HLA class II immunogenic mutation burden predicts response to immune checkpoint blockade.

BACKGROUND: Whereas human leukocyte antigen (HLA) class I mutation-associated neoantigen burden has been linked with response to immune checkpoint blockade (ICB), the role of HLA class II-restricted neoantigens in clinical responses to ICB is less studied. We used computational approaches to assess HLA class II immunogenic mutation (IMM) burden in patients with melanoma and lung cancer treated with ICB. PATIENTS AND METHODS: We analyzed whole-exome sequence data from four cohorts of ICB-treated patients with melanoma (n = 110) and non-small-cell lung cancer (NSCLC) (n = 123). MHCnuggets, a neural network-based model, was applied to estimate HLA class II IMM burdens and cellular fractions of IMMs were calculated to assess mutation clonality. We evaluated the combined impact of HLA class II germline genetic variation and class II IMM burden on clinical outcomes. Correlations between HLA class II IMM burden and density of tumor-infiltrating lymphocytes were computed from expression data. RESULTS: Responding tumors harbored a significantly higher HLA class II IMM burden for both melanoma and NSCLC (P ≤ 9.6e-3). HLA class II IMM burden was correlated with longer survival, particularly in the NSCLC cohort and in the context of low intratumoral IMM heterogeneity (P < 0.001). HLA class I and II IMM landscapes were largely distinct suggesting a complementary role for class II IMMs in tumor rejection. A higher HLA class II IMM burden was associated with CD4+ T-cell infiltration and programmed death-ligand 1 expression. Transcriptomic analyses revealed an inflamed tumor microenvironment for tumors harboring a high HLA class II IMM burden. CONCLUSIONS: HLA class II IMM burden identified patients with NSCLC and melanoma that attained longer survival after ICB treatment. Our findings suggest that HLA class II IMMs may impact responses to ICB in a manner that is distinct and complementary to HLA class I-mediated responses.

MicroRNA-145 inhibits proliferation and promotes apoptosis of HepG2 cells by targeting ROCK1 through the ROCK1/NF-κB signaling pathway.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a malignant cancer with a high fatality rate, and the expression of microRNA-145 (miR-145) is significantly low in HCC tissue. Therefore, the effect of miR-145 on HCC was explored. PATIENTS AND METHODS: Primary hepatocellular carcinoma samples and corresponding normal samples, and HepG2 cells were analyzed using flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Real-time quantitative reverse transcription-polymerase chain reaction, Western blotting, and dual-luciferase reporter assay. RESULTS: miR-145 expression was significantly downregulated in HCC tissue and HepG2 cells as compared to normal liver tissue. After HepG2 cells were transfected with miR-145 mimics, miR-145 expression was recovered, accompanied by a significantly lower cell number, inhibition of the G1/S phase transition, and promotion of the apoptosis of HepG2 cells, as well as changes in levels of G1/S-specific cyclin-E1 (CCNE1) and activated caspase-3. Furthermore, the rho-associated protein kinase 1 (ROCK1) levels were opposite the levels of miR-145 expression in vivo and in vitro, and additional experiments with co-transfection of miR-145 mimics and pEGFP-N3-3'UTR provided the direct evidence that the ROCK1 gene is a target of miR-145. Moreover, a significant decrease or increase in the expression of ROCK1 was associated with nuclear factor-kB (NF-κB)(p65) activity, and lipopolysaccharide (LPS) significantly increased NF-κB(p65) activity, accompanied by recovery of the reduction in the number of HepG2 cells for miR-145 mimics. The NF-κB activity and cell number were significantly (p < 0.05, p < 0.01) increased in response to the overexpression of the ROCK1 gene in HepG2 cells. CONCLUSIONS: We showed that miR-145 can target and downregulate ROCK1 expression, and it controls HCC by inhibiting the cell cycle and activating apoptosis via the ROCK1/NF-κB signaling pathway. Our findings will provide a new perspective for the therapy of HCC.

GAS5 modulated autophagy is a mechanism modulating cisplatin sensitivity in NSCLC cells.

