Activation of porcine oocytes via an exogenously introduced rat muscarinic M1 receptor.

Thirty hours after the beginning of in vitro maturation, porcine oocytes were microinjected with mRNA coding for the rat muscarinic M1 receptor. They were then incubated for 15 h to allow sufficient time for completing maturation, translation of the mRNA, and insertion of the receptor into the plasma membrane. They were then treated with acetylcholine, the receptor's agonist, and its effect on inducing various activation-related changes was examined. Acetylcholine treatment triggered the release of Ca2+ from internal stores that could be blocked by atropine, the receptor's antagonist. The Ca2+ release was probably mediated via a G protein, since prior injection of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) totally inhibited the effect of the agonist. Pertussis toxin (PT) had no effect on the Ca2+ transients induced by acetylcholine, suggesting that the signal transduction pathway involved a PT-insensitive G protein. Electron microscopy revealed that in the injected oocytes, acetylcholine induced cortical granule exocytosis. The oocytes were released from meiotic arrest as evidenced by the decrease in H1 kinase activity measured in the oocytes during the histone H1 kinase assay. After resuming meiosis they entered interphase: 58.8% of the injected oocytes formed pronuclei after incubation with the agonist. Injection without subsequent acetylcholine treatment, or acetylcholine incubation without prior injection with the receptor mRNA, did not cause these changes. The results provide further evidence that the components of a G protein-mediated signal transduction pathway exist in porcine oocytes and that the activation of this pathway via an exogenously supplied G protein-coupled receptor results in a full complement of oocyte activation events. Whether this pathway transduces the activating signal at sperm-induced oocyte activation requires further examination.

[Diagnostic value of thyroid antibodies]. / A pajzsmirigy elleni autoantitestek diagnosztikus értéke.

Thyroid antibodies against thyroglobulin, the microsomal fraction and TSH receptor have been investigated in 150 patients (41 of them with Graves'disease, 24 of them with Hashimoto thyroiditis, 21 of them with subacute thyroiditis and 34 cases with treated hypothyroidism). 30 patients having thyroid disease without immune etiology served as a control group. As compared with the control group, significant differences were detected in the titers of microsomal and TSH-receptor antibodies in Graves' disease, microsomal and thyroglobulin antibodies in Hashimoto thyroiditis, while significantly higher antibody titers were measured against the microsomal fraction antibodies in hypothyroidism. In subacute thyroiditis, no significant elevation of any of the three antibodies was demonstrated. The diagnostic values of antibodies were investigated; in Graves disease, the antibody against TSH receptor is the most sensitivity method (70%), while in Hashimoto thyroiditis the highest sensitivity (87,5%) is exhibited by the determination antibody against the microsomal fraction. The relationship between the thyroid hormone values and the antibody titer was investigated too. In Hashimoto thyroiditis with hypothyroid state significantly elevated microsomal antibodies were found, as compared with the euthyroid state. It may be concluded that in cases suspect of Graves disease, determination of antibody against of TSH receptor is recommended. In case of normal value, finding the antibody against microsomal fraction can confirm the diagnosis. In further conclusion, antibody measurements are rarely informative in subacute thyroiditis. In cases suspect of other thyroid diseases of immune origin, measurement of the antibodies against the microsomal fraction may be helpful, while the discriminative value of the thyroglobulin antibody is more limited.

Differentiation-dependent expression of the brown adipocyte uncoupling protein gene: regulation by peroxisome proliferator-activated receptor gamma.

Uncoupling protein (UCP) is expressed only in brown adipocytes and is responsible for the unique thermogenic properties of this cell type. The novel brown preadipocyte cell line, HIB-1B, expresses UCP in a strictly differentiation-dependent manner. Transgenic mice studies have shown that a region from kb -2.8 to -1.0 of the marine UCP gene is required for brown adipocyte-specific expression. Subsequent analysis identified a potent 220-bp enhancer from kb -2.5 to -2.3. We show that this enhancer is active only in differentiated HIB-1B adipocytes, and we identify a peroxisome proliferator-activated receptor gamma (PPARgamma) response element, referred to as UCP regulatory element 1 (URE1), within the enhancer. URE1 has differentiation-dependent enhancing activity in HIB-1B cells and is required for enhancer action, since mutations of URE1 that block protein binding abolish enhancer activity. We also show that PPAR gamma antibodies block binding to URE1 of nuclear extracts from cultured brown adipocytes and from the brown adipose tissue of cold-exposed mice. Protein binding to URE1 increases substantially during differentiation of HIB-1B preadipocytes, and PPAR-gamma mRNA levels increase correspondingly. Although forced expression of PPAR gamma and retinoid X receptor alpha activates the enhancer in HIB-1B preadipocytes, these receptors are not capable of activating the enhancer in NIH 3T3 fibroblasts. Our results show that PPAR gamma is a regulator of the differentiation-dependent expression of UCP and suggest that there are additional factors in HIB-1B cells required for brown adipocyte-specific UCP expression.

