In vivo competition between a metallothionein regulatory element and the SV40 enhancer.

The human metallothionein-IIA (hMT-IIA) gene contains an enhancer element within its 5' regulatory region. This enhancer element can compete with the SV40 enhancer for one or more cellular factors in vivo. The competition between the two elements is modulated by cadmium, an inducer of hMT-IIA transcription. The data presented are consistent with a model in which heavy metal ions control the ability of the hMT-IIA enhancer to bind a positive factor, leading to increased transcription. The same factor is required for maximal activity of the SV40 enhancer, which suggests that viruses utilize factors that have a normal role in cellular gene expression to control their own genes.

Metal-responsive elements act as positive modulators of human metallothionein-IIA enhancer activity.

The human metallothionein IIA (hMT-IIA) gene contains two enhancer elements whose activity is induced by heavy-metal ions such as Cd2+. To determine the nature of the relationship between the metal-responsive elements and the element(s) responsible for the basal activity of the enhancers, the basal-level enhancer element(s), the hMT-IIA enhancers were subjected to mutational analysis. We show that deletion of the metal-responsive elements had no effect on the basal activity of the enhancer but prevented further induction by Cd2+. On the other hand, replacement of the basal-level enhancer element with linker DNA led to inactivation of the enhancer both before and after treatment with Cd2+. Therefore, the metal-responsive elements seems to act as a positive modulator of enhancer function in the presence of heavy-metal ions. In addition to the two enhancers, the hMT-IIA promoter contained one other element, the GC box, required for its basal expression. Unlike deletion of the basal-level enhancer element, replacement of the GC box with linker DNA had no effect on the ability of the promoter to be induced by Cd2+.

Discriminant power of combined cerebrospinal fluid tau protein and of the soluble interleukin-6 receptor complex in the diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) still can only be definitively diagnosed with certainty by examination of brain tissue. There is a great need for a noninvasive, sensitive and specific in vivo test for AD. We combined cerebrospinal fluid analyses of tau protein (levels were significantly increased in AD patients [p=0.0001]), a putative marker of neuronal degeneration, with components of the soluble interleukin-6 receptor complex (sIL-6RC: IL-6, soluble IL-6 receptor and soluble gp130), putative markers of neuroregulatory and inflammatory processes in the brain. A stepwise multivariate discriminant analysis revealed that tau protein and soluble gp130 (levels were significantly reduced in AD subjects [p=0.007]), the affinity converting and signal-transducing receptor of neuropoietic cytokines, maximized separation between the investigated groups. The discriminant function predicted 23 of 25 clinically diagnosed AD patients (sensitivity 92%) with mild to moderate dementia correctly as having AD. Furthermore, 17 of 19 physically and cognitively healthy age-matched control subjects (specificity 90%) were accurately distinguished by this test. Later predicting with the jackknife procedure each case in turn through the remaining patient group, the discriminant function remained stable. Our data suggest that multivariate discriminant analysis of combined CSF tau protein and sIL-6RC components may add more certainty to the diagnosis of AD, however, the method will need to be extended to an independent group of patients, comparisons and control subjects to assess the true applicability.

Interleukin-6 and the soluble IL-6 receptor are decreased in cerebrospinal fluid of geriatric patients with major depression: no alteration of soluble gp130.

Interleukin-6 (IL-6) is hypothesized to play an important role in the interaction between immune mechanisms and the central nervous system. We investigated whether cerebrospinal fluid (CSF) concentrations of interleukin-6 (IL-6), the soluble IL-6 receptor (sIL-6R) and the soluble form of the signal transducing and affinity converting receptor gp130 (sgp130) are altered in geriatric patients with major depression (MD). In 20 geriatric patients with MD and 20 age-matched healthy control subjects CSF concentrations of the three components of the sIL-6R complex were analyzed by enzyme-linked immunosorbent assays (ELISA). All patients except one were treated with psychotropic drugs. We found statistically significant decreased CSF concentrations of IL-6 (P<0.001) and of the sIL-6R (P<0.001) of patients with MD. Levels of sgp130 showed no statistically significant difference between patients and controls.

