An evaluation of patient performance of and their satisfaction with various rapid blood glucose measurement systems.

We evaluated the performance of 50 insulin-dependent diabetic patients in the measurement of their own capillary blood glucose concentrations using Chemstrip bG, Dextrostix-Dextrometer, and Stat Tek systems. With all systems, patient performance was suboptimal when compared with the accuracy of paramedical personnel. The percentage of patient determinations that differed from the laboratory value by more than 20% was 37%, 30%, and 14% for the Chemstrip bG, Dextrostix-Dextrometer, and Stat Tek systems, respectively. Only 39 of the patients (78%) could perform accurately with any system. Youth, lack of a higher education, and lower income status contributed significantly to the patients' inaccuracy with the Chemstrip bG technique, whereas these factors had no effect on patient performance with the reflectance meter techniques. Nearly all of the patients were enthusiastic about the value of home glucose monitoring as a means to assess their glycemic control. However, only 30% of the patients selected for home use a technique at which they were suitably adept. In part, this selection error appeared to be due to the greater cost and inconvenience of the reflectance meter techniques compared with the Chemstrip bG technique. These data indicate that unless proper instruction is provided, home glucose monitoring should only be used by a fraction of insulin-requiring diabetic patients and the choice of a particular system for use by an individual patient should be predicated upon his or her demonstrated proficiency with that system.

Noradrenergic activation of immediate early genes in rat cerebral cortex.

Previous studies have shown that stimulation of adrenergic receptors in the brain increases the expression of the immediate early gene (IEG), c-fos, in vivo (Mol. Brain Res., 6(1989) 39-45). The present study was undertaken to determine whether this also holds for other IEGs which have been shown to be activated in brain cell culture by adrenergic agonists. Both yohimbine injection and stressful stimulation, two treatments causing brain norepinephrine (NE) release, were found to cause a parallel, transient activation of at least 5 IEGs (c-fos, nur77, tis-7, zif-268 and tis-21) in the rat cortex. Genes that are not immediate early (beta-actin, NGF and HSP70) were found not to be affected in the interval used (6 h). The responses were mediated predominantly by beta-adrenoceptors with some contribution from alpha 1 receptors. The parallel activation of multiple genes by noradrenergic receptors may enable the coding of different biochemical responses to the activation of different receptors.

Distribution of glutamic acid decarboxylase messenger RNA-containing nerve cell populations of the male rat brain.

The distribution of glutamic acid decarboxylase (GAD) mRNA was investigated throughout the rat brain by means of in situ hybridization. Hybridization was carried out with a 35S-radiolabeled cRNA probe transcribed from a cDNA from cat occipital cortex and cloned in a SP6-T7 promoter-containing vector. Fixed tissue sections were hybridized with 35S GAD probe (0.6 kb length). Signal was detected by means of film or emulsion autoradiography. The autoradiograms were semiquantitatively evaluated by means of computer-assisted image analysis. The results obtained with this evaluation were correlated with the results of the semiquantitative analysis of GAD immunoreactivity performed by Mugnaini and Oertel. Specific labeling was only observed in neuronal cell bodies, whereas no labeling was found over neuropil, glial and endothelial cells. The highest labeling was found in the bulbus olfactorius (internal plexiform and granular layers) and in the caudal magnocellular nucleus of the hypothalamus. Strong labeling was observed in the Purkinje layer of the cerebellar cortex, the interpeduncular nucleus, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch and the suprachiasmatic nucleus. Intermediate or low levels of GAD mRNA were present in various brain nuclei, where gamma-aminobutyric acid (GABA)-containing cell bodies had been observed with other techniques. Interestingly, a low level of GAD mRNA was found in the caudate-putamen and nucleus accumbens, where the vast majority of nerve cells is known to contain GAD immunoreactivity. Only a poor correlation was found between the present semiquantitative measurements of GAD mRNA content and previous analyses of the number of GAD-immunoreactive cell bodies. The present study demonstrates that there exists a differential regional expression of GAD mRNA. The comparison with cell counts performed by immunocytochemistry suggests that some brain areas, such as caudate-putamen and nucleus accumbens, contain a large number of GAD-immunoreactive cell bodies which express a low level of GAD mRNA. The opposite seems to be true for other nuclei, such as the globus pallidus, the zona reticulata of the substantia nigra and the inferior collicle, where few GAD-immunoreactive cell bodies contain high levels of GAD mRNA. In conclusion, the present study gives a low magnification map of GAD mRNA levels in the adult male rat brain. Marked biochemical heterogeneities may be present among GABA neuronal populations based on their expression of GAD mRNA. The comparison between the present in situ hybridization and previous immunocytochemical studies suggests that there may exist at least two populations of GABA neurons in the brain, having high and low levels respectively of both GAD mRNA and GAD enzyme.

