Fed-batch cultures of recombinant Rchia pastoris were conducted for production of angiostatin. The whole fermentation included a growth phase on glycerol and an expression phase on methanol. When ammonium hydroxide solution was used to adjust pH, the cell growth during the expression phase was inhibited and the highest angiostatin concentration was 9.08 mg/L. Shake-flask cultures were carried out in media containing different quantities of ammonia. The results showed that ammonia had an obvious inhibition effect on the cell growth during the expression phase. Therefore KOH solution was used to adjust pH, and during the expression phase cells were able to grow and the highest angiostatin concentration reached 20 mg/L.

OBJECTIVETo investigate aging-related ionic remodeling of L-type voltage dependent calcium channel (LVDCC) in left atria of canine.

METHODSSeven adult (2.0 - 2.5 years) and 10 aged (> 8 years) dogs were used. The current of LVDCC was recorded by patch clamp technique in the whole cell mode. The action potential duration (APD(90)), amplitude of action potential plateau (APA), I(Ca-L) peak current density of LVDCC were recorded. The mRNA and protein expressions of α1c subunit (Ca(V1.2)), sarcoplasmic reticulum Ca(2+)-ATPase (SECRA(2)), Calpain-I, ryanodine receptor (RYR(2)) were detected by quantitative RT-PCR and Western blot, respectively.

RESULTSI(Ca-L) peak current density [(-8.11 ± 0.54) pA/pF vs. (-14.04 ± 0.82) pA/pF, P < 0.05] was significantly reduced and action potential duration to 90% repolarization (APD(90)) significantly prolonged [(340.5 ± 10.1) ms vs. (320.0 ± 7.9) ms, P < 0.05] in aged group than in adult group. The mRNA gene expression level of Ca(V1.2) was significantly lower (0.90 ± 0.35 vs. 2.38 ± 0.40, P < 0.05) while mRNA expression of RYR(2) was significantly higher (4.39 ± 4.68 vs. 1.49 ± 1.69, P < 0.05) in the aged dogs than in the adult dogs. mRNA expression of SECRA(2) and Calpain-I was similar between the two groups. Similarly, the protein expression level of Ca(V1.2) was significantly lower (0.13 ± 0.10 vs. 0.29 ± 0.12, P < 0.05) while the protein expression level of RYR(2) was significantly higher (0.18 ± 0.21 vs. 0.08 ± 0.36, P < 0.05) in the aged dogs than in the adult dogs. Again, protein expression of SECRA(2), PLN(1) and Calpain-I was similar between the two groups.

CONCLUSIONThese data suggest that aging could induce mRNA and protein expression changes of Ca(V1.2) and RYR(2) of LVDCC which might serve as the molecular basis of I(Ca-L) remodeling in aged dogs and might be linked to the increased likelihood of developing atrial fibrillation (AF) in aged dogs.

Action Potentials ; Age Factors ; Animals ; Atrial Fibrillation ; metabolism ; physiopathology ; Calcium Channels, L-Type ; metabolism ; Dogs ; Heart Atria ; metabolism ; physiopathology ; Myocytes, Cardiac ; metabolism ; Patch-Clamp Techniques ; Ryanodine Receptor Calcium Release Channel ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

OBJECTIVETo study the diagnosis and treatment of lumbar internal disc disruption (IDD).

METHODSThirty-six patients with chronic disabling low back pain proved by discography as IDD were treated with disc excision and lumbar interbody fusion, and 8 cases were treated with PLIF, 28 with ALIF. The clinical results were evaluated by pre- and post-operative VAS, and the fusion results were evaluated by X-ray studies of the lumbosacral spine.

RESULTSThe average period of follow-up was 18 months, ranging from 6 to 26 months. Six patients treated with PLIF basically disappeared low back pain, 2 complained of mild back pain; the VAS after operation was decreased significantly in comparison with that of pre-operation (P < 0.01). Twenty-seven treated with ALIF basically disappeared low back pain; the VAS after operation was decreased significantly in comparison with that of pre-operation (P < 0.01). Fusion rate was 88% in patients who were treated with PLIF, and 97% in patients with ALIF.

