Segregation of Tay-Sachs and Sandhoff alleles in a non-Jewish family.

A non-Jewish family is presented in which the genes for Tay-Sachs disease and Sandhoff disease are segregating. Individuals heterozygous for both alleles have low serum and white cell total hexosaminidase levels together with a proportion of heat-labile hexosaminidase A (HEX A) which falls in the normal range. The individuals would not be detected as carriers of Tay-Sachs disease or Sandhoff disease in a population screening program.     

Purification and characterization of an (Na++K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain

Highly purified lamb kidney (Na⁺+K⁺)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na⁺+K⁺)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (γ1 and γ2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that γ1 is an aggregated form of γ2. The total yield was 0.8–1.5 mol of γ component per mol of catalytic subunit recovered. This indicates that the γ component is present in stoichiometric amounts in the (Na⁺+K⁺)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.     

Mesenchyme cells degrade epithelial basal lamina glycosaminoglycan

We investigated whether turnover of basal lamina glycosaminoglycan (GAG), an active process during epithelial morphogenesis, involves the mesenchyme. Fixed, prelabeled, isolated mouse embryo submandibular epithelia were prepared retaining radioactive surface components, as determined by autoradiographic and enzymatic studies, and a basal lamina, as assessed by electron microscopy. Recombination of mouse embryo submandibular mesenchyme with these epithelia stimulates the release of epithelial radioactivity when the labeled precursor is glucosamine or glucose but not when it is amino acid. The release is linear with time during 150 min incubation. Augmented release of epithelial label requires living mesenchyme which must be close proximity with the epithelia. Although heterologous mesenchymes, including lung, trachea, and jaw, stimulate the release of submandibular epithelial label, epithelial tissues do not. The label released by intact submandibular mesenchyme from prelabeled epithelia is in GAG and in two unique fractions: heterogeneous materials of tetrasaccharide or smaller size and N-acetylglucosamine. Enzymatic treatment of the heterogeneous materials revealed the presence of glycosaminoglycan-derived oligosaccharides. These unique products were not obtained by incubating prelabeled epithelia with a mesenchymal cell extract, suggesting that intact mesenchymal cells are required. N-Acetylglucosamine was also released when mesenchyme was recombined with living prelabeled epithelia which contained labeled basal laminar GAG. Our results establish that submandibular epithelial basal lamina GAGs are degraded by submandibular mesenchyme. We propose that one mechanism of epithelial-mesenchymal interaction is the degradation of epithelial basal laminar GAG by mesenchyme.     

Yeast RNA polymerase II subunit RPB9 is essential for growth at temperature extremes

The Saccharomyces cerevisiae RNA polymerase II subunit gene RPB9 was isolated and sequenced. RPB9 is a single copy gene on chromosome VII. The RPB9 sequence predicts a protein of 122 amino acids with a molecular mass of 14,200 Da. The yeast RPB9 subunit is similar in size and sequence to a protein encoded by DNA adjacent to the suppressor of the Hairy Wing gene in Drosophila melanogaster. Deletion of the RPB9 gene produced cells that were heat- and cold-sensitive. The RPB9 subunit, like the previously described RNA polymerase II subunit RPB4, is not essential for synthesis of mRNA, but is required for normal cell growth over a wide temperature range.     

Homologies between members of the germin gene family in hexaploid wheat and similarities between these wheat germins and certain Physarum spherulins