Immunohistochemical studies of noradrenergic-induced expression of c-fos in the rat CNS.

Previous studies have shown that stimulation of adrenergic receptors in the rat brain causes increased levels of mRNA of the immediate early gene, c-fos. The present studies were undertaken to determine if this stimulation also induces increased levels of c-fos immunoreactivity in the central nervous system (CNS). Rats were treated with the alpha-2 adrenoceptor blockers, yohimbine or atipamezole, or with restraint stress to activate central noradrenergic activity and were perfused 2 h later for immunohistochemical analysis of the cerebral cortex. Yohimbine, atipamezole and restraint stress each was found to cause increases in c-fos-like immunoreactivity (c-fos-li). Western blot analysis revealed increased c-fos protein in the cortex after yohimbine treatment. The c-fos-li response to yohimbine was blocked by prior administration of the beta receptor antagonist, dl-propranolol, and to a lesser degree by the alpha-1 antagonist, prazosin. It is concluded that adrenergic receptor stimulation in the cortex causes increased production of c-fos or fos related antigens and that this (these) immediate early gene product(s) may play a role in noradrenergic function in the CNS.

Effect of locus coeruleus lesion on c-fos expression in the cerebral cortex caused by yohimbine injection or stress.

The injection of the alpha-2 adrenoceptor antagonist, yohimbine, has been shown to increase c-fos immunoreactivity in the rat cerebral cortex. To determine the extent to which this response is mediated by the central noradrenergic system, the present studies examined it in rats previously given unilateral 6-OHDA lesions of the locus coeruleus. The lesions were found to produce a significant attenuation of the response. A similar effect on the c-fos immunoreactive response to restraint stress was found. It is concluded that the noradrenergic system plays a necessary role in the above c-fos responses in the cortex to yohimbine and to stress. The c-fos protein therefore appears to be involved in the effects of noradrenergic neurotransmission in the CNS.

Regulation and molecular characterization of dopamine D2 receptors in a prolactin-secreting 7315a anterior pituitary tumor.

The regulation and molecular properties of the dopamine (DA) D2 receptors were compared in the prolactin-secreting 7315a anterior pituitary tumor with those in the striatum of rats. Chronic treatment with haloperidol increases the maximal binding for [3H]spiroperidol in tumor and striatum, but the percent increase is much higher in tumor than in striatum. Photoaffinity labelling of DA D2 receptors with N-(p-azido-m-[125I]iodophenethyl)spiperone ([125I]N3-NAPS) yielded a major specifically labeled peptide with the Mr of 32-34 kDa in tumor, and two specifically labeled peptides with Mr of 32-34 and 92-94 kDa in striatum. The analysis of DA D2 receptor mRNA shows that the size is similar in tumor and striatum. The DA D2 receptor mRNA in tumor is very low and chronic treatment with haloperidol produces a considerable increase of the specific mRNA. It is postulated that the reported defect in regulation of prolactin release by DA agonists might be due to posttranslational changes in the tumor DA D2 receptor.

Duplication of the tuf gene, which encodes peptide chain elongation factor Tu, is widespread in Gram-negative bacteria.

The tuf gene which encodes peptide chain elongation factor Tu was found to be duplicated in nine enteric and four nonenteric gram-negative bacteria, but present only in one copy in two gram-positive genera. In two of the nonenteric gram-negative genera, Pseudomonas and Caulobacter, the duplicate tuf genes were found to be very close together on the chromosome, which contrasts with the situation in Escherichia coli, where they are more than 660 kilobases apart.

Identification by sequence analysis of a second rat brain cDNA encoding the dopamine (D2) receptor.

A rat brain cDNA library constructed in lambda ZAP II was screened with three oligonucleotide probes based on the reported coding region of the D2 receptor gene, RGB-2. A complete cDNA clone, D2(8)-1, showing positive signals with the three probes was subsequently identified by restriction analysis and dideoxy sequence analysis to be a variant of the RGB-2 gene. Comparison of the two genes revealed almost complete homology except that D2(8)-1 contains an 87 bp insert within the protein coding region and 265 additional nucleotides 5' upstream from the 5' end reported for RGB-2. It is suggested that at least two mRNA species encoding for D2 receptors exist in rat brain, possibly resulting from alternative splicing of RNA.

