Effects of individual and partner factors on anxiety and depression in Taiwanese prostate cancer patients: A longitudinal study.

Studies exploring the mediating and predictive factors of anxiety and depression for prostate cancer patients in Eastern countries are scant. Guided by the transactional model of stress and coping, this study determined the predictors and mediators of anxiety and depression in prostate cancer patients. The participants comprised 115 prostate cancer patients and 91 partners. The patients and partners completed questionnaires regarding physical symptoms, disease appraisals, coping behaviours, anxiety and depression in the period before confirmation of treatment decisions and 1, 3, 6 and 12 months after treatment. The results revealed that partner anxiety engendered a stressful situation and aggravated patient anxiety. Patients' threat appraisals and affective-oriented coping behaviours mediated the relationships between their anxiety levels and those of their partners. The patients' most recent prostate-specific antigen (PSA) levels and hormonal symptoms were key predictors of their anxiety and depression levels. The patients' harm appraisals mediated the relationships between their most recent PSA levels and hormonal symptoms and depression. Their threat appraisals and affective-oriented coping behaviours mediated the relationships between their hormonal symptoms and anxiety and depression. To manage those key factors, reframing, appraising disease and improving coping behaviours may reduce anxiety and depression levels in prostate cancer patients.

Development of the hybrid Sleeping Beauty: baculovirus vector for sustained gene expression and cancer therapy.

Antiangiogenesis is an appealing anticancer approach but requires continued presence of the antiangiogenic agents, which can be remedied by gene therapy. Baculovirus is an emerging gene delivery vector but only mediates transient expression (<7 days); thus, this study primarily aimed to develop a hybrid baculovirus for sustained antiangiogenic gene expression and cancer therapy. We first constructed plasmids featuring adeno-associated virus inverted terminal repeats (AAV ITRs), oriP/Epstein-Barr virus-expressed nuclear antigen 1 (EBNA1) or Sleeping Beauty (SB) transposon and compared their efficacies in terms of persistent expression. In human embryonic kidney (HEK293) cells, AAV ITR failed to prolong the expression while oriP/EBNA1 moderately extended the expression to 35 days. In contrast, the SB system led to stable expression beyond 77 days even without antibiotic selection. Given this finding, we constructed a hybrid SB baculovirus expressing the SB transposase and harboring the transgene cassette flanked by inverted repeat/direct-repeat (IR/DR) elements recognizable by SB. The hybrid SB baculovirus efficiently transduced mammalian cells and mediated an expression duration longer than that by conventional baculoviruses, thanks to the transgene persistence and integration. The SB baculovirus (Bac-SB-T2hEA/w) expressing the antiangiogenic fusion protein comprising endostatin and angiostatin (hEA) also enabled prolonged hEA expression. With sustained hEA expression, Bac-SB-T2hEA/w repressed the angiogenesis in vivo, hindered the growth of two different tumors (prostate tumor allografts and human ovarian tumor xenografts) in mice and extended the life span of animals. These data altogether implicated the potential of the hybrid SB-baculovirus vector for prolonged hEA expression and for the treatment of multiple types of angiogenesis-dependent tumors.

Mifepristone acts as progesterone antagonist of non-genomic responses but inhibits phytohemagglutinin-induced proliferation in human T cells.

