Left ventricular outflow tract pressure gradient changes after carvedilol-disopyramide cotherapy in a cat with hypertrophic obstructive cardiomyopathy.

Disopyramide reduces the left ventricular outflow tract (LVOT) pressure gradient and improves symptoms in humans with hypertrophic obstructive cardiomyopathy (HOCM). However, the efficacy of disopyramide in cats has not been reported. We treated a cat with HOCM with carvedilol and disopyramide cotherapy and monitored the changes in LVOT flow velocity and N-terminal pro B-type natriuretic peptide (NT-proBNP) concentration. A 10-month-old neutered male Norwegian Forest cat was referred with a moderate systolic cardiac murmur. Echocardiography revealed thickening of the left ventricular wall, systolic anterior motion of the mitral valve leaflets, and turbulent aortic flow in the LVOT at systole. The LVOT flow velocity was 5.6 m/s. The plasma NT-proBNP concentration exceeded 1,500 pmol/L. The cat was diagnosed with HOCM and the ß-blocker carvedilol was started and gradually increased to 0.30 mg/kg, bid. After 57 days, the LVOT flow velocity (4.8 m/s) and plasma NT-proBNP concentration (870 pmol/L) had decreased but remained elevated. Therefore, disopyramide was added at 5.4 mg/kg po bid and increased to 10.9 mg/kg po bid after 22 days. After 141 days of carvedilol and disopyramide treatment, the systolic anterior motion of the mitral valve leaflets had disappeared and the LVOT flow velocity and plasma NT-proBNP concentration had decreased to 0.7 m/s and 499 pmol/L, respectively. No adverse effect has been observed during the follow-up. Disopyramide might relieve feline LVOT obstruction after only partial response to a beta-blocker. Further large-scale studies are required to investigate the efficacy and safety of disopyramide use in cats with moderate to severe HOCM.

A systematic review of cultural orientation and perinatal depression in Latina women: are acculturation, Marianismo, and religiosity risks or protective factors?

Latinas in the USA and Spanish-speaking countries experience elevated rates of perinatal depression (PND) because of high psychosocial stressors. Latinas are heterogeneous and have varying cultural practices. It is unclear whether specific cultural orientations have differential risks for PND. This systematic review aimed to determine whether degree of acculturation, Marianismo, and religiosity are risks or protective factors for PND in Latina women living in the USA, Latin America, and other countries. The review included PubMed, CINAHL, PsycINFO, PsycARTICLES, Academic Search Ultimate (EBSCO), and Social Services Abstracts, and used Boolean combined keywords. English and Spanish language articles were considered. The review was conducted between July 2017 and February 2018, with no boundaries on publication dates. Ten studies were selected for inclusion. Of those, two studies were conducted in Mexico and most studies conducted in the USA included women of Mexican descent. Degree of acculturation (adoption of mainstream values) was inconsistently directly associated with PND; evidence suggest indirect associations. Marianismo, the traditional female role of virtue, passivity, and priority of others over oneself, was inconsistently correlated with risk for depression in pregnancy, but significantly and indirectly associated with postpartum depression. Two of three studies found religiosity to be protective postpartum. Further research on protective and risk factors of specific cultural orientations, particularly degree of acculturation and Marianismo, for PND in Latinas in the USA and abroad is needed. Attention to specific perinatal periods is necessary given the inconsistent findings.

Orthotopic Kidney Transplantation in an Elderly Patient With Various Severe Comorbid Conditions: A Case Report.

In recent years, the frequency of high-risk kidney transplantations has increased. We report a case in which a 72-year-old man with various severe comorbidities (prostate cancer, diabetes mellitus, complete atrioventricular block, coronary artery stenosis, severe stenosis of the popliteal arteries, and severe calcification of the iliac arteries) who received an orthotopic kidney transplantation. To prevent the occurrence of acute limb ischemia due to the steal phenomenon (caused by the kidney graft), we decided that a heterotopic kidney transplantation involving the iliac arteries was not an appropriate option. Therefore, as an alternative, left native nephrectomy was performed followed by an orthotopic kidney transplantation to the native renal artery and renal vein through a left subcostal incision. Postoperative ureteral stenosis occurred, and so stent exchange was required every 6 months. Despite the ureteral complication, the patient's serum creatinine level was 1.5 mg/dL at 2 years after the procedure.