OBJECTIVE: In this study, we investigated the association between lncRNA GAS5 and cisplatin (DDP) resistance in NSCLC and further studied the regulative effect of GAS5 on autophagy and DDP resistance. PATIENTS AND METHODS: GAS5 expression in cancerous and adjacent normal tissues from 15 NSCLC patients received neoadjuvant chemotherapy and the following surgery were measured using qRT-PCR analysis. GAS5 gain-and-loss study was performed using A549 and A549/DDP cells as an in-vitro model to investigate the effect of GAS5 on autophagy and cisplatin sensitivity. RESULTS: NSCLC tissues had a substantially lower expression of GAS5 than adjacent normal tissues. The NSCLC tissues from patients with progressive disease (PD) had even lower GAS5 expression. GAS5 knockdown increased DDP IC50 of A549 cells, while GAS5 overexpression decreased DDP IC50 of A549/DDP cells. A549/DDP cells had significantly higher basal autophagy than A549 cells. GAS5 knockdown resulted in decreased autophagy in A549 cells, while GAS5 overexpression led to increased autophagy in A549/DDP cells. Treatment with 3-MA, an autophagy inhibitor, significantly decreased DDP IC50 and promoted DDP-induced cell apoptosis in A549 cells. In addition, 3-MA also partly reversed the effect of GAS5 knockdown. In A549/DDP cells, GAS5 showed the similar effect as 3-MA in reducing DPP IC50 and promoting DDP-induced apoptosis and also presented synergic effect with 3-MA. CONCLUSIONS: GAS5 downregulation is associated with cisplatin resistance in NSCLC. GAS5 can inhibit autophagy and therefore enhance cisplatin sensitivity in NSCLC cells.

Cholinergic neurotransmission in the preBötzinger Complex modulates excitability of inspiratory neurons and regulates respiratory rhythm.

We investigated whether there is endogenous acetylcholine (ACh) release in the preBötzinger Complex (preBötC), a medullary region hypothesized to contain neurons generating respiratory rhythm, and how endogenous ACh modulates preBötCneuronal function and regulates respiratory pattern. Using a medullary slice preparation from neonatal rat, we recorded spontaneous respiratory-related rhythm from the hypoglossal nerve roots (XIIn) and patch-clamped preBötC inspiratory neurons. Unilateral microinjection of physostigmine, an acetylcholinesterase inhibitor, into the preBötC increased the frequency of respiratory-related rhythmic activity from XIIn to 116+/-13% (mean+/-S.D.) of control. Ipsilateral physostigmine injection into the hypoglossal nucleus (XII nucleus) induced tonic activity, increased the amplitude and duration of the integrated inspiratory bursts of XIIn to 122+/-17% and 117+/-22% of control respectively; but did not alter frequency. In preBötC inspiratory neurons, bath application of physostigmine (10 microM) induced an inward current of 6.3+/-10.6 pA, increased the membrane noise, decreased the amplitude of phasic inspiratory drive current to 79+/-16% of control, increased the frequency of spontaneous excitatory postsynaptic currents to 163+/-103% and decreased the whole cell input resistance to 73+/-22% of control without affecting the threshold for generation of action potentials. Bath application of physostigmine concurrently induced tonic activity, increased the frequency, amplitude and duration of inspiratory bursts of XIIn motor output. Bath application of 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 2 microM), a M3 muscarinic acetylcholine receptor (mAChR) selective antagonist, increased the input resistance of preBötC inspiratory neurons to 116+/-9% of control and blocked all of the effects of physostigmine except for the increase in respiratory frequency. Dihydro-beta-erythroidine (DH-beta-E; 0.2 microM), an alpha4beta2 nicotinic receptor (nAChR) selective antagonist, blocked all the effects of physostigmine except for the increase in inspiratory burst amplitude. In the presence of both 4-DAMP and DH-beta-E, physostigmine induced opposite effects, i.e. a decrease in frequency and amplitude of XIIn rhythmic activity. These results suggest that there is cholinergic neurotransmission in the preBötC which regulates respiratory frequency, and in XII nucleus which regulates tonic activity, and the amplitude and duration of inspiratory bursts of XIIn in neonatal rats. Physiologically relevant levels of ACh release, via mAChRs antagonized by 4-DAMP and nAChRs antagonized by DH-beta-E, modulate the excitability of inspiratory neurons and excitatory neurotransmission in the preBötC, consequently regulating respiratory rhythm.

One-third of term babies after perinatal hypoxia-ischaemia have transient hearing impairment: dynamic change in hearing threshold during the neonatal period.

