The monoclonal antibodies Trop-4 and 4F2 detect the same membrane antigen that is expressed at an early stage of lymphocyte activation and is retained on secondary lymphocytes

The monoclonal antibodies Trop-4 and 4F2 recognize cell surface antigens present on peripheral blood monocytes, activated lymphocytes, and on continuous cell lines, but not on resting lymphocytes in blood. The membrane antigens detected by antibodies Trop-4 and 4F2 were compared by serial immunoprecipitations from membrane lysates of surface labeled T lymphoid cells and by parallel polyacrylamide gel electrophoretic analysis. It is shown that both to these antibodies recognize the heavy subunit of the heterodimeric membrane complex of an 85,000 m.w. glycoprotein disulphide linked to a light subunit of 41,000 m.w. The kinetics of the expression of the antigen was studied by indirect immunofluorescence on peripheral blood T lymphocytes during blast transformation induced by concanavalin A in vitro and during reversion of the lymphocytes back to small "secondary" lymphocytes. Upon activation of T lymphocytes with concanavalin A, the first blast cells staining with the antibodies appear within 6 hr after the initiation of the culture. After 18 hr, all blast cells displayed strong expression of the antigen. Inhibition of DNA synthesis by hydroxyurea treatment did not affect the early blast transformation and the expression of the antigen. When the mitogen-induced blast cells reverted back to small secondary lymphocytes during prolonged culturing for up to 18 days, these cells retained the expression of the antigen detected by antibodies Trop-4 and 4F2, whereas another membrane marker of activation, the transferrin receptor, rapidly disappeared. These findings demonstrate a phenotypical difference between primed, secondary T lymphocytes and resting, unstimulated cells.     

On the sex chromosome trivalent in some Lepidoptera females

In four of the moth species investigated, viz. Witlesia murana, Scoparia arundinata (Pyraloidea), Bactra furfurana and B. lacteana (Tortricoidea) the metaphase plates of the first meiotic division of their oocytes show a trivalent in addition to the normal bivalents. It evidently has its rise in a transverse break in one of the conjugated chromosomes. Two sex chromatin bodies can be seen in the female somatic cells of three of these species, whereas other species with a normal XY bivalent have only one. These two sex chromatin bodies are unequal in size, and their sizes bear approximately the same relation to each other as do those of the two smaller chromosomes of the trivalent. The broken chromosome is evidently the Y chromosome. The sex chromosome designation for the four above-mentioned species is thus XY₁Y₂ for the females and XX for the males. The sex chromosomes of the four species are among the biggest of the respective complements. This supports the view that the big chromosome to be found in several Lepidoptera species is the sex chromosome. It seems that in animals with holokinetic chromosomes an excessive fragmentation is hindered, at least in the case of the sex chromosomes, by its deleterious effect on the balance of sex-determining genes.Dedicated to Doctor Sally Hughes-Schrader on the occasion of her seventy-fifth birthday.     

Activation of latent human neutrophil collagenase by reactive oxygen species and serine proteases

The ability of various reactive oxygen species and serine proteases to activate latent collagenase (matrix metalloproteinase-1) purified from human neutrophils was examined. Latent 70-75 kD human neutrophil collagenase (HNC) was efficiently activated by known non-proteolytic activators phenylmercuric chloride (an organomercurial compound) and gold thioglucose (Au(I)-salt). Corresponding degree of activation was achieved by reactive oxygen species including hypochlorous acid (HOCl), hydrogen peroxide (H2O2) and hydroxyl radical generated by hypoxanthine/xanthine oxidase (HX/XAO). The presence of trace amounts of iron and EDTA were necessary and even enhanced H2O2 induced activation of latent HNC. This activation could be abolished by an iron chelator desferrioxamine and a hydroxyl radical scavenger mannitol. HOCl induced activation of latent HNC was not affected by desferrioxamine and mannitol. Thus, these compounds do not inhibit the active/activated form of HNC. Latent HNC could also be activated by trypsin and chymotrypsin but not by plasmin and plasma kallikrein. The ability of mannitol and desferrioxamine to inhibit the H2O2-induced activation of HNC suggests the transition metal dependent Fenton reaction to be responsible for localized and/or site-specific generation of hydroxyl radical/hydroxyl radical -like oxidants to act as the activating oxygen species. Our results support the ability of myeloperoxidase derived HOCl to act as a direct oxidative activator of HNC and further suggest the existence of a new/alternative oxidative activation pathway of HNC involving hydroxyl radical.     

