Evidence for functional hemizygosity at the Emtr locus in CHO cells through segregation analysis

The hypothesis of functional hemizygosity at the emetine-resistant (Emt^r, a non-X-linked recessive marker) locus in Chinese hamster ovary (CHO) cells has been examined by segregation analysis. The frequencies and the rates of segregation of the Emt^r and Thg^r (thioguanine-resistant, an X-linked recessive mutation) markers were determined from hybrids constructed between an Emt^r-Thg^r CHO cell line and various other Chinese hamster lines (V79, M3-1, CHO, GM7S, CHW and CHL). Thg^r segregants were obtained at similar frequencies (10^?2–10^?3) from all the hybrids. The frequency of segregation of the Emt^r marker, however, was similar to that of Thg^r only in the CHO × CHO hybrids and was much lower (10^?4–10^?6) than the CHO × other Chinese hamster hybrids. Similar results were obtained when the segregation rates for the two markers from various hybrids were determined. These results are consistent with the hypothesis that in CHO cells, the gene responsible for Emt^r is present in a single (functional) copy, whereas two copies of this gene are present in other Chinese hamster lines examined.     

The chemistry of human transcortin. The effects of pH, urea, salt, and temperature on the binding of cortisol and progesterone.

The interaction of cortisol and progesterone with pure transcortin was investigated. The temperature dependence of cortisol and progesterone binding is a result of the predominantly negative enthalpy of binding which suggests a very good fit between ligand and protein such that the bonds formed are of the van der Waals type. The optimal pH of cortisol (8.0) and progesterone (8.5) binding suggests involvement of cysteine, histidine, and/or tyrosine residues in the binding process. Transcortin is irreversibly denatured at pH 4.0. The effect of sodium chloride on the binding of both steroids is small. At lower sodium chloride concentrations (less than 0.15 m), binding decreases somewhat with decreasing salt concentration. Urea produces a progressive decrease in the association constants of both steroids which is completely reversible up to 2.0 m and 30% reversible at 3.0 m. Scatchard analysis of cortisol binding in the presence of a constant amount of progesterone and vice versa confirms earlier data obtained on plasma that cortisol and progesterone do not bind at two independent sites. It is not possible, however, to decide whether they bind at the same site or at two interdependent (interacting) sites.     

Thyrotoxicosis: relations between clinical state and biochemical changes during carbimazole treatment.

The relation between clinical and biochemical changes in thyrotoxicosis were studied in 12 patients with Graves''s disease who were being treated with carbimazole. Clinical assessment (using the Crooks-Wayne index) was combined with the measurement of free thyroxine and triiodothyronine indices (FT4I and FT3I) and the assessment of two tissue markers of thyroid hormone action--sex-hormone-binding globulin (SHBG) levels and the thyrotrophin responses to TRH. In general the FT4I and FT3I fell rapidly once treatment was started, and returned to normal in one to four weeks, followed shortly by SHBG levels. The thyrotrophin response returned at this time in two patients, who still had borderline high levels of FT3I and SHBG. The clinical score fell more slowly and variably and was less closely related to any of the biochemical indices than these were to each other. During the early phase of treatment with antithyroid drug the clinical evaluation may be an unreliable indicator of persisting thyroid hormone excess, and when the patient seems clinically but not biochemically thyrotoxic the symptoms should be treated on their own merits with beta-blocking drugs and not with increased doses of antithyroid drugs.     

Polyamine metabolism in McCoy cells: III. Comparative studies of the metabolic fate of exogenous putrescine in human fibroblasts and McCoy cultures

The fate of exogenously added 14C-putrescine following incubation for 24 hours with McCoy and human skin fibroblast cultures was examined. The nature of the polyamine derivatives found were quite different indicative of a difference in the cellular metabolism of polyamines. Exogenously added putrescine (PUT) was metabolized by both McCoy and human skin fibroblast cultures to form spermidine (SPD), spermine (SPM), gamma-aminobutyric acid (GABA) and some unidentified compounds. Within the experimental period of observation, human cultured fibroblasts metabolized PUT more efficiently than McCoy cells and converted more than 50% of it into SPD, SPM, GABA and unknown compounds. Monoacetyl putrescine (MAP) was formed by human skin fibroblasts. It was mainly identified in the culture medium. No MAP was detectable either intracellularly or extracellularly in McCoy cultures. The percentage of 14C-radioactivity found as PUT in the culture medium was greater in McCoy cells (86.0%) than in human fibroblasts (53.9%). The reverse was true for the percentage distribution of 14C-radioactivity as PUT inside the cells. No low Mr conjugates of SPD or SPM were found in the medium or intracellularly with either culture type. Some low Mr putrescine conjugates were found in the culture media; these were identified by the liberation of PUT upon acid hydrolysis.     

Direct effect of glucose on the preproinsulin mRNA level in isolated pancreatic islets

The effects of glucose on the preproinsulin mRNA level and the rate of (pro)insulin biosynthesis were examined in isolated mouse pancreatic islets. Relative concentrations of preproinsulin mRNA were quantitated by a RNA-dot hybridization procedure. The level of preproinsulin mRNA in islets incubated for up to 7 days at 20 mM glucose remained constant. In islets incubated at 3.3 mM glucose the preproinsulin mRNA level decreased and was after 24 h reduced to one tenth of the level at 20 mM glucose. Subsequent incubation at 20 mM glucose completely restored the preproinsulin mRNA level but only after 3 days of culture, while the insulin release was restored within 24 h. The insulin-biosynthetic activity of the islets was correlated to the variation in the level of the preproinsulin mRNA. These results suggest that glucose does have a direct influence on the level of preproinsulin mRNA and that the rate of (pro)insulin biosynthesis is limited by the level of the preproinsulin mRNA.     

Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.     

Synthesis and assembly of the synaptic cleft protein S-laminin by cultured cells.

S-laminin, a homologue of the B1 chain of laminin, is concentrated in a subset of basal laminae (BLs), including the BL at the skeletal neuromuscular junction and bears an adhesive site for motoneuron-like cells. Here, we have begun to characterize the native form of the protein. We show that several muscle- and glia-like cell lines synthesize and secrete S-laminin as well as the A, B1, and B2 subunits of the conventional laminin trimer. Experiments using subunit-specific antibodies showed that S-laminin is complexed with the A and B2 subunits of laminin but not with B1, suggesting that S-laminin replaces B1 to form a novel laminin-like trimer. Comparison of material precipitated by different antibodies provided evidence for two immunochemically distinct forms of S-laminin, both of which associate with B2 and A-like subunits. Analysis of tunicamycin-treated cells indicated that N-linked glycosylation is required neither for the selective association of S-laminin with B2 and A subunits nor for the distinction between two forms of S-laminin. Finally, a full-length S-laminin cDNA was constructed and transfected into muscle and non-muscle cells. S-laminin was detected intracellularly in both cell types, in extracellular matrix of muscle cells, and in two immunochemically distinct forms. Thus, the cDNA contains sufficient information to permit assembly, secretion, and post-translational modification of S-laminin.