AIM: To examine the process of change in hearing threshold during the neonatal period after perinatal hypoxia-ischaemia. METHODS: The threshold of brainstem auditory evoked responses (BAER) was measured serially during the first month after birth in 92 term babies who suffered hypoxia-ischaemia. RESULTS: The mean BAER threshold in these babies was significantly increased on day 1 (ANOVA p < 0.001). The elevated threshold decreased progressively on days 3 and 5, but was still significantly higher than that in normal controls (p < 0.01). The elevation continuously decreased more slowly on days 10 and 15, and to a near normal level on day 30. Threshold elevation was seen in 31.7% of the babies on day 1, and 34.5% during the first three days. The rate of elevation decreased progressively thereafter. On day 30, 10.6% of the subjects still had increased thresholds. Moderate to severe elevation occurred mainly during the first week and severe elevation occurred predominately on day 1. Threshold elevation starting after days 3-5 is likely to be due to middle-ear disorders. As a whole, during the first month, 44.6% (41/92) of the babies studied had threshold elevation. BAER threshold was correlated weakly with the stage of hypoxic-ischaemic encephalopathy on days 1 and 3. The threshold was significantly higher in babies with severe encephalopathy than in those with mild or moderate encephalopathy during the first 3 d of life. CONCLUSION: Hearing threshold is elevated in about one-third of term babies after hypoxia-ischaemia. The elevated threshold decreases progressively after birth, and returns to normal by one month in most babies. The threshold correlates weakly with the severity of encephalopathy.

RT-PCR reveals muscarinic acetylcholine receptor mRNA in the pre-Bötzinger complex.

Muscarinic receptors mediate the postsynaptic excitatory effects of acetylcholine (ACh) on inspiratory neurons in the pre-Bötzinger complex (pre-BötC), the hypothesized site for respiratory rhythm generation. Because pharmacological tools for identifying the subtypes of the muscarinic receptors that underlie these effects are limited, we probed for mRNA for these receptors in the pre-BötC. We used RT-PCR to determine the expression of muscarinic receptor subtypes in tissue punches of the pre-BötC taken from rat medullary slices. Cholinergic receptor subtype M(2) and M(3) mRNAs were observed in the first round of PCR amplification. All five subtypes, M(1)-M(5), were observed in the second round of amplification. Our results suggest that the majority of muscarinic receptor subtypes in the pre-BötC are M(2) and M(3), with minor expression of M(1), M(4), and M(5).

Mechanisms underlying regulation of respiratory pattern by nicotine in preBötzinger complex.

Cholinergic neurotransmission plays a role in regulation of respiratory pattern. Nicotine from cigarette smoke affects respiration and is a risk factor for sudden infant death syndrome (SIDS) and sleep-disordered breathing. The cellular and synaptic mechanisms underlying this regulation are not understood. Using a medullary slice preparation from neonatal rat that contains the preBötzinger Complex (preBötC), the hypothesized site for respiratory rhythm generation, and generates respiratory-related rhythm in vitro, we examined the effects of nicotine on excitatory neurotransmission affecting inspiratory neurons in preBötC and on the respiratory-related motor activity from hypoglossal nerve (XIIn). Microinjection of nicotine into preBötC increased respiratory frequency and decreased the amplitude of inspiratory bursts, whereas when injected into XII nucleus induced a tonic activity and an increase in amplitude but not in frequency of inspiratory bursts from XIIn. Bath application of nicotine (0.2--0.5 microM, approximately the arterial blood nicotine concentration immediately after smoking a cigarette) increased respiratory frequency up to 280% of control in a concentration-dependent manner. Nicotine decreased the amplitude to 82% and increased the duration to 124% of XIIn inspiratory bursts. In voltage-clamped preBötC inspiratory neurons (including neurons with pacemaker properties), nicotine induced a tonic inward current of -19.4 +/- 13.4 pA associated with an increase in baseline noise. Spontaneous excitatory postsynaptic currents (sEPSCs) present during the expiratory period increased in frequency to 176% and in amplitude to 117% of control values; the phasic inspiratory drive inward currents decreased in amplitude to 66% and in duration to 89% of control values. The effects of nicotine were blocked by mecamylamine (Meca). The inspiratory drive current and sEPSCs were completely eliminated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the presence or absence of nicotine. In the presence of tetrodotoxin (TTX), low concentrations of nicotine did not induce any tonic current or any increase in baseline noise, nor affect the input resistance in inspiratory neurons. In this study, we demonstrated that nicotine increased respiratory frequency and regulated respiratory pattern by modulating the excitatory neurotransmission in preBötC. Activation of nicotinic acetylcholine receptors (nAChRs) enhanced the tonic excitatory synaptic input to inspiratory neurons including pacemaker neurons and at the same time, inhibited the phasic excitatory coupling between these neurons. These mechanisms may account for the cholinergic regulation of respiratory frequency and pattern.

