Biomedical subjects
M L Wu
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Images in pathology. Placenta: 6 - 4 = 2.
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Atypical hairy cell leukemia.
The morphologic differential diagnosis of mature B-cell neoplasms with cytoplasmic projections includes splenic lymphoma with villous lymphocytes and hairy cell leukemia. Although the classification of hairy cell leukemia is not universally recognized, 3 variants have been described, namely, classic, variant, and Japanese variant, each of which has different clinical and immunophenotypic features. Classic hairy cell leukemia is virtually always CD11c(+), CD25(+), and CD103(+). Variant and Japanese variant hairy cell leukemias are usually CD11c(+), always CD25(-), and occasionally CD103(+). Each variant is characteristically CD10(-). We present a case of hairy cell leukemia with a unique immunoprofile in that the cells were CD10(+), CD25(+), and CD103(-), and we review the criteria helpful in differentiating "hairy" B-cell neoplasms. This case emphasizes the variability of hairy cell leukemia and the need to correlate all clinical and pathologic data in reaching a diagnosis.
Cl--dependent and Cl--independent Na+/ HCO3- acid extrusion in cultured rat cerebellar astrocytes.
It is still uncertain whether the Na+-dependent Cl--HCO3- exchanger (NCBE) is expressed in mammalian astrocytes. Using fluorescent indicators to monitor the intracellular pH (pHi) and intracellular Na+ or Cl- levels, the NCBE in cultured rat cerebellar astrocytes was examined in detail. In nominally bicarbonate-free (Hepes-buffered) medium, a marked pHi recovery from internal acid load was seen which could be blocked completely by 30 microM HOE 694, a specific Na+-H+ exchanger isoform 1(NHE-1) inhibitor, at a pHi above 6.9. These conditions were therefore used to block NHE activity in CO2/HCO3-buffered media when the NCBE was being studied at pHi above 6.9. After internal acid loading in completely Cl--free bicarbonate-buffered medium (containing HOE 694), the rates of pHi recovery and transient Na+ influx were considerably slowed, and the Cl--dependent acid extrusion was both Na+- and 4,4-diisothiocyano-stilbene-disulphonic acid (DIDS)-sensitive. Moreover, a HCO3-dependent Cl- efflux during internal acid injection was seen. These results suggest that the NCBE is present in astrocytes. Following repetitive internal acid loading by addition of 5% CO2 to internal Cl- depleted cells, a similar rate of pHi recovery was consistently seen, suggesting Cl--independent pHi regulation also occurred in astrocytes. Moreover, this pHi recovery was completely blocked in the absence of sodium or on addition of DIDS, confirming that the Na+-HCO3 cotransporter (NBC) is present. Thus, the present study provides evidence that both the NCBE and NBC play important roles in acid extrusion in cultured mammalian astrocytes.
Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease.
The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.
Early metabolic inhibition-induced intracellular sodium and calcium increase in rat cerebellar granule cells.
1. Possible mechanisms responsible for the increases in intracellular calcium ([Ca2+]i) and sodium ([Na+]i) levels seen during metabolic inhibition were investigated by continuous [Ca2+]i and [Na+]i measurement in cultured rat cerebellar granule cells. An initial small mitochondrial Ca2+ release was seen, followed by a large influx of extracellular Ca2+. A large influx of extracellular Na+ was also seen. 2. The large [Ca2+]i increase was not due to opening of voltage-dependent or voltage-independent calcium channels, activation of NMDA/non-NMDA channels, activation of the Na+i-Ca2+o exchanger, or inability of plasmalemmal Ca2+-ATPase to extrude, or mitochondria to take up, calcium. 3. The large [Na+]i increase was not due to activation of the TTX-sensitive Na+ channel, the Na+i-Ca2+o exchanger, the Na+-H+ exchanger, or the Na+-K+-2Cl- cotransporter, or an inability of Na+-K+-ATPase to extrude the intracellular sodium. 4. Phospholipase A2 (PLA2) activation may be involved in the large influx, since both were completely inhibited by PLA2 inhibitors. Moreover, melittin (a PLA2 activator) or lysophosphatidylcholine or arachidonic acid (both PLA2 activation products) caused similar responses. Inhibition of PLA2 activity may help prevent the influx of these ions that may result in serious brain injury and oedema during hypoxia/ischaemia.
Cloning and complete nucleotide sequence of Acinetobacter radioresistens CMC-1 AglyA gene encoding serine hydroxymethyltransferase.
