Nuclear envelope‐associated dynein drives prophase centrosome separation and enables Eg5‐independent bipolar spindle formation

The microtubule motor protein kinesin‐5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti‐cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5‐independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)‐associated dynein that drives centrosome separation in prophase. This NE‐dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5‐dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin‐5 inhibitors.     

Distribution and altitudinal structuring of phlebotomine sand flies (Diptera: Psychodidae) in southern Anatolia, Turkey: their relation to human cutaneous leishmaniasis.

The two Old World genera, Phlebotomus and Sergentomyia, were both recorded in southern Anatolia in Turkey. Phlebotomus species predominated and comprised about 93% of the entire collection (3,172 specimens). Out of the sixteen species identified, two belonged to the genus Sergentomyia: S. dentata and S. theodori. The remaining fourteen species in the genus Phlebotomus were grouped under four subgenera including some species that are elsewhere known to act as vectors of human cutaneous leishmaniasis. Most of the Phlebotomus were P. tobbi (32.5%), but P. papatasi, P. transcaucasicus, P. halepensis, P. galilaeus, P. sergenti, P. syriacus, P. neglectus, P. simici, P. alexandri, P. similis, P jacusieli, P. perfiliewi, and P. brevis were also identified. There were two associations of sand fly fauna with altitudinal gradient; the first one at relatively higher altitudes and the second one at lower altitudes. The transition between these two assemblages was within the range of 800-1,000 m. It is likely that Adana and Hatay provinces are transitional areas between western and eastern Anatolia. Mountains do not appear to be important geographical barriers for sand fly distribution. We also found that the proven vector P. sergenti is a widely distributed species throughout southern Anatolia and this species, together with its closely related species P. similis, shows sympatry in Konya Province.     

Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity

The N-terminal part sequences of pituitary growth hormone, N-acetyl-hGH 7–13 and hGH 6–13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

ELECTRON MICROSCOPIC OBSERVATIONS OF RAT AND MOUSE CEREBELLUM IN TISSUE CULTURE

Closely ordered stages of myelin formation in cultures of newborn rat and mouse cerebellum, selected by direct light microscopy, were studied with the electron microscope. Electron micrographs of these cultures reveal the presence of neurons, axons, neuroglia, microglia, and ependymal cells. The appearance of the neuron is identical to that previously described in vivo. The neuroglial cell has long, branching processes, and its cytoplasm is characterized by packets of long, narrow fibrils. During myelin formation, a glial cell process surrounds the axon. This process may form an internal mesaxon and may spiral for several turns around the axon. Other glial cell processes may interdigitate with or overlay the innermost process to contribute to the multilamellated structure. The glial processes flatten and the cytoplasmic surfaces of the cell membrane come into contact to form the lamellae of the myelin sheath. These adhesions may be temporarily incomplete as evidenced by sequestered islands of glial cytoplasm among the myelin lamellae. Ultimately, a compact, apparently spiral, myelin sheath is formed. These findings are discussed in relation to in vivo central myelin formation.     

Heat shock stimulates the release of arachidonic acid and the synthesis of prostaglandins and leukotriene B4 in mammalian cells

Heat shock has a profound influence on the metabolism and behavior of eukaryotic cells. We have examined the effects of heat shock on the release from cells of arachidonic acid and its bioactive eicosanoid metabolites, the prostaglandins and leukotrienes. Heat shock (42-45 degrees) increased the rate of arachidonic acid release from human, rat, murine, and hamster cells. Arachidonate accumulation appeared to be due, at least partially, to stimulation of a phospholipase A2 activity by heat shock and was accompanied by the accumulation of lysophosphatidyl-inositol and lysophosphatidylcholine in membranes. Induction of arachidonate release by heat did not appear to be mediated by an increase in cell Ca++. Stimulation of arachidonate release by heat shock in hamster fibroblasts was quantitatively similar to the receptor-mediated effects of alpha thrombin and bradykinin. The effects of heat shock and alpha thrombin on arachidonate release were inhibited by glucocorticoids. Increased arachidonate release in heat-shocked cells was accompanied by the accelerated accumulation of cyclooxygenase products prostaglandin E2 and prostaglandin F2 alpha and by 5-lipoxygenase metabolite leukotriene B4. Elevated concentrations of arachidonic acid and metabolites may be involved in the cytotoxic effects of hyperthermia, in homeostatic responses to heat shock, and in vascular and inflammatory reactions to stress.     

Trace amines and Tourette's syndrome

There has been considerable interest in recent years in possible neurochemical abnormalities in Tourette's Syndrome (TS). In studies combining neuropsychological and neurochemical measurements, we have investigated the possible roles of trace amines in this disorder. Urinary levels of free -phenylethylamine (PEA) and plasma levels of its precursor amino acid phenylalanine were decreased in TS patients when compared to values in normal children. These urinary PEA levels in TS patients were inversely related to several scores from the Tourette's Syndrome Global Scale (TSGS). Further investigation of the group of subjects with low urinary PEA indicated that they also had low levels of MHPG, normetanephrine, 5-HT andm- andp-tyramine. Patients with low PEA were also compared on an extensive battery of neuropsychological measures and observed to perform significantly worse than TS patients with normal urinary PEA levels. Biochemical measurements also suggest a possible abnormality in tryptamine turnover in TS since urinary levels of indole-3-acetic acid (IAA; the acid metabolite of tryptamine) are significantly lower in TS patients than in normal controls.