BACKGROUND: Progesterone is an endogenous immunomodulator that suppresses T cell activation during pregnancy. The stimulation of membrane progesterone receptors (mPRs) would seem to be the cause of rapid non-genomic responses in human peripheral T cells, such as an elevation of intracellular calcium ([Ca(2+)](i)) and decreased intracellular pH (pH(i)). Mifepristone (RU486) produces mixed agonist/antagonist effects on immune cells compared with progesterone. We explored whether RU486 is an antagonist to mPRs and can block rapid non-genomic responses and the induction by phytohemagglutinin (PHA) of cell proliferation. METHODS: Human male peripheral T cell responses in terms of pH(i) and [Ca(2+)](i) changes were measured using the fluorescent dyes, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and fura-2, respectively. Expression of mPR mRNA was determined by RT-PCR analysis. Cell proliferation and cell toxicity were determined by [(3)H]-thymidine incorporation and MTT assay, respectively. RESULTS: The mRNAs of mPRalpha, mPRbeta and mPRgamma were expressed in T cells. RU486 blocked progesterone-mediated rapid responses including, the [Ca(2+)](i) increase and pH(i) decrease, in a dose related manner. RU486 did not block, but enhanced, the inhibitory effect of progesterone on PHA induced cell proliferation. RU486 alone inhibited proliferation induced by PHA and at >25 microM seems to be cytotoxic against resting T cells (P < 0.01). CONCLUSIONS: RU486 is antagonistic to the rapid mPR-mediated non-genomic responses, but is synergistic with progesterone with respect to the inhibition of PHA-induced cell proliferation. Our findings shine new light on RU486's clinical application and how this relates to the non-genomic rapid physiological responses caused by progesterone.

Antiproliferative effects of 2-methoxyestradiol alone and in combination with chemotherapeutic agents on human endometrial cancer cells.

OBJECTIVE: 2-methoxyestradiol (2-ME), an endogenous estradiol metabolite, has potent antiproliferative effects on cancer cells. However, its usefulness for treating endometrial cancer has not yet been fully explored. We investigated for the first time whether in vitro combinations of 2-ME with various chemotherapeutic agents might result in a synergistic inhibitory effect on the proliferation of human endometrial cancer cells. METHODS: As a model, two different human endometrial cancer cell lines, HEC-1-A and RL95-2, were used. These cells were treated with 2-ME alone or in combination with paclitaxel, cisplatin, or doxorubicin. Measurements to detect an antiproliferative effect were performed after 24, 48, and 72 hours using the MTT assays. RESULTS: In both endometrial cancer cell lines a significant synergistic effect of 2-ME with paclitaxel was observed. The combination of 2-ME and cisplatin was not synergistic and provided only additive effects. The antiproliferative effect of 2-ME was somewhat antagonized by doxorubicin. CONCLUSIONS: Our study shows that 2-ME has a direct antiproliferative effect on endometrial cancer cells. Our results also show a potential anticancer synergy between 2-ME and paclitaxel in vitro. On the other hand, no remarkable synergistic actions were observed between 2-ME and doxorubicin, suggesting that 2-ME may selectively enhance the anticancer actions of certain chemotherapeutic agents in human endometrial cancer. Therefore, combination therapy should be investigated further as an additional therapeutic option for advanced or recurrent endometrial cancer.

Effective Decision-Making and Decisional Regret in Living Kidney Donors of Taiwan.

BACKGROUND: While most living kidney donors are satisfied with their decision and do not regret donating, few studies have been conducted on the determinants related to the effectiveness and regret of the decision. This study aims to explore the relationship between basic attributes, quality of life, positive affect, negative affect, effectiveness of decision-making, and regret in living kidney donors. METHODS: In this cross-sectional study, living kidney donors were recruited from urology and kidney transplant outpatient services. The structured questionnaire used to collect the data included the Positive and Negative Affect Schedule, Medical Outcomes Study 12-Item Short-Form Health Survey, Decision Conflict Scale, and Decision Regret Scale. RESULTS: The findings indicate that living donors with better health status, 24-hour creatinine clearance, physical health-related quality of life (HRQOL), and positive affect experienced greater feelings of effective decision-making. Moreover, women and donors with better physical HRQOL, positive affect, and decision effectiveness were less regretful about the decision of kidney donation. CONCLUSION: Health status, physical HRQOL, and positive affect are related to decision validity and regret of living donors. Therefore, clinical care providers should regularly assess the mood and health of living kidney donors. Furthermore, activities promoting their health should be encouraged, especially for men.

Positive and Negative Affects in Living Kidney Donors.