A gene (AglyA) encoding serine hydroxymethyltransferase of Acinetobacter radioresistens CMC-1 was cloned and sequenced. Nucleotide sequence analysis of AglyA predicted a single open reading frame (ORF) of 1251 bp encoding a 417-amino acid polypeptide. Two putative MetR-like binding sites (5'-TGAAACATGAGCT) and (5'-TGAGCAAAGTTCA), centered at bp -123 and -95 relative to the +1 translation start site were found, which have six out of nine and eight out of nine nucleotides that match to the consensus sequence of Escherichia coli (5'-TGAANNT/ANNTTCA), respectively. The enzyme also showed a high level of homology to other sources of serine hydroxymethyltransferase proteins.
Agricultural avermectins: an uncommon but potentially fatal cause of pesticide poisoning.
STUDY OBJECTIVE: Avermectins have been used in the control of parasites and insects; however, human data concerning poisoning are lacking. This study investigated the clinical spectrum of avermectin poisoning. METHODS: A retrospective study was conducted to evaluate patients with avermectin poisoning reported to a poison center from September 1993 through December 1997. RESULTS: Eighteen patients with abamectin (Agri-Mek; 2% wt/wt abamectin) exposure and 1 with ivermectin (Ivomec; 1% wt/vol ivermectin) ingestion were identified. There were 14 male and 5 female patients, ranging in age from 15 to 83 years. Most patients were exposed as a result of attempted suicide (14). Oral ingestion (15) was the most common route of exposure. Four patients were asymptomatic, and 8 had minor symptoms after a mean ingestion of 23 mg/kg abamectin (4.2 to 67 mg/kg), or after dermal and inhalation contact. Seven patients manifested severe symptoms, such as coma (7), aspiration with respiratory failure (4), and hypotension (3), after a mean ingestion of 100.7 mg/kg avermectin (15.4 mg/kg for ivermectin and 114.9 mg/kg for abamectin). All 7 patients received intensive supportive care; 1 patient died 18 days later as a result of multiple organ failure. CONCLUSION: Ingestion of a large dose of avermectin may be associated with life-threatening coma, hypotension, and subsequent aspiration.
Modulation of volume-sensitive Cl - channels and cell volume by actin filaments and microtubules in human cervical cancer HT-3 cells.
Hypotonicity activates volume-sensitive Cl- currents, which are implicated in the regulatory volume decrease (RVD) responses and transport of taurine in human cervical cancer HT-3 cells. In this study, the role of cytoskeleton in the regulation of volume-sensitive Cl- channels and RVD responses in HT-3 cells was studied. Cells were incubated with various compounds, which depolymerized or polymerized cytoskeletal elements, i.e. actin filaments and microtubules. The hypotonicity-induced changes in Cl- conductance and in cell volume were measured by whole-cell voltage clamping and cell size monitoring, respectively. Our results show that in HT-3 cells hypotonicity activated an outward rectified Cl- current that was abrogated by Cl- channel blockers. Cytochalasin B, an actin-depolymerizing compound, induced a substantial increase in Cl- conductance under isotonic condition and potentiated the expression of Cl- currents in hypotonic stress. Phorbol 12-myristate 13-acetate (PMA) significantly inhibited the cytochalasin B-induced activation of Cl- conductance under isotonic condition. On the other hand, treatment with cytochalasin B significantly prolonged the RVD responses. Phalloidin, a stabilizer of actin polymerization, did not change the basal currents under isotonic condition, but completely abolished the increase in whole-cell Cl- conductance elicited by hypotonicity and retarded the cell volume recovery. Colchicine, a microtubule-assembly inhibitor, had no effect on either basal Cl- conductance or volume-sensitive Cl- current and was unable to inhibit the RVD responses. Taxol, a microtubule-stabilizing compound, did not alter the basal Cl- conductance, but inhibited the activation of volume-sensitive Cl- channels as well as the process of RVD in a dose-dependent manner. These data support the notion that functional integrity of actin filaments and microtubules plays critical roles in maintaining the RVD responses and activation of Cl- channels in human cervical cancer HT-3 cells.
A novel effect of cyclic AMP on capacitative Ca2+ entry in cultured rat cerebellar astrocytes.