CONCLUSIONDisc excision and interbody fusion is an effective method for the treatment of IDD, but the operation indications should be known well.

Adolescent ; Adult ; Female ; Follow-Up Studies ; Humans ; Internal Fixators ; Intervertebral Disc ; diagnostic imaging ; Low Back Pain ; etiology ; Lumbar Vertebrae ; diagnostic imaging ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Spinal Diseases ; complications ; diagnosis ; surgery ; Spinal Fusion ; instrumentation ; methods ; Tomography, X-Ray Computed ; Treatment Outcome ; Young Adult

The present study aimed to investigate the mRNA expression levels of collagen, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) in the left and right atria in patients with chronic atrial fibrillation (CAF). Forty five patients with valvular heart disease were sampled in this study, including 18 patients with sinus rhythm (SR), 27 patients with CAF. Clinical data of these patients were collected, and the left and right atrial appendages were obtained from these patients during heart valvular replacement surgery. The mRNA levels of collagen type I, collagen type III, MMP1, MMP9, TMP1 of the atria were then measured by semi-quantitative RT-PCR. The results obtained were as follows. Compared to those in SR group, in atria of CAF group, the mRNA levels of collagen type I, MMP1 and MMP9 increased (P<0.05), while the mRNA level of TMP1 decreased (P<0.01). There were no significant differences in the mRNA levels of collagen type I, collagen type III, MMP1, MMP9 and TMP1 between the left and right atria of SR group (P>0.05). In CAF group, the mRNA level of MMP1 in the right atrium was higher than that in the left atrium (P<0.05), however, the mRNA level of MMP9 in the left atrium was higher than that in the right atrium (P<0.01). In both the left and right atria, the mRNA of collagen type I was positively correlated with the corresponding atrial diameter; the mRNA of MMP1 and MMP9 was positively correlated with the mRNA of collagen type I, and was negatively correlated with the mRNA of TMP1. These results suggest that the increased level of collagen type I associated with selective upregulation of MMP1, 9 and downregulation of TMP1, 9 in the atrium might be the molecular basis of atrial interstitial fibrosis in patients with CAF. Moreover, during CAF development, there is difference in the expression of MMPs between the left and right atria.
Aged ; Atrial Fibrillation ; physiopathology ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Heart Atria ; metabolism ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; RNA, Messenger ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

A recombinant strain of Pichia pastoris with a phenotype of Muts was used to produce angiostatin in a 5-L fermentor. The methanol utilization ability of the present strain was weak, which resulted in extremely low growth rate and angiostatin productivity during the expression phase with methanol as the sole carbon source. To enhance the cell density and angiostatin expression level, mixed-carbon-source of glycerol-methanol was used in the expression phase. The methanol concentration was well controlled at 5 g/L by a methanol sensor and control system, and glycerol was continuously fed into the fermentor to achieve a higher cell density. 120 g/L of cells and 39 mg/L of angiostatin were reached at the end of fermentation which lasted 110 h. The mean specific cell growth rate in the expression phase was 0.01 h(-1), and the mean specific angiostatin productivity was 0.006 mg/(g x h). According to the data obtained in several runs of fermentation in which glycerol was fed at different rates, a higher mean specific angiostatin productivity was reached at the mean specific cell growth rate of 0.012 h(-1). To avoid the repression of angiostatin expression caused by residual glycerol and ethanol accumulation due to overfeeding of glycerol, glycerol addition was controlled to produce continuous oscillations in dissolved oxygen, because the change of dissolved oxygen concentration could deliver the information of available carbon source in the fermentation broth. Controlled glycerol feeding also avoided the problem of oxygen limitation brought by high cell density, and thus decreased the cooling requirement of the fermentor. Cell density reached 150 g/L at the end of fermentation, and angiostatin level reached 108 mg/L after an expression period of 96 h when the mean specific growth rate was maintained at 0.012 h(-1) by using the glycerol feeding strategy to result in the oscillations in dissolved oxygen. The mean specific angiostatin productivity was improved to 0.02 mg/(g x h). The apparent cell yield on glycerol and methanol were respectively 0.69 g/g and 0.93 g/g, higher than those in the fermentation without using the feeding strategy with dissolved oxygen as the indicator of metabolism.
Angiostatins ; genetics ; metabolism ; physiology ; Biotechnology ; methods ; Carbon ; metabolism ; Fermentation ; physiology ; Glycerol ; metabolism ; Methanol ; metabolism ; Oxygen ; metabolism ; Pichia ; genetics ; metabolism

OBJECTIVETo study the pathogenesis of the pain of discography and the discogenic low back pain.

