Comparison of urinary incontinence following three different prostate apex disconnection techniques in transurethral thulium laser prostatectomy.

OBJECTIVE: This study investigates the incidence of urinary incontinence following transurethral thulium laser prostatectomy with three different prostate apex disconnection techniques: semi-separation, pre-separation, and post-separation. The findings aim to provide references for clinical treatment. PATIENTS AND METHODS: A retrospective analysis was conducted on 74 patients treated with transurethral thulium laser prostatectomy for prostatic hyperplasia from April 2022 to March 2023. Complete clinical and follow-up data were available for 52 patients. Clinical and follow-up data were collected for these patients. A comparison was made of urinary incontinence following the three different types of prostate apex disconnection in transurethral thulium laser prostatectomy. RESULTS: In this study, the immediate postoperative urinary incontinence rate for transurethral thulium laser prostatectomy was 9.62% (5/52), the short-term incontinence rate was 11.54% (5/52), and the long-term incontinence rate was 9.62% (5/52). The immediate postoperative incontinence rates for semi-separation, pre-separation, and post- separation were 8.33% (1/12), 8.33% (2/24), and 12.5% (2/16), respectively. The short-term incontinence rates for semi-separation, pre-separation, and post-separation were 8.33% (1/12), 8.33% (2/24), and 18.75% (3/16), respectively. The long-term incontinence rates for semi-separation, pre-separation, and post-separation were 8.33% (1/12), 8.33% (2/24), and 12.5% (2/16), respectively. CONCLUSIONS: The incidence of urinary incontinence following transurethral thulium laser prostatectomy was lower with semi-separation and pre-separation compared to post-separation.

[Prognostic value of the age-adjusted Charlson comorbidity index in patients over 60 years old with laryngeal squamous cell carcinoma].

Objective: To evaluate the value of the age-adjusted Charlson comorbidity Index (ACCI) in predicting the prognosis and guiding the clinical treatment of laryngeal squamous cell carcinoma (LSCC) in patients over 60 years old. Methods: Retrospective analysis of 249 cases of LSCC in Shanxi Provincial Cancer Hospital and First Hospital of Shanxi Medical University from 2008 to 2015 was performed. There were 234 males and 15 females, aged from 60 to 88 years. The clinical characteristics, treatment information and follow-up data were collected. ACCI was used to score the comorbidities of the patients. Receiver operating characteristic (ROC) curve was drawn and the patients were divided into high ACCI group and low ACCI group according to the cut-off value of ACCI. Prognostic factors were analyzed. Kaplan-Meier method was used for survival analysis, rank sum test was used for comparison between groups, χ2 test was used for enumeration data. Results: Overall survival (OS) was 54.6%, progression-free survival (PFS) was 59.4%, and cancer-specific survival (CSS) was 58.6%. Both the median survival time and PFS time were 60 months. The best cutoff point of the ACCI group was 5. Cox multivariate analysis showed that ACCI was an independent risk factor for OS, PFS and CSS (OR=1.553, 1.499 and 1.534,respectively, all P<0.05). In the high ACCI group, OS (χ2=4.120 and 4.115,P<0.05) and CSS (χ2=4.510 and 5.009,P<0.05) of patients treated with surgery plus radiotherapy and patients with radiotherapy alone were better than those of patients with surgery alone (P<0.05). But in the low ACCI group, there was no significant difference in prognosis among the three treatment regimens (P>0.05). Conclusion: High ACCI offors important prognostic information for LSCC in patients over 60 years old, and can guide clinical treatment options.

[Association of hyperuricemia-induced renal damage with sirtuin 1 and endothelial nitric oxide synthase in rats].

