Protein crystal growth and the International Space Station.

Protein structural information plays a key role in understanding biological structure-function relationships and in the development of new pharmaceuticals for both chronic and infectious diseases. The Center for Macromolecular Crystallography (CMC) has devoted considerable effort studying the fundamental processes involved in macromolecular crystal growth both in a 1-g and microgravity environment. Results from experiments performed on more than 35 U.S. space shuttle flights have clearly indicated that microgravity can provide a beneficial environment for macromolecular crystal growth. This research has led to the development of a new generation of pharmaceuticals that are currently in preclinical or clinical trials for diseases such as cutaneous T-cell lymphoma, psoriasis, rheumatoid arthritis, AIDS, influenza, stroke and other cardiovascular complications. The International Space Station (ISS) provides an opportunity to have complete crystallographic capability on orbit, which was previously not possible with the space shuttle orbiter. As envisioned, the x-ray Crystallography Facility (XCF) will be a complete facility for growing protein crystals; selecting, harvesting, and mounting sample crystals for x-ray diffraction; cryo-freezing mounted crystals if necessary; performing x-ray diffraction studies; and downlinking the data for use by crystallographers on the ground. Other advantages of such a facility include crystal characterization so that iterations in the crystal growth conditions can be made, thereby optimizing the final crystals produced in a three month interval on the ISS.

Differential effects of the intraventricular administration of 6-hydroxydopamine on the induction of type II beta-tubulin and tyrosine hydroxylase mRNA in the locus coeruleus of the aging Fischer 344 rat.

Noradrenergic neurons of the locus coeruleus have been shown to respond to injury by increasing the synthesis of neurotransmitter (via the activation and induction of tyrosine hydroxylase, the rate-limiting catalyst in the production of catecholamines) and initiating compensatory axonal sprouting. However, this laboratory has recently described a significant deficit in the activation of tyrosine hydroxylase in the aged Fischer 344 rat, in contrast to the young and mature rat, following partial damage to cortical and hippocampal noradrenergic terminals induced by the neurotoxin 6-hydroxydopamine. To extend these observations, we measured changes in the relative levels of neuron-specific type II beta-tubulin and tyrosine hydroxylase mRNA in locus coeruleus neurons of 2, 12, and 24-month-old Fischer 344 rats following intraventricular infusions of 6-hydroxydopamine by using in situ hybridization histochemistry. These measures were used as markers of the responsiveness of these neurons to injury. 6-Hydroxydopamine treatment induced a persistent increase (at least 10 days) in the expression of type II beta-tubulin mRNA only in 2-month-old animals; this marker decreased in the 12 and 24-month-old animals. Relative levels of tyrosine hydroxylase mRNA increased in 2 and 12-month-old lesioned animals both 3 and 10 days post-treatment. In contrast, the induction of tyrosine hydroxylase mRNA in 24-month-old animals, seen three days post-treatment, was attenuated by 10 days. These data indicate that the capacity of locus coeruleus neurons to compensate for injury by either initiating a potential sprouting response or increasing their capacity to synthesize neurotransmitter is reduced in older animals.

Protein crystal growth in microgravity-temperature induced large scale crystallization of insulin.

One of the major stumbling blocks that prevents rapid structure determination using x-ray crystallography is macromolecular crystal growth. There are many examples where crystallization takes longer than structure determination. In some cases, it is impossible to grow useful crystals on earth. Recent experiments conducted in conjunction with NASA on various Space Shuttle missions have demonstrated that protein crystals often grow larger and display better internal molecular order than their earth-grown counterparts. This paper reports results from three Shuttle flights using the Protein Crystallization Facility (PCF). The PCF hardware produced large, high-quality insulin crystals by using a temperature change as the sole means to affect protein solubility and thus, crystallization. The facility consists of cylinders/containers with volumes of 500, 200, 100, and 50 ml. Data from the three Shuttle flights demonstrated that larger, higher resolution crystals (as evidenced by x-ray diffraction data) were obtained from the microgravity experiments when compared to earth-grown crystals.

Elastin repeat peptides as chemoattractants for bovine aortic endothelial cells.