Cerebrospinal fluid tau protein shows a better discrimination in young old (<70 years) than in old old patients with Alzheimer's disease compared with controls.

Tau protein is consistently reported to be elevated in cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD). CSF tau alone, however, is not a clinically useful diagnostic marker due to its relatively low diagnostic specificity. Therefore, efforts are under way to combine tau measurements with other criteria in order to improve diagnostic applicability. We investigated whether age could serve as an useful criterion to increase diagnostic accuracy. CSF levels of tau were measured in young old (<70 years) and old old (> or =70 years) patients with probable AD, elderly patients with major depression (MD), and age-matched healthy controls (HC). In AD patients, CSF tau levels were significantly elevated compared with MD patients and HC (P < 0.001). Based on a previously established cut-off of 260 pg/ml, the discriminative power was higher in the young old than in the old old subjects. Similarly, receiver operating characteristics analysis revealed a statistically significant higher correct classification rate in the young old. Our findings indicate that the discriminative power of CSF tau is higher in the young than in the old old. We suggest that the effect of age should be considered in studies investigating CSF tau as a diagnostic marker for neurodegenerative disorders.

Glucose-induced fibronectin expression in endothelial cells is mediated by protein kinase C.

One of the crucial pathophysiological changes in diabetic angiopathy might be the glucose-induced synthesis of components of the extracellular matrix-like fibronectin. This effect has been described for endothelial and mesangial cells. In order to gain further insight into the mechanisms how glucose-induced fibronectin expression is regulated, confluent monolayers of human umbilical vein endothelial cells (HUVEC) were incubated with varying concentrations of D-glucose (5-30 mM) in a time-dependent manner. Using Westernblotting techniques with a monoclonal antibody, a 3 +/- 0.2-fold increase of the 220 kDa-signal corresponding to human fibronectin was visible after an incubation time of 6 h indicating de-novo synthesis. Using incubation times of 6 or 10 days, glucose-mediated fibronectin overexpression was still visible, reaching a maximum at 15 mM D-glucose. The corresponding maximum values were 2.4 +/- 0.3 (6 d) and 2.3 +/- 0.2 (10 d). There was a concomitant glucose-dependent onset of expression of Protein Kinase C (PKC)-isoforms PKC-delta (2.5 +/- 0.3-fold), PKC-epsilon (2 +/- 0.2-fold) and PKC-zeta (1.8 +/- 0.2-fold), which reached a maximum after 12 h and was still visible during long-term culture. Induction of fibronectin expression was also obtained using the PKC-activating phorbolester Phorbol-12-myristat-13-acetat (PMA) which mimicks glucose-induced PKC activation. Glucose-induced fibronectin expression was decreased by the PKC-inhibitor H-7 (1- [5-isoquinolinylsulfonyl]-2-methylpiperazine). These experiments suggest that: 1. Short-term induction of fibronectin expression mediated by glucose is probably PKC-triggered since concomitant induction of PKC-isoforms PKC-delta, PKC-epsilon and PKC-zeta was observed. 2. Long-term incubation with D-glucose leads to an ongoing concentration-dependent coexpression of fibronection and various PKC-isoforms. If glucose-induced, PKC-mediated de-novo synthesis of components of the connective tissue or the extracellular matrix may be important in the pathomechanism of diabetic angiopathy, has to be proven.

[Euthanasia--limits of therapy in oncology. From the viewpoint of the attorney]. / Euthanasie--Grenzen der Therapie in der Onkologie. Aus der Sicht des Juristen.