Long-term follow-up of collateral pathways established after lymphadenectomy in rats.

A collateral pathway established after lymphadenectomy could play an important role in long-term lymphedema treatment. The present study investigated alterations of lymph dynamics 1 year after lymphadenectomy using indocyanine green fluorescent lymphography to determine if a collateral pathway may be used for long-term lymphedema treatment. Wistar rats were anaesthetized and lymph nodes were excised at the inguinal and popliteal fossae. The treated hind limb was evaluated by fluorescent lymphography between 10 weeks and 6 months and between 6 months and 1 year postoperatively. Fluorescent lymphography demonstrated a lymphatic pathway to the ipsilateral axillary fossa in all rats 1 year after lymphadenectomy. Some capillary branches in the paths leading to the ipsilateral axillary fossa were dilated and tortuous. In addition, areas in which a fluorescent signal was not visible were increased in the thigh. In conclusion, the collateral pathway did not appear to be only for temporary use to compensate for drainage from the edematous limb but appears more stable as a component of a compensating lymphatic system. These new dilated vessels, although functional at this point, may still be susceptible to disturbance by further alteration to the lymph vessels.

Serial magnetic resonance imaging study of posterior cruciate ligament reconstruction or augmentation using hamstring tendons.

PURPOSE: The purpose of this study was to analyze serial changes in the magnetic resonance imaging (MRI) signals of autograft hamstrings single bundle posterior cruciate ligament (PCL) reconstruction and the effects of remnant preservation (augmentation). MATERIAL AND METHODS: Twenty-two isolated PCL injuries were arthroscopically reconstructed or augmented with hamstring tendons. MRI scans were obtained at 3, 6, and 12 months, and prior to the second-look arthroscopy (average 20.7 months). The patients were divided into 2 groups by remnant preservation: five PCL reconstructions after PCL remnant resection (Group Rec) (23%), and 17 reconstructions preserving the remnant (Group Aug) (77%). The 22 patients were also divided in two groups depending on the location of the PCL tear. There were 9 knees with proximal tear (Type P) (41%) and 13 knees with distal tear (Type D) (59%). The signal intensity and fiber continuity of 4 zones (proximal, middle, distal intra-articular and tibial tunnel zones) were evaluated by the Mariani score. RESULTS: The average MRI evaluation score gradually increased from 6 months through the final MRI. The intra-articular part of the graft exhibited slower maturation (12 months - final scan) as compared with the tibial tunnel (6-12 months). The distal zone underwent better maturation than the proximal or middle zones at all points. In the proximal zone, the score for Group Aug was significantly higher than Group Rec. In the proximal zone, the Type D score with a proximally-preserved remnant was significantly higher than Type P without a proximal remnant. CONCLUSIONS: The hamstring tendons require more than 1 year to achieve low-signal intensity. PCL remnant has a beneficial effect on the maturation of the hamstring graft. LEVEL OF EVIDENCE IV: therapeutic case series.

Alterations of lymph flow after lymphadenectomy in rats revealed by real time fluorescence imaging system.

Secondary lymphedema is one of the sequella of cancer treatment that in inadequately understood. The purpose of the present study is to investigate lymphedema formation and to explore the escape routes for excess interstitial fluid using lymphadenectomy in a rat model. In twelve Wistar rats, lymph nodes in the right inguinal and popliteal fossas were completely removed and lymph vessels carefully ligated. After operation, treated hind limbs were evaluated by indocyanine green lymphography and circumferential measurement. Both evaluations were performed from day 3 to ten weeks. Approximately 2 to 3 weeks after operation, a network-like pattern of the fluorescent signal appeared around the surgical site which then transitioned into a linear pattern in the lower abdomen. Videorecordings identified fluorescent flow moving from the lower abdomen to the ipsilateral axillary lymph node and in some rats, the network-like pattern was also observed to pass transversely over the suprapubic region to the contralateral inguinal lymph nodes. The network-like pattern on the lower abdomen and the linear pattern to the axillary fossa were seen continuously to the end of observation. Circumferential measurements of the treated hind limbs increased initially and then declined over time. This imaging system may be useful to detect early changes in lymphatic flow before swelling occurs and further study is needed.