OBJECTIVE: This study aimed to identify the factors influencing the positive and negative affects and the health-related quality of life (HRQOL) of living kidney donors. METHODS: With the use of a cross-sectional study design and a structured questionnaire, information on the basic characteristics, positive affect, negative affect, and HRQOL of 41 living kidney donors were compared. RESULTS: The negative affect in living kidney donors was similar to that of the general population, but the positive affect was slightly lower. The physical HRQOL of living kidney donors was slightly higher than that of the general population, and the mental HRQOL was similar. Female donors showed a greater positive affect than male donors. The donors who were siblings of the recipients showed a more negative affect. Donors without chronic disease and with good perceived physical health showed improved positive affect, negative affect, and mental HRQOL. Furthermore, living kidney donors with better positive and negative affects showed improved physical and mental HRQOLs. CONCLUSIONS: Clinical health providers should evaluate and determine the positive affect, negative affect, and quality of life of living kidney donors, especially in men, siblings of the recipients, those with chronic disease, and those with poorer perceived physical health. Moreover, psychosocial interventions should be provided to improve these factors.

Calcitonin inhibits testosterone and luteinizing hormone secretion through a mechanism involving an increase in cAMP production in rats.

Effects of calcitonin peptides, including human calcitonin (hCT), salmon calcitonin (sCT), and calcitonin gene-related peptide (CGRP), on the secretion of testosterone and luteinizing hormone (LH) in male rats were studied. Male rats were injected intravenously with human chorionic gonadotropin (hCG), calcitonin peptides, or hCG plus calcitonin peptides. Blood samples were collected at several intervals following hormone challenge. In an in vitro experiment, testis blocks were incubated with hCG (0, 0.05, 0.5, or 5 IU/ml) or hCG (0.5 IU/ml) plus calcitonin peptides (0-10(-9) or 10(-6) M) at 34 degrees C for 30 minutes. Both medium and plasma samples were extracted by ether and analyzed for testosterone by radioimmunoassay (RIA). The concentration of calcium in each plasma sample was measured by an automatic calcium analyzer. The anterior pituitary gland (AP) was incubated with or without calcitonin peptides (0-10 nM) at 37 degrees C for 30 minutes. They were then incubated with gonadotropin releasing hormone (GnRH, 10 nM) for a further 30 minutes. The concentration of LH in AP medium was measured by RIA. The accumulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in both testicular tissues and APs were measured by RIA. A single intravenous injection of calcitonin peptides decreased the basal and hCG-stimulated levels of plasma testosterone gradually from 60 to 180 or 360 minutes after challenge. The plasma calcium was not altered by the injection of calcitonin peptides and/or hCG. Administration of calcitonin peptides in vitro resulted in a dose-dependent inhibition of both basal and hCG-stimulated release of testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 immunotherapy in children.

Immunotherapy with interleukin (IL)-2 possesses great potential in the treatment of immune-mediated diseases and cancers. However, only a few reports on a small number of children have appeared in the literature. From March 1988 to March 1989, 11 children and adolescents were treated with IL-2. They included 1 patient with hepatocellular carcinoma, 1 with hepatoblastoma, 6 with childhood atopic dermatitis, and 3 with juvenile rheumatoid arthritis. The dosages ranged from 10,000 to 50,000 U/kg every 8 hours by intravenous drip. The following side effects were observed: anorexia, fever, and chillness (100%), general malaise (82%), irritability (64%), diarrhea (100%), nausea and vomiting (73%), weight gain (82%), edema (82%), abdominal distension (73%), oliguria (82%), cough (91%), dyspnea (27%), pleural effusion (40%), hypotension (82%), skin eruption (82%), oral ulcer (18%), enlarged liver (73%) liver function abnormalities (82%), renal function impairment (36%), electrolyte imbalance (73%), anemia (91%), thrombocytopenia (54%), leukopenia (18%), and eosinophilia (73%). Immunologically, numbers of natural killer cells were increased and natural killer and lymphokine-activated killer cell activities were augmented after IL-2 treatment. There was a tendency for serum levels of IL-2 and receptor IL-2 to decrease, especially in patients with atopic eczema. Ten patients (91%) completed one course (9 to 12 days) of therapy, and the remaining patient interrupted the treatment because of intolerable adverse effects. Clinically, complete remission for 3 months was obtained in 1 juvenile rheumatoid arthritis patient, transient improvement (2 to 6 weeks) in all atopic dermatitis patients, minor response in the hepatoblastoma patient, and no response in the patient with hepatocellular carcinoma.

