Congenital adrenal hypoplasia, myopathy, and glycerol kinase deficiency: Molecular genetic evidence for deletions

Glycerol kinase deficiency (GKD) is an X-linked recessive trait that occurs in association with congenital adrenal hypoplasia (AH) and developmental delay with or without congenital dystrophic myopathy. Several such patients have recently been reported to have cytological deletions of chromosome region Xp21 and/or of DNA markers that map near the locus for Duchenne muscular dystrophy (DMD) in band Xp21. We have examined the initial family reported in the literature and, using prometaphase chromosome studies and Southern blot analysis with 13 different DNA probes derived from band Xp21, have found no deletions within this region of the X chromosome. When DNA samples from six other unrelated affected males were analyzed, four of them were found to have different-size deletions within Xp21. Thus, the form of GKD associated with AH and dystrophic myopathy exhibits significant genetic heterogeneity at the DNA level. No deletions were detected in two patients with isolated GK deficiency. Comparison of our molecular studies of unrelated patients with deletions of DNA segments allows us to define the region of Xp21 (between probes J-Bir and L1.4) that most likely contains the genes for GKD and AH. This location is distal to the DMD locus. The patients with progressive muscular dystrophy tended to have larger deletions that include markers known to derive from the DMD locus, while GKD/AH/dystrophic-myopathy patients without current evidence of deletion seemed to have a milder, nonprogressive form of congenital myopathy.     

Developmental Change in the Amount of Polysialosyl Glycopeptides Isolated from the Rat Brain

Polysialosyl glycopeptides were coisolated with glycosaminoglycans by Pronase digestion of the whole brains of perinatal rats and could be separated from known glycosaminoglycans by two-dimensional electrophoresis on cellulose acetate film. The polysialosyl glycopeptides could not be obtained from fetal rat brain on day 13 of gestation, but began to be detected on day 14. The amount of polysialosyl glycopeptides was estimated from the dye concentration of the Alcian blue-stained spot in the electrophoretogram. The glycopeptide content increased almost linearly, on the basis of brain DNA, up to 10 days after birth. Thereafter, the content decreased rapidly, and hardly any polysialosyl glycopeptides could be isolated from the brain at approximately 30 days. This developmental change may be involved in morphogenesis and maturation of the brain. The polysialosyl glycopeptides could be isolated from the cerebellum, from the cerebrum, or from the brainstem of the neonatal rat. However, each region of the brain had a postnatal developmental change in glycopeptide content different from those of the other regions.     

Developmental Change in the Glycosaminoglycan Composition of the Rat Brain

Abstract: Glycosaminoglycans (GAGs) were isolated from the brains of pre- and postnatal rats. The GAG content of the brain, based on the amount of DNA, was constant during the period from day 13 to day 15 of gestation. After day 15, the GAG content began to increase and reached a plateau by 10 days after birth. Hyaluronate (HA) was the main GAG (> 60% of the total) in the fetal rat brain, and the relative amount of HA decreased after birth. Conversely, the relative amount of chondroitin sulfate increased with development and reached the adult level by 20 days after birth. Heparan sulfate (HS) was the major sulfated GAG in the fetal rat brain at early developmental stages, but HS accounted for approximately 10% of the total GAG in the postnatal brains. In addition to these GAGs, a polysialosyl glycoconjugate was isolated from rapidly growing brains of the rat. These three GAGs could be isolated either from the cerebellum, cerebrum, or brainstem of the newborn rat. A closely similar age-related change in the GAG composition was observed in each of these different regions of the brain. The developmental change could be implicated in morphogenesis or maturation of the brain.     

Two classes of Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

Summary Six mutant strains of Bacillus subtilis hypersensitive to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were shown to be deficient in the adaptive response to MNNG and termed ada mutants (Morohoshi and Munakata 1985). All the mutations mapped between the attSPO2 and lin loci on the chromosome. The mutant and wild-type (ada ⁺) cells contained similar constitutive levels of O⁶-methylguanine-DNA methyltransferase activity. Pretreatment with low concentrations of MNNG increased the activity about nine-fold in the ada ⁺ cells, while it uniformly decreased the activity in the ada cells. The pretreatment of three mutants (ada-3, ada-4, and ada-6) as well as ada ⁺, augumented the activity of methylpurine-DNA glycosylase and rendered the cells resistant to the lethal and mutagenic effects of N-propyl- or N-butyl-N-nitro-N-nitrosoguanidine. With the rest of the mutant strains (ada-1, ada-2, and ada-5), neither of such responses was elicited by the pretreatment. Thus, the former ada strains seem to have a defect in the gene specifically involved in the induction of the methyltransferase, while the latter ada strains have a defect in the gene controlling the adaptive response as a whole.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - ENNG N-ethyl-N-nitro-N-nitrosoguanidine - PNNG N-propyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - MMS methyl methanesulphonate     

Gene expression of foreign metaphase chromosomes introduced into cultured mammalian cells?