[Midterm restenosis of freestyle valved conduit in an adult tetralogy of Fallot].

We report a case of midterm restenosis of freestyle valved conduit implanted between the right ventricle and the pulmonary artery. A 69-year-old woman visited our hospital with dyspnea and general fatigue and was diagnosed as tetralogy of Fallot. Total corrective surgery including patch closure of ventricular septal defect was performed using the freestyle valve with the Hemashield prothesis to reconstruct the right ventricle to the pulmonary artery. The catheterization revealed almost normal right ventricular pressure on 40th postoperative day, but the opening of the valve was limited. Twenty-two months later she visited our hospital with severe right heart failure. The pressure gradient over the valve was 122 mmHg and the leaflet of the freestyle valve was markedly thickened causing severe stenosis. Use of the freestyle valve for the right ventricular out flow tract reconstruction has to be further studied.

Leukocytapheresis treatment for pyoderma gangrenosum.

A 42-year-old man presented with painful erythema with pustules and multiple small ulcers on the shins. He had suffered from ulcerative colitis (UC) and received oral glucocorticosteroid and salicylazosulfapyridine therapies for 7 years. Biopsy of the lesion demonstrated mixed cellular infiltrates with dominant neutrophils. The patient was diagnosed with pyoderma gangrenosum (PG) and underwent leukocytapheresis (LCAP), an extracorporeal leucocyte removal therapy, once a week for 5 weeks without changing the doses of the oral medications. The skin lesions as well as clinical signs of UC rapidly improved after LCAP, and no recurrence was seen during a follow-up period. There were no major complications during LCAP. LCAP will provide an effective and safe tool for the treatment of PG.

A novel mechanism associated with idiopathic ventricular fibrillation (IVF) mutations R1232W and T1620M in human cardiac sodium channels.

Two mutations associated with idiopathic ventricular fibrillation (IVF) are localized within extracellular loops between segments DIIIS1-S2 (R1232W) and DIVS3-S4 (T1620M) of the human cardiac sodium channel (hNav1.5) alpha-subunit. We studied wild-type hNav1.5 channels and hNav1.5 channels with the R1232W/T1620M double mutation expressed in Xenopus oocytes using the cell-attached macropatch technique. We demonstrate that these mutations destabilize the fast-inactivated state (described with a two-state first-order reaction model) by decreasing reaction valence, accelerating recovery, and slowing the onset of fast inactivation, collectively resulting in delayed decay of macroscopic currents. R1232W/T1620M mutations in hNav1.5 channels also significantly increase steady-state channel availability, indicating that mutated channels occupy the slow inactivated state less than hNav1.5 channels. Under the stress of repetitive depolarizing pulses, R1232W/T1620M channels demonstrate less use-dependent current reduction compared to wild-type channels. We propose that increased channel availability coupled with destabilized fast inactivation contributes to the pathological effect of R1232W/T1620M mutations, and leads to increased excitability of cardiac tissue in vivo.

A single residue differentiates between human cardiac and skeletal muscle Na+ channel slow inactivation.

Slow inactivation determines the availability of voltage-gated sodium channels during prolonged depolarization. Slow inactivation in hNa(V)1.4 channels occurs with a higher probability than hNa(V)1.5 sodium channels; however, the precise molecular mechanism for this difference remains unclear. Using the macropatch technique we show that the DII S5-S6 p-region uniquely confers the probability of slow inactivation from parental hNa(V)1.5 and hNa(V)1.4 channels into chimerical constructs expressed in Xenopus oocytes. Site-directed mutagenesis was used to test whether a specific region within DII S5-S6 controls the probability of slow inactivation. We found that substituting V754 in hNa(V)1.4 with isoleucine from the corresponding position (891) in hNa(V)1.5 produced steady-state slow inactivation statistically indistinguishable from that in wild-type hNa(V)1.5 channels, whereas other mutations have little or no effect on slow inactivation. This result indicates that residues V754 in hNa(V)1.4 and I891in hNa(V)1.5 are unique in determining the probability of slow inactivation characteristic of these isoforms. Exchanging S5-S6 linkers between hNa(V)1.4 and hNa(V)1.5 channels had no consistent effect on the voltage-dependent slow time inactivation constants [tau(V)]. This suggests that the molecular structures regulating rates of entry into and exit from the slow inactivated state are different from those controlling the steady-state probability and reside outside the p-regions.