Cultured bovine aortic endothelial cells migrate toward a concentration gradient of repeating elastin peptides, specifically the repeating nonamers Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro and Gly-Leu-Gly-Val-Gly-Ala-Gly-Val-Pro and the repeating hexamer Val-Gly-Val-Ala-Pro-Gly. Dose-response experiments demonstrate that the peak of activity occurs at 8 x 10(-8) M for the nonapeptides and 1 x 10(-8) M for the hexapeptide. Checkerboard assays establish that the movement is chemotaxis and not chemokinesis. Because of the concentration difference in the responsiveness between the nonapeptide and the hexapeptide, the cells can differentiate between the two types of repeats. The positive control for the chemotaxis studies was fibronectin.

A synthetic polypentapeptide of elastin for initiating calcification.

A polypentapeptide (PPP) of tropoelastin having a repeating amino acid sequence of (Val-Pro-Gly-Val-Gly)n was evaluated for its potential to initiate in vivo calcification and to enhance bone formation in nonhealing calvarial wounds (8.0 mm) in 396 adult Walter Reed rats. There were four configurations of the PPP (molecular weight range of 50-100K dalton) consisting of 1-dry PPP; 2-coacervate PPP; 3-gamma irradiated, cross-linked PPP; 4-calcified, gamma irradiated, cross-linked PPP. These four iterations plus a control group of animals constituted the five treatment classes that were evaluated at days 1, 3, 7, 21, 42, and 147. Seventy two rats were used for each treatment and 36 rats for the control. Following euthanatization, specimens were placed into 70% ethanol, embedded in polymethyl methacrylate, sectioned at 3.5 micrometers, and alternating sections were stained with Masson-Goldner trichrome and von Kossa stains. Histomorphometric analysis was accomplished using a Zeiss Universal microscope (250 X) and Videoplan Image Analysis System to evaluate five random histologic fields from margin to margin of the craniotomy. Trabecular bony volume and area of calcification islands were quantitated. A Student's t test for unpaired data to determine treatment differences (within the same temporal groups) revealed that there was no significant difference between treatments and control for trabecular bony volume; however, there was a significant difference between experimentals and control for calcification islands (P less than 0.05) such that calcifications islands for the experimentals were greater than the control. There was not a significant difference between experimental treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemotaxis of fibroblasts toward nonapeptide of elastin.

Bovine ligamentum fibroblasts, which produce elastin, migrate towards a positive chemical gradient of human platelet-derived growth factor and of the tropoelastin repeat hexapeptide Val-Gly-Val-Ala-Pro-Gly, as previously shown. They are also responsive to two permutations of a nonapeptide that repeats in tropoelastin, i.e., Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly and Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro. Concentration curves and checkerboard assays prove that the nonapeptides are chemoattractants. The component pentapeptide, Gly-Phe-Gly-Val-Gly, is chemotactic, while the component tetrapeptide Ala-Gly-Val-Pro is not. The hexapeptide competitively suppresses the nonapeptide chemotaxis suggesting the involvement of a common cell receptor. The results support the concept that elastin has multiple cell recognition sites as measured by the chemotactic response and that among the hydrophobic repeating sequences of elastin chemotacticity is selectively and multiply localized.

Cell attachment and chemotaxis can utilize the same peptide sequence of fibronectin.

Fibronectin and fragments of the parent molecule are involved in cell-substrate attachment, cell migration and chemotaxis. The cell attachment sequence, Gly-Arg-Gly-Asp-Ser-Pro, was synthesized and tested for its ability to effect ligamentum nuchae fibroblast chemotaxis. This hexapeptide and fibronectin itself both caused directed cell migration, with maximal activity in the 10(-10) to 10(-9) M range. These data demonstrate that the fibronectin cell binding site and chemotaxis site share a common sequence.

Polytetrapeptide of elastin. Temperature-correlated elastomeric force and structure development.