Transcriptional activation of c-myc proto-oncogene by estrogen in human ovarian cancer cells.

NIH:OVCAR-3 is a human ovarian cancer cell line that expresses a moderate amount of estrogen receptors, 28 fmol/mg protein. We have found that estrogen at a concentration of 10(-7) M induced a 2.3-fold increase in the growth of NIH:OVCAR-3 cells after 48 h stimulation. A 4-fold increase in c-myc mRNA expression at 30 min and a 7.5-fold increase at 50 min post-induction with estradiol were observed. Nuclear run-on analysis indicated that c-myc transcripts increased 4-fold within 10 min of estrogen addition. The half-life of c-myc mRNA was 64 min +/- 5 min and was not affected by estrogen. Antisense oligonucleotide to c-myc specifically inhibited the estrogen stimulated c-myc protein expression as well as the growth of NIH:OVCAR-3 cells. A control ovarian cancer cell line OC-3-VGH that had few estrogen receptors (1 fmol/mg protein) did not respond to estrogen in growth; however, these cells respond to estrogen with a 1.5-fold increase in c-myc mRNA. The stability of c-myc mRNA of these cells was not affected by estrogen. Our results indicate that transcriptional induction of c-myc expression by estrogen plays a critical role in the proliferation of NIH:OVCAR-3 cells.

The protective effect of hypervolemic hemodilution in experimental heatstroke.

The authors tested the hypothesis in a rat model that hypervolemic hemodilution during heatstroke affected the mean arterial pressure (MAP), striatal dopamine (DA) release, and local cerebral blood flow and neuronal damage score in different brain structures. The heatstroke was induced by exposing the urethane-anesthetized rats to an ambient temperature of 42 degrees C. Hypervolemic hemodilution was produced by intravenous administration of 10% human albumin. Relative and absolute blood flow in the corpus striatum were determined using the laser Doppler flowmetry and the autoradiography diffusible tracer technique, respectively. The DA release in the striatum was estimated using the in vivo microdialysis technique. After onset of heatstroke, animals with hypervolemic state alone, produced by saline or heparinized blood injection, displayed higher values of DA release, as well as neuronal damage score in the striatum, hypothalamus, or cortex, but lower values of MAP and blood flow in the striatum, hypothalamus, or cortex compared to normothermic controls. However, the heatstroke-induced arterial hypotension, cerebral ischemia, increased striatal DA overload, and increased neuronal damage score were attenuated by induction of both hypervolemic and hemodilution state with 10% albumin either before or after the onset of heatstroke. In addition, constant infusions of a vasopressor agent phenylephrine (2 microg kg(-1) min(-1)) after the onset of heatstroke failed to maintain appropriate levels of MAP and resulted in no protection against heatstroke. Thus, it appears that the observed benefit of the 10% albumin is secondary to hemodilution and/or maintenance of MAP.

Point mutation of the ras oncogene in human ovarian cancer.

The ras oncogene exists in a variety of human cancers, including carcinomas of bladder, breast, colon, kidney, liver, lung, ovary, pancreas, and stomach. The ras genes acquire transforming activity either by enhanced expression or by a single point mutation. A single base-pair mutation at specific sites within ras genes endows them with the capacity to transform certain cell lines in vitro. In this study, we showed the patterns of point mutations in codons 12, 13, and 61 of ras genes in human ovarian cancer. The experimental procedures were isolation of genomic DNA from normal ovary and ovarian cancer tissue specimens, amplification of a genomic DNA segment (about 100 bp) using different 5' and 3' extension primers in the polymerase chain reaction (PCR), labeling and purification of synthetic mutation-specific oligonucleotide probes, slot-blot hybridization, and autoradiography. The three reaction steps for the PCR cycle were: 96 degrees C for 1 min in step 1, 56 degrees C for 1 min in step 2, and 74 degrees C for 1 min in step 3. The PCR reaction was repeated totally for 30 cycles. In 28 tissue specimens of human ovarian cancer examined, one specimen was found with a c-Ha-ras point mutation at codon 12, two had a c-Ki-ras mutation at codon 12, and one had a c-Ki-ras mutation at codon 13.