Objective: To investigate the association of hyperuricemia-induced renal damage with sirtuin 1 (SIRT1) and endothelial nitric oxide synthase (eNOS) in rats. Methods: Using the random number table method, 32 Sprague-Dawley rats were randomly divided into 4 groups: control group, model A group (the model was generated using oxonic acid potassium salt alone), model B group (hyperuricemia model was generated using oxonic acid potassium salt combined with uric acid) and resveratrol group, with 8 rats in each group. The experiment lasted 12 weeks. Serum uric acid and cystatin C levels were monitored regularly. In week 12, serum creatinine and urea nitrogen levels were measured, and the kidneys were extracted. The expression of SIRT1 and eNOS in renal tissues was measured and determined by immunohistochemistry, quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blotting. Immunohistochemistry of alpha-smooth muscle actin combined with Masson staining was employed to evaluate the degree of renal fibrosis, and pathological changes were observed based on hematoxylin and eosin staining. Results: In week 12, the uric acid levels in both the model A and model B groups were higher than those in the control group [(316±43) µmol/L, (297±40) µmol/L vs (118±44) µmol/L, both P<0.05]. The levels of cystatin C in the model A, model B, and resveratrol groups were all higher than those in the control group [(156±20) ng/ml, (143±29) ng/ml, (128±26) ng/ml vs (62±18) ng/ml, all P<0.05]. Creatinine levels were higher in the model A and model B groups than those in the control group [(68.5±10.3) µmol/L, (64.5±13.9) µmol/L vs (43.2±10.6) µmol/L, both P<0.05]. The levels of uric acid, cystatin C and creatinine in the resveratrol group were lower than those in the model A group (all P<0.05). Immunohistochemistry, RT-qPCR, and Western blotting for renal SIRT1 and eNOS showed that the expression in the model A and model B groups was inhibited, while the expression in the resveratrol group was not significantly inhibited, compared with that in the control group. Microscopically, obvious abnormalities were not found in the renal tissue of the control group. Renal inflammatory cell aggregation and edema occurred, and interstitial fibrosis was obvious in both the model A and model B groups, while these lesions in the resveratrol group were significantly improved. Conclusions: Hyperuricemia may cause renal injury by inhibiting the expression of SIRT1 and eNOS.

[The efficacy of sublingual immunotherapy for allergic rhinitis and the predictive role of cytokines in its therapeutic effect].

Objective:The study aimed to investigate the efficacy of sublingual immunotherapy(SLIT) in treatment of adult allergic rhinitis, and to explore the predictive role of baseline serum cytokine levels in its therapeutic effect.Method:Sixty patients with moderatesevere perennial AR sensitized with house dust mites were treated for 2 years. The SLIT group(n=30) were treated with standardized dust mite vaccine SLIT and conventional drugs, and the control group(n=30) were treated with placebo and conventional drugs. The combined symptom and medication score(CSMS) were compared between the two groups to evaluate the efficacy at baseline and 2 year endpoint. According to therapeutic effect, the SLIT group and the control group were divided into subgroups respectively, and the baseline IFN-γ and IL4, IL10, IL17 levels were compared between the effective group and the ineffective group in each group. The ROC curve was drawn to find the best predictive index and the best cut-off value was calculated. Result:①There was no significant difference between the SLIT group and the control group at baseline CSMS(P>0.05). There was significant difference between the two groups at 2year endpoint CSMS(P<0.05). ②In the SLIT group, there was no significant difference between the effective group and the ineffective group with the IFN-γ and IL17(P>0.05). The IL4 level in the effective group was significantly higher than the ineffective group while the IL10 level was significantly lower(P<0.05). In the control group, there were no significant differences in the levels of IFN-γ, IL4, IL10 and IL17 between the two subgroups(P>0.05). ③Baseline IL4/IL10 has higher predictive value than IL4 and IL10 alone. The best cut-off value is 2.04, and the sensitivity and specificity of predictive value were 72.7% and 73.7% respectively. Conclusion: SLIT combined with conventional drug therapy is more effective than conventional drug therapy alone. IL4/IL10 has a better predictive role in SLIT effect than IL4 or IL10 alone. The higher the ratio, the better therapeutic effect is.

[Features of cognitive function in patients with laryngeal carcinoma].

