Anticonvulsant effect of FS-1 subfraction isolated from roots of Delphinim denudatum on hippocampal pyramidal neurons

The effects were investigated of a partially purified subfraction (FS-1) isolated from Delphinium denudatum on sustained repetitive firing (SRF) of cultured neonatal rat hippocampal pyramidal neurons. The blockade of sustained repetitive firing is one of the basic mechanisms of antiepileptic drugs at the cellular level. Using the whole cell current-clamp technique, sustained repetitive firing was elicited in pyramidal neurons under study by a depolarizing pulse of 500 ms duration, 0.3 Hz and 0.1-0.6 nA current strength. FS-1 (0.01-0.06 mg/mL) reduced the number of action potentials per pulse in a dose-dependent manner until no action potentials were elicited for the remainder of the pulse. There was a corresponding use-dependent reduction in amplitude and Vmax of action potentials. The Vmax of action potential 1 exhibited a dose-dependent reduction. At a dose of 0.06 mg/mL FS-1 reduced Vmax to 29%-38% and amplitude to 16%-20 % of the control values. The blockade of sustained repetitive firing by FS-1 was reversed by hyperpolarization of the membrane potential (-65 to -75 mV) while depolarization of the membrane potential (-53 mV to -48 mV) potentiated the block. The results suggest that FS-1 blocks sustained repetitive firing in hippocampal neurons in a use-dependent and voltage-dependent manner similar to the prototype anticonvulsant drug, phenytoin. However, unlike phenytoin, which binds preferably to the inactive state, the compounds present in FS-1 also interacted with the resting state of the Na+ channels by reducing Vmax of action potential 1. The results indicate that the partially purified FS-1 subfraction of Delphinium denudatum contains a potent anticonvulsant compound.     

Renal tubular function in β-thalassemia

Studies of the renal involvement in thalassemic syndromes have been varied and few. This study was designed to define the renal abnormalities associated with β-thalassemia and to correlate the renal findings with clinical parameters. One hundred and four β-thalassemic children with various disease severity were studied. The patients were divided into three groups: 48 with severe anemia [hematocrit (Hct) <25%], 31 on a hypertransfusion program and desferrioxamine treatment, and 25 with moderate anemia (Hct >25%). The results were compared with 15 normal children. Significantly higher levels of proteinuria and low molecular weight proteinuria were found in all patients compared with normal children. Aminoaciduria was detected in one-third of patients. Thalassemic patients had significantly lower morning urine osmolarity, higher urine N-acetyl-β-D-glucoseminidase and malondialdehyde (MDA, an indicator of lipid peroxidation). Patients with severe anemia had significantly higher low-molecular weight proteinuria and MDA, and lower urine osmolarity than those with moderate anemia. Our data confirmed the high frequency of renal abnormalities in β-thalassemia patients and indicated some degree of proximal tubular dysfunction. Severity of the abnormalities correlated with the degree of anemia and were least severe in patients on hypertransfusion and desferrioxamine therapy. This suggested that the damage might be caused by anemia and increased oxidation induced by excess iron deposits. Received April 5, 1997; received in revised form November 6, 1997; accepted November 12, 1997     

Inhibition of HeLa cells metastasis by bioactive compounds in crocodile (Crocodylus siamensis) white blood cells extract

Matrix metalloproteinases (MMPs) play a key role in cancer progression, including cell invasion, metastasis, cell growth, apoptosis, angiogenesis, and cell adhesion. Thus, suppression of the MMPs activities is crucial for inhibiting cancer cells metastasis. Herein, bioactive agents from crocodile (Crocodylus siamensis) leukocyte extracts (WBCex) showed the anticancer activity with HeLa cells and inhibited the migration and invasion process by reducing gelatinases (MMP‐2, MMP‐9) activity and their protein levels. This mechanism is regulated via interfering Ras and p38 signal transduction. Moreover, disrupting VEGF and integrin‐signaling cascade by bioactive agents are the predictable mechanisms that cause the decreasing of MMP‐2 and MMP‐9 activity. Hence, bioactive substances in WBCex may play the mode of action similar with MMPs inhibitor due to HeLa cell metastasis being suppressed in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1329–1336, 2016.     

Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts

Four novel antibacterial peptides, Leucrocin I-IV from Siamese crocodile white blood cell extracts were purified by reverse phase high performance liquid chromatography (RP-HPLC). Leucrocins exhibit strong antibacterial activity towards Staphylococcus epidermidis, Salmonella typhi and Vibrio cholerae. The peptides were 7-10 residues in length with different primary structure. The amino acid sequence of Leucrocin I is NGVQPKY with molecular mass around 806.99 Da and Leucrocin II is NAGSLLSGWG with molecular mass around 956.3 Da. Further, the interaction between peptides and bacterial membranes as part of their killing mechanism was studied by fluorescence and electron microscopy. The outer membrane and cytoplasmic membrane was the target of action of Leucrocins as assayed in model membrane by release of β-galactosidase due to the membrane permeabilization. Finally, the hemolytic effect was tested against human red blood cell. Leucrocin I, III and IV showed less toxicity against human red blood cells than Leucrocin II.     