METHODS19 specimens of lumbar intervertebral discs from 17 patients with discogenic low back pain during posterior lumbar interbody fusion, and 12 physiologically aging discs and 10 normal control discs were collected to investigate the morphologic features and innervation containing neuropeptides substance P (SP), neural filament (NF), and vasoactive-intestinal peptide (VIP).

RESULTSThe distinct morphologic characteristic of the disc from the patient with discogenic low back pain was the formation of the strip zone of vascularized granulation tissue from the nucleus pulposus to the outer part of the annulus fibrosus in which there was one or several fissures. The structure of annulus fibrosus beyond the strip zone of granulation tissue was basically normal. The structures of the aging discs and the control discs showed the age-related changes. The innervation of SP, NF and VIP immunoreactive nerve fibers in the painful discs was more extensive compared with the aging discs and the control discs. The nerve in growth deep into annulus fibrosus and nucleus pulposus was observed mainly along the strip zone of granulation tissue in the painful discs.

CONCLUSIONSFindings indicate that the strip zone of granulation tissue with extensive innervation in the posterior part of the painful disc is the original site of the pain of discography and the discogenic low back pain. The strip zone of granulation tissue might originate from the injury and subsequent reparation of the margin of annulus fibrosus. The difference of the aging disc and painful disc which can not be differed each other on MRI is the formation of the strip zone of granulation tissue along tear histologically in posterior part of the painful disc.

Adult ; Female ; Humans ; Intervertebral Disc ; chemistry ; innervation ; pathology ; Low Back Pain ; etiology ; metabolism ; pathology ; Lumbar Vertebrae ; Male ; Middle Aged ; Neurofilament Proteins ; analysis ; Substance P ; analysis ; Vasoactive Intestinal Peptide ; analysis

OBJECTIVETo explore the methods of diagnosis and surgical treatment for patients with back pain originating from endplate.

METHODSAll patients received examinations of radiography, CT, and MR imaging. Pain level of disc was decided by discography in each patient. The principal outcome judgment were pain and disability, and the efficacy of surgical treatment was assessed by visual analog scale (VAS) for pain, and the Oswestry disability index (ODI) for functional recovery.

RESULTSAll patients with a diagnosis of back pain originating from endplate according to discography were treated with anterior or posterior fusion surgery. The mean follow-up period was three years and five months (from 2 to 6 years). Among of the 21 patients, 20 (95%) reported a disappearance or marked alleviation of low back pain, and experienced a definite improvement in physical function. A statistically significant and clinically meaningful improvement on the ODI and the VAS scores were obtained in the patients with chronic low back pain originating from endplate (P = 0.0001) after treatment.

CONCLUSIONSThe study suggests that the discography and fusion surgery may be very effective methods for the diagnosis and treatment of chronic back pain originating from endplate respectively.

Adult ; Female ; Follow-Up Studies ; Humans ; Low Back Pain ; diagnosis ; etiology ; surgery ; Male ; Middle Aged ; Spinal Fusion ; methods ; Spinal Injuries ; complications ; Treatment Outcome

To study the impact of the enterovirus 71(EV71) on the nuclear transport mechanism,The pGFP-NLS vector with nuclear location signal(NLS) was constructed, RD cells transfected by the pGFP-NLS vector were inoculated with the EV71 or cotransfected by EV71-2A vector. The results showed that GFP protein with NLS was expressed in the cytoplasm due to the inhibition of nuclear transport. In order to further study the mechanism of the EV71 to prevent nuclear transport,Nup62 was detected by Western blotting after RD cells were infected with EV71 or transfected by EV71-2A vector. The results showed that decreased expression of Nup62 could be detected after infection with EV71 and transfection by EV71-2A vector. This study demonstrates that the cleavage of Nup62 by EV71 2A protease may be the mechanism of nuclear transport inhibition.
Active Transport, Cell Nucleus ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Enterovirus A, Human ; enzymology ; genetics ; metabolism ; Enterovirus Infections ; virology ; Gene Expression Regulation, Viral ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Humans ; Membrane Glycoproteins ; metabolism ; Nuclear Localization Signals ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Peptide Hydrolases ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Transfection