Objective:To study the differences in cognitive function between patients with laryngeal carcinoma and healthy volunteers. Method:Patients with laryngeal carcinoma who have been first diagnosed with laryngeal carcinoma, but not received treatment at the Department of Otolaryngology in two hospitals in Shanxi Province and healthy volunteers of the same age, gender-matched and similar education were studied for the purpose to evaluate the cognitive status by using the Wechsler memory scale.Result:No significant difference of age, gender and educational level was found between both groups（P>0.05）.The score of Memory Quotient was significantly lower in the laryngeal carcinoma group than that in healthy control group (P<0.05). There were significant differences in the results of Wechsler memory scale except for Experience, Orientation and Association test (P<0.05).Conclusion:The memory, attention and computing power of patients in the laryngeal carcinoma group were not as good as those of patients in the healthy control group. Patients with laryngeal carcinoma have cognitive impairment or lower ability , so we need to pay more attention to the patients during their rehabilitation. The early detection of cancer-related cognitive impairments can help patients improve their cognitive function early, reduce the burden on their families and society, and promote better return of patients to society.

[Research progress on the changes of emotional and cognitive functions in patients with laryngeal cancer].

At present, laryngeal cancer is more common in otolaryngology and head and neck surgery malignancies. Patients such as hoarseness, difficulty swallowing, ear pain, cough or cough, phlegm, dyspnea and other symptoms. which brings severe physical and psychological trauma to the patients and brings a heavy burden to the families and families of patients.Laryngeal cancer patients often take surgery, radiotherapy and other treatment methods, but these methods often cause patients with speech and speech disorders,patients with adverse psychological effects.With the continuous improvement of clinical diagnosis and treatment, patient survival gradually extended, the quality of their lives are increasingly valued.This basic indicator is the normal function of the throat recovery,preoperative and postoperative mood and cognitive status are also important aspects of quality of life (QOL).This article reviews the progress of preoperative and postoperative mood and cognitive changes in patients with laryngeal cancer..

[Analysis of early failure of the PHILOS in proximal humerus fractures].

OBJECTIVE: To analyze the reasons of early failure of the PHILOS in proximal humerus fractures. METHODS: From Nov. 2010 to Nov. 2014, there were 117 patients with humerus fractures treated with PHILOS locking plate in Department of Orthopaedics, Xuanwu Hospital. All of the patients were treated with the plate by open reduction internal fixation, and we analyzed these cases retrospectively. After the operation, we removed the drainage tube within 48 h, and the patients were allowed to do the passive motion 3 days after the surgery if the X-Ray showed the plate and screws were reliable. Eight cases failed within 4 weeks after the operation. We analyzed the reasons of the failure. RESULTS: The rate of the failed cases was 6.83%(8/117). The average age was 72.4(66-82) years. In the 8 failed cases, 3 were on the right side, and the other 5 on the left side. As for the reason of the fractures, 2 cases were because of car accidents, and the other 6 because of daily life injury. According to the Neer classification, 3 cases were 2-part fractures, and the other 5 3-part fractures. Three cases were total failure, and the other 5 partial failure. All the 8 failed cases failed within 4 weeks after the operation, of which 1 was on the sixth day after surgery, the other 7 2 to 4 weeks after the surgery.The 3 totally failed cases were treated by removing the screws and plates, the other 5 by conservative methods. All of the cases were malunion at the end. CONCLUSION: The early failure of the PHILOS locking plate in proximal humerus fractures is related to the bad reduction during the operation, the loss of medial cortex support, the limitation of screw length, the osteoporosis and the improper rehabilitation after operation.It is very important to do good preoperative plan for a surgeon. During the operation, we should try our best in the fracture reduction, use the appropriate plate and screws, and then pay attention to the rehabilitation after the operation. After all of this, the rate of failure may be decreased.

[Expression of Jagged1 mRNA in human epithelial ovarian carcinoma tissues and effect of RNA interference of Jagged1 on growth of xenograft in nude mice].

