Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate transcriptional response.

Pyridostigmine, a carbamate acetylcholinesterase (AChE) inhibitor, is routinely employed in the treatment of the autoimmune disease myasthenia gravis. Pyridostigmine is also recommended by most Western armies for use as pretreatment under threat of chemical warfare, because of its protective effect against organophosphate poisoning. Because of this drug's quaternary ammonium group, which prevents its penetration through the blood-brain barrier, the symptoms associated with its routine use primarily reflect perturbations in peripheral nervous system functions. Unexpectedly, under a similar regimen, pyridostigmine administration during the Persian Gulf War resulted in a greater than threefold increase in the frequency of reported central nervous system symptoms. This increase was not due to enhanced absorption (or decreased elimination) of the drug, because the inhibition efficacy of serum butyryl-cholinesterase was not modified. Because previous animal studies have shown stress-induced disruption of the blood-brain barrier, an alternative possibility was that the stress situation associated with war allowed pyridostigmine penetration into the brain. Here we report that after mice were subjected to a forced swim protocol (shown previously to simulate stress), an increase in blood-brain barrier permeability reduced the pyridostigmine dose required to inhibit mouse brain AChE activity by 50% to less than 1/100th of the usual dose. Under these conditions, peripherally administered pyridostigmine increased the brain levels of c-fos oncogene and AChE mRNAs. Moreover, in vitro exposure to pyridostigmine increased both electrical excitability and c-fos mRNA levels in brain slices, demonstrating that the observed changes could be directly induced by pyridostigmine. These findings suggest that peripherally acting drugs administered under stress may reach the brain and affect centrally controlled functions.

Insulin-sensitive tyrosine kinase is increased in livers of adult obese Zucker rats: correction with prolonged fasting.

Adult obese Zucker rats (fa,fa) are hyperinsulinemic and insulin resistant. Specific insulin binding to crude membranes prepared from livers was 2.8% (per mg protein) in fatty animals compared with 7.9% in homozygous lean (Fa,Fa) and 9.0% in heterozygous lean (Fa,fa) animals. Insulin binding increased in liver membranes from fatty animals after a 72-h fast to 6.4%. The reduced insulin binding in livers from fatty rats was associated with elevated insulin-sensitive tyrosine kinase activity, which fell towards control values after the fast. The elevated tyrosine kinase activity was associated with an increased maximum velocity (Vmax) without a change in Michaelis-Menten constant (Km) for its substrates, ATP and poly(Glu,Tyr)4:1. These findings suggest that, in adult fatty rats, insulin-sensitive tyrosine kinase has increased intrinsic activity. Further, the effect of the prolonged fast on both insulin binding and kinase activity, suggest that in this model environmental factors, and not necessarily a genetic abnormality, may regulate liver insulin receptors and their kinase. Whether the inverse relationship of the kinase and insulin receptor number is the result of a compensatory mechanism remains to be elucidated.

Tissue-specific transcription start site usage in the leader exons of the rat insulin-like growth factor-I gene: evidence for differential regulation in the developing kidney.

The production of insulin-like growth factor-I (IGF-I) in extrahepatic tissues supports both autocrine and paracrine functions and is regulated differently from that in liver, which supports endocrine function. In rat liver, transcription initiation primarily occurs at four distinct, widely separated sites in exon 1 of the IGF-I gene, whereas in exon 2, transcription initiation occurs at a cluster of sites. To understand the molecular basis for tissue-specific regulation of IGF-I gene expression, we have mapped transcription start site usage in the following extrahepatic tissues: testes, lung, kidney, heart, brain, muscle, and stomach, with liver serving as a control. In adult rats, kidney and brain exhibited a pattern of exon 1 transcription similar to that seen in liver, i.e. roughly equivalent use of start sites 2 and 3. In contrast, testes and lung preferentially used start site 3, while stomach, heart, and muscle predominantly used start site 3. Start sites 1 and 4 were used in all tissues at extremely low levels. In those tissues studied in which exon 2 transcripts are expressed (testes, lung, stomach, and kidney), the pattern of exon 2 transcription initiation was identical to that in adult rat liver. During postnatal development, the use of all transcription start sites in exons 1 and 2 was coordinate in lung and stomach. Selection of transcription start sites in the kidney, on the other hand, was subject to regulation during postnatal development. Specifically, within exon 1, start site 3 was expressed constitutively throughout peri- and postnatal development. In contrast, the usage of start site 2 was not detected at late fetal or early postnatal stages, but appeared and rapidly increased only at the stage of weaning. Exon 2 transcripts in kidney also did not appear until the postnatal period. These data suggest tissue-specific and developmentally regulated transcription factors regulating IGF-I promoter activity or, alternatively, tissue-specific and developmental stage-dependent differences in the stability of IGF-I mRNAs resulting from the use of different transcription start sites. These different mRNAs may be of significance in the differential regulation of IGF-I production for autocrine or paracrine function.