Neuronal connections between the auricular skin and the sympathetic pre- and postganglionic neurons of the dog as studied by using pseudorabies virus.

Pseudorabies virus (PrV) as a neuronal tracer was microinjected into the concave surface of the puppy's left pinna to establish the morphological basis of somato-visceral linkage. The virus infected neurons were detected by FITC conjugated with polyclonal swine anti-PrV serum. Labelled neurons were localized in: (1) the trigeminal, geniculate and superior vagal ganglia; (2) the subnucleus caudalis of the spinal trigeminal nucleus; (3) the intermediolateral column (IML) of the thoracolumbar segments and (4) the sympathetic chain ganglia. Present results suggest that when injected into the peripheral nerves, PrV was retrogradely transported to the nerve cell bodies located in the respective sensory ganglia. From the first order sensory neurons, the virus would self-replicate and was transported trans-synaptically via the brainstem nuclei and IML to reach the neurons in the sympathetic ganglia.

Differential expression of beta-amyloid precursor and Bcl-2 proto-oncogene proteins in the developing dog retina.

Previous studies of rat retinas have not only provided evidence that beta-amyloid precursor (APP) and B-cell lymphoma proto-oncogene (Bcl-2) proteins are colocalized in retinal Müller glial cells, but have also indicated that common mechanisms regulate their expression in these cells (Chen, S.T., Garey, L.J., Jen, L.S., 1994. Bcl-2 proto-oncogene protein immunoreactivity in normally developing and axotomised rat retinas. Neurosci. Lett. 172, 11 14; Chen, S.T., Gentleman, S.M., Garey, L.J., Jen, L.S., 1996. Distribution of beta-amyloid precursor and B-cell lymphoma proto-oncogene proteins in the rat retina after optic nerve transection or vascular lesion. J. Neuropathol. Exp. Neurol. 55, 1073-1082; Chen, S.T., Garey, L.J., Jen, L.S., 1997. Expression of beta-amyloid precursor protein immunoreactivity in the retina of the rat during normal development and after neonatal optic tract lesion. NeuroReport 8, 713-717). This investigation attempts to resolve whether or not the pattern observed in rats also applies to other higher mammalian species by examining the expression of immunoreactivity to APP and Bcl-2 in developing as well as mature dog retinas using immunocytochemical methods. Experimental results indicate that the immunoreactivity of both APP and Bcl-2 is located primarily in the inner retina, particularly in the ganglion cells and their axons in late fetal and neonatal stages. From the second postnatal week (the time of eye opening) onwards, immunoreactivity to APP, but not Bcl-2, is localized primarily in the endfeet and proximal part of the radial process of retinal Müller glial cells. Although the findings show both APP and Bcl-2 are expressed in ganglion cells and their processes suggest that the molecules have a role in the differentiation of neurons in the central nervous tissue, the lack of Bcl-2 in the Müller glial cells in dog retinas further suggests that the two molecules may have different biological roles with respect to glial function.

The role of bcl-2 in the progression of the colorectal adenoma-carcinoma sequence.

BACKGROUND: The bcl-2 proto-oncogene is a known inhibitor of apoptosis that may allow the accumulation and propagation of cells containing genetic alterations. METHODS: An immunohistochemical study was performed to examine the role of BCL-2 protein expression in normal colonic mucosa, adenoma, and adenocarcinoma. RESULTS: BCL-2 was present in the crypt base of normal mucosal glands, while diffuse expression of protein product was observed in 69 cases (65.1%) of adenoma and 29 cases (60%) of adenocarcinoma (p > 0.5). A diffuse expression pattern was often noted in adenomas of the tubular type, solitary lesions, small lesions (< 1 cm), and those with mild glandular dysplasia (p < 0.05, respectively). There was, however, no apparent difference as to location, Yamada type of gross appearance, and gender of patients (p > 0.05, respectively). Patterns of BCL-2 expression did not correlate with the biologic indicators of adenocarcinoma (p > 0.1, respectively). CONCLUSIONS: Our results support that bcl-2 may play an important role in the early stage of the adenoma-carcinoma sequence. Down-regulation of bcl-2 is associated with the risk of malignant transformation for colorectal adenoma.