The novel antiepileptic drug carisbamate (RWJ 333369) is effective in inhibiting spontaneous recurrent seizure discharges and blocking sustained repetitive firing in cultured hippocampal neurons

This study was initiated to investigate effects of the novel neuromodulator carisbamate (RWJ 333369) in the hippocampal neuronal culture model of status epilepticus and spontaneous epileptiform discharges. Whole-cell current clamp techniques were used to determine the effects of carisbamate on spontaneous recurrent epileptiform discharges (SREDs, in vitro epilepsy), depolarization-induced sustained repetitive firing (SRF) and low Mg²⁺-induced continuous high frequency spiking (in vitro status epilepticus). This in vitro model is an important tool to study the effects of anticonvulsant drugs (AEDs) on SREDs that occur for the life of the neurons in culture. Carisbamate dose dependently blocked the expression and reoccurrence of SREDs. The ED₅₀ value for its antiepileptic effect was 58.75 ± 2.43 μM. Inhibition of SRF is considered a common attribute of many AEDs. Carisbamate (100 μM) significantly decreased SRF in hippocampal neurons. All these effects of carisbamate were reversed during a 5 to 30 min drug washout period. When exposed to low Mg²⁺ medium cultured hippocampal neurons exhibit high frequency spiking. This form of in vitro status epilepticus is not effectively blocked by conventional AEDs that are known to be effective in treating status epilepticus in humans. Carisbamate, like phenytoin and phenobarbital, had little or no effect on low Mg²⁺-induced continuous high frequency spiking. These results characterize the effects of carisbamate in the hippocampal neuronal culture model of epileptiform discharges and suggest that the ability of carisbamate to inhibit depolarization-induced SRF may account in part for some of it's anticonvulsant effect.     

Traumatic brain injury causes a long-lasting calcium (Ca2+)-plateau of elevated intracellular Ca levels and altered Ca2+ homeostatic mechanisms in hippocampal neurons surviving brain injury

Traumatic brain injury (TBI) survivors often suffer chronically from significant morbidity associated with cognitive deficits, behavioral difficulties and a post-traumatic syndrome and thus it is important to understand the pathophysiology of these long-term plasticity changes after TBI. Calcium (Ca2+) has been implicated in the pathophysiology of TBI-induced neuronal death and other forms of brain injury including stroke and status epilepticus. However, the potential role of long-term changes in neuronal Ca2+ dynamics after TBI has not been evaluated. In the present study, we measured basal free intracellular Ca2+ concentration ([Ca2+](i)) in acutely isolated CA3 hippocampal neurons from Sprague-Dawley rats at 1, 7 and 30 days after moderate central fluid percussion injury. Basal [Ca2+](i) was significantly elevated when measured 1 and 7 days post-TBI without evidence of neuronal death. Basal [Ca2+](i) returned to normal when measured 30 days post-TBI. In contrast, abnormalities in Ca2+ homeostasis were found for as long as 30 days after TBI. Studies evaluating the mechanisms underlying the altered Ca2+ homeostasis in TBI neurons indicated that necrotic or apoptotic cell death and abnormalities in Ca2+ influx and efflux mechanisms could not account for these changes and suggested that long-term changes in Ca2+ buffering or Ca2+ sequestration/release mechanisms underlie these changes in Ca2+ homeostasis after TBI. Further elucidation of the mechanisms of altered Ca2+ homeostasis in traumatized, surviving neurons in TBI may offer novel therapeutic interventions that may contribute to the treatment and relief of some of the morbidity associated with TBI.     

Carisbamate prevents the development and expression of spontaneous recurrent epileptiform discharges and is neuroprotective in cultured hippocampal neurons

Purpose: Although great advances have been made in the development of treatments for epilepsy, acquired epilepsy following brain injury still comprises approximately 50% of all the cases of epilepsy. Thus, development of drugs that would prevent or decrease the onset of epilepsy following brain injury represents an important area of research. Methods: Here, we investigated effects of carisbamate (RWJ 333369) on the development and expression of spontaneous recurrent epileptiform discharges (SREDs) and its neuroprotective potential in cultured hippocampal neurons. This model utilizes 3 h of low Mg²⁺ treatment to mimic status epilepticus (SE‐like) injury in vitro. Following the injury, networks of neurons manifest synchronized SREDs for their life in culture. Neuronal cultures were treated with carisbamate (200 μM) for 12 h immediately after the SE‐like injury. The drug was then removed and neurons were patch clamped 24 h following drug washout. Results: Treatment with carisbamate after neuronal injury prevented the development and expression of epileptiform discharges. In the few neurons that displayed SREDs following carisbamate treatment, there was a significant reduction in SRED frequency and duration. In contrast, phenytoin and phenobarbital, when used in place of carisbamate, did not prevent the development and expression of SREDs. Carisbamate was also effective in preventing neuronal death when administered after SE‐like injury. Conclusions: Carisbamate prevents the development and generation of epileptiform discharges and is neuroprotective when administered following SE‐like injury in vitro and may offer a novel treatment to prevent the development of epileptiform discharges following brain injuries.