Aspartokinase of Myxococcus xanthus: "feedback stimulation" by required amino acids.

The aspartokinase activity found in extracts of the bacterium Myxococcus xanthus was subject to feedback inhibition and feedback repression by l-threonine and l-lysine. Both types of inhibition were essentially additive. The required amino acids, l-isoleucine and l-methionine, caused considerable increase in the activity of the enzyme. This phenomenon is referred to as "feedback stimulation." The polyamine, spermidine, exerted strong enhancement of the activity even at 0.1 mM. Meso-diaminopimelate, although not inhibitory by itself, abolished the activation exerted by either l-isoleucine or l-methionine. The possible physiological significance of interactions between the various effectors is discussed.

Aspartokinase activity and the developmental cycle of Myxococcus xanthus.

The relationship between aspartokinase activity and fruiting body formation in Myxococcus xanthus was investigated. Two required amino acids, methionine and isoleucine, which stimulated the enzyme in vitro also inhibited fruiting body formation when added to 0.1% Casitone agar. Threonine, a potent feedback inhibitor of the aspartokinase, completely reversed the effects of methionine and isoleucine both on enzyme activity and fruiting body formation. A mutant, M. xanthus FB-S, which had the unusual property of forming fruiting bodies on 1.0% Casitone agar, also exhibited an altered regulation of aspartokinase activity. Spermidine, which is a strong stimulator of the enzyme in vitro, interfered with the developmental cycle of both M. xanthus FB and FS-S. During glycerol induction of myxospores the level of aspartokinase dropped more than 75% during the first hour. These data indicate a strong correlation between aspartokinase activity and the induction of the developmental cycle in M. xanthus. It is suggested that the decrease in aspartokinase activity results in diaminopimelic acid starvation, blockage of cell wall growth, and subsequent induction of the developmental cycle.

Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis.

We have investigated the role of serine 40 (Ser-40) in tyrosine hydroxylase (TH) catalysis of basal and activated enzymes by protein kinase A (PKA)-mediated phosphorylation. Wild type and mutant TH were transiently and stably expressed in AtT-20 cells, and the enzymatic activities of the recombinant enzymes were analyzed. The specific enzymatic activity of transiently expressed TH mutants Ser-40-->leucine or-->tyrosine (Leu-40m or Tyr-40m) was higher than that of the wild type enzyme or of other mutants in which Ser-8, -19, and -31 were replaced by leucine. The kinetic studies carried out with the stably expressed TH show that the Km for the cofactor 6-methyltetrahydropterine is lower and the Ki for dopamine is higher when the enzymatic hydroxylation is catalyzed by the Leu-40m or Tyr-40m than by the wild type enzyme. The kinetic parameters and the pH profile of the enzymatic hydroxylation catalyzed by the Leu-40m or Tyr-40m are similar to the enzyme activated by PKA-mediated phosphorylation. We suggest that Ser-40 in TH exerts an inhibitory influence on the enzymatic activity, and its replacement with another amino acid by site-directed mutagenesis or its modification by phosphorylation leads to a change in conformation with an increased enzymatic activity. The importance of Ser-40 in the activation of TH by PKA-mediated phosphorylation was investigated by comparing the activation of the wild type enzyme with that of Leu-40m or Tyr-40m. The findings that the enzymatic activity is increased by PKA-mediated phosphorylation of the wild type enzyme, but not of the Leu-40m or Tyr-40m, demonstrate that phosphorylation at Ser-40 is essential for activation of TH by PKA. The findings that addition of ATP plus cAMP to homogenates from transfected AtT-20 cells stimulates the recombinant wild type TH activity indicate that these cells contain endogenous cAMP-dependent protein kinase.

Myxospore coat synthesis in Myxococcus xanthus: in vivo incorporation of acetate and glycine.