Transfer of genetic information from isolated hamster chromosomes to mouse cells is described. Metaphase chromosomes isolated from Chinese hamster diploid cells were incubated with mouse Cl. 1-d cells deficient in thymidine kinase activity. Two viable colonies appeared from the treated mouse cells after HAT selection with a frequency of about 10⁻⁸. The first colony isolated (Cl. 1) failed to grow, however. The second colony isolated (Cl. 2) grew well in HAT medium and was subcultured for more than 70 generations. Cl. 2 cells possessed an elevated tetrahydrofolate dehydrogenase activity of molecular species resembling that of Chinese hamster cells, as shown by disc electrophoresis. The cell line also expressed surface antigen(s) specific to hamster species, as shown by mixed hemadsorption test and immune cell electrophoresis. This latter phenotype disappeared after prolonged cultivation (59 generations) of the cells in non-selective medium. The karyotype of Cl. 2 cells corresponded to that of the mouse species and was quite different from that of hamster cells. Hamster chromosomes could not be identified in any of the cell clones by detailed analysis by the banding method (Q- and C-band). Not one revertant cell was obtained among 4.2×10⁸ Cl. 1-d cells in the control.     

Identification of the Carboxyl-Terminal Processing Protease for the D1 Precursor Protein of the Photosystem II Reaction Center of Spinach

The enzyme involved in the carboxyl-terminal processing of theD1 precursor protein (pD1) of the photosystem II reaction centerwas purified from extracts of sonicated spinach thylakoids bya method that included chromatography on quaternary aminoethylanion-exchange, hydroxylapatite, copper-chelating affinity andgel-filtration columns. The enzyme was identified, from itschromatographic behavior, to be a monomeric protein of about45 kDa. The sequence of the amino-terminal 27 amino acids ofthis protein was determined directly, which exhibited low butappreciable (37%) homology to that deduced from a gene (ctpA)in Synechocystis sp. PCC 6803 that was proposed recently toencode the processing protease from results of genetic complementationanalysis. ³Present address: Asahi Kasei Chem. Co.     

Resting eggs of the marine rotifer Brachionus plicatilis Müller: development,and effect of irradiation on hatching

Diapause and hatching of Brachionus plicatilis Müller resting eggs were examined through histological and optical approaches. Compound microscope observations on 1% toluidine blue-stained embryo sections suggests that the total number of nuclei in an embryo during the internal diapause period increased from 22 on Day 2 to 39 (each n = 1) on Day 6. The outer layer of embryo membrane gradually thickens from 1.2 (Day 0) to 4.0 µm (Day 8) (each n = 10).Resting eggs that have completed maturation and are in the external diapause period require light for hatching. The threshold of light (halogen lamp) intensity for hatching was estimated to be 4400 lux for 30 min. Hatching rate decreased with longer wavelength irradiation (mercury lamp). Irradiation at more than 350 nm caused 1–25% hatching, but it reached 50–60% at 250–310 nm light. The addition of hydrogen peroxide or prostaglandins (E ₁, E ₂ or F ₂) caused resting egg hatching even in darkness. The production of peroxide in seawater caused by light as well as the oxidation of fatty acid to prostaglandins inside the embryo is a possible mechanism of resting egg hatching.     

Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases

IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.     

Mechanism of the protective immunity against murine typhoid: persistence of salmonella L forms in the liver after immunization with live-cell vaccines

Abstract Live-cell vaccines of Salmonella typhimurium , either a sub-lethal dose of a wild-type (strain LT2) or a high dose of its two-heptose Rd₁ mutant (strain SL1004), induced acquired resistance to murine typhoid, which remained 180 days after immunozation. Growth of S. typhimurium as a bacillary form ceased between days 30 and 60 of immunization, but L forms of this bacterium colonized the liver (the mean number of L forms in the liver: 600 L-forming units) even at 180 days post-immunization. In contrast, a high inoculum of either a Ra mutant (strain TV148) of strain LT2 or S. schottmülleri 8006 sharing the same O antigenic components with those of S. typhimurium induced only a short-lived protection in proportion to the number of L forms in the liver, and the protective immunity was lost before day 180. However, there was no significant difference in the salmonella-specific T-cell responses among groups of immunized mice on day 180 of immunization. A lethal infection with strain LT2 in mice which had been immunized 75 days previously with living cells of strain SL1004 resulted in a rapid clearance of the challenge inoculum, together with a rapid elevation of anti- S. typhimurium antibody responses. Thus, the present data suggest that the long-lived immunity conferred upon live S. typhimurium vaccines is attributable to the colonization of this bacterium in the liver as L forms and the ability to colonize the liver as L forms is independent of the brain length of salmonella O-antigens.     

Construction of yeast secretion vectors designed for production of mature proteins using the signal sequence of yeast invertase

Summary Two kinds of yeast secretion vectors were constructed by site-directed mutagenesis of the invertase signal sequence and ligation of synthetic oligonucleotides coding appropriate signals. Each has a cloning site for a foreign gene preceded by a sequence encoding either the signal peptide cleavage site or a Lys-Arg sequence which is a cleavage site for the product of the KEX2 gene. Both vectors were able to direct the expression and secretion of mouse amylase. One of them has a SalI site within the signal sequence, and an attempt to clone sequences enhancing secretion of amylase with this vector is reported.