Confidence is the major prerequisite for a doctor to be able to help his seriously ill patient. The patient's expectations of this confidence are: the doctor will not act without the patient's consent and the doctor will not undertake any measure to shorten the patient's life. The patient is strengthened in this confidence by Austrian law in as much as any treatment without the patient's consent is punishable, even when performed secundum artem (section 110 StGB). This principle prohibits necessary and life-sustaining treatment if refused by the patient after appropriate clarification. Alternatively, this principle gives the patient the right to a merciful death by refusing life-prolonging treatment which is of no further benefit. Even more important is the legal prohibition of intentional hastening of death (euthanasia). Even the patient's own repeated request does not exculp the doctor if he directly acts to end the life of the patient before the disease has run its natural course (sections 77, 78 StGB). A "crisis of confidence", which can be found sometimes in the elderly and severe ill patients, can only by overcome by inspiring confidence and by a direct personal doctor to patient relationship. Law and ethics dictate: intentional shortening of life by doctors: never; humanitarian care for the dying: always.

[Marriage without a marriage certificate]. / Ehe ohne Trauschein

PIP: This article is concerned with the development of non-marital cohabitation in Austria. Recent trends in Austrian nuptiality and fertility are first discussed, and an overview of the increasing incidence of non-marital cohabitation in Europe and the United States is presented. Data from a study of the family formation process among the 1974 and 1977 Austrian marriage cohorts are then analyzed, with particular reference to the extent and average duration of premarital cohabitation as well as to regional and social differences. The demographic consequences of this phenomenon are also examined.^ieng

[Illegitimate births in Austria: historical and regional patterns]. / Uneheliche Geburten in Osterreich: historische und regionale Muster

PIP: Historical trends and regional differences in illegitimacy in Austria are examined using official data for the nineteenth and twentieth centuries as well as results of a 1978 survey. A high rate of illegitimacy and the persistence of regional differences are noted. Multivariate analysis is also used to examine socioeconomic factors affecting illegitimacy. (summary in ENG)^ieng

Upstream promoter element of the human metallothionein-IIA gene can act like an enhancer element.

Initiation of transcription by RNA polymerase II in eukaryotes is strongly increased by cis-acting genetic elements, known as activators or enhancers. Enhancers, first detected in simian virus 40 (SV40), were subsequently also found to control the expression of several cellular genes. The human metallothionein-IIA (hMT-IIA) gene, although inducible by heavy metals and glucocorticoids, is widely expressed in most cell types in the absence of inducers. Here we show that the high basal level of transcription of the hMT-IIA gene is due to the presence of an enhancer element within the hMT-IIA promoter region. The structural and functional organization of this cellular enhancer element in two direct repeats is strikingly similar to that of the enhancer element of SV40. This suggests a possible functional and evolutionary relationship between enhancers and upstream promoter elements.

[Seizures and driver's licence]. / Anfall und Führerschein.

Legislation leaves a wide margin of freedom of judgement to the medical expert in the forensic assessment of a person's suitability for holding a driver's license. We have to make use of this freedom very cautiously with regard to social aspects as well as to the presently accepted state of scientific knowledge. In cases of first manifestations of epilepsy we must postulate--as a rule of thumb--a seizure-free period of two years with regular medication and clinical and electro-encephalographic checkups. The (epileptic or non-epileptic) "accidental seizure" is characterized by the releasing influence of powerful external noxious factors. In these cases, the patients should be seizure-free for at least six months before driving can be resumed. In all cases, the evaluation depends on the continuous observance of the patients, taking into account any underlying primary illness (alcoholism, cerebral vascular disease, conditions following brain surgery or trauma). The organic brain syndrome is an essential additional factor, together with the feasibility of treatment and the patient's compliance. Evaluation of the clinical picture is of foremost importance, support by EEG-findings, serum levels and psychological tests. Knowledge of cumulative factors, elimination and side-effects of anticonvulsive drugs is essential. According to the present view, medication should be continued over a period of many years. Curative medicine and medical assessment are not mutually exclusive, but require a distinct differentiation in every single case.

The effect of heat-shocking on batch fermentation by Clostridium beijerinckii NRRL B592.