Pretibial cyst formation after anterior cruciate ligament reconstruction using auto hamstring grafts: two case reports in a prospective study of 89 cases.

Eighty-nine cases after anterior cruciate ligaments (ACL) reconstruction were followed prospectively with magnetic resonance imaging (MRI). The patients were examined using axial and sagittal MRI at least twice during the postoperative evaluation of reconstructed ACL. Two cases of pretibial cyst formation were observed. At the time of cyst formation, neither patient had any subjective or objective evidence of knee instability. The cyst of one case communicated with the intra-articular. The minimum follow-up period after the surgical excision was 9 months, with no evidence of recurrence. We might speculate that the critical period for cyst formation in both patients occurred at less than 12 months after their ACL reconstruction. We concluded that the cyst formation was most likely due to incomplete graft tendon incorporation within the osseous tunnel.

The delay in recovery from fast inactivation in skeletal muscle sodium channels is deactivation.

1. Using macropatch techniques, we tested the assumption that deactivation underlies the observed delay in the onset to recovery from fast inactivation by comparing open-state deactivation to recovery delay for rat skeletal muscle mutations R1441C and R1441P. 2. Deactivation kinetics from the open state were determined from the exponential decay of tail currents. R1441C and R1441P prolonged open-state deactivation, with the greatest effect produced by R1441P. 3. Delays in the onset to recovery from fast inactivation for R1441P and for R1441C were abbreviated compared to those for rSkM1. Recovery delay was longer in R1441P than R1441C at voltages more negative than -110 mV. Recovery from inactivation exhibited a voltage dependence which, unlike delay, saturated at depolarized voltages. Recovery rate constants were increased to a similar extent for R1441C and R1441P at -150 to -120 mV compared to rSkM1. 4. These results indicate that the delay in the onset to recovery from fast inactivation in skeletal muscle sodium channels is due to deactivation. Lessening of charge immobilization for R1441C and R1441P may contribute to observed biophysical defects underlying the hyperexcitability of muscle fibers containing paramyotonia congenita mutations. The second stage of recovery from fast inactivation may be affected differentially by these mutations.

Effect of cerebrospinal fluid shunting on experimental syringomyelia: magnetic resonance imaging and histological findings.

The histological changes associated with syringomyelia after reduction of the syrinx size were investigated after cerebrospinal fluid shunting in experimental syringomyelia in the rabbit. Five weeks after syringomyelia was induced by the injection of kaolin into the cisterna magna in Japanese white rabbits, ventriculosubgaleal shunting or syringoepidural shunting were performed. After 1 week magnetic resonance (MR) imaging and histological examination were then carried out. Five of 11 shunted animals showed postoperative reduction of syrinx size on MR imaging. Grossly, some specimens showed cavity collapse and parenchymal healing, and others showed a small residual syrinx in the dorsal horn. The most dramatic histological changes occurred in the gray matter. Specimens with syrinx collapse showed rarefaction and tearing of the gray matter, with mild glial reaction. The edematous gray matter showed both degeneration and regeneration, with neuronal processes surrounded by edema fluid. Reactive astrocytes were observed mainly at the margin of the residual syrinx. Some astrocytic processes invested the extraaxonal space and gray matter lacked supportive tissue. Greater reduction of the syrinx after shunting operation was correlated with more regeneration and less degeneration, and the white matter was edematous and histological changes were milder. Syrinx shrinkage occurred after shunting in this experimental model of syringomyelia. The selective vulnerability of gray matter even after shunting may explain discrepancies between imaging findings and clinical features in this disease. The study supports the potential benefit from early treatment, considering the associated morphological findings of regeneration.

Evaluation of the internal thoracic arterial graft patency by the transthoracic Doppler method under continuous intravenous infusion of adenosine triphosphate disodium.