Electrical coupling and excitatory synaptic transmission between rhythmogenic respiratory neurons in the preBötzinger complex.

Breathing pattern is postulated to be generated by brainstem neurons. However, determination of the underlying cellular mechanisms, and in particular the synaptic interactions between respiratory neurons, has been difficult. Here we used dual recordings from two distinct populations of brainstem respiratory neurons, hypoglossal (XII) motoneurons, and rhythmogenic (type-1) neurons in the preBötzinger complex (preBötC), the hypothesized site for respiratory rhythm generation, to determine whether electrical and chemical transmission is present. Using an in vitro brainstem slice preparation from newborn mice, we found that intracellularly recorded pairs of XII motoneurons and pairs of preBötC inspiratory type-1 neurons showed bidirectional electrical coupling. Coupling strength was low (<0.10), and the current that passed between two neurons was heavily filtered (corner frequency, <10 Hz). Dual recordings also demonstrated unidirectional excitatory chemical transmission (EPSPs of approximately 3 mV) between type-1 neurons. These data indicate that respiratory motor output from the brainstem involves gap junction-mediated current transfer between motoneurons. Furthermore, bidirectional electrical coupling and unidirectional excitatory chemical transmission are present between type-1 neurons in the preBötC and may be important for generation or modulation of breathing rhythm.

Maximum length sequence brainstem auditory evoked responses in term neonates who have perinatal hypoxia-ischemia.

Maximum length sequence brainstem auditory evoked response (BAER) was studied within the first week after birth in 28 term neonates who had perinatal hypoxia-ischemia, or asphyxia. In the BAER recorded using conventional averaging techniques (click rate 21/s), the only abnormality was a slight increase in III-V interval, in addition to an increase in wave latencies when including those who had an elevated threshold (t test, all p<0.05). In the maximum length sequence BAER, however, both the III-V and I-V intervals in the asphyxiated infants were significantly increased at all the 91/s, 227/s, 455/s, and particularly 910/s click rates (p<0.05-0.001). The I-III interval was also increased significantly at 455/s and 910/s click rates (both p< 0.05). Wave V amplitude was significantly reduced at all the click rates used (ANOVA, p<0.05-0.001), particularly at 910/s, which sometimes was the only abnormality indicative of brain damage. Both the amplitude ratios V/I and V/III were significantly decreased at 455/s and 910/s click rates (p<0.01 or 0.001). A general trend was that BAER abnormalities after hypoxia-ischemia became more prominent as click rate was increased. Significant abnormalities occurred mainly at very high click rates (455/s and 910/s), which can be achieved using the maximum length sequence technique but not by using conventional averaging techniques. Thus, this technique, which can be used at the cribside, appears to be a better method for the early detection of brain damage after hypoxia-ischemia than using conventional averaging techniques, enhancing the diagnostic value of the BAER.

Acetylcholine modulates respiratory pattern: effects mediated by M3-like receptors in preBötzinger complex inspiratory neurons.