Transcriptional organization and expression of noIXWBTUV, a locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257.

Rhizobium fredii is a nitrogen-fixing bacterial symbiont of soybean and a number of other legume species. We have studied the transcriptional organization of a Sym plasmid locus that restricts the host range of R. fredii USDA257 at both the host species and cultivar level. The genes of this host-specificity locus, noIXWBTUV, are transcribed from three promoters. Two of these, which are upstream of noIW and noIBTUV, are oriented face to face and initiate transcription at sites that are 14 bp apart. The third lies upstream from noIX. The noIW promoter is constitutive, whereas the noIB and noIX promoters are inducible by flavonoid signals. We have attempted to express genes from this locus in Escherichia coli systems, both in vivo and in vitro. We detected the insert- and orientation-specific expression of two genes, noIX and noIW, but we were unable to obtain expression of noIBTUV. Antiserum raised against NoIT nevertheless detected an abundantly expressed polypeptide of the predicted size in protein extracts of USDA257. This observation, as well as RNA dot blot data from a series of mutants, indicates that noIBTUV is expressed as a single transcriptional unit in R. fredii. Immunological detection of NoIT, and of a second protein, NoIX, was strictly dependent on flavonoid induction. The NoIX protein was larger than the size predicted from the previously published nucleotide sequence, and this led to resequencing and revision of the open reading frame.

[Clinical significance of the ultrasensitive TSH assay]. / A TSH ultrasensitiv-assay alkalmazásának klinikai jelentösége.

The authors attempted to answer the question whether the low thyroid stimulating hormone (TSH) levels measurable by the TSH ultrasensitive DELFIA kit have any clinical significance and whether they are more informative than the results obtained by the supersensitive TSH assay. No measurable TSH was detected in 111 sera among 896 random specimens, by using a supersensitive fluorimetric kit. These 111 sera were further investigated, TSH was measured by an ultrasensitive assay, in addition, the levels of the peripheral hormones (total T4, total T3, T3-uptake, free T4, free T3), were also determined. On basis of the latter, the patients were classified as having subclinical (n = 28) or manifest (n = 80) hyperthyroidism. The TSH levels of the patients affected by manifest hyperthyroidism were found significantly (p < 0.0001) lower than those encountered in subclinical hyperthyroidism. The groups were then further divided to homogeneous clinical subgroups (patients treated with thyrostatic drugs, untreated patients, toxic adenoma, Graves' disease) and the results were analyzed. It can be stated that the ultrasensitive test safely distinguishes manifest and subclinical disease in all subgroups (range of sensitivity: 90.0-94.7%). Specificity for the diagnosis of subclinical hyperthyroidism was 66.7% for the untreated subgroups, irrespective of aetiology, while in treated patients the value of specificity was 10%. In Graves' disease, specificity was 100%, in toxic adenoma 0% (the number of patients, however, was very small in these homogeneous subgroups). These results suggest that although the ultrasensitive method furnishes more information than the supersensitive test, its exclusive application would not be appropriate in characterizing thyroid function because of the broad range of individual scatter.

Mapping and genetic organization of pTiChry5, a novel Ti plasmid from a highly virulent Agrobacterium tumefaciens strain.

Agrobacterium tumefaciens Chry5, a wild-type strain originally isolated from chrysanthemum, is unusually tumorigenic, particularly on soybean. We have mapped the Chry5 Ti plasmid by genomic walking and restriction endonuclease analysis, and have located its virulence, T-DNA, plasmid incompatibility, and L,L-succinamopine utilization loci. Southern analysis has revealed that about 85% of the Chry5 Ti plasmid is highly homologous to another Ti plasmid, pTiBo542. Although all the functions that we have located on pTiChry5 are encoded by pTiBo542-homologous regions, the two Ti plasmids differ in their genetic organization. The overall patterns of restriction sites in the plasmids also differ, with the exception of an approximately 12 kb segment of the virulence region, where the BamHI sites appear to be conserved. Complementation analysis has shown that deletion of a DNA segment which flanks the oncogenic T-DNA results in severe attenuation of virulence. This region also contains a sequence that is repeated in the Chry5 genome outside the Ti plasmid, and that is widely distributed in the Rhizobiaceae.