Usefulness of the Doppler method under continuous infusion of adenosine triphosphate disodium (ATP) for improvement of accuracy in the diagnosis of the left internal thoracic arterial graft (LITA) patency was examined using transthoracic ultrasonic echocardiography. 1) Influence of ATP on the Doppler velocity in a graft was examined in 7 patients with good LITA grafts using physiological saline as the control. In the ATP group, 80 mg of ATP was dissolved in 20 ml physiological saline and continuously infused at 0.14 mg/kg/min. In the saline group, an equal volume of physiological saline was administered and the blood flow velocity in the LITA was recorded continuously by the transthoracic Doppler method from the supraclavicular fossa approach. Results; ATP administration increased the blood flow velocity in the LITA and the rate of increase was 48.3% for systolic peak velocity, 111% for diastolic peak velocity, 64.4% for systolic time velocity integral and 99% for diastolic time velocity integral indicating particularly high rates of increase in diastolic components. The diastolic/systolic peak velocity ratio or diastolic fraction did not increase significantly. In the saline group, none of the parameters showed a change. 2) Angiographic findings of the LITA were compared with the measurement values of the diastolic components by the Doppler method to examine usefulness of diastolic component measurement with ATP infusion for diagnosis of LITA patency. Subjects were 19 patients with good LITA (group A) and 8 patients with bad LITA (group B). Results; while there were significant differences in the mean baseline diastolic peak velocity, mean diastolic time velocity integral and mean diastolic fraction between the groups, overlapping was seen in individual cases. However, the inter-group differences were more distinct by ATP infusion and the borderline values were 30 cm/sec for diastolic peak velocity and 10 for diastolic time velocity integral. 3) Reliability of the diagnosis for LITA patency by measuring the diastolic components using the Doppler method with ATP infusion was examined and compared with the angiographic findings as the gold standard. Subjects were 27 patients and the diagnostic criteria for good LITA were set at 30 cm/sec for diastolic peak velocity and 10 for diastolic time velocity integral. Results; sensitivity and specificity of the Doppler method with ATP infusion were 100% for diagnosis of LITA patency by measuring the diastolic components. Conclusion, in diagnosis of LITA patency by the transthoracic ultrasonic cardiography, diagnostic accuracy was improved by measuring the diastolic parameters under continuous infusion of ATP.

Beneficial effect of basic fibroblast growth factor on the repair of full-thickness defects in rabbit articular cartilage.

The effects of exogenous basic fibroblast growth factor (bFGF) on the repair of full-thickness cartilage defects were examined. Four-millimeter diameter, cylindrical defects were made in rabbit articular cartilage and were filled with human recombinant bFGF. The addition of bFGF to the defect induced the formation of a thick cartilage layer composed of chondrocytes and a metachromatic-stained matrix after 6 weeks. The score of the bFGF-treated tissue, as evaluated by a semiquantitative histological scale, was significantly higher than that of the untreated tissue. At 24 weeks, the cartilage-like matrix that contained the proteoglycans and type II collagen was thicker in the bFGF-treated tissue than in the untreated tissue. Immunohistochemical analysis of the tissues at 6-12 weeks with an anti-bFGF monoclonal antibody suggested that a single application of bFGF increased the number of differentiating chondrocytes that synthesized bFGF at a high level. In contrast, immunostaining of the tissues at 6-12 weeks with a monoclonal antibody against proliferating cell nuclear antigen showed that the number of proliferating cells in the bFGF-treated tissue was fewer than in the untreated tissue. These findings suggest that administration of bFGF into cartilagenous defects promotes the differentiation of chondrocytes and their matrix synthesis, and that this growth factor is useful for improving cartilage repair.

Appearance of anterior cruciate ligament autografts in their tibial bone tunnels on oblique axial MRI.