Regulation of insulin-like growth factor (IGF) binding protein-5 in the T47D human breast carcinoma cell line by IGF-I and retinoic acid.

The T47D human breast carcinoma cell line has been shown to synthesize insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) and IGF-I receptors, and to exhibit a mitogenic response to exogenous IGF-I. We have used T47D cells to investigate the regulation of IGFBPs by IGF-I and retinoic acid (RA), agents that affect cell proliferation and have been shown to regulate IGFBP levels in other cell types. Exposure of T47D cells to IGF-I resulted in the appearance of IGFBP-2, -4, and -5 in conditioned medium but had no effect on the levels of IGFBPs in Triton X-100-extracted cells. This effect was most pronounced for IGFBP-5 and was also elicited by an IGF-I analog that retains affinity for IGFBPs but not by insulin or IGF analogs that have decreased affinity for IGFBPs. Additionally, this effect was not associated with a change in IGFBP-5 messenger RNA (mRNA) levels; however, the appearance of IGFBP-5 in the conditioned medium was inhibited by an anti-IGF-I receptor antibody (alpha IR-3). RA decreased IGFBP-5 mRNA levels and cell-associated IGFBP-5 in both the presence and absence of IGF-I and inhibited the IGF-I-stimulated secretion of IGFBP-5 into T47D cell conditioned medium. These results suggest that IGF-I increases IGFBP-5 levels in the T47D cell line both through direct interaction with IGFBP-5 as well as through a receptor-mediated process that does not require direct interaction with IGFBPs. The latter results are consistent with an effect of IGF-I on a factor that may modulate an IGFBP protease activity. The inhibitory effect of RA, on the other hand, appears to be due primarily to regulation of IGFBP-5 mRNA levels. Thus, IGFBP-5 accumulation appears to be positively regulated by IGF-I, potentially at the level of susceptibility to proteolysis, and negatively regulated at the level of gene expression by RA.

Regulation of insulin-like growth factor-I receptor gene expression by tumor necrosis factor-alpha and interferon-gamma.

The insulin-like growth factor-I receptor (IGF-l-R) plays a critical role in normal and pathological growth processes. The expression of the IGF-l-R gene is regulated by various stimuli, including hormones and growth factors. We have investigated the molecular mechanisms by which two inhibitory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), regulate IGF-l-R gene expression. TNF-alpha and IFN-gamma reduced the proliferation rates of the osteogenic sarcoma cell line, Saos-2, and the human salivary gland cell line, HSG, in a dose- and time-dependent fashion. This effect was associated with significant reductions in the levels of IGF-l-R mRNA and protein, and with inhibition of IGF-l-R promoter activity, suggesting that TNF-alpha and IFN-gamma affect IGF-l-R gene expression at the transcriptional level. In addition, TNF-alpha significantly decreased IGF-l-R mRNA stability. Combined cytokine treatment inhibited cellular proliferation and promoter activity in an additive manner. Taken together, these results suggest that a novel potential mechanism by which TNF-alpha and IFN-gamma affect cellular proliferation involves suppression of IGF-l-R promoter activity, as well as destabilization of IGF-l-R transcripts.

Insulin and IGF-I stimulate phosphorylation of their respective receptors in intact neuronal and glial cells in primary culture.