Lactate and the effects of exercise on testosterone secretion: evidence for the involvement of a cAMP-mediated mechanism.

The effects of swimming and lactate on the release of testosterone were examined in male rats. During in vivo experiments, male rats were catheterized via the right jugular vein and blood was collected at 0, 10, 15, 30, and 60 min following the exercise, or they were catheterized via the right jugular vein and the left femoral vein and blood was collected at 0, 2, 5, 10, 15, 30, 60, and 120 min after a 10-min infusion at lactate (13 mg.kg-1.min-1). Trunk blood and blood from the testicular vein were also collected after 10 min of swimming or water immersion. In an in vitro experiment, testicular fragments were challenged with lactate (0.01-10 mM) and/or human chorionic gonadotropin (hCG; 0.5 IU.mL-1), and the mediobasal hypothalamus (MBH) was challenged with lactate (8 mM). The post-exercise levels of plasma lactate and testosterone at 10, 15, and 30 min were higher than resting levels. Plasma luteinizing hormone (LH) was increased following 30 min of swimming. Administration of lactate or hCG increased in a dose dependent manner testicular cyclic adenosine 3':5' monophosphate (cAMP) and testosterone release. Plasma testosterone increased after swimming and lactate infusion. Incubation of MBH with lactate increased the gonadotropin-releasing hormone (GnRH) level in the medium. These results suggest that the increased plasma testosterone levels in male rats during exercise is at least partially a result of a direct and LH-independent stimulatory effect of lactate on the secretion of testosterone by increasing testicular cAMP production. Swim-elevated plasma LH may be a result of a rise of GnRH caused by lactate.

Cell cycle analysis and detection of proliferative cell nuclear antigen of the endometrium after hormone replacement therapy.

BACKGROUND: To understand the effect of sequential combined hormone replacement therapy (HRT) on the postmenopausal endometrium. METHODS: Sonographic endometrial thickness, endometrial histopathology, flow cytometric cell cycle analysis and the level of proliferative cell nuclear antigen (PCNA) were studied. RESULTS: One hundred and thirty-eight postmenopausal women were enrolled in this study. Among which, 97 women had their endometrium being adequately obtained; the most frequent type of histopathology was normal endometrium (91.8%). Endometrial hyperplasia was found in seven patients (7.2%), including typical simple hyperplasia (n=1, 1%), focal simple hyperplasia (n=5, 5.2%) and complex hyperplasia without atypia (n=1, 1%). The proliferative fractions (PF; S plus G2-M phase) of cells from normal and hyperplastic endometrium of menopausal women after HRT were 8.18 and 8.95%, respectively, which were lower than those from 29 premenopausal women without HRT. The level of PCNA of normal and hyperplastic endometrium in postmenopausal women after HRT was about 80 and 84%, respectively, of that from premenopausal endometrium. CONCLUSIONS: Our study showed the PF of the cell cycle and the level of PCNA were not increased in the menopausal endometrium under HRT as compared to the premenopausal controls.

Multiple colonic ulcers caused by Churg-Strauss syndrome in a 15-year-old girl.

Churg-Strauss syndrome (CSS), or allergic granulomatous angiitis, is an uncommon vasculitic syndrome that is found mainly in middle-aged adults. We describe a 15-year-old girl with CSS, diagnosed by histological findings and characteristic clinical features. The patient experienced two episodes of catastrophic gastrointestinal vasculitis, resulting in resection of 150 cm of small intestine and right hemicolectomy. Colonoscopic examination showed multiple colonic ulcers with active bleeding. The clinical course of the patient was grave and refractory to the therapy of steroid and cytotoxic drugs. In the world literature only two patients with multiple colonic ulcers caused by CSS have been reported, and very rare cases of childhood-onset CSS have been published. We reviewed CSS in children and found that the prognosis was poorer than that in adults.