One of the most important intracellular Ca2+ regulatory mechanisms in nonexcitable cells, "capacitative Ca2+ entry" (CCE), has not been adequately studied in astrocytes. We therefore investigated whether CCE exists in cultured rat cerebellar astrocytes and studied the roles of cyclic AMP (cAMP) and protein kinase C (PKC) in CCE. We found that (1) at least two different intracellular Ca2+ stores, the endoplasmic reticulum and mitochondria, are present in cerebellar astrocytes; (2) CCE does exist in these cells and can be inhibited by Ni2+, miconazole, and SKF 96365; (3) CCE can be directly enhanced by an increase in intracellular cAMP, as 8-bromoadenosine 3',5'-cyclic monophosphate (8-brcAMP), forskolin, and isobutylmethylxanthine have stimulatory effects on CCE; and (4) neither of the two potent protein kinase A (PKA) inhibitors, H8 and H89, nor a specific PKA agonist, Sp-adenosine 3',5'-cyclic monophosphothioate, had a significant effect on cAMP-enhanced Ca2+ entry. The [Ca2+]i increase was not due to a release from calcium stores, hyperpolarization of the membrane potential, inhibition of calcium extrusion, or a change in pHi, suggesting that cAMP itself probably acts as a novel messenger to modulate CCE. We also conclude that activation of PKC results in an increase in CCE. cAMP and PKC seem to modulate CCE by different pathways.
Diverse manifestations of oral methylene chloride poisoning: report of 6 cases.
OBJECTIVE: Methylene chloride is a solvent used in domestic and industrial preparations, such as paint removers and degreasing agents. Although it is considered of low toxicity, acute toxic manifestations have been reported following inhalation of methylene chloride, mainly from working in an enclosed environment. Oral ingestion of methylene chloride, however, remains rare and its consequences are less clearly understood. CASE REPORTS: The various clinical manifestations of 6 patients with oral ingestion of methylene chloride are reported. Central nervous system depression, tachypnea, and corrosive gastrointestinal injury were the most common presentations following ingestion. An elevated carboxyhemoglobin level was documented in only 2 patients (35% and 8.4% carboxyhemoglobin, respectively). Due to a frequent misleading history of "chloroform" ingestion and unawareness of probable carboxyhemoglobin production in these patients, carboxyhemoglobin was not routinely measured. Renal failure, hepatic failure, and acute pancreatitis occurred in the 2 most severe cases of methylene chloride ingestion. CONCLUSIONS: Ingestion of methylene chloride can result in diverse manifestations, including a high carboxyhemoglobin level. Corrosive gastrointestinal injury is common in oral poisoning and needs further therapeutic consideration. A high index of suspicion and appropriate laboratory studies are needed in those patients who allegedly ingest "chloroform" but do not present the incriminating solvent to their treating physicians. The presence of an elevated carboxyhemoglobin level suggests the diagnosis of methylene chloride poisoning. Symptomatic and supportive measures remain the mainstay in the treatment of patients with oral methylene chloride poisoning.
Novel role of the Ca(2+)-ATPase in NMDA-induced intracellular acidification.
The mechanism involved in N-methyl-D-glucamine (NMDA)-induced Ca(2+)-dependent intracellular acidosis is not clear. In this study, we investigated in detail several possible mechanisms using cultured rat cerebellar granule cells and microfluorometry [fura 2-AM or 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM]. When 100 microM NMDA or 40 mM KCl was added, a marked increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and a decrease in the intracellular pH were seen. Acidosis was completely prevented by the use of Ca(2+)-free medium or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca(2+). The following four mechanisms that could conceivably have been involved were excluded: 1) Ca(2+) displacement of intracellular H(+) from common binding sites; 2) activation of an acid loader or inhibition of acid extruders; 3) overproduction of CO(2) or lactate; and 4) collapse of the mitochondrial membrane potential due to Ca(2+) uptake, resulting in inhibition of cytosolic H(+) uptake. However, NMDA/KCl-induced acidosis was largely prevented by glycolytic inhibitors (iodoacetate or deoxyglucose in glucose-free medium) or by inhibitors of the Ca(2+)-ATPase (i.e., Ca(2+)/H(+) exchanger), including La(3+), orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca(2+)-ATPase is involved in NMDA-induced intracellular acidosis in granule cells. We also provide new evidence that NMDA-evoked intracellular acidosis probably serves as a negative feedback signal, probably with the acidification itself inhibiting the NMDA-induced [Ca(2+)](i) increase.
Poisoning by Datura leaves used as edible wild vegetables.