OBJECTIVE: To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. METHODS: (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. RESULTS: (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (P<0.01). (2) The tumor volume was (491± 68) mm(3) and tumor mass was (2.6±0.4) g in Jagged1 interference group, which were significantly lower than that in the blank plasmid group [(842±88) mm(3) and (4.4±0.8) g, respectively] and that in the control group [(851±90) mm(3) and (4.5±0.9) g, respectively; P<0.05], the tumor inhibition rate was 42.2% in Jagged1 interference group, which was significantly higher than that in the blank plasmid group and that in the control group (2.2% and 0, respectively), the differences were statistically significant (P<0.05). The relative mRNA and protein expression of Jagged1, Hes1 and Hey1 in xenograft tissues of nude micein Jagged1 interference group were lower than that in the other two groups, the differences were statistically significant (P<0.05). There were no differences of relative mRNA and protein expression of Notch1 in xenograft tissues of nude mice among the three groups (P>0.05). CONCLUSIONS: Jagged1 is highly expressed in epithelial ovarian carcinoma. Jagged1 gene interference in xenograft tumor can inhibit ovarian cancer cell growth and improve tumor suppressor rate, which probably play roles by inhibiting Notch1 signaling pathway.

Bio-Root and Implant-Based Restoration as a Tooth Replacement Alternative.

We previously reported that dental stem cell-mediated bioengineered tooth root (bio-root) regeneration could restore tooth loss in a miniature pig model. As a potential new method for tooth restoration, it is essential to compare this method with the widely used commercial dental implant-based method of tooth restoration. Tooth loss models were created by extracting mandibular incisors from miniature pigs. Allogeneic periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs) were isolated and cultured. A PDLSC sheet was prepared by adding 20.0 µg/mL vitamin C to the culture medium; in addition, a hydroxyapatite tricalcium phosphate (HA/TCP)/DPSC graft was fabricated and cultured in a 3-dimensional culture system. A total of 46 bio-root implantations and 9 dental implants were inserted, and crown restorations were performed 6 mo after implantation. Histological, radiological, biomechanical, and elemental analyses were used to evaluate and compare tissue-engineered bio-roots and dental implants to the natural tooth roots. After 6 mo, both computed tomography scans and histological examinations showed that root-like structures and dentin-like tissues had formed. Three months after crown restoration, clinical assessments revealed that tooth function was equivalent in the regenerated bio-root and the dental implant. Biomechanical testing showed that the bio-roots were similar to natural tooth roots in compressive strength, modulus of elasticity, and torsional force; however, these properties were significantly higher in the dental implants. Elemental analysis revealed a higher similarity in elemental composition between bio-roots and natural tooth roots than between bio-roots and dental implants. However, the dental implant success rate was 100% (9 of 9) and the bio-root success rate was only 22% (10 of 46). Taken together, we showed that an allogeneic HA/TCP/DPSC/PDLSC sheet could successfully build a bio-root with structure and function similar to the natural tooth root; however, tissue engineering procedures must be optimized further to improve the success rate.

[Analysis of early failure of the PHILOS in proximal humerus fractures].

OBJECTIVE: To analyze the reasons of early failure of the PHILOS in proximal humerus fractures. METHODS: From Nov. 2010 to Nov. 2014, there were 117 patients with humerus fractures treated with PHILOS locking plate in Department of Orthopaedics, Xuanwu Hospital. All of the patients were treated with the plate by open reduction internal fixation, and we analyzed these cases retrospectively. After the operation, we removed the drainage tube within 48 h, and the patients were allowed to do the passive motion 3 days after the surgery if the X-Ray showed the plate and screws were reliable. Eight cases failed within 4 weeks after the operation. We analyzed the reasons of the failure. RESULTS: The rate of the failed cases was 6.83%(8/117). The average age was 72.4(66-82) years. In the 8 failed cases, 3 were on the right side, and the other 5 on the left side. As for the reason of the fractures, 2 cases were because of car accidents, and the other 6 because of daily life injury. According to the Neer classification, 3 cases were 2-part fractures, and the other 5 3-part fractures. Three cases were total failure, and the other 5 partial failure. All the 8 failed cases failed within 4 weeks after the operation, of which 1 was on the sixth day after surgery, the other 7 2 to 4 weeks after the surgery.The 3 totally failed cases were treated by removing the screws and plates, the other 5 by conservative methods. All of the cases were malunion at the end. CONCLUSION: The early failure of the PHILOS locking plate in proximal humerus fractures is related to the bad reduction during the operation, the loss of medial cortex support, the limitation of screw length, the osteoporosis and the improper rehabilitation after operation.It is very important to do good preoperative plan for a surgeon. During the operation, we should try our best in the fracture reduction, use the appropriate plate and screws, and then pay attention to the rehabilitation after the operation. After all of this, the rate of failure may be decreased.