Experiments in intensity guided range sensing recognition of three-dimensional objects.

With the advent of devices that can directly sense and determine the coordinates of points in space, the goal of constructing and recognizing descriptors of three-dimensional (3-D) objects is attracting the attention of many researchers in the image processing community. Unfortunately, the time required to fully sense a range image is large relative to the time required to sense an intensity image. Conversely, a single intensity image lacks the depth information required to construct 3-D object descriptors. This paper presents a method of combining the two sensory sources, intensity and range, such that the time required for range sensing is considerably reduced. The approach is to extract potential points of interest from the intensity image and then selectively sense range at these feature points. After the range information is known at these points, a graph structure representing the object in the scene is constructed. This structure is compared to the stored graph models using an algorithm for partial matching. The results of applying the method to both synthetic data and real intensity/range images are presented.

Small neuraminidase gene of Clostridium perfringens ATCC 10543: cloning, nucleotide sequence, and production.

The small nanH gene encoding a neuraminidase from Clostridium perfringens ATCC 10543 was cloned in JM109 using pUC19 as a vector. Sequence analysis revealed an ORF, nt 310-1455, encoding 382 amino acids that was proceeded by a typical Shine-Dalgarno sequence, GGACGAGA. The nt sequence in the 15-402 region had in vivo promoter activity in an Escherichia coli promoter probe plasmid pKK232-8, which suggested that the small nanH promoter is functional in E. coli. Four regions of amino acids demonstrated great similarity to the "Asp boxes" (-Ser-X-Asp-X-Gly-X-Thr-Trp-) of other bacterial nanH proteins. The small nanH expressing clone, pCPN-1, which was cultured under aerobic conditions resulted in NanH activity which was 203-fold in culture and 211-fold in intracellular fraction compared to that of C. perfringens which has to be cultured under anaerobic conditions. Production of small NanH was also induced by adding sialyllactose to the culture medium of JM109 [pCPN-1]. The enzyme activity could be detected in the periplasmic fraction and the culture medium of JM109 [pCPN-1] after culturing to the stationary phase. The molecular weight, K(m), and optimum pH and pI of the cloned enzyme are identical to those of the parent strain. The cloned, small nanH could be used to study the structure-functional relationship of nanH, while the pCPN-1 clone could be used in the aerobic production of neuraminidase.

Site-directed mutations of the catalytic and conserved amino acids of the neuraminidase gene, nanH, of Clostridium perfringens ATCC 10543.

The small nanH gene encoding the neuraminidase from Clostridium perfringens ATCC 10543 was cloned in JM109 using pUC19 as a vector. Sequence analysis revealed an ORF encoding 382 amino acids without a signal peptide sequence. Four regions of amino-acid sequence, 71-82, 140-151, 208-219, and 255-266 constituted four repeated and conserved sequence motifs-Ser-X-Asp-X-Gly-X-Thr-Trp-, the "Asp boxes." When compared, the nanH polypeptides of C. perfringens ATCC 10543 and Salmonella typhimurium LT12 shared 33% sequence identity and 60% similarity if conservative replacements were included. The homology-modeled structure of C. perfringens NanH showed the same folding topology as the x-ray three-dimensional structure of NanH in S. typhimurium LT12. Amino acid residues Arg37, Arg56, Asp62, His63, Asp100, Glu230, Asp247, Tyr347, and Glu362 located around the pocket of modeled C. perfringens small nanH were superimposed with the active-site pocket of S. typhimurium LT12, nanH. The catalytic amino-acid residues as well as the role of the "Asp boxes" have not been characterized for C. perfringens and S. typhimurium. In this study, Asp100, Glu230, and Asp62 were found to be involved in the catalytic activity of C. perfringens small nanH with immunoreactive properties and site-directed mutagenesis analysis. Four "Asp-box" motifs were found remote from the active-site pocket. Mutational and immunoreactive analysis of the highly conserved amino acids located in the "Asp boxes" suggest that these highly conserved residues are important in maintaining the tertiary structure of NanH. The results of this study provide some knowledge for the design of new inhibitors of small neuraminidase.