The causes of Datura intoxication include medication overdose, misuse of edible vegetables, deliberate abuse as a hallucinogen, homicidal or robbery and accidental intoxication from contaminated food. We report an incident of 14 people with Datura intoxication caused by ingesting wild Datura suaveolans for food. The incubation period was 15 to 30 min. The symptoms/signs were dizziness, dry mouth, flushed skin, palpitation, nausea, drowsiness, tachycardia, blurred vision, mydriasis, hyperthermia, disorientation, vomiting, agitation, delirium, urine retention, hypertension and coma. Three patients were hospitalized for 2-3 days. Thirteen persons received supportive fluid therapy. One patient did not receive medical therapy, he induced vomiting and drank a lot of water. Four patients presented with delirium/coma and 3 received physostigmine therapy with good response. One patient was intubated because of coma and respiratory depression. Three persons needed Foley catheterization for urine retention or coma status. One patient had a complication of urinary tract infection and antibiotic management. All patients recovered with no sequelae.
Hemodialysis as adjunctive therapy for severe acetaminophen poisoning: a case report.
Acetaminophen overdose is a common intoxication in daily practice the standard treatment is N-acetylcysteine (NAC) antidotal therapy for possible poisoning. However, dialysis procedures can remove the drug from the body effectively. We describe a case of acetaminophen overdose that was treated with both hemodialysis (HD) and NAC due to severe intoxication and slow drug clearance. A 37-year-old woman attempted suicide by ingestion of 100 tablets (500 mg each) of acetaminophen, and presented with vomiting, hematemesis and abdominal pain. The patient had elevated liver enzymes, coagulation defects, thrombocytopenia a high serum acetaminophen level (201 mg/l at 12 hours post-ingestion) with a prolonged half-life. Oral NAC was given; however, it was ineffective due to severe vomiting and hematemesis. HD as adjunctive therapy was initiated at 19 hours post-ingestion. HD reduced the serum acetaminophen level from 102.77 to 35.77 mg/l. Severe hepatic injury, bacteremia and pancytopenia were noted in the following days. The patient later recovered after treatment with NAC, HD and intensive supportive care. HD removed 66% of the total acetaminophen body burden during a single four-hour session, increased the clearance by 2.75-fold and shortened the half-life from 7.2 hours to 2.6 hours during HD. Through NAC therapy is the standard regimen for acetaminophen poisoning, in the severely poisoned patient who cannot tolerate NAC therapy, HD may be used as adjunctive therapy to enhance the elimination of acetaminophen.
A rapidly evolving homeobox at the site of a hybrid sterility gene.
The homeodomain is a DNA binding motif that is usually conserved among diverse taxa. Rapidly evolving homeodomains are thus of interest because their divergence may be associated with speciation. The exact site of the Odysseus (Ods) locus of hybrid male sterility in Drosophila contains such a homeobox gene. In the past half million years, this homeodomain has experienced more amino acid substitutions than it did in the preceding 700 million years; during this period, it has also evolved faster than other parts of the protein or even the introns. Such rapid sequence divergence is driven by positive selection and may contribute to reproductive isolation.
Regulation of presynaptic NMDA responses by external and intracellular pH changes at developing neuromuscular synapses.
NMDA receptors play important roles in synaptic plasticity and neuronal development. The functions of NMDA receptors are modulated by many endogenous substances, such as external pH (pHe), as well as second messenger systems. In the present study, the nerve-muscle cocultures of Xenopus embryos were used to investigate the effects of both external and intracellular pH (pHi) changes on the functional responses of presynaptic NMDA receptors. Spontaneous synaptic currents (SSCs) were recorded from innervated myocyte using whole-cell recordings. Local perfusion of NMDA at synaptic regions increased the SSC frequency via the activation of presynaptic NMDA receptors. A decrease in pHe from 7.6 to 6.6 reduced NMDA responses to 23% of the control, and an increase in pHe from 7.6 to 8.6 potentiated the NMDA responses in increasing SSC frequency. The effect of NMDA on intracellular Ca2+ concentration ([Ca2+]i) was also affected by pHe changes: external acidification inhibited and alkalinization potentiated [Ca2+]i increases induced by NMDA. Intracellular pH changes of single soma were measured by ratio fluorometric method using 2,7-bis (carboxyethyl)-5, 6-carboxyfluorescein (BCECF). Cytosolic acidification was used in which NaCl in Ringer's solution was replaced with weak organic acids. Acetate and propionate but not methylsulfate substitution caused intracellular acidification and potentiated NMDA responses in increasing SSC frequency, intracellular free Ca2+ concentration, and NMDA-induced currents. On the other hand, cytosolic alkalinization with NH4Cl did not significantly affect these NMDA responses. These results suggest that the functions of NMDA receptors are modulated by both pHe and pHi changes, which may occur in some physiological or pathological conditions.