Perturbations of cholinergic neurotransmission in the brain stem affect respiratory motor pattern both in vivo and in vitro; the underlying cellular mechanisms are unclear. Using a medullary slice preparation from neonatal rat that spontaneously generates respiratory rhythm, we patch-clamped inspiratory neurons in the preBötzinger complex (preBötC), the hypothesized site for respiratory rhythm generation, and simultaneously recorded respiratory-related motor output from the hypoglossal nerve (XIIn). Most (88%) of the inspiratory neurons tested responded to local application of acetylcholine (ACh) or carbachol (CCh) or bath application of muscarine. Bath application of 50 microM muscarine increased the frequency, amplitude, and duration of XIIn inspiratory bursts. At the cellular level, muscarine induced a tonic inward current, increased the duration, and decreased the amplitude of the phasic inspiratory inward currents in preBötC inspiratory neurons recorded under voltage clamp at -60 mV. Muscarine also induced seizure-like activity evident during expiratory periods in XIIn activity; these effects were blocked by atropine. In the presence of tetrodotoxin (TTX), local ejection of 2 mM CCh or ACh onto preBötC inspiratory neurons induced an inward current along with an increase in membrane conductance under voltage clamp and induced a depolarization under current clamp. This response was blocked by atropine in a concentration-dependent manner. Bath application of 1 microM pirenzepine, 10 microM gallamine, or 10 microM himbacine had little effect on the CCh-induced current, whereas 10 microM 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the current. The current-voltage (I-V) relationship of the CCh-induced response was linear in the range of -110 to -20 mV and reversed at -11.4 mV. Similar responses were found in both pacemaker and nonpacemaker inspiratory neurons. The response to CCh was unaffected when patch electrodes contained a high concentration of EGTA (11 mM) or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (10 mM). The response to CCh was reduced greatly by substitution of 128 mM Tris-Cl for NaCl in the bath solution; the I-V curve shifted to the left and the reversal potential shifted to -47 mV. Lowering extracellular Cl(-) concentration from 140 to 70 mM had no effect on the reversal potential. These results suggest that in preBötC inspiratory neurons, ACh acts on M3-like ACh receptors on the postsynaptic neurons to open a channel permeable to Na(+) and K(+) that is not Ca(2+) dependent. This inward cation current plays a major role in depolarizing preBötC inspiratory neurons, including pacemakers, that may account for the ACh-induced increase in the frequency of respiratory motor output observed at the systems/behavioral level.

Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Bötzinger complex: differential roles of glycinergic and GABAergic neural transmission.

A key distinction between neural pacemaker and conventional network models for the generation of breathing rhythm in mammals is whether phasic reciprocal inhibitory interactions between inspiratory and expiratory neurons are required. In medullary slices from neonatal rats generating respiratory-related rhythm, we measured the phasic inhibitory inputs to expiratory neurons with the use of whole cell patch clamp in the hypothesized rhythm generation site, the pre-Bötzinger complex (pre-BötC). Expiratory neurons, which generate tonic impulse activity during the expiratory period, exhibited inhibitory postsynaptic potentials (IPSPs) synchronized to the periodic inspiratory bursts of the hypoglossal nerve root (XIIn). Bath application of the glycine receptor antagonist strychnine (STR; 5-10 microM) reversibly blocked these inspiratory-phase IPSPs, whereas the gamma-aminobutyric acid-A (GABA(A)) receptor antagonist bicuculline (BIC; 10-100 microM) had no effect on these IPSPs. Replacing the control in vitro bathing solution with a Cl(-)-free solution also abolished these IPSPs. Respiratory-related rhythmic activity was not abolished when inspiratory-phase IPSPs were blocked. The frequency and strength of XIIn rhythmic activity increased and seizurelike activity was produced when either STR, BIC, or Cl(-)-free solution was applied. Inspiratory-phase IPSPs were stable after establishment of whole cell patch conditions (patch pipettes contained 7 mM Cl-). Under voltage clamp, the reversal potential of inspiratory-phase inhibitory postsynaptic currents (IPSCs) was -75 mV. The current-voltage (I-V) curve for IPSCs shifted to the right when extracellular Cl- concentration was reduced by 50% (70 mM) and the reversal potential was reduced to -60 mV, close to the new Cl- Nernst potential. In tetrodotoxin (0.5 microM) under voltage clamp (holding potential = -45 mV), local application of glycine (1 mM) over pre-BötC induced an outward current and an increase in membrane conductance in expiratory neurons. The effect was blocked by bath application of STR (0.8-1 microM). Local application of the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 1 mM) induced an outward current and an increase in membrane conductance that was blocked by BIC (10-100 mM). Under voltage clamp (holding potential = -45 mV), we analyzed spontaneous IPSCs during expiration in expiratory neurons. Bath application of BIC (10 microM) reduced the IPSC frequency (from 2.2 to 0.3 per s), whereas the inspiratory-phase IPSCs did not change. Bath application of STR (8-10 microM) abolished both IPSCs. These results indicate that 1) reciprocal inhibition of expiratory neurons is glycinergic and mediated by a glycine-activated Cl- channel that is not required for respiratory-related rhythm generation in neonatal rat medullary slices; 2) endogenous GABA and glycine modulate the excitability of respiratory neurons and affect respiratory pattern in the slice preparation; 3) both glycine and GABA(A) receptors are found on pre-BötC expiratory neurons, and these receptors are sensitive to STR and BIC, respectively; 4) glycine and GABA(A) inhibitory mechanisms play different functional roles in expiratory neurons: both glycine and GABA(A) receptors modulate neuronal excitability, whereas glycinergic transmission alone is responsible for reciprocal inhibition; and 5) intracellular Cl- concentration in these neonatal expiratory neurons is similar to that in adults.