Myxospore coat synthesis in Myxococcus xanthus was studied by incorporation of [(14)C]acetate into intermediates in the biosynthesis of coat polysaccharide and into acid-insoluble material during vegetative growth and after glycerol induction of myxospores. During short labeling periods at 27 degrees C, the radioactivity was shown to be located primarily in N-acetyl groups rather than sugar moieties. Two hours after glycerol induction, the pools of N-acetylglucosamine 6-phosphate and uridine 5'-diphosphate-N-acetylgalactosamine (UDPGalNAc) plus uridine 5'-diphosphate-N-glucosamine increased about twofold and were labeled at twice the rate measured for vegetative cells. The increased rate of synthesis of UDPGalNAc and its precursors could be correlated with increased enzyme activities measured in vitro. Controlled acid hydrolysis revealed that the galactosamine portion of the myxospore coat was N-acetylated. After glycerol induction, the incorporation of acetate into acid-insoluble material increased threefold. This enhanced incorporation was sensitive to neither penicillin nor d-cycloserine. In contrast, bacitracin inhibited the incorporation of [(14)C]acetate into acid-insoluble material more effectively 2 h after myxospore induction than during vegetative growth. Chloramphenicol added to cells 90 min after induction blocked further increase in the rate of [(14)C]acetate incorporation. Since the myxospore coat contains glycine, polymer synthesis was also measured by chloramphenicol-insensitive [(14)C]glycine incorporation into acid-insoluble material. Although protein synthesis decreased after glycerol induction, glycine incorporation increased. Two hours after induction, glycine incorporation was only 75% inhibited by chloramphenicol and rifampin. The chloramphenicol-insensitive rate of incorporation of [(14)C]glycine increased during the first hour after myxospore induction and reached a peak rate after 2 to 3 h. The chloramphenicol-resistant incorporation of [(14)C]glycine was resistant to penicillin but sensitive to bacitracin.

Myxospore coat synthesis in Myxococcus xanthus: enzymes associated with uridine 5'-diphosphate-N-acetylgalactosamine formation during myxospore development.

Activities of the enzymes glutamine synthetase (EC 6.3.1.2.), glucosamine 6-phosphate acetyltransferase (EC 2.3.1.4.), uridine 5'-diphosphate (UDP)-N-acetylglucosamine pyrophosphorylase (EC 2.7.23.), UDP-N-acetylglucosamine 4-epimerase (EC 5.1.3.7.), fructose 1,6-diphosphate phosphatase (EC 3.13.11.), L-glutamine-fructose 6-phosphate transamidase (EC 5.3.1.19.), alkaline phosphatase (EC 3.1.3.1.), and malic dehydrogenase (EC 1.1.1.37) were assayed in partially purified extracts prepared at different stages of myxospore formation and germination in liquid cultures of Myxococcus xanthus. The specific activities of the first six of these enzymes increased 4.5- to 7.5-fold after 2 h of induction with 0.5 M glycerol or 0.2 M dimethyl sulfoxide. The increase in specific activities of these six enzymes was not observed in a mutant unable to be induced with glycerol. During the first 2 to 4 h of induction and during the first hour of germination, the level of these enzymes decreased to the level characteristic of vegetative cells. It is suggested that the six enzymes are responsible for the increased conversion of fructose 1,6-diphosphate to UDP-N-acetylgalactosamine, the major precursor of the myxospore coat.

The conservation of DNA sequences over very long periods of evolutionary time. Evidence against intergeneric chromosomal transfer as an explanation for the presence of Escherichia coli tuf gene sequences in taxonomically-unrelated prokaryotes.

In the present study we tried to determine whether the presence of DNA sequences homologous to the Escherichia coli tuf gene (encodes peptide chain elongation factor Tu) in many taxonomically-unrelated prokaryotes is due to selective pressure for these sequences or due to the transfer of chromosomal material subsequent to the divergence of the genera from their progenitors. We found that the degree of sequence homology to the DNA immediately adjacent to the E. coli tuf A gene is either nonexistent or much less than that found for the tuf gene. Furthermore, the tuf-homologous sequences present in one prokaryote were found to be in large part the same as or a subset of those present in others. That is, various prokaryotes share a common subset of tuf-homologous sequences. These findings suggest that strong selective pressure and not recent intergeneric chromosomal transfer is responsible for the ubiquitous presence of certain tuf-homologous sequences. Because the genetic code is degenerate, DNA sequence need not be conserved to conserve protein sequence. Therefore, if the only function of these sequences is to encode protein, their persistence must mean that in some instances codon sequence is selected for.

Ovine corticotrophin-releasing factor in dogs: dose-response relationships and effects of dexamethasone.