Characterization of a novel human putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing proteins 3 and 4

We report here a novel human gene, hMRS3/4, encoding a putative mitochondrial transporter structurally and functionally homologous to the yeast mitochondrial RNA splicing proteins 3 and 4. These proteins belong to the family of mitochondrial carrier proteins (MCF) and are likely to function as solute carriers. hMRS3/4 spans approximately 10 kb of genomic DNA on chromosome 10q24 and consists of four exons that encode a 364-aa protein with six transmembrane domains. A putative splice variant, encoding a 177-aa protein with three transmembrane domains, was also identified. hMRS3/4 has a well-conserved signature sequence of MCF and is targeted into the mitochondria. When expressed in yeast, hMRS3/4 efficiently restores the mitochondrial functions in mrs3(o)mrs4(o) knock-out mutants. Ubiquitous expression in human tissues and a well-conserved structure and function suggest an important role for hMRS3/4 in human cells.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

GRACILE syndrome,a lethal metabolic disorder with iron overload,is caused by a point mutation in BCS1L

GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death) syndrome is a recessively inherited lethal disease characterized by fetal growth retardation, lactic acidosis, aminoaciduria, cholestasis, and abnormalities in iron metabolism. We previously localized the causative gene to a 1.5-cM region on chromosome 2q33-37. In the present study, we report the molecular defect causing this metabolic disorder, by identifying a homozygous missense mutation that results in an S78G amino acid change in the BCS1L gene in Finnish patients with GRACILE syndrome, as well as five different mutations in three British infants. BCS1L, a mitochondrial inner-membrane protein, is a chaperone necessary for the assembly of mitochondrial respiratory chain complex III. Pulse-chase experiments performed in COS-1 cells indicated that the S78G amino acid change results in instability of the polypeptide, and yeast complementation studies revealed a functional defect in the mutated BCS1L protein. Four different mutations in the BCS1L gene have been reported elsewhere, in Turkish patients with a distinctly different phenotype. Interestingly, the British and Turkish patients had complex III deficiency, whereas in the Finnish patients with GRACILE syndrome complex III activity was within the normal range, implying that BCS1L has another cellular function that is uncharacterized but essential and is putatively involved in iron metabolism.     

Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-mediated nuclear targeting of virus

Nuclear targeting of adenovirus is mediated by the microtubule-dependent, minus-end-directed motor complex dynein/dynactin, in competition with plus- end-directed motility. We demonstrate that adenovirus transiently activates two distinct signaling pathways to enhance nuclear targeting. The first pathway activates integrins and cAMP-dependent protein kinase A (PKA). The second pathway activates the p38/MAP kinase and the downstream MAPKAP kinase 2 (MK2), dependent on the p38/MAPK kinase MKK6, but independent of integrins and PKA. Motility measurements in PKA-inhibited, p38-inhibited or MK2-lacking (MK2(-/-)) cells indicate that PKA and p38 stimulated both the frequency and velocity of minus-end-directed viral motility without affecting the perinuclear localization of transferrin-containing endosomal vesicles. p38 also suppressed lateral viral motilities and MK2 boosted the frequency of minus-end-directed virus transport. Nuclear targeting of adenovirus was rescued in MK2(-/-) cells by overexpression of hsp27, an MK2 target that enhances actin metabolism. Our results demonstrate that complementary activities of PKA, p38 and MK2 tip the transport balance of adenovirus towards the nucleus and thus enhance infection.     

Assignment of the disease locus for lethal congenital contracture syndrome to a restricted region of chromosome 9q34, by genome scan using five affected individuals.

Lethal congenital contracture syndrome (LCCS) is an autosomal recessive disease leading to death before the 32d gestational week. It is characterized by the fetal akinesia phenotype, with highly focused degeneration of motoneurons in the spinal cord as the main neuropathological finding. We report here the assignment of the LCCS locus to a defined region of chromosome 9q34, between markers D9S1825 and D9S1830. The initial genome scan was performed with the DNA samples of only five affected individuals from two unrelated LCCS families. The conventional linkage analysis performed with 20 affected individuals and their families was focused on those chromosomal regions in which the affected siblings were identical by descent in the initial scan. One core haplotype of 3 cM was observed in LCCS alleles, supporting the assumption of one major mutation underlying LCCS, and linkage disequilibrium analysis restricted the critical chromosomal region to <100 kb in the vicinity of marker D9S61. Two genes, NGAL (neutrophil gelatinase-associated lipocalin and NOTCH 1, were excluded as causative genes for LCCS