Previous studies have shown that insulin and IGF-I bind to their respective receptors and stimulate autophosphorylation of the receptor beta subunits in detergent extracts of neuronal and glial cells. In the present study, intact neuronal and glial cells in primary culture have been utilized to characterize insulin- and IGF-I-stimulated phosphorylation of their receptors. Following [32P]orthophosphate labelling and stimulation by insulin or IGF-I, the cells were solubilized and the phosphorylated receptors were partially purified on wheat germ agglutinin--agarose columns, and immunoprecipitated using anti-phosphotyrosine or anti-insulin receptor antibodies. Insulin stimulated the phosphorylation of its receptor beta subunit (95 kD phosphoprotein) in a dose-dependent manner, within at least 20 seconds in both neuronal and glial cells. Additionally, a 102-kD phosphoprotein was observed in insulin-stimulated neuronal cells. Maximal stimulation of receptor phosphorylation occurred at 1 minute for the glial cells, and 10 minutes for the neuronal cells. IGF-I stimulated the phosphorylation of two phosphoproteins in intact neuronal and glial cells; a 95-kD protein and a 102-kD protein, in a dose-dependent manner. These observations demonstrate that both insulin and IGF-I stimulate the phosphorylation of the beta subunits of their respective receptors in brain cells in a similar fashion to their effects on receptors from nonneural tissues.

Seronegative spondyloarthropathy in familial Mediterranean fever.

To define a possible association between familial Mediterranean fever (FMF) and seronegative spondyloarthropathy (SNSA) and to study features of SNSA in FMF patients, we screened for the presence and manifestations of SNSA in 3,000 FMF patients attending the National Center for FMF in our institution. This population included 160 patients with chronic arthritis, most who suffered from SNSA. Patients were considered to suffer from SNSA if they had chronic arthritis, inflammatory back/neck pain, and sacroiliitis. Patients who had other diseases associated with SNSA were excluded. Eleven patients, nine men and two women, with chronic monoarthritis or oligoarthritis, grade 2 (four patients) or grades 3 to 4 (seven patients), sacroiliitis, and inflammatory back pain met the criteria for diagnosis of SNSA of FMF. These patients were rheumatoid factor (RF) and HLA-B27 negative. In seven patients, spondyloarthropathy developed while they received colchicine, and in four before colchicine. Most patients responded to treatment with nonsteroidal antiinflammatory drugs, but three required second-line agents. These findings suggest that SNSA is one of the musculoskeletal manifestations of FMF that may occur despite colchicine therapy and requires specific treatment.

Colchicine in the treatment of AA and AL amyloidosis.

Colchicine is an effective medication in the prevention and treatment of amyloidosis of familial Mediterranean fever. Its therapeutic effect depends on the stage of renal disease and the drug dose. To evaluate colchicine effect in AA amyloidosis of other diseases and in primary AL amyloidosis, the literature was reviewed. Findings were that (1) the effect of colchicine in reactive amyloidosis has not been methodically studied, but anecdotal reports suggest it may be beneficial; and (2) the results of studies and case reports on the effect of colchicine in primary amyloidosis are conflicting. Because a therapeutic effect of colchicine in primary and reactive amyloidosis has been shown in sporadic cases, a prospective, controlled, multicenter study assessing the effect of colchicine in all types of amyloidosis appears to be justified. Until such a study is available, the addition of colchicine in an appropriate dose to any therapeutic regimen of patients with AA or AL amyloidosis should be considered.

The occurrence, recrudescence, and worsening of asthma in a population of young adults: impact of varying types of occupation.

OBJECTIVE: To describe the rates of exacerbation of existing asthma and incidence of new disease in Israeli men during military service. DESIGN: All 17-year-old Israeli nationals are obliged by law to appear at the Israel Defense Forces (IDF) recruiting office for medical examination. The medical history of army recruits was noted during the 30-month period after their induction into the IDF, and medical examinations were performed by pulmonary specialists in all suspected cases of asthma. The duty status of the soldiers in combat units (CUs), maintenance units (MUs), and clerical tasks was related to their asthma status. RESULTS: Of a total of 59,058 recruits, 1.0% developed asthma during the 30 months of this study; of those in CUs, 1.2% developed asthma; of those in MUs, 0.8% developed asthma; and of those performing clerical tasks, 0.6% developed asthma. The relative risk for developing or worsening of asthma was related to both the preexisting asthma status of the recruit and the environment in which he carried out his military service. The annual incidence of occupational-related asthma in MUs was found to be 800/million: five to six times the rates reported elsewhere. CONCLUSIONS: Service in CUs was associated with an increased frequency of exacerbation of asthma among recruits with previous disease and with the appearance of disease de novo. "Normal" conscripts with a history of childhood asthma are at a higher risk of developing overt asthma when compared to subjects with no such history. We found a 25% relative excess of incident cases of asthma in soldiers posted in MUs compared to those performing clerical tasks [(0.8 to 0.6%)/0.8%]. This difference is probably attributed to the difference in occupational hazards in these categories. Further studies are needed to determine if this represents the elicitation of underlying preexisting airway lability by new work demands or other environmental conditions, or if this represents a new development of airway lability because of specific immune or nonimmune factors.