Parametric survival analysis for gating kinetics of single potassium channels.

The gating mechanism and the role of the S4 region in the activation of Shaker potassium channel was studied by statistical analysis on the wild type and mutant channels which have mutations in the S4 region. Single channel currents were recorded with the patch-clamp technique. The first latency time was analyzed with a parametric survival time regression model in which the generalized gamma distribution for the error term was assumed. Discrimination among Weibull, gamma, log-normal and exponential distributions was done with the likelihood ratio test. The three-parameter generalized gamma distribution was shown to be appropriate for the data set. The multiple regression function provides the statistical tests and the quantitative descriptions for the relationships between the mutations and the voltage dependence of the gating process. These results on statistical relations support the hypothesis that the S4 region plays an important role in sensing transmembrane voltage in the gating process, but the gating mechanism is not solely accounted for by the electrostatic interaction between the charged amino acids and the transmembrane voltage field. This work demonstrates that the survival analysis procedures can be useful tools for analyzing neurophysiological signals.

Measure and statistical test for cross-correlation between paired neuronal spike trains with small sample size.

Recent development of multi-unit recording techniques such as optical recording and multi-electrode arrays makes it possible to record neuronal activities from tens or hundreds of neurons simultaneously. To analyze functional connections between these neurons, cross-correlation analysis has been most commonly applied to the hundreds to thousands of pairs of these neurons. However, conventional cross-correlation data needs statistical tests for significance especially when the sample size of recorded spike trains is small. Here, a multiple hypergeometric model based on a transformation of the cross-correlogram data to a 2 x J table has been suggested. The exact p value for significance can be obtained by the generalized Fisher's method with small sample size and a cross-correlation coefficient for the strength of cross-correlation can be obtained based on the R-square analogue for nominal data. For large sample size, chi 2 test can be applied based on the same transformation. Examples of real spike train data set and simulation show that the methods are applicable to the data of multi-unit activity with only tens of spikes. These methods are especially useful when thousands of cross-correlograms need to be screened quickly and automatically.

Electrostatic interactions of S4 voltage sensor in Shaker K+ channel.

The S4 segment comprises part of the voltage sensor in Shaker K+ channels. We have used a strategy similar to intragenic suppression, but without a genetic selection, to identify electrostatic interactions of the S4 segment that may be important in the mechanism of voltage-dependent activation. The S4 neutralization mutations K374Q and R377Q block maturation of the protein, suggesting that they prevent proper folding. K374Q is specifically and efficiently rescued by the second site mutations E293Q and D316N, located in putative transmembrane segments S2 and S3, respectively. These results suggest that K374, E293, and D316 form a network of strong, local, electrostatic interactions that stabilize the structure of the channel. Some other double mutant combinations result in inefficient suppression, identifying weak, presumably long-range electrostatic interactions. A simple structural hypothesis is proposed to account for the effects of the rescued double mutant combinations on the relative stabilities of open and closed channel conformations.

Computed tomography in predicting the efficacy of oral cholelitholysis with bile acids.

The efficacy of oral cholelitholytic therapy with chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) in 137 patients with gallstones was studied in relation to their CT patterns. The best dissolving results were obtained in patients with the stones in isodensity and faint category (< 50 Hu) on CT. The stones with high density or heterogeneous calcification on CT were insoluble, and therefore, were contraindicated for oral cholelitholytic therapy. The attenuation value of stones was classified as complete dissolution ranged from -2 to 35 Hu (14 +/- 12 Hu, n = 13), and their upper limit of 95 percentiles was 33.4 Hu. CT analysis improved the predictability of dissolving gallstones in comparison with plain abdominal radiography or oral cholecystography (OCG). The complete dissolving rate increased from 9.49% (13/137) in patients selected by classic X-ray to 40.7% (11/27) in isodensity category on CT. Besides, radiolucent gallstones, which showed no obvious filling defect on OCG but distinct echo and shadow on B-type ultrasonography, were also insoluble.