The objective of this study was to observe the changing appearance of human anterior cruciate ligament (ACL) grafts in their tibial bone tunnels by MRI using oblique axial images. One-hundred and eight knees in 75 patients were studied by MRI at 1-33 months after arthroscopic ACL reconstructions using double-looped, autogenous semitendinosus and/or gracilis tendons. Knees with poor stability were excluded from this study. The examinations were performed at 0.2T with spin echo proton density and T2-weighted oblique axial images. Appearances of grafts were mainly described on spin echo proton density images based upon time after surgery. The grafts appeared as homogeneous, low signal intensity areas in the bone tunnels at 1 month after the surgery. Ring-shaped low signal intensity areas were observed along the wall of the bone tunnels in the 2- to 3-month group. In many grafts from this group, each tendinous bundle appeared as a low signal area separated by a high signal intensity area. In all cases in the 4- to 6-month group, the thickness of the ring-shaped low signal intensity area had increased, whereas the thickness of the high signal intensity area had decreased. In almost all of the cases, the interior of the bone tunnel gradually became a homologous low signal intensity region by 7 to 12 months after the surgery. According to these results, it is suggested that the maturation of the tendon-bone interface was completed from 6 to 12 months after the ACL reconstruction.

Differential effects of homologous S4 mutations in human skeletal muscle sodium channels on deactivation gating from open and inactivated states.

1. The outermost charged amino acid of S4 segments in the alpha subunit of human skeletal muscle sodium channels was mutated to cysteine in domains I (R219C), II (R669C), III (K1126C), and IV (R1448C). Double mutations in DIS4 and DIVS4 (R219C/R1448C), DIIS4 and DIVS4 (R669C/R1448C), and DIIIS4 and DIVS4 (K1126C/R1448C) were introduced in other constructs. Macropatch recordings of mutant and wild-type (hSkM1-wt) skeletal muscle sodium channels expressed in Xenopus oocytes were used to measure deactivation kinetics from open or fast inactivated states. 2. Conductance (voltage) curves (G (V)) derived from current (voltage) (I (V)) relations indicated a right-shifted G (V) relationship for R669C and for R669C/R1448C, but not for other mutations. The apparent valency was decreased for all mutations. Time-to-peak activation at -20 mV was increased for R1448C and for double mutations. 3. Deactivation kinetics from the open state were determined from the monoexponential decay of tail currents. Outermost charge-to-cysteine mutations in the S4 segments of domains III and IV slowed deactivation, with the greatest effect produced by R1448C. The deactivation rate constant was slowed to a greater extent for the DIII/DIV double mutation than that calculated from additive effects of single mutations in each of these two domains. Mutation in DIIS4 accelerated deactivation from the open state, whereas mutation in DIS4 had little effect. 4. Delays in the onset to recovery from fast inactivation were determined to assess deactivation kinetics from the inactivated state. Delay times for R219C and R669C were not significantly different from those for hSkM1-wt. Recovery delay was increased for K1126C, and was accelerated for R1448C. 5. Homologous charge mutations of S4 segments produced domain-specific effects on deactivation gating from the open and from the fast inactivated state. These results are consistent with the hypothesis that translocations of S4 segments in each domain during deactivation are not identical and independent processes. Non-identical effects of these mutations raise several possibilities regarding deactivation gating; translocation of DIVS4 may constitute the rate-limiting step in deactivation from the open state, DIVS4 may be part of the immobilizable charge, and S4 translocations underlying deactivation in human skeletal muscle sodium channel may exhibit co-operativity.

Characterization of a novel Ras-binding protein Ce-FLI-1 comprising leucine-rich repeats and gelsolin-like domains.

Ras proteins are conserved from yeasts to mammals and implicated in regulation of the actin cytoskeleton. The flightless-1 (fli-1) gene of Drosophila melanogaster and its homologs in Caenorhabditis elegans and humans encode proteins (FLI-1) comprising a fusion of a leucine-rich repeats (LRRs) domain and a gelsolin-like domain. This LRRs domain is highly homologous to those of three proteins involved in Ras-mediated signaling; Saccharomyces cerevisiae adenylyl cyclase, C. elegans SUR-8, and mammalian RSP-1. Here we report that the LRRs domain of C. elegans FLI-1 (Ce-FLI-1) associates directly with Ras (Kd = 11 nM) and, when overexpressed, suppresses the heat shock sensitive phenotype of yeast cells bearing the activated RAS2 gene (RAS2(Val-19)). Further, the gelsolin-like domain of Ce-FLI-1 is shown to possess a Ca2+-independent G-actin-binding activity as well as F-actin-binding and -severing activities. FLI-1 may be involved in regulation of the actin cytoskeleton through Ras.

Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation.

Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.