Dose-response relationships between iv bolus injections (0, 0.1, 1 or 10 micrograms/kg) of synthetic ovine corticotropin-releasing factor (oCRF) and plasma immunoreactive (i) ACTH and cortisol concentrations were examined in healthy, conscious dogs. All doses of oCRF resulted in elevated plasma iACTH and cortisol levels over those of the controls. Maximum (or Peak) plasma iACTH concentrations were generally observed 20-30 min after oCRF and the magnitude of these peaks was a linear function (P less than 0.001) of the logarithm of the oCRF dose. The time of peak cortisol concentrations was more variable but the peak cortisol level was also linearly related (P less than 0.001) to the logarithm of the oCRF dose. An estimate for the response areas for both hormones demonstrated a quadratic (P less than 0.05) relationship with the logarithm of the oCRF dose. The relationship between oCRF and the iACTH response suggested a progressively greater response at increasing oCRF doses while a maximally effective oCRF dose was predicted in the cortisol response area relationship. Graded (0, 0.01, 0.1 or 1 mg/kg) bolus doses of dexamethasone produced a dose-dependent (P less than 0.03) decline in baseline plasma iACTH levels and a non-dose-dependent suppression in baseline plasma cortisol. Pretreatment with 0.001 mg dexamethasone/kg 4 or 8 h before injection of 1 microgram oCRF/kg did not alter the plasma iACTH or cortisol response; however, 0.1 mg dexamethasone/kg administered at these times totally abolished the responses to oCRF.(ABSTRACT TRUNCATED AT 250 WORDS)

Reproducibility of leucocyte migration from agarose microdroplets.

The 18-hour migration of leucocytes from 31 healthy persons was measured in 33 experiments by the agarose microdroplet method using 15-26 replicate droplets in separate culture wells in each experiment. A comparison of the measurement of the migration of leucocytes by a formula derived from two diameters of the droplets and by planimetry of the projected image of droplets, demonstrated the same wide range of coefficients of variation for the two methods and therefore no important difference in their accuracy. In using a ratio, the migration index, to express the size of differences between experimental groups of cultures, the correct choice of cut-off value demands knowledge of the distribution of that ratio amongst cultures no inhibited by the inclusion of "antigen" or other agent. This could not be ascertained from the observed data because of the practical limitation on the number of samples obtainable in each test. A computer simulation of the sampling process was used to determine this distribution and to evaluate statistical cut-off points. The size of the sample required for conventional degrees of accuracy is shown to vary according to the size of the mean migration area and its standard deviation in unstimulated control cultures. In the study 25/33 (76%) experiments would have been adequately assessed if 30 replicates had been used in every case; if 5 replicates had been used only 2/33 (6%) experiments would have been controlled. In the literature upon leucocyte migration, only 3 or 4 replicates of the control and test cultures are generally described and a cut-off migration index of 0.8 is usually accepted arbitrarily. Our observations indicate that this is an inefficient way of assessing data in such a variable system.

Variability of in vitro migration of human leucocytes.

A comparison of the capillary method (CM) and the agarose microdroplet method (AMM), using the same materials, for measurement of human leucocyte migration in vitro yielded similar large degrees of variability. Between fifteen and twenty-six replicate cultures were established for each method in fourteen subjects The range of the coefficients of variation were .7-28.8% (CM) and 7.2-19.3% AMM). Most of 115 publications reviewed describe only three or four replicates. Their results relating to host responses in the fields of cancer, tissue transplantation and several chronic diseases should be interpreted with regard to the variability of the system. The use of human leucocyte migration in vitro for any purpose should incorporate more stringent statistical control of sample size than has been customary. The use of two standard statistical formulae is proposed as this control.

Tyrosine 3-hydroxylase in rat brain and adrenal medulla: hybridization histochemistry and immunohistochemistry combined with retrograde tracing.

Rat brain and adrenal gland were analyzed by hybridization histochemistry using an RNA probe complementary to mRNA for tyrosine 3-hydroxylase (TyrOHase; tyrosine 3-monooxygenase, EC 1.14.16.2), by immunohistochemistry using TyrOHase antiserum, and by retrograde tracing using the fluorescent compound Fast blue. Cell bodies in the ventral mesencephalon contained mRNA for TyrOHase, and these cells were also TyrOHase immunoreactive. After injection of Fast blue into the striatum, such double-labeled cells in addition contained the retrograde tracer, showing that these cells send axonal projections to the injection site. These results show that hybridization histochemistry can be used to identify transmitter-specific neuron populations and that their projections can be established.