High molecular weight polytetrapeptide of elastin, (L.Val1-L.Pro2-Gly3-Gly4)n, was synthesized using activation of the (GGVP) permutation for polymerization. The temperature-dependence of aggregation was characterized as a function of concentration and the circular dichroism spectra were obtained in the 20 degrees to 70 degrees C temperature range. The latter showed an inverse temperature transition centered near 50 degrees C in which polypeptide order increased on raising the temperature. A concentration of 0.6 g of polytetrapeptide in 1 g of water was gamma irradiation cross-linked (20 Mrad) to form an elastomeric matrix. A study of the temperature-dependence of elastomeric force demonstrated a transition toward increased force on raising the temperature with a midpoint of the transition near 50 degrees C. Thus, there is a correlation between increase in intramolecular order and elastomeric force development. These results are compared to previous results on the polypentapeptide of elastin, (VPGVG)n and on an analog, (IPGVG)n, to demonstrate that the temperature of the transition is proportional to the hydrophobicity of the repeating unit. The point is noted that the elastomeric force development correlates better with intramolecular ordering than with intermolecular processes.

Temperature dependence of dielectric relaxations in alpha-elastin coacervate: evidence for a peptide librational mode.

The dielectric permittivity of alpha-elastin coacervate is reported over the frequency range of 1 MHz to 1000 MHz and the temperature dependence from 6.8 degrees C to 70 degrees C is also reported. A temperature-dependent simple Debye-type relaxation is observed with a correlation time of 8 nsec (40 degrees C) which is similar to that of the polypentapeptide of elastin (i.e. 7 nsec at 40 degrees C) where the band has been assigned to a peptide librational mode. By analogy this allows for the first assignment of a peptide librational mode in a naturally occurring polypeptide or protein. The strong spectrally localized band indicates a regularity of structure. The low temperature dependence of the correlation time, giving a 1.7 kcal/mole enthalpy of activation, is consistent with torsional motions associated with a peptide librational mode.

Synthesis of polypeptide models of elastin. Synthesis and properties of a cross-linked polytetrapeptide.

Synthesis of two copolymers, H-(phi-Pro-Gly-Gly)n-Val-OMe and H-(phi'-Pro-Gly-Gly)n-Val-OMe, where phi is Val or Lys and phi' is Val or Glu is described. Cross-linking between the two copolymers is achieved by a coupling reaction between the epsilon-amino groups of the lysine containing copolymer and delta-carboxyls of the glutamic acid of the other copolymer. The cross-linking reaction was performed during a temperature elicited phase separation with flow orientation of the copolymers. Coacervation of the intermediate polymers is presented, as is the scanning electron micrograph of the insoluble cross-linked product and its calcifiability as determined with electron probe microanalysis. The purity of the key intermediates and polymers is demonstrated by the usual analytical methods. Carbon-13 magnetic resonance spectra of the intermediate monomer units are included to validate the purity of their synthesis.

Irradiation crosslinking of the polytetrapeptide of elastin and compounding to dacron to produce a potential prosthetic material with elasticity and strength.

The poly(tetra peptide), H-(L . Val1-L . Pro2-Gly3-Gly4)n-L . Val-OMe, which is a recurring sequence in tropoelastin the precursor protein of the elastic fiber, has been irradiation crosslinked to produce an elastomeric material with limited strength. When a material such as a Dacron fabric is impregnated by the coacervate phase of the poly(tetrapeptide) prior to irradiation crosslinking at 50 Mrad, the crosslinked product exhibits stress-strain curves with good elastomeric properties and high strength. In addition to the stress-strain curves, the material is characterized by scanning electron microscopy.

Compounding of elastin polypentapeptide to collagen analogue: a potential elastomeric prosthetic material.

The polypentapeptide, H-(L . Val1-L . Pro2-Gly3-L . Val4-Gly5)n-L . Val-OMe, which is the most common recurring sequence within the elastic fiber, is demonstrated to be elastomeric when irradiation cross-linked but to have limited strength. On irradiation compounding with a collagen analogue, such as Dacron, stress-strain studies show the product to have an elastic modulus greater than that of fibrous aortic elastin and similar to that of aortic wall. In addition, the compounded product has the requisite strength. Of the 40, 50 and 60 MRAD cross-linked polypentapeptide-Dacron products, those derived from the larger doses of 50 and 60 MRAD exhibited somewhat better elastomeric properties. The unstretched and stretched products were characterized by scanning electron microscopy which demonstrated the importance of a fabric weave with a uniform extension. In general irradiation cross-linking has the advantage of being able to produce larger quantities of elastomeric material and compounding to a collagen analogue provides the required strength.