Localization of the McLeod locus (XK) within Xp21 by deletion analysis.

The McLeod phenotype is an X-linked, recessive disorder in which the red blood cells demonstrate acanthocytic morphology and weakened antigenicity in the Kell blood group system. The phenotype is associated with a reduction of in vivo red cell survival, but the permanent hemolytic state is usually compensated by erythropoietic hyperplasia. The McLeod phenotype is accompanied by either a subclinical myopathy and elevated creatine kinase (CK) or X-linked chronic granulomatous disease (CGD). Seven males with the McLeod red-blood-cell phenotype and associated myopathy but not CGD, one male with the McLeod phenotype associated with CGD, and two males known to possess large deletions of the Duchenne muscular dystrophy (DMD) locus were studied. DNA isolated from each patient was screened for the presence or absence of various cloned sequences located in the Xp21 region of the human X chromosome. Two of the seven males who have only the McLeod phenotype and are cousins exhibit deletions for four Xp21 cloned fragments but are not deleted for any portion of either the CGD or the DMD loci. Comparison of the cloned segments absent from these two McLeod cousins with those absent from the two DMD boys and the CGD/McLeod patient leads to the submapping of various cloned DNA segments within the Xp21 region. The results place the locus for the McLeod phenotype within a 500-kb interval distal from the CGD locus toward the DMD locus.     

Biosynthesis of lipoprotein: location of nascent apoAI and apoB in the rough endoplasmic reticulum of chicken hepatocytes

Our previous studies showed that in hepatic RER of young chickens, nascent apoAI is not associated with lipoprotein particles and only becomes part of these lipoprotein structures in the Golgi. In this study, we have used three different methodologies to determine the locations of apoAI and apoB in the RER and compared them to that of albumin. Immunoelectron microscopic examination of the RER cell fractions showed that both apoAI and apoB were associated only with the RER membrane whereas albumin was located both within the lumen and on the limiting membrane of the vesicles. To examine the possibility of membrane integration of nascent apoAI and apoB in the RER, we administered L-[3H]leucine to young chickens for 10 min, isolated RER, treated this cell fraction with buffers of varying pH, and measured the release of radioactive albumin, apoAI, and apoB. The majority of nascent apoAI (64%), nascent apoB (100%), and nascent albumin (97%) was released from RER vesicles at pH 11.2, suggesting that, like albumin, apolipoproteins are not integrated within the membrane. To determine if nascent apoproteins are exposed to the cytoplasmic surface, we administered L-[3H]leucine to young chickens and at various times isolated RER and Golgi cell fractions. Radioactive RER and Golgi cell fractions were treated with exogenous protease and the percent of nascent apoAI and apoB accessible to proteolysis was determined and compared to that of albumin. At 5, 10, and 20 min of labeling, 35-56% of nascent apoAI and 60-75% of apoB in RER were degraded, while albumin was refractive to this treatment. At all times both apolipoproteins and albumin present in Golgi cell fractions were protected from proteolysis. These biochemical and morphological findings indicate that apoAI and apoB are associated with the rough microsomal membrane and are partially exposed to the cytoplasmic surface at early stages of secretion. They may later enter the luminal side of the ER and, on entering the Golgi, form lipoprotein particles.     

ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules

Correct orientation of cell division is considered an important factor for the achievement of normal brain size, as mutations in genes that affect this process are among the leading causes of microcephaly. Abnormal spindle orientation is associated with reduction of the neuronal progenitor symmetric divisions, premature cell cycle exit, and reduced neurogenesis. This mechanism has been involved in microcephaly resulting from mutation of ASPM, the most frequently affected gene in autosomal recessive human primary microcephaly (MCPH), but it is presently unknown how ASPM regulates spindle orientation. In this report, we show that ASPM may control spindle positioning by interacting with citron kinase (CITK), a protein whose loss is also responsible for severe microcephaly in mammals. We show that the absence of CITK leads to abnormal spindle orientation in mammals and insects. In mouse cortical development, this phenotype correlates with increased production of basal progenitors. ASPM is required to recruit CITK at the spindle, and CITK overexpression rescues ASPM phenotype. ASPM and CITK affect the organization of astral microtubules (MT), and low doses of MT‐stabilizing drug revert the spindle orientation phenotype produced by their knockdown. Finally, CITK regulates both astral‐MT nucleation and stability. Our results provide a functional link between two established microcephaly proteins.     

