Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation

Bright nucleolar immunofluorescence was observed in HeLa S3 cells by immunostaining with a monoclonal antibody to the nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1). After 48 h of incubation in a serum-free medium, the nucleolar fluorescence was diminished and a general nuclear immunofluorescence was observed. This change in localization of fluorescence indicated that protein B23 had migrated out of the nucleoli. No gross morphological change in nucleoli was observed by light microscopy and the immunolocalization of another nucleolar phosphoprotein, C23, was unaffected by serum deprivation. Relocation of protein B23 in nucleoli was observed after refeeding with serum-containing medium. This re-entry process was not observed after treatment with actinomycin D (50 ng/ml-5 micrograms/ml), but the process was unaffected by cycloheximide (0.2 mM). Quantitation of protein B23 in the nucleoli of the control (fed) or starved HeLa cells was done by ELISA immunoassay. A marked decrease in the amount of protein B23 occurred in the nucleoli of the starved cells (11.8 micrograms B23/mgDNA) as compared with the control nucleoli (20.8 micrograms B23/mgDNA). The amount of protein B23 in the nucleoplasm (excluding nucleoli) was 70% higher in the starved cells. Protein B23 was analysed by one- and two-dimensional PAGE. Three components of protein B23 with slightly different molecular weights and pIs (37 kD/5.1, 35 kD/5.1 and 35 kD/5.3) were observed in nucleoli. The lower molecular weight components were predominantly found in the nucleoplasm.     

A histochemical study of the changes occurring in the protein-carbohydrate composition of the cumulus-oocyte complex and zona pellucida in immature mice in response to gonadotrophin stimulation

Summary A histochemical account is presented of the changes that occur in the protein—carbohydrate composition of the cumulus—oocyte complex in immature mice after gonadotrophin treatment. The distribution and nature of the glycosaminoglycans (GAG) present was established by enzymic digestion of tissue sections with testicular orStreptomyces hyaluronidase prior to staining with periodic—acid Schiff (PAS) or Alcian Blue. Treatment with exogenous gonadotrophins [pregnant mare's serum and human chorionic gonadotrophin (hCG)] induced gross changes in the appearance of the zona pellucida (and in the histochemical staining of the cumulus—oocyte complex). A reduction was observed in the amount of PAS-positive material present within the zona pellucida of oocytes located in large Graafian follicles examined 40 h after stimulation with pregnant mare's serum. After the injection of hCG, the zona pellucida was further depleted of PAS-positive naterial. Most of the PAS-positive material became confined to the plasma membrane of the oocyte, while the oocyte itself also became increasingly PAS-positive. All the GAGs disappeared from zona pellucida within 4 h of hCG stimulation. The changes observed in the protein—carbohydrate composition of the zona pellucida in preovulatory oocytes immediately prior to ovulation may be a prerequisite for successful sperm-egg interactions.     

The kinetic equation for the chloride transport cycle of band 3. A 35Cl and 37Cl NMR study

The nature of a transmembrane transport process depends largely on the identity of the reaction that is rate-limiting in the transport cycle. The one-for-one exchange of two chloride ions across the red cell membrane by band 3 can be decomposed into two component reactions: 1) the binding and dissociation of chloride at the transport site, and 2) the translocation of bound chloride across the membrane. The present work utilizes 35 Cl NMR and 37 Cl NMR to set lower limits on the rates of chloride binding and dissociation at the saturated inward- and outward-facing band 3 transport sites (greater than or equal to 10(5) events site-1 s-1 in all cases). At both 0-3 and 37 degrees C, the NMR data specify that chloride binding and dissociation at the saturated transport sites are not rate-limiting, indicating that translocation of bound chloride across the membrane is the slowest step in the overall transport cycle. Using these results, it is now possible to describe many features of the kinetic equation for the ping-pong transport cycle of band 3. This transport cycle can be decomposed into two half-reactions associated with the transport of two chloride ions in opposite directions across the membrane, where each half-reaction is composed of sequential binding, translocation, and dissociation events. One half-reaction contains the rate-limiting translocation event that controls the turnover of the transport cycle; in this half-reaction, translocation must be slower than binding and dissociation. The other half-reaction contains the non-rate-limiting translocation event that in principle could be faster than binding or dissociation. However, when the following sufficient (but not necessary) condition is satisfied, both translocation events are slower than binding and dissociation: if the non-rate-limiting translocation rate is within a factor of 10(2) (0-3 degrees C) or 2 (37 degrees C) of the overall turnover rate, then translocation is rate-limiting in each saturated half-reaction. Thus, even though chloride appears to migrate through a channel that leads from the transport site to solution, the results support a picture in which the binding, dissociation, and channel migration events are rapid compared to the translocation of bound chloride across the membrane. In this case, chloride binding to the transport site can be described by a simple dissociation constant (KD = kappa OFF/kappa ON) rather than by a Michaelis-Menten constant (KM = (kappa OFF + kappa TRANSLOCATION)/KAPPA ON).     