By screening approximately 10(6) plaques in a wheat DNA library with a "full-length" germin cDNA probe, two genomic clones were detected. When digested with EcoRI, one clone yielded a 2.8-kilobase pair fragment (gf-2.8) and the other yielded a 3.8-kilobase pair fragment (gf-3.8). By nucleotide sequencing, each of gf-2.8 and gf-3.8 was found to encode a complete sequence for germin and germin mRNA, and to contain appreciable amounts of 5'- and 3'-flanking sequences. The "cap" site in gf-2.8 was determined by primer extension and the corresponding site in gf-3.8 was deduced by analogy. The mRNA coding sequences in gf-2.8 and gf-3.8 are intronless and 87% homologous with one another. The 5'-flanking regions in gf-2.8 and gf-3.8 contain recognizable sites of what are probably cis-acting elements but there is otherwise little if any significant similarity between them. In addition to putative TATA and CAAT boxes in the 5'-flanking regions of gf-2.8 and gf-3.8, there are AT-rich inverted-repeats, GC boxes, long purine-rich sequences, two 19-base pair direct-repeat sequences in gf-2.8, and a remarkably long (200-base pair) inverted-repeat sequence (approximately 90% homology) in gf-3.8. An 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is reflected by a corresponding 7% difference between the corresponding 201-residue proteins. Most significantly, the same 8% difference between the mature-protein coding regions in gf-2.8 and gf-3.8 is allied with no change whatever in a central part (61-151) of the encoded polypeptide sequences. It seems likely that this central, strongly conserved core in the germins is of first importance in the biochemical involvements of the proteins. When an equivalence is assumed between like amino acids, the gf-2.8 and gf-3.8 germins show significant (approximately 44%) similarity to spherulins 1a and 1b of Physarum polycephalum, a similarity that increases to approximately 50% in the conserved core of germin. Near the middle (87-96) of the conserved core in the germins is a rare PH(I/T)HPRATEI decapeptide sequence which is shared by spherulins (1a and 1b) and germins (gf-2.8 and gf-3.8). These similarities are discussed in the context of evidence which can be interpreted to suggest that the biochemistry of germins and spherulins is involved with cellular, perhaps cell-wall responses to desiccation, hydration, and osmotic stress.(ABSTRACT TRUNCATED AT 400 WORDS)     

Primary mesenchyme cell migration requires a chondroitin sulfate/dermatan sulfate proteoglycan

Primary mesenchyme cell migration in the sea urchin embryo is inhibited by sulfate deprivation and exposure to exogenous beta-D-xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary mesenchyme cells of a very large, cell surface chondroitin sulfate/dermatan sulfate proteoglycan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective cells in vitro. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary mesenchyme cell migration since treatment of actively migrating cells with chondroitinase ABC reversibly inhibited their migration in vitro.     

A comparison of the intracellular distribution of mu-calpain, m-calpain, and calpastatin in proliferating human A431 cells.

Little is known about the relative intracellular localizations of the calcium-dependent proteases, calpains, and their naturally occurring inhibitor, calpastatin. In the present study, the intracellular localization of mu-calpain, m-calpain, and calpastatin was studied at the light microscopic level in proliferating A431 cells. Highly specific antibodies against the three antigens revealed distinct staining patterns in interphase and mitotic cells. Most notably, calpastatin in interphase cells was localized near the nucleus in tube-like, or large granular structures, while the calpains were more uniformly distributed through the cytoplasm in either a fibrillar form (mu-calpain) or a diffuse or fine granular form (m-calpain). The distribution patterns of the two calpain isozymes were distinctly different during mitosis. m-Calpain was concentrated at the mitotic spindle poles and midbody, while mu-calpain appeared to accumulate at the cell membrane and the spindles. Four other human cell lines as well as normal human monocytes were examined to determine if the calpains-calpastatin segregation patterns are common to other cells or are unique to the A431 line. With the exception of abundant nuclear mu-calpain in the C-33A cervical carcinoma, the segregation of the proteins was similar to that of A431. These studies indicate that calpains may be localized at regions which are relatively poor in calpastatin content. Proteins at these sites may be susceptible to calpain-catalyzed cleavage.     

Inhibition or down-regulation of protein kinase C attenuates late phase p70s6k activation induced by epidermal growth factor but not by platelet-derived growth factor or insulin.

The late phase of the time-dependent epidermal growth factor (EGF)-induced biphasic activation of the p70s6k is selectively attenuated by the specific PKC inhibitor, CGP 41,251, a staurosporine derivative. At a 40-fold lower concentration than CGP 41,251, staurosporine inhibits both phases of S6 kinase activation to the same extent, whereas the inactive staurosporine derivative CGP 42,700 shows no effect on either phase. Platelet-derived growth factor (PDGF) and insulin also induce biphasic S6 kinase activation, but in neither case is either phase of activation affected by the presence of CGP 41,251. This finding was unexpected in the case of PDGF, which is a potent activator of PKC and whose receptor directly interacts with phospholipase C gamma 1. However, similar results were obtained following down-regulation of PKC by prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Therefore, even though EGF and PDGF induce PKC activation, PDGF, unlike EGF, does not appear to use this signaling pathway for late phase p70s6k activation.