[New strategy for thyroid function testing]. / Uj stratégia a pajzsmirigyfunkció diagnosztikájában.

The authors used a new protocol, based upon a supersensitive TSH assay, to examine the thyroid status of 1720 patients. Based upon the serum hormone levels, the patients were divided into different clinical groups. The biochemical relationship between the different hormone levels, and the rate of occurrence of various thyroid diseases were studied. 76.1% of the new patients hadn't received any previous treatment. 15.5% of those patients who had received treatment had hyperthyroidism, while 8.4% of those had hypothyroidism. 76% of the new patients, 38.3% of those who had hyperthyroidism, and only 29.7% of those who had hypothyroidism, were euthyroid. Undetectable TSH levels (< 0.03 mU/L) where observed in 51.8% of the new hyperthyroid patients, and in 33.8% of those who had subclinical hyperthyroidism. Similar results were obtained with those who had been previously treated for hyperthyroidism. The new protocol has the following advantages: it's more convenient to the patients, it's quick, it's economical. With this method it is possible to reduce the assays per patient by 31%. The algorithm was supplemented with results of free hormone levels. By doing this the authors were able to measure free-T4 and T3 hormone levels of 150 more patients. According to the authors, the free-T4 test is more informative than the free-T4-index, especially in the border-line cases and in treated hyperthyroidism. Primarily the free-T3 test is most necessary when examining patients treated with methimasol.

[Diagnostic value of CA 19-9 and CEA in gastrointestinal pathology]. / A CA 19-9 és a CEA tumorantigén diagnosztikus értéke gastrointestinalis kórképkben.

The diagnostic values of CA 19-9 and CEA were evaluated in 187 cases (including 31 gastric, 41 colorectal, 12 pancreatic, 7 hepatobiliar and 5 hepatocellular carcinomas). These tumor markers were compared to the other laboratory parameters [hemoglobin, erythrocyte sedimentation rate, serum bilirubin, ASAT (aspartate amino transferase), ALAT (alanine amino transferase) GGT (gamma glutamil transpeptidase), ALP (alkaline phosphatase)]. The specificity of CA 19-9 was 89.5%, while the sensitivity of this tumor markers was 91.7% in pancreatic carcinoma, 54.8% in gastric carcinoma and 43.9% in colorectal carcinoma. The sensitivity of CEA only in colorectal patients was higher than that of CA 19-9 (specificity 73.9%, sensitivity 64.5%). Although the CA 19-9 and CEA are not known to give any cross-reaction with each other, simultaneous measurement and evaluation of these two tumor antigens did not result in a better diagnostic sensitivity. After undergoing a gastrointestinal carcinoma operation, CA 19-9 indicated the appearance of tumor recidiva with a 62% sensitivity. Calculated together with CEA the sensitivity elevated to 88.9%. In most of the patient with benign cholostasis, the CA 19-9 and CEA values were out of the normal range (53.3% and 36.4% respectively), so these tumor markers are not suitable to differentiate between benign and malign cholostasis. According to the authors, CA 19-9 is the most useful diagnostic tool to differentiate between pancreatic carcinoma and pancreatitis chronica (both group without cholostasis), as well as for monitoring the patients after surgery of a gastrointestinal cancer.

Role of prolactin-opiate interactions in the central regulation of pain threshold.

Endogenous hyperprolactinaemia induced by anterior pituitary transplantation under the kidney capsule has been found to reduce the behavioural responsiveness to electrical footshock and to increase morphine-induced analgesia. The apparent analgesic effect of prolactin has been related to the stimulation of nigro-striatal dopaminergic transmission, as suggested by the increase in striatal dopamine turnover observed in hyperprolactinaemic rats. It seems likely that central opiate system is involved in the behavioural effects of prolactin. Thus, naloxone prevents the effects of hyperprolactinaemia on footshock responsiveness and heroin self-administration is decreased in hyperprolactinaemic rats.