S4 mutations alter the single-channel gating kinetics of Shaker K+ channels.

The S4 sequence comprises at least part of the voltage sensor in Shaker K+ channels. Two site-directed mutations in the Shaker S4 sequence, R368Q and R377K, which decrease the voltage dependence of the whole-cell current, alter voltage-dependent gating at the single-channel level. Compared with the wild-type channel, they increase the latency to first opening, destabilize the open state, and alter the equilibria of voltage-dependent transitions, so that some of the charge movement occurs after the first opening. Whether these changes reflect a new mechanism of activation is a key question. Although the mutations alter the kinetics of many steps in the gating process, we conclude that the mutant channels are likely to activate using the same fundamental mechanism as wild-type channels.

[Pathological changes in adrenal cortex in rats after phosphorus burn (stereological and histochemical changes)].

The experiment dealt with stereological and histochemical study on the submicroscopic structure of adrenal cortex after phosphorus burn within 15 days. The main findings were as follows: (1). The increased dense lysosome-like bodies in the adrenal cortical cells after burn were not all lysosomes, and it was conjectured they were hormone carrier-protein which might come from the Golgi complex; (2). The ZR cells developed better and had more abundant organelles and higher enzymic activity than other zonal cells. These differences were more obvious after burn. We speculated that ZR was not a degenerated zone, but an important functional area of synthesis and secretion of steroid hormone; (3) Different cellular functional conditions in adrenal cortex were related to the secretory rhythm of the cells.

Activation and desensitization of the 5-HT3 receptor in a rat glioma x mouse neuroblastoma hybrid cell.

1. Tight-seal voltage-clamp techniques were used to study the 5-HT3 receptor of differentiated NG108-15 cells. 2. The inward current caused by 5-HT was dependent on the 5-HT concentration: the apparent dissociation constant was 3.3 microM and the Hill coefficient was 1.8. 3. Immediately after establishing a recording, sustained application of a saturating concentration of 5-HT caused the response to decline with a half-time of 0.57 s (at a membrane potential of -70 mV). The time course of desensitization was best fitted by a sum of two exponentials. 4. Desensitization became slower during the first 10 min of recording in the whole-cell configuration, with the half-time for response decay increasing to 1.8 s. The deceleration of desensitization may result from wash-out of a cytoplasmic regulator of the receptor. 5. Desensitization declined less during whole-cell recordings when patch pipettes contained non-hydrolysable analogues of adenosine 5'-triphosphate. 6. Desensitization developed more rapidly following the addition of forskolin, prostaglandin E1, cholera toxin or 1,9-dideoxyforskolin to the recording medium. Non-hydrolysable adenosine 5'-phosphate analogues had no effect on the enhancement of desensitization induced by forskolin.

Differentiation of NG108-15 cells alters channel conductance and desensitization kinetics of the 5-HT3 receptor.

1. NG108-15 cells undergo morphological differentiation in response to appropriate culture conditions. We have used patch clamp techniques to compare responses mediated by the 5-HT3 receptor in differentiated and undifferentiated NG108-15 cells. 2. In differentiated cells, desensitization of 5-hydroxytryptamine (5-HT) responses was much slower than in undifferentiated cells. Desensitization in differentiated cells was also highly variable, with half-times varying by greater than 40-fold. Rapidly desensitized responses in differentiated cells were qualitatively similar to the responses of undifferentiated cells. 3. In outside-out patches from undifferentiated cells, single channel currents could be seen after 5-HT application. These channels had a conductance of 12 pS. The 5-HT-activated channels in differentiated cells were too small to observe at the single-channel level. Noise analysis indicated that the channel conductance was approximately 4 pS. In differentiated cells, both rapidly and slowly desensitized responses were generated by channels with essentially the same conductance. 4. The 5-HT responses of differentiated cells were also distinguished from those of undifferentiated cells on the basis of the voltage dependence of desensitization and the curvature of the current-voltage curve. 5. NG108-15 cells can produce different receptor subtypes, which may be expressed in different tissues or at different stages of development. These variations in receptor behavior suggest that there are at least two distinct mechanisms for regulation of the 5-HT3 receptor.