A major single-stranded DNA binding protein from ovaries of the frog, Xenopus laevis, is lactate dehydrogenase

The most abundant single-stranded DNA binding protein (SSB) found in ovaries of the frog, Xenopus laevis, was purified to electrophoretic homogeneity. Under physiological conditions, the purified SSB lowered the Tm of poly[d(A-T)] and stimulated DNA synthesis by the homologous DNA polymerase DNA primase alpha complex on single-stranded DNA templates. These properties are characteristic of a bona fide single-stranded DNA binding protein. The Stokes radius of native SSB was calculated to be 45 A, corresponding to a molecular mass of about 140 kDa. On SDS polyacrylamide gels, the SSB migrated as a single band with a molecular mass of 36 kDa. We assumed, therefore, that the SSB was a tetramer of 36 kDa subunits. We subsequently discovered that the SSB was LDH, D-lactate dehydrogenase, EC 1.1.1.28. Purified SSB has high LDH specific activity. Following electrophoresis on SDS polyacrylamide gels, the 36 kDa subunits were renatured and exhibited LDH activity. The amino-acid composition of X. laevis SSB/LDH was similar to that of LDH from other species and to other reported single-stranded DNA binding proteins. Mammalian SSB/LDH also preferentially bound single-stranded DNA. Mammalian SSB/LDH bound to RNA as demonstrated by affinity chromatography on poly(A)-agarose and by its effect on translation of mRNA in vitro.     

Cleavage-site motifs in mitochondrial targeting peptides

Although mitochondrial targeting peptides lack a common consensus sequence, a certain bias in the positional distribution of amino acids has recently been found. These patterns seem to be associated with cleavage of the precursor proteins by matrix processing proteases. We have extended the previous studies and found new sequence motifs that are conserved within subgroups of mitochondrial targeting peptides. These motifs have certain common themes, indicating that they are associated with cleavage by one single protease. Two of the conserved patterns have a high predictive value, but even for sequences that do not possess these patterns, a fairly accurate prediction of the cleavage site is shown to be possible. We also suggest that a well-conserved RXY decreases (S/A) pattern may be used to engineer efficiently recognized cleavage sites into uncleaved or artificial mitochondrial targeting peptides.     

Neuron-specific and state-specific differences in calcium homeostasis regulate the generation and degeneration of neuronal architecture

Many stimuli (e.g., neurotransmitters and electrical activity) regulate neuromorphogenesis by changing intracellular calcium. The ionophore A23187 was employed as a receptor-independent method to investigate neuronal calcium homeostasis. Distinctive neuron-specific (B5 versus B19) and state-specific (growing versus non-growing) differences in calcium homeostasis were observed in cultured identified Helisoma neurons. Fura-2 studies revealed that A23187 induced a transient rise in intracellular calcium in growing neurons B5 but a sustained rise in growing neurons B19. In stable-state (non-growing) cells A23187 evoked only a transient calcium rise. Both neuron-specific and state-specific differences in calcium homeostasis were dependent on extracellular sodium. Morphological studies also indicated that such differences in calcium-regulatory capacity can have profound consequences on the generation and degeneration of neuronal architecture.     

Organization of hindbrain segments in the zebrafish embryo

To learn how neural segments are structured in a simple vertebrate, we have characterized the embryonic zebrafish hindbrain with a library of monoclonal antibodies. Two regions repeat in an alternating pattern along a series of seven segments. One, the neuromere centers, contains the first basal plate neurons to develop and the first neuropil. The other region, surrounding the segment boundaries, contains the first neurons to develop in the alar plate. The projection patterns of these neurons differ: those in the segment centers have descending axons, while those in the border regions form ventral commissures. A row of glial fiber bundles forms a curtain-like structure between each center and border region. Specific features of the individual hindbrain segments in the series arise within this general framework. We suggest that a cryptic simplicity underlies the eventual complex structure that develops from this region of the CNS.     

Immunocytochemical detection of prolactin or prolactin-like immunoreactivity in epididymis of mature male mouse

Summary Prolactin (PRL) binds to the testis of mice and rats where it increases the number of luteinizing hormone receptors, increases the binding of human chorionic gonadotropin (hCG) to LH receptors, and enhances testosterone synthesis and secretion. PRL also binds to the prostate and seminal vesicles of rats and humans where it increases organ weight and stimulates growth and uptake of testosterone. PRL binds to the epididymis of rats but the effect of PRL on this organ is unknown. In the present study, a standard immunoperoxidase (PAP) technique was used to detect the binding of endogenous and exogenous PRL or PRL-like peptides to the epididymis of the mature mouse. Throughout the epididymal duct, a positive reaction for peroxidase, suggesting PRL or PRL-like binding, occurred in the Golgi area of principal cells. In segment 1, positive reactions were also visualized in the perinuclear area and in the region located between the Golgi area and the apical surface of the principal cells (supra-Golgi area). In the corpus and cauda epididymidis, scattered entire principal cells were also positive. Throughout the epididymal duct, the reactions indicating the binding of exogenous PRL were slightly stronger than those testing for binding of endogenous peptides. The significance of such binding to the epididymis is uncertain but PRL may perform the same functions in epididymal principal cells as it does in the testis, prostate, and seminal vesicles.     

Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation.

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.