Establishing a clinic-based pancreatic cancer and periampullary tumour research registry in Quebec.

BACKGROUND: Enrolling patients in studies of pancreatic ductal adenocarcinoma (pdac) is challenging because of the high fatality of the disease. We hypothesized that a prospective clinic-based study with rapid ascertainment would result in high participation rates. Using that strategy, we established the Quebec Pancreas Cancer Study (qpcs) to investigate the genetics and causes of pdac and other periampullary tumours (pats) that are also rare and underrepresented in research studies. METHODS: Patients diagnosed with pdac or pat were introduced to the study at their initial clinical encounter, with a strategy to enrol participants within 2 weeks of diagnosis. Patient self-referrals and referrals of unaffected individuals with an increased risk of pdac were also accepted. Family histories, epidemiologic and clinical data, and biospecimens were collected. Additional relatives were enrolled in families at increased genetic risk. RESULTS: The first 346 completed referrals led to 306 probands being enrolled, including 190 probands affected with pdac, who represent the population focus of the qpcs. Participation rates were 88.4% for all referrals and 89.2% for pdac referrals. Family history, epidemiologic and clinical data, and biospecimens were ascertained from 91.9%, 54.6%, and 97.5% respectively of patients with pdac. Although demographics and trends in risk factors in our patients were consistent with published statistics for patients with pdac, the qpcs is enriched for families with French-Canadian ancestry (37.4%), a population with recurrent germ-line mutations in hereditary diseases. CONCLUSIONS: Using rapid ascertainment, a pdac and pat research registry with high participation rates can be established. The qpcs is a valuable research resource and its enrichment with patients of French-Canadian ancestry provides a unique opportunity for studies of heredity in these diseases.

Malus hupehensis NPR1 induces pathogenesis-related protein gene expression in transgenic tobacco.

Most commercially grown apple cultivars are susceptible to fungal diseases. Malus hupehensis has high resistance to many diseases affecting apple cultivars. Understanding innate defence mechanisms would help to develop disease-resistant apple crops. Non-expressor of pathogenesis-related genes 1 (NPR1) plays a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). MhNPR1 cDNA, corresponding to genomic DNA and its 5' flanking sequences, was isolated from M. hupehensis. Sequence analysis showed that the regulatory mechanism for oligomer-monomer transition of the MhNPR1 protein in apple might be similar to that of GmNPR1 in soybean, but different from that of AtNPR1 in Arabidopsis. No significant differences in MhNPR1 expression were found in M. hupehensis after infection with Botryosphaeria berengeriana, showing that MhNPR1 might be regulated by pathogens at the protein level, as described for Arabidopsis and grapevine. SA treatment significantly induced MhNPR1 expression in leaves, stems and roots, while methyl jasmonate (MeJA) treatment induced MhNPR1 expression in roots, but not in leaves or stems. The expression of MhNPR1 was highly increased in roots, moderately in leaves, and did not change in stems after treatment with 1-aminocyclopropane-1-carboxylic acid (ACC). SAR marker genes (MhPR1 and MhPR5) were induced by SA, MeJA and ACC in leaves, stems and roots. Overexpression of MhNPR1 significantly induced the expression of pathogenesis-related genes (NtPR1, NtPR3 and NtPR5) in transgenic tobacco plants and resistance to the fungus Botrytis cinerea, suggesting that MhNPR1 orthologues are a component of the SA defence signalling pathway and SAR is induced in M. hupehensis.

Dendritic cells transfected with lentiviral vector-encoding human granulocyte-macrophage colony-stimulating factor augment anti-tumour T-cell response in vitro.