Nucleotide sequence and gene organization of ColE1 DNA

The primary structure of the plasmid ColE1 DNA has been determined. The plasmid DNA consists of 6646 base pairs (molecular mass of 4.43 MDa) and is 48.46% in GC content. The phi 80 trp insert of the composite plasmid of ColE1, pVH51, has also been determined. The determination of the nucleotide sequence of ColE1 DNA provides the basis for examining the relationships between the DNA sequence and the gene organization of the plasmid. The focus of this paper is to use this sequence data coupled with a review of the literature and our own work to examine the nine known functional regions of ColE1: imm (colicin E1 immunity), rep (replication function), inc (plasmid incompatibility and copy number control), bom (basis of mobility), rom (modulator of inhibition of primer formation by RNA I), mob (plasmid mobilization), cer (determinant for conversion of plasmid multimers to monomers), exc (plasmid entry exclusion), cea (structural gene for colicin E1), and kil (structural gene for the Kil protein).     

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 mol m^–2 s^–1 PAR during incubation with the leaf base in 2 mM ¹⁵NH₄Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. ¹⁵N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO₂ assimilation (8.3 and 17.4 mol m^–1 s^–1 at 150 and 1350 mol m^–2 s^–1 PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% ¹⁵N. In the high light regime the amino acids contained up to 15% ¹⁵N, whereas in low light ¹⁵N enrichments were small (up to 6%). The kinetics of ¹⁵N incorporation indicated that NH₃ was firstly assimilated into glutamine and then into glutamate. After 15 min ¹⁵N was also found in glycine, serine and alanine. At high light intensity nearly half of the ¹⁵N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant ¹⁵N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.     

Epidemiological trends of dermatophytoses and dermatophytes in Jerusalem between 1954 and 1981

Data for dermatophyte infections analysed for five 3-year periods between 1954 to 1981 led to the following conclusions: (1) Tinea pedis, tinea cruris and tinea manuum showed an increase in the 50's and 60's and declined in the 70's; (2) Tinea unguium and tinea corporis showed an increase during the whole period; (3) At all these sites, the percentage of Trichophyton rubrum, the main etiologic agent, increased steadily over the periods while the percentage of Trichophyton mentagrophytes, the secondary etiological agent, decreased. Epidermophyton floccosum, the third etiological agent in these sites, showed no sharp fluctuations; (4) These three dermatophytes which show similar microclimatic requirements and favour the same microecological niches, were called glabrohydrophilic. In tinea corporis they form a definite subset, their percentage being similar to that at other glabrous sites; (5) Tinea capitis was at its peak in the 50's, decreased sharply until the second half of the 70's, its main etiological agent being Trichophyton violaceum. Since 1979, an increase of tinea capitis occurred due to the newly introduced Microsporum canis; (6) Dermatophytes favouring scalp hair were called trichophilic. In tinea corporis they form a definite subset, their percentage being similar to that of tinea capitis; (7) A comparison with other studies from this country shows that macroclimate (i.e. humid warm coastal climate compared with dry cooler inland-mountain climate) is not an important factor in the etiology of tinea.     

Insertion of a foreign nucleotide sequence into mitochondrial DNA causes senescence in Neurospora intermedia

The kalilo variants of Neurospora contain a cytoplasmic genetic factor that causes senescence. This factor is a 9.0 kb transposable element (kalDNA) that lacks nucleotide sequence homology with mtDNA and is inserted into the mitochondrial chromosome, often at sites located within the open reading frame in the intron-DNA of the mitochondrial 25S-rRNA gene. Genomes containing the "foreign" DNA insert accumulate during growth, and death occurs as the cells become deficient in functional large and small subunits of mitochondrial ribosomes. The kalDNA transposon may be an "activator" element that causes breaks in mtDNA. Nonsenescing [+] strains of Neurospora do not contain kalDNA.     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglycemic (db/db) mice. Effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-3H]glucose, uptake of 3-O-[methyl-3H]glucose (methylglucose) and [2-14C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 +/- 18 and 180 +/- 9 microliter/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V- of methylglucose transport was decreased with no change in Km in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and 3H2O production from [5-3H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Intergenic suppression of spoO phenotypes by the Bacillus subtilis mutation rvtA

Summary A collection of intergenic suppressors of the Bacillus subtilis spoOF221 mutation has been isolated. One of these suppressors, rvtA, has been mapped between lys-1 and aroD. The rvtA suppressor restores spoOF sporulation to wild type levels and substantially improves the sporulation efficiencies of spoOB and spoOE strains. The rvtA gene does not affect the Spo phenotype of spoOH, spoOJ or spoOK mutants. The rvtA gene also prevents the induction by aliphatic alcohols of SpoO phenocopies in wild type B. subtilis cells.