Biological and structural properties of MIP-1 alpha expressed in yeast.

The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).     

An apparatus to measure in vivo biomechanical behavior of dorsi- and plantarflexors of mouse ankle.

We developed an apparatus to quantify the biomechanical behavior of the dorsi- and plantarflexor muscles of the ankle of an anesthetized mouse. When the dorsi- or plantarflexor muscle group is activated by electrical stimulation of either the peroneal or tibial nerve, the apparatus measures the moment developed about the ankle during isometric, isovelocity shortening, or isovelocity lengthening contractions. Displacements may be performed over the full 105 degrees range of ankle motion with an angular resolution of 0.09 degrees. Bidirectional isovelocity ramps in ankle angle up to 1,100 degrees/s are possible and are equivalent to maximum velocities of 2.3 fiber lengths/s (Lf/s) for the fibers in tibialis anterior muscle and 11.9 Lf/s for those in gastrocnemius muscle. During single contractions of the dorsi- and plantarflexor muscle groups at 37 degrees C and with both knee and ankle joint at 90 degrees neutral position, the isometric tetanic force developed was 1.4 and 3.3 N, power output at 2.2 Lf/s was 3.1 and 5.9 mW, and power absorption at 0.5 Lf/s was 4.9 and 9.0 mW, respectively. These values are in reasonable agreement with data from the same muscle groups tested in situ. We conclude that the apparatus provides valid measurements of force and power of the dorsi- and plantarflexor muscle groups.     

Relation between D-glucose and L- and D-alanine in the initiation of germination of Bacillus subtilis spore

The rate of L-alanine-initiated germination of Bacillus subtilis spore was measured by both loss of heat resistance and loss of turbidity, and the effect of glucose on the germination response to a wide range of concentrations of the germinant was analyzed in the presence and absence of D-alanine, an inhibitor. Glucose stimulated L-alanine germination by means of a cooperative effect: glucose increased the affinity of L-alanine by about 3-fold and the rate of germination by about 1.3-fold. However, glucose had little effect on the binding affinity of D-alanine. The apparent binding constant of L-alanine to the spore, which was determined by the next measurable event in the trigger reaction, was 1.2 X 10(-5), that of D-alanine was 6 X 10(-6), and that of glucose was 5 X 10(-5). The relation between the binding site for glucose and those for L- and D-alanine on the spore is discussed. Effect of glucose analogs was also examined.     

The biological effects in animals related to the accident at the Chernobyl Atomic Electric Power Station. 1. The experimental model. The radiation loads on animals kept continuously under external and internal radiation exposure in the area of the Chernobyl Atomic Electric Power Station]

Irradiation conditions in which laboratory animals were kept in experimental laboratories of Chernobyl and Kiev after the accident at the Chernobyl A.P.S. are described. The data are presented on the spectral structural and activity of radionuclides in the diet as well as in the organs and tissues of the animals. The radiation loads have been estimated with regard to an external gamma component and the internal one contributed by the incorporated radionuclides. It has been shown that radiation doses received by the animals during their lifetime due to these contributions do not exceed units of cGy.     

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.