Colchicine inhibition of plasma protein release from rat hepatocytes

Colchicine, both in vitro and in vivo, inhibits secretion of albumin and other plasma proteins. In vitro, secretion by rat liver slices is inhibited at 10-minus 6 M with maximal effect at 10-minus 5 M. Inhibition of secretion is accompanied by a concomitant retention of nonsecreted proteins within the slices. Colchicine does not inhibit protein synthesis at these concentrations. Vinblastine also inhibits plasma protein secretion but lumicolchicine, griseofulvin, and cytochalasin B do not. Colchicine also acts in vivo at 10-25 mumol/100 g body weight. Inhibition of secretion is not due to changes in the intracellular nucleotide phosphate levels. Colchicine, administered intravenously, acts within 2 min and its inhibitory effect lasts for at least 3 h. Colchicine has no effect on transport of secretory proteins in the rough or smooth endoplasmic reticulum but it causes these proteins to accumulate in Golgi-derived secretory vesicles.     

THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN : Localization of Newly Formed Proteins within the Endoplasmic Reticulum

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the ¹⁴C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.     

Cryopreserved human fetal pancreas: a source of insulin-producing tissue?

Human fetal pancreata (HFP) were obtained from dilatation and extraction aborted fetuses of 11-18 weeks' gestation. The pancreas was excised under sterile conditions and kept in culture medium at 4 degrees C, prior to stepwise digestion into 50- to 150-micron fragments. The fragmented pieces were allowed to sediment by gravity, then transferred to tissue culture for 24-48 h, and cryopreserved. The freeze-thaw protocol used stepwise equilibration with dimethyl sulfoxide, nucleation of the sample at -10 degrees C, and a slow cooling rate of 0.25 degrees C/min to -40 degrees C, followed by submersion in liquid nitrogen (-196 degrees C). Rapid thawing at 300 degrees C/min from -196 degrees C was employed. Both fresh and frozen-thawed HFP fragments appeared viable as judged by light and electron microscopy, and secreted insulin in a perifusion system upon stimulation with glucose (28 mM) and theophylline (10 mM) or glucose (2.8 mM) and theophylline (10 mM). Six patients with Type I insulin-dependent diabetes mellitus, already requiring immunosuppression for a kidney transplant, had intraportal injection of 20 cryopreserved-thawed and pooled HFP fragments. Up to the 1-year post-transplant follow-up, there has been no evidence of in vivo insulin or C-peptide production. The usefulness of cryopreserved human fetal pancreata as a source of insulin-producing tissue for diabetic patients, therefore, remains to be demonstrated.     

Cocaine Induces Alterations in Mitochondrial Membrane Potential and Dual Cell Cycle Arrest in Rat C6 Astroglioma Cells

Investigations with astroglial cells carry more prominence in drug abuse studies. However, due to earlier perception that astroglial cells were only passive bystanders in neural signal transmission, not many investigations were conducted on the toxicity of various abused drugs, like cocaine. The present study was aimed to discern the effect of cocaine on rat astroglioma cells and analyzed qualitatively for morphological features as well as vacuolation by phase contrast microscope, quantitatively for cytotoxicity, mitochondrial membrane potential by rhodamine- 123 fluorometric assay, and cell cycle analysis by flow cytometry. Based on population cell doubling time studies, glial cells were grown in 10% FBS in RPMI 1640 medium and treated with cocaine for 24 or 48 h. Microscopic assessments clearly demonstrated massive vacuolation and significant disruption at general architecture of glial cell morphology with cocaine. Chronic cocaine treatment (24 or 48 h) caused significant loss of cell viability. The sublethal dose of cocaine was found to be 4.307 and 3.794 mM at 24 and 48 h, respectively. Cocaine reduced the mitochondrial membrane potential in a dose dependent manner with ED₅₀ of 4 mM after 24 h. Cell cycle analysis suggested dual inhibition at G0/G1 and G2/M phases after 24 and 48 h, respectively. In summary, our findings suggest that cocaine toxicity was due to loss of mitochondrial membrane potential, vacuolation, and dual inhibition of cell cycle phases. These results may shed light in understanding the onset of some early key events in cocaine-induced toxicity in glial cells.