Quinine alone versus quinine plus a pyrimethamine-sulfadoxine combination in the treatment of Plasmodium faliciparum cerebral malaria.

Fifty-two patients with severe chloroquine resistant Plasmodium falciparum malaria were treated in a randomized double blind study with either quinine and a single dose of pyrimethamine-sulfadoxine (Fansidar) or quinine alone. Although no statistically significant differences were observed, the 25 patients who received both drugs responded faster and had a more favorable outcome (no deaths) when compared to the 27 who received quinine alone (2 deaths).

The interaction of brain insulin receptors with wheat germ agglutinin.

Brain insulin receptors adsorb to and are recoverable from wheat germ agglutinin-agarose (WGA) columns. Similar results are obtained using dissuccinimidyl suberate (DSS)-crosslinked receptors or photo-affinity labeled receptors. WGA can be used for partial purification of brain insulin receptors provided the appropriate WGA preparation is chosen and the optimal ratio of receptor protein to lectin is achieved.

Comparison of the counterregulatory hormone response to semisynthetic human insulin and purified porcine insulin in normal subjects and patients with type I diabetes mellitus.

The counterregulatory hormone responses to semisynthetic human insulin and purified porcine insulin were compared in 20 healthy volunteers (ten men and ten women) and 16 patients (8 men and 8 women) with type I diabetes mellitus (IDDM). In both groups blood glucose fell to similar levels following insulin administration; no difference in counterregulatory hormone response or hypoglycemic awareness was noted when comparing human to porcine insulin. However, when men were compared to women, significant differences were noted in basal glucagon, cortisol, and growth hormone levels, as well as in norepinephrine, prolactin, and cortisol responses to hypoglycemia. These differences could not be attributed to insulin species, different doses of insulin, or degree of hypoglycemia. These findings suggest that hormonal response to and awareness of hypoglycemia are similar in healthy subjects and patients with IDDM following administration of human and porcine insulin and that hormonal responses in men and women should be studied separately to avoid confusion in interpreting results arising from differences in sex.

Tumor necrosis factor alpha and interferon gamma--induced cell growth arrest is mediated via insulin-like growth factor binding protein-3.

Retinoic acid (RA), and the combination of TNFalpha and Interferon (IFN-gamma) inhibit human salivary gland tumor (HSG) cell growth with the combination of all three being even more inhibitory (P<0. 05). Previous studies have demonstrated that these inhibitory effects of RA, and the combination of TNFalpha and IFN-gamma are associated with increased accumulation of IGFBP-3 in the culture medium of HSG cells. Therefore, we set out to determine if the increase in IGFBP-3 was due to increased production of IGFBP-3 by the cells and whether IGFBP-3 played a causative role in the inhibition of cellular proliferation. TNFalpha and IFN-gamma induced a rise in IGFBP-3 mRNA levels between 4 and 8 h, which returned to control levels after 24 h. IGFBP-3 was shown to inhibit HSG cell growth at concentrations of >/=75 U (P<0.05). When antibodies to IGFBP-3 were used with TNFalpha and IFN-gamma, the inhibitory effect of the cytokines on cell growth was diminished. Retinoic acid with TNFalpha and IFN-gamma had a marked inhibitory effect (P<0.05) which was similarly reversed by increasing concentrations of IGFBP-3 antibody. The present data support the hypothesis that the combination of TNFalpha and IFN-gamma with retinoic acid exert their anti-proliferative effect on HSG cells by reducing the mitogenic effect of IGF-I due to a shift in IGF-I from the free to the IGFBP-3-bound form.