In spite of the large-scale industrial use of the acetone-butanol fermentation process earlier this century (until 1983 in South Africa), very little has been published on the inoculum preparation techniques required for successful fermentation using these bacteria. In particular, heat-shocking has often been referred to as "useful" but no quantitative data are available. Data presented in this paper demonstrate and quantify the effect of heat-shocking on batch fermentation yields using one organism capable of this fermentation.

Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40.

Genetic analysis of eukaryotic transcriptional promoters has revealed that protein-coding genes often contain a complex array of cis-control elements consisting of upstream activator sequences and enhancer elements. The metallothionein genes provide a useful example for dissecting the action of multiple interspersed control elements that govern both basal level and regulated expression in animal cells. The human metallothionein (hMTIIA) promoter has been analysed in detail and found to contain no less than five distinct control elements in the 5' flanking regions of the gene that mediate specificity and regulation of transcription. These different control elements can be functionally subdivided into two categories: basal and induced elements. There are several distinct basal recognition sequences, which include a TATA-box, a GC-box, and at least two basal level enhancer (BLE) sequences, that function like classical enhancer elements. The hMTIIA gene also responds to induction by heavy metals and by steroid hormones through the action of metal regulatory elements (MRE) and glucocorticoid responsive elements (GRE). Here we report the identification of two cellular DNA-binding proteins that interact selectively with sequences governing the basal level expression of hMTIIA. One of these factors is a novel activator protein (AP1) that interacts with sequences in the BLE of hMTIIA and also binds to a site within the 72-base pair (bp) repeats of the simian virus 40 (SV40) enhancer region. The second protein has been purified to homogeneity and shown to be transcription factor Sp1 which recognizes and binds to a single GC-box element within the hMTIIA promoter.

Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5'-flanking DNA.

Human metallothionein-IIA (hMT-IIA) gene expression is regulated by heavy metals and glucocorticoids. When the cloned hMT-IIA gene or its 5'-flanking DNA structure fused to herpes simplex virus thymidine kinase (HSV-TK) structural gene sequences were transferred into TK- Rat 2 fibroblasts, both genes were inducible by Cd++ and/or dexamethasone. Placement of the hMT-IIA gene 5'-flanking region, either intact of deleted in its TATA box and cap site, upstream of the HSV-TK gene promoter rendered the latter both glucocorticoid- and heavy metal-inducible. Thus the structure that mediates both Cd++ and glucocorticoid responsiveness is present in the hMT-IIA gene 5'-flanking DNA, does not require its TATA box or cap site, and can activate a heterologous promoter.

Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast.

Copper resistance in yeast is controlled by the CUP1 locus. The level of resistance is proportional to the copy number of this locus, which can be found in up to 15 tandemly iterated copies. To elucidate the molecular mechanisms controlling the amplification and expression of the CUP1, locus, we determined its full nucleotide sequence. We have also identified and mapped two transcription units within the basic amplification unit of CUP1 in laboratory yeast strains. One of those transcription units is inducible by copper and encodes a low molecular weight copper binding protein--copper chelatin. The increased production of chelatin, due to both gene amplification and induction of transcription, leads to increased resistance of yeast cells to copper ions.

Transcriptional control mechanisms which regulate the expression of human metallothionein genes.

The human genome contains at least 12 distinct metallothionein genes. The expression of these genes is regulated at the transcriptional levels by heavy metal ions, steroid hormones, interferon, interleukin 1 and phorbol esters. Comparisons of metallothionein gene sequences reveals a higher level of heterogeneity in the 5' flanking control regions than in the coding regions. This is due to the presence of distinct promoter elements on each of the genes. These elements are responsible for inducer and tissue specificity of metallothionein gene expression.

Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene.

Deletion experiments have defined two stretches of DNA (genetic elements), lying close to the promoter for a human gene for metallothionein, that separately mediate the induction of the gene by heavy metal ions, particularly cadmium, and by glucocorticoid hormones. The element responsible for induction by cadmium is duplicated, yet a single copy is fully functional; the element responsible for induction by glucocorticoid hormones is coincident with the DNA-binding site for the glucocorticoid hormone receptor.