Dendritic cells (DC) are professional antigen-presenting cells that can actively taken up and present tumour-derived proteins to induce a tumour-specific immune response. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a pivotal role in the generation, sensitization, maturation and survival of DC. We charged the peripheral blood monocyte cell-derived DC with tumour lysate, and then transfected the DC with lentiviral vector-encoding human GM-CSF (hGM-CSF). The antigen-presenting capacity of the hGM-CSF-transfected DC was tested by means of the mixed lymphocyte reaction and cytotoxic T-lymphocyte assay using wild-type DC as the control. The Lenti-hGM-CSF-transfected DC was able to stimulate the proliferation of naive allogeneic T lymphocytes and to generate tumour-specific cytotoxic T lymphocytes more efficiently than the wild-type DC. This data indicates that Lenti-hGM-CSF-transfected DC could potentially be used as an effective clinical approach for cancer immunotherapy.

Identification of casein kinase I substrates by in vitro expression cloning screening.

Casein kinase I (CKI) is a widely expressed protein kinase family implicated in diverse processes including membrane trafficking, DNA repair, and circadian rhythm. Despite the large number of CKI genes, few biologically relevant substrates have been identified. As an approach to better defining the spectrum of CKI substrates, we extended a recently described in vitro expression cloning (IVEC) strategy. Polypeptides pools were screened for kinase-dependent electrophoretic mobility shifts. Ten putative CKI substrates were isolated from an initial sample of 3000 random cDNA clones. Candidate substrates include proteins involved in RNA metabolism (a putative RNA helicase, the nucleolar protein hNOP56, and hnRNP A1, and ribosomal proteins L4, L8, and L13), as well as keratin 17, a necdin-related protein, and the calcium-binding proteins desmoglein 2 and annexin II. The same pools were also screened with active ERK2, and four substrates identified: aldolase, NSD-like protein, uracil-DNA glycosylase, and HHR23A. IVEC is an effective method to identify novel protein kinase substrates.

Calcineurin B- and calmodulin-binding preferences identified with phage-displayed peptide libraries.

Calcineurin B (CnB) and calmodulin (CaM) are two structurally similar but functionally distinct 'EF-hand' Ca2+-binding proteins. CnB is the regulatory subunit of the CaM-stimulated protein phosphatase, calcineurin. CaM is a unique multifunctional protein that interacts with and modulates the activity of many target proteins. CnB and CaM are both required for the full activation of the phosphatase activity of calcineurin and are not interchangeable. The two proteins recognize distinct binding sites on calcineurin A subunit (CnA) and perform different functions. Phage-displayed peptide libraries (pIII and pVIII libraries) were screened with CnB and CaM to isolate peptides that could then be compared to determine if there were binding preferences of the two proteins. The Ca2+-dependent binding of phage-displayed peptides to CnB and CaM is specifically blocked by synthetic peptides derived from the CnB-binding domain of CnA and the CaM-binding domain of myosin light chain kinase respectively. Both CnB- and CaM-binding peptides have a high content of tryptophan and leucine, but CnB-binding peptides are more hydrophobic than CaM-binding peptides. CnB-binding peptides are negatively charged with clusters of hydrophobic residues rich in phenylalanine, whereas the CaM-binding peptides are positively charged and often contain an Arg/Lys-Trp motif. The binding preferences identified with peptide libraries are consistent with the features of the CnB-binding domains of all CnA isoforms and the CaM-binding domains of CaM targets.

Lymphocyte responsiveness to glucocorticoids, cyclosporine, or both.