BACKGROUNDSmall noncoding microRNAs regulate gene expression in cardiac development and disease and have been implicated in the aging process and in the regulation of extracellular matrix proteins. However, their role in age-related cardiac remodeling and atrial fibrillation (AF) was not well understood. The present study was designed to decipher molecular mechanisms underlying age-related atrial structural remodeling and AF.

METHODSThree groups of dogs were studied: adult and aged dogs in sinus rhythm and with persistent AF induced by rapid atrial pacing. The expressions of microRNAs were measured by quantitative real-time polymerase chain reaction. Pathohistological and ultrastructural changes were tested by light and electron microscopy. Apoptosis index of myocytes was detected by TUNEL.

RESULTSSamples of atrial tissue showed the abnormal pathohistological and ultrastructural changes, the accelerated fibrosis, and apoptosis with aging and/or in AF dogs. Compared to the adult group, the expressions of microRNAs-21 and -29 were significantly increased, whereas the expressions of microRNAs-1 and -133 showed obvious downregulation tendency in the aged group. Compared to the aged group, the expressions of microRNAs-1, -21, and -29 was significantly increased in the old group in AF; contrastingly, the expressions of microRNA-133 showed obvious downregulation tendency.

CONCLUSIONThese multiple aberrantly expressed microRNAs may be responsible for modulating the transition from adaptation to pathological atrial remodeling with aging and/or in AF.

Age Factors ; Animals ; Apoptosis ; Atrial Fibrillation ; etiology ; Atrial Remodeling ; Connective Tissue Growth Factor ; physiology ; Dogs ; Electrocardiography ; Fibrosis ; In Situ Nick-End Labeling ; MicroRNAs ; analysis ; physiology ; Myocardium ; pathology ; ultrastructure

BACKGROUNDRecent studies have suggested an association between elevated pelvic incidence (PI) and the development of lumbar spondylolysis. However, there is still lack of investigation for Han Chinese people concerning the normal range of spinopelvic parameters and relationship between abnormal sagittal parameters and lumbar diseases. The objective of the study was to investigate sagittal lumbosacral parameters of adult lumbar spondylolysis patients in Han Chinese population.

METHODSA total of 52 adult patients with symptomatic lumbar spondylolysis treated in the General Hospital of Armed Police Force (Beijing, China) were identified as the spondylolysis group. All the 52 patients were divided into two subgroups, Subgroup A: 36 patients with simple lumbar spondylolysis, and Subgroup B: 16 patients with lumbar spondylolysis accompanying with mild lumbar spondylolisthesis (slip percentage <30%). Altogether 207 healthy adults were chosen as the control group. All patients and the control group took lumbosacral lateral radiographs. Seven sagittal lumbosacral parameters, including PI, pelvic tilt (PT), sacral slope (SS), lumbar lordosis (LL), L5 incidence, L5 slope, and sacral table angle (STA), were measured in the lateral radiographs. All the parameters aforementioned were compared between the two subgroups and between the spondylolysis group and the control group with independent-sample t- test.

RESULTSThere were no statistically significant differences of all seven sagittal lumbosacral parameters between Subgroup A and Subgroup B. PI, PT, SS, and LL were higher (P < 0.05) in the spondylolysis group than those in the control group, but STA was lower (P < 0.001) in the spondylolysis group.

CONCLUSIONSCurrent study results suggest that increased PI and decreased STA may play important roles in the pathology of lumbar spondylolysis in Han Chinese population.

Adolescent ; Adult ; China ; Female ; Humans ; Lumbosacral Region ; anatomy & histology ; Male ; Middle Aged ; Radiography ; Spondylolysis ; pathology ; Young Adult