly speaking, each ossicle is nothing but the calcified vacuolar space of a single syncytium of sclerocytes. In fully grown parts, however, the continuous sheath may split open and the matrix-coated mineral may come into contact with the extracellular space. According to biochemical analyses the matrix consists of insoluble components, but most (95%) of its constituents are soluble in EDTA or weak acids. If routine transmission electron microscope methods are used the soluble components are lost and the matrix at best looks electron light. If tannic acid is added to the fixative the soluble matrix components are preserved and reveal further ultrastructural details of the biomineralization process in echinoderms. The matrix coat looks extremely electron dense. Further soluble material is to be found within the vacuolar space or attached to the vacuolar surface of the cytoplasmic sheath. The results lead to the opinion that the matrix coat consists of a hydrophobic framework of insoluble components that contains soluble components which guide the Ca through pores in the hydrophobic layers into the interior of the matrix-coated space. It is only within this space that the mineral is deposited.     

Function of a heterologous muscarinic receptor in T cell antigen receptor signal transduction mutants

Previously we have described a system of somatic cell genetics (J.CaM1 and J.CaM2) for analyzing signal transduction via the T cell antigen receptor complex (CD3/Ti). Here we describe a third mutant, J.CaM3, which also expresses high levels of receptors that are functionally impaired. Like J.CaM1, J.CaM3 demonstrates partial signal transduction via CD3/Ti to only certain stimuli. J.CaM1, J.CaM2, and J.CaM3 define three non-Ti complementation groups involved in receptor function. To evaluate the mutations further we have introduced a heterologous receptor, the human muscarinic receptor 1 (HM1), into the parental Jurkat and mutant cell lines. This receptor demonstrates signal transduction competence in all these hosts, indicating that 1) T cells express the necessary apparatus for the coupling of HM1 to second messenger generation and 2) the mutations in the J.CaM family all affect molecules that are specific to CD3/Ti, and not HM1, function. Finally, the HM1 receptor exhibits partial sensitivity to cholera toxin in Jurkat cells, in contrast to the virtually complete sensitivity of CD3/Ti to cholera toxin.     

Isolated iron-molybdenum cofactor of nitrogenase exists in multiple forms in its oxidized and semi-reduced states

Electrochemical and EPR spectroscopic experiments demonstrate that the isolated iron-molybdenum cofactor from the molybdenum-iron protein of nitrogenase from Azotobacter vinelandii exists in multiple forms in both its oxidized and semi-reduced states. The particular forms found in either oxidation state appear to be a function of the acid/base status of the solvent, N-methylformamide. In "alkaline" N-methylformamide, a single, detectable form of iron-molybdenum cofactor is observed for both oxidized and semi-reduced states. The semi-reduced form, termed R(s-r), is the one previously recognized with an S = 3/2 EPR spectrum with apparent g values of 4.6, 3.4, 2.0. Its oxidized counterpart, termed B(ox), is characterized electrochemically by a differential pulse voltammetric reduction peak at -0.37 V versus the normal hydrogen electrode. In "acidic" solvent, two distinct, previously unrecognized redox pairs of iron-molybdenum cofactor forms exist. The two semi-reduced forms, N(s-r) and W(s-r), are characterized by EPR spectra with g = 4.5, 3.6, 2.0 and g = 4.9, 3.1, 1.9, respectively. Their oxidized counterparts, A(ox) and C(ox), have differential pulse voltammetric reduction peaks at -0.32 and -0.43 V versus the normal hydrogen electrode, respectively. Manipulations of either the isolation protocol or the sample conditions affects both the type and distribution of forms present. Each form likely corresponds to a biologically significant state of the cofactor cluster within the protein.     

Active and inactive forms of the transition-state analog protease inhibitor leupeptin: explanation of the observed slow binding of leupeptin to cathepsin B and papain

Leupeptin and similar peptide argininal (arginine aldehyde) transition-state analog protease inhibitors exist in three covalent forms in aqueous solution, the leupeptin hydrate (IH), a cyclic carbinolamine form (IC) generated by the addition of the guanidino epsilon N to the aldehydic carbon, and the free aldehyde form (IA). 1H NMR in D2O show their equilibrium concentrations to be 42, 56, and 2% for IH, IC (R and S enantiomers), and IA. The rates of conversion of (formula; see text) were determined by 1H NMR in D2O by trapping IA with semicarbazide. Application of a deuterium isotope effect of 2.8 led to rate constants in H2O for kC of 0.092 min-1 and kD of 0.73 min-1. The equilibrium concentration of IA and rates for kC and kD are then used to explain the lag phase in the inhibition of cathepsin B and papain by leupeptin. Two circumstances are observed. (i) At micromolar concentrations of leupeptin and papain the binding of leupeptin is biphasic with rate constants identical to kD and kC. (ii) At more dilute nanomolar concentrations of total leupeptin and proteases, the observed lag phase for approach to steady-state inhibition (with rate constant k') is now explained by the low values of the koff rate constants (0.072 min-1 for cathepsin B and 0.024 min-1 for papain) together with the extremely low concentrations of the active inhibitor form IA, with k' = kon[IA] + koff. While kon[IA] is slow, the second-order rate constant kon is found to be quite fast, 1.2 x 10(7) M-1 s-1 for cathepsin B and 1.8 x 10(7) M-1 s-1 for papain. Thus, the binding of leupeptin to cathepsin B and papain may show a lag phase, but this is not due to slow binding.     

Modulation of Ganglioside Biosynthesis in Primary Cultured Neurons

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.     

Activation of the c-H-ras proto-oncogene by retrovirus insertion and chromosomal rearrangement in a Moloney leukemia virus-induced T-cell leukemia.

A rearrangement of the c-H-ras locus was detected in a T-cell line (DA-2) established from a Moloney leukemia virus-induced tumor. This rearrangement was associated with the high-level expression of H-ras RNA and the H-ras gene product, p21. DNA from DA-2 cells transformed fibroblasts in DNA transfection experiments, and the transformed fibroblasts contained the rearranged H-ras locus. The rearrangement involved one allele and was present in tissue from the primary tumor from which the cell line was isolated. Cloning and sequencing of the rearranged allele and comparison with the normal allele demonstrated that the rearrangement was complex and probably resulted from the integration of a retrovirus in the H-ras locus between a 5' noncoding exon and the first coding exon and a subsequent homologous recombination between this provirus and another newly acquired provirus also located on chromosome 7. These events resulted in the translocation of the coding exons of the H-ras locus away from the 5' noncoding exon region to a new genomic site on chromosome 7. Sequencing of the coding regions of the gene failed to detect mutations in the 12th, 13th, 59th, or 61st codons. The possible reasons for the complexity of the rearrangement and the significance of the activation of the H-ras locus to T-cell transformation are discussed.     

Regulated production of recombinant echistatin by yeast

Summary The -mating factor pre-pro-leader sequence under the regulation of theGAL10 promoter was used to direct the secretion of echistatin by recombinant yeasts. Optimization of the culture medium and host strain increased the productivity of shake flask cultures twenty-fold to 8 mg/L. In fermentors, the production of echistatin was greater than 40 mg/L.