The reason why some patients with glomerular diseases respond to steroid treatment and others do not remains obscure, and it is not possible to prospectively evaluate the probability of response in individual patients. One factor that might contribute to the clinical response to treatment could be the relative sensitivity of a patient's immune system to the suppressive effects of steroids or other immunosuppressive agents. To evaluate this possibility, phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) from 16 patients with various biopsy-proven glomerulopathies were cultured with prednisolone or methylprednisolone in final concentrations of 10(-5) to 10(-8) mol/L. From the dose-response curves, the concentration of steroid required to cause 50% inhibition (IC50) of the PHA-induced proliferative response was determined. The PBMC from 10 patients also were cultured with 400 ng/mL cyclosporine both alone and with 10(-7) mol/L steroid, and the inhibitory effects were calculated. There was considerable heterogeneity in the sensitivities of individual patients to steroid inhibition, and the mean +/- SEM IC50 was significantly lower for methylprednisolone than for prednisolone. Cyclosporine caused 50% or greater inhibition in 6 of the 10 patients but had < 10% inhibitory effect in 2 patients. In most patients studied, cyclosporine plus steroid was significantly more inhibitory than cyclosporine alone, but the combination was usually no more effective than 10(-7) mol/L methylprednisolone alone. These results are consistent with the hypothesis that differences in the sensitivity of individual patient's immune systems to the immunosuppressive effects of steroids and cyclosporine might contribute to differences in their clinical responsiveness to treatment.

Photoaffinity labeling of a peptide substrate to myosin light chain kinase.

The substrate binding properties of skeletal muscle myosin light chain kinase were investigated with a synthetic peptide containing the photoreactive amino acid p-benzoylphenylalanine (Bpa) incorporated amino-terminal of the phosphoacceptor serine (BpaKKRAARATSNVFA). When photolyzed at 350 nm, the peptide was cross-linked stoichiometrically to myosin light chain kinase in a Ca2+/calmodulin-dependent manner. Peptide incorporation into kinase inhibited light chain phosphorylation, and the loss of kinase activity was proportional to the extent of peptide incorporated. After peptide I was incorporated into myosin light chain kinase, it was partially phosphorylated in the absence of Ca2+/calmodulin. The extent of phosphorylation increased in the presence of Ca2+/calmodulin. The cross-linked photoadduct was digested, labeled peptides were purified by high performance liquid chromatography, and sites of covalent modification were determined by amino acid sequencing and analysis. The covalent modification in the catalytic core occurred on Ile-373 (66%) and in a peptide containing residues Asn-422 to Met-437 (14%), respectively. Lys-572 in the autoinhibitory region accounted for 20% of the incorporated label. The coincident covalent modification of the autoinhibitory domain suggests that it is located near the catalytic site. Based upon a model of the catalytic core, the substrate peptide is predicted to bind in the cleft between the two lobes of the kinase. The orientation of the substrate peptide on myosin light chain kinase is similar to the orientation of the substrate recognition fragment, but not the high affinity binding fragment, of inhibitor peptide of cAMP-dependent protein kinase in the catalytic subunit of the cAMP-dependent protein kinase.

Activation of four enzymes by two series of calmodulin mutants with point mutations in individual Ca2+ binding sites.

Activation of four target enzymes by two series of calmodulin Ca2+ binding site mutants has been examined. In each mutant, the conserved bidentate glutamate of one of the Ca2+ binding sites is mutated to glutamine or lysine. The enzymes studied were smooth and skeletal muscle myosin light chain kinases, adenylylcyclase, and plasma membrane Ca(2+)-ATPase. For the first three enzymes, the activation patterns with the two mutant series were very similar: mutation of site 4 was most deleterious, then site 2, site 3, and site 1. This ranking was observed previously in Ca2+ binding and Ca(2+)-induced conformational studies of these mutants. Thus the response of these enzymes is probably determined by the extent to which each mutant's competence to interact with target binding regions has been compromised. In contrast, for Ca(2+)-ATPase, mutants of sites 3 and 4 were much poorer activators than those of sites 1 and 2. Events beyond calmodulin binding and related to enzyme activation probably dictate this unusual activation pattern and also the anomalously poor activation of skeletal muscle myosin light chain kinase by site 1 mutant B1Q. Site 1 mutant B1K showed wild type activation of all four enzymes suggesting that in site 1, the lysine substitution can evoke the conformational changes associated with Ca2+ binding.

Autophosphorylation of skeletal muscle myosin light chain kinase.

Ca2+/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca2+/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of 32P/mol of kinase and a slower rate of incorporation up to 1.52 mol of 32P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low Km value for myosin light chain kinase. Autophosphorylation at both rates was concentration-independent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended "tail" region of the enzyme can fold into the active site of the kinase.