Effect of nifedipine on the contractile responses of the isolated rat bladder

The effect of the calcium channel blocker nifedipine on the motor transmission in isolated preparations of rat detrusor smooth muscle has been studied. Nifedipine blocked the major part (75 to 80%) of the contractile response to electrical field stimulation, while atropine only blocked 20 to 25%. In preparations pretreated with atropine, the response to electrical field stimulation was completely abolished by nifedipine. The converse was also true; in preparations pretreated with nifedipine the response was fully blocked by atropine. The nifedipine-resistant response was greatly potentiated by the anticholinesterase eserine. The blocking action of nifedipine on motor transmission was partially antagonised by raising Ca2(+)-concentration. Acetylcholine concentration-response curve was shifted to the right by nifedipine. It is concluded that the non-cholinergic motor neurotransmitter evokes contraction of the rat detrusor smooth muscle by activating external Ca2(+)-transport channels whereas the cholinergic contraction is mediated partly or wholly by alternative mechanisms.     

A persisting secular trend for body measurements in Dutch children. The Oosterwolde 11 study

To investigate whether the secular trend for growth in Dutch children still exists, the Oosterwolde I study of 1980 was repeated in 1989. A persisting secular trend was visible for height while the z scores of body proportions show no change during the past 10 years, which suggests that there is no change in the timing of puberty.     

The role of TNF-alpha in fever: opposing actions of human and murine TNF-alpha and interactions with IL-beta in the rat.

1. The role of tumour necrosis factor-alpha (TNF-alpha) in fever is controversial. Some studies have indicated that TNF-alpha acts as a cryogen to inhibit fever, while others suggest that TNF-alpha is an endogenous pyrogen which mediates fever. The majority of studies in experimental animals supporting a cryogenic action have been conducted using human (h)TNF-alpha, which has been shown to bind only to one (p55) of the two TNF-alpha receptors in rodents. 2. The aim of the present investigation was to study the role of TNF-alpha in fever by comparing effects of hTNF-alpha, which binds only to the p55 receptor, with those of murine (m) TNF-alpha, which binds to both p55 and p75 TNF-alpha receptors, and to investigate the relationship between TNF-alpha and interleukin-1 (IL-1), an important endogenous pyrogen. 3. Injection of hTNF-alpha (0.3-10 micrograms kg-1, i.p.) had no effect on core temperature in conscious rats (measured by remote radiotelemetry), whereas mTNF-alpha (3 micrograms kg-1) induced fever which was maximal 1 h after the injection (38.2 +/- 0.2 degrees C compared to 37.3 +/- 0.1 degrees C in controls). Intracerebroventricular (i.c.v.) administration of either form of TNF-alpha elicited dose-dependent fever at doses higher than 0.12 microgram kg-1. 4. Peripheral injection of hIL-1 beta (1 microgram kg-1) resulted in fever (38.3 +/- 0.2 degree C compared to 37.2 +/- 0.1 degrees C in controls at 2 h), which was significantly attenuated (P < 0.01) by co-administration of a sub-pyrogenic dose of hTNF-alpha (1 microgram kg-1), but was unaffected by co-administration of mTNF-alpha (0.1 or 0.3 microgram kg-1, i.p.). In contrast, intracerebroventricular (i.c.v.) co-administration of a sub-pyrogenic dose (0.12 microgram kg-1) of hTNF-alpha did not attenuate fever induced by intraperitoneal (i.p.) injection of IL-1 beta, and sub-pyrogenic dose (0.12 microgram kg-1, i.c.v.) of mTNF-alpha significantly prolonged the febrile response to IL-1 beta. Pretreatment of animals with anti-TNF-alpha antiserum (i.c.v.) did not affect the febrile response to systemic IL-1 beta. 5. Animals injected i.p. with a pyrogenic dose of mTNF-alpha developed fever (38.2 +/- 0.2 degrees C compared to 37.3 +/- 0.1 degrees C in controls 2 h after the injection) that was completely abolished by peripheral administration of IL-1ra (2 mg kg-1, P < 0.001), while i.c.v. administration of IL-1ra (400 micrograms/rat) did not affect mTNF-alpha-induced fever. 6. These data indicate that endogenous TNF-alpha is probably a pyrogen and that previous results suggesting cryogenic actions of TNF-alpha resulted from the use of a heterologous protein in the rat. The markedly contrasting effects of mTNF-alpha and TNF-alpha could result from different interactions with the two TNF-alpha receptor subtypes. The data also suggest that fever induced by exogenous TNF-alpha is mediated via release of IL-1 beta in peripheral tissues, but not in the brain.     

