Small heat shock/alpha-crystallin proteins function as molecular chaperones, protecting other proteins from irreversible denaturation by an energy-independent process. The brine shrimp, Artemia franciscana, produces a small heat shock/alpha-crystallin protein termed p26, found in embryos undergoing encystment, diapause, and metabolic arrest. These embryos withstand long-term anoxia and other stresses normally expected to cause death, a property likely dependent on molecular chaperone activity. The association of p26 with tubulin in unfractionated cell-free extracts of Artemia embryos was established by affinity chromatography, suggesting that p26 chaperones tubulin during encystment. To test this possibility, both proteins were purified by modifying published protocols, thereby simplifying the procedures, enhancing p26 yield about 2-fold, and recovering less tubulin than before. The denaturation of purified tubulin as it "aged" and exposed hydrophobic sites during incubation at 35 degrees C was greatly reduced when p26 was present; however, tubulin polymerization into microtubules was reduced. On incubation at 35 degrees C, centrifugation in sucrose density gradients demonstrated the association of purified p26 with tubulin. This is the first study where the relationship between a small heat shock/alpha-crystallin protein and tubulin from the same physiologically stressed organism was examined. The results support the proposal that p26 binds tubulin and prevents its denaturation, thereby increasing the resistance of encysted Artemia embryos to stress. Additional factors are apparently required for release of tubulin from p26 and restoration of efficient assembly, events that would occur as embryos resume development and the need for microtubules is established.

The aim of this study was to approach the question of neuronal dependence on neurotrophins during embryonic development in mice in a way other than gene targeting. We employed amyogenic mouse embryos and fetuses that develop without any skeletal myoblasts or skeletal muscle and consequently lose motor and proprioceptive neurons. We hypothesized that if, in spite of the complete inability to maintain motor and proprioceptive neurons, the remaining spinal and dorsal root ganglia tissues of amyogenic fetuses still contain any of the neurotrophins, that particular neurotrophin alone is not sufficient for the maintenance of motor and proprioceptive neurons. Moreover, if the remaining spinal and dorsal root ganglia tissues still contain any of the neurotrophins, that particular neurotrophin alone may be sufficient for the maintenance of the remaining neurons (i.e., mostly non-muscle- and a few muscle-innervating neurons). To test the role of the spinal cord and dorsal root ganglia tissues in the maintenance of its neurons, we performed immunohistochemistry employing double-mutant and control tissues and antibodies against neurotrophins and their receptors. Our data suggested that: (a) during the peak of motor neuron cell death, the spinal cord and dorsal root ganglia distribution of neurotrophins was not altered; (b) the distribution of BDNF, NT-4/5, TrkB and TrkC, and not NT-3, was necessary for the maintenance of the spinal cord motor neurons; (c) the distribution of BDNF, NT-4/5 and TrkC, and not NT-3 and Trk B, was necessary for the maintenance of the DRG proprioceptive neurons; (d) NT-3 was responsible for the maintenance of the remaining neurons and glia in the spinal cord and dorsal root ganglia (possibly via TrkB).

Two species of DNA polymerase alpha free of primase activity were identified in extracts of Ehrlich mouse cells that had been infected with minute virus of mice. Primase-free forms of DNA polymerase alpha eluted with 150 and 180 mM NaCl during ion-exchange chromatography on DEAE-cellulose columns, exhibited sedimentation coefficients of 11 S and 8.2 S, respectively, and were inhibited by aphidicolin, N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate, and 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)adenine 5'-triphosphate. The ratio of primase-free DNA polymerase alpha to the DNA polymerase alpha-primase complex increased from 1.5 to greater than 100 during the course of infection, and free primase was produced during the MVM replicative cycle.

The effect of tamoxifen (ICI-46474) (alpha-[4-(beta-N-dimethylamino ethoxy)-phenyl]alpha'-ethyl trans stilbene on estrogen cytosolic receptor was observed in different parts of fallopian tube and uterus of rabbits during ovum transport. Tubal segments viz. ampulla (A), ampullary isthmic junction (AIJ), isthmus (I), uterine isthmic junction (UIJ) and uterus (U) were studied during the passage of ovum. Significant differences were observed in normal, drug treated and pregnant animals at 14, 24, 34, 48, 72, 144 and 168 h. In treated animals during 14 h post-coitum (p.c.) to 34 h p.c. the ampullary cytosol receptor concentration increased and suddenly decreased at 48 h p.c. whereas from 72 h p.c. to 144 h p.c. it increased gradually. Tamoxifen accelerated the rate of egg transport decreasing tubal cytosolic receptors at 48 h p.c. However, beyond 48 h p.c. receptors increased till 144 h p.c. Results suggest that tamoxifen modulated tubal cytosolic estrogen receptors during egg transport and prevents pregnancy.

The effect of clomiphene on nuclear estrogen receptors of the Fallopian tube during ovum transport in the rabbit has been studied. Nuclear binding capacity was observed in ampulla (A), ampullary-isthmic junction (AIJ), isthmus (I), uterine-isthmic junction (UIJ) and uterus (U). Receptor concentration decreased in all segments of the tube after administration of clomiphene in mated animals. The bindings are of high affinity and low capacity. Important alterations were observed during transport when compared to that of 14, 24, 34, 48, 72, 144 and 168 hr post-coitum (p.c). At 24 hr p.c binding increased only in I and decreased in A and AIJ. Retention of eggs at I at 24 hr p.c showed as increase in binding at I. Egg transport was accelerated and eggs reached prematurely in the uterus due to the influence of clomiphene. Binding in I remained constant from 48 hr p.c to 144 hr p.c but concurrently the binding level increased in U from 34 hr p.c. The elevation of nuclear estrogen receptor level was maximum at 24 hr p.c which coincided with increased plasma estrogen level. The result of such study showed that clomiphene depleted nuclear estrogen receptor complex in the fallopian tube before transfer to the uterus. Further, observation indicated that clomiphene acted directly on the rate of egg transport because of the variations in estrogen receptors during different time periods. Thus, clomiphene reduced the quantity of estrogen receptor i.e., insensitiveness to estrogen. The variations in estrogen binding to its receptor and plasma level at different post-coital periods are modulated by clomiphene resulting in the acceleration of egg transport and prevention of pregnancy.