A retrospective study to determine the risk of red cell alloimmunization and transfusion during pregnancy

A retrospective review of post-delivery antibody records was performed at a teaching hospital and a community hospital to determine the frequency of new red cell alloantibody production and transfusion during pregnancy. If alloantibody was undetected at delivery, it was assumed that alloimmunization had not occurred. When antibody was detected, a chart review was performed to determine if the antibody was present at the beginning of the pregnancy or was newly produced during the pregnancy. A total of 17,568 pregnancies were reviewed. Antibody was detected at delivery in 948 (5.4%) cases, of which 89.5 percent (848/948) involved passive anti-D or clinically insignificant antibodies. The remaining 100 pregnancies involved clinically significant IgG antibodies. In 58 pregnancies, the antibody was detected in the first trimester, and in 42, new antibody production occurred during the pregnancy. Thus, the prevalence of new antibody production during pregnancy was 0.24 percent (95% confidence interval [CI], 0.17-0.32). Transfusion records indicated that the prevalence of transfusions during pregnancy was 0.09 percent (95% CI, 0.04-0.14). None of the women with new alloantibody formation during their pregnancies required transfusion; hence, new alloantibody production and the need for transfusion appear to be independent events. The probability of these events occurring together was 2.1 × 10(-6), or 1 in 500,000 deliveries.     

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5–10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular condition that afflicts as many as 1 of 350 males and 420 females over the age of 18 (1). In ALS, degeneration of upper and lower motor neurons causes progressive muscle paralysis and spasticity, affecting mobility, speech, swallowing, and respiration (2). Half of affected individuals die within 3 y, and less than 20% survive for more than 5 y (3); 90–95% of ALS cases are sporadic (SALS) in which some apparently facilitating gene mutations, such as repeat expansions in the gene that encodes ataxin-2 (4), have been identified. The remaining 5–10% of ALS cases are familial (FALS) and predominantly associated with Mendelian-inherited mutations in the genes encoding Cu/Zn superoxide dismutase (SOD1), TAR-DNA–binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/TLS), C9ORF72, and other genes (reviewed in ref. 3).Despite the profusion of functionally diverse genes implicated in FALS and SALS, clinical and pathological similarities between all forms of ALS suggest the existence of a common pathogenic pathway that could be united by a single gene/protein (5). One of the mechanisms by which a mutant or wild-type (WT) protein can dominate pathogenesis of phenotypically diverse diseases is by propagated protein misfolding, such as that underpinning the prion diseases, which has been increasingly implicated in other neurodegenerative and systemic disorders (6, 7). A role for propagated protein misfolding in ALS is supported by the prion-like spatiotemporal progression of disease through the neuroaxis (8, 9). However, given the disparity in protein inclusion pathology between subtypes of ALS, a single unifying prion-like protein that could explain such a progression remains obscure.Whereas it is generally accepted SOD1 is not found in large perikaryal cytoplasmic inclusions outside of SOD1 FALS cases, misfolded SOD1 has been increasingly identified in SALS and non-SOD1 FALS (5, 10, 11). Indeed, we have reported that misfolded human wild-type SOD1 (HuWtSOD1) can be detected by spinal cord immunohistochemistry (IHC) in FALS secondary to FUS mutation, and in SALS patients with cytosolic WT TDP-43 accumulation (11). Moreover, in cell models, overexpression of WTTDP-43, or expression of mutant FUS or TDP-43, is associated with HuWtSOD1 misfolding (11). Collectively, these data are consistent with SOD1 being a molecular common denominator for all types of ALS. Furthermore, prion-like activity has been described for the cell-to-cell transmission of misfolding of mutant SOD1 (12), and we have reported that mutant SOD1 can confer its misfold on HuWtSOD1 (13). However, mutant SOD1 cannot explain propagation in SALS.To test if HuWtSOD1 participates in cell-to-cell transmission of protein misfolding, we make use of previously developed mouse mAb probes for misfolded/oxidized SOD1, recognizing either full-length human mutant or WT SOD1, generated against regions that are antibody-inaccessible in natively folded SOD1 (13–15). Misfolded SOD1 mAbs used in this work are 10E11C11 and 3H1, directed against an unstructured electrostatic loop [disease-specific epitope-2 (DSE2)], and 10C12, directed against a C-terminal dimer interface peptide in which the cysteine at position 146 is substituted by a cysteic acid residue to mimic oxidation of this residue (DSE1a) (13). The use of such antibody probes have enabled us to unambiguously determine the role of misfolded mutant G127X in the induced misfolding of HuWtSOD1, which upon misfolding acquires a marked increase in sensitivity to protease digestion, consistent with global loosening of structure (13). The finding that misfolded endogenous HuWtSOD1 was observed long after transfected G127X-SOD1 was degraded suggested that HuWtSOD1, once misfolded, is capable of triggering an intracellular propagated misfolding reaction (13). We now report for the first time that misfolded HuWtSOD1 can transit cell to cell both via exosomes, and release of protein aggregates and subsequent uptake in neuronal cells. In addition, misfolded HuWtSOD1 can sustain intercellular propagated misfolding in vitro and is detectable in the spinal cord of all ALS patients tested, regardless of the genetic etiology of the disease. Collectively, these data indicate that HuWtSOD1 is competent to participate in propagated misfolding, suggesting a common pathogenic mechanism linking FALS and SALS.