Adenosine triphosphate (ATP) regeneration systems are essential for efficient biocatalytic phosphoryl transfer reactions. Polyphosphate kinase (PPK) is a versatile enzyme that can transfer phosphate groups among adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, and polyphosphate (Poly P). Utilization of PPK is an attractive solution to address the problem of ATP regeneration due to its ability to use a variety of inexpensive and stable Poly P salts as phosphate group donors. This review comprehensively summarizes the structural characteristics and catalytic mechanisms of different types of PPKs, as well as the variations in enzyme activity, catalytic efficiency, stability, and coenzyme preference observed in PPKs from different sources. Moreover, recent advances in PPK-mediated ATP regeneration systems and protein engineering of wild-type PPK are summarized.
Adenosine Triphosphate/metabolism* ; Adenosine Monophosphate ; Polyphosphates/metabolism* ; Catalysis ; Regeneration

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg²⁺), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.
Sphingobacterium/metabolism* ; Phosphotransferases (Phosphate Group Acceptor)/metabolism* ; Amino Acids ; Adenosine Triphosphate ; Regeneration ; Polyphosphates/metabolism*

OBJECTIVE: To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7. METHODS: The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains. RESULTS: At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion. CONCLUSIONS We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.
Escherichia coli O157 ; Escherichia coli Proteins ; Polyphosphates

Calcium pyrophosphate (CPP) crystals can present as acute inflammatory arthritis which is known as an acute CPP crystal arthritis. Although monocytes/macrophages have been shown to play a role in the initiation of crystal-mediated inflammatory responses, differences in their phenotypes between acute CPP crystal arthritis and acute gouty arthritis have not yet been investigated. We examined the immunological characteristics of synovial monocytes/macrophages in patients with acute CPP crystal and acute gouty arthritis. CD14⁺CD3⁻CD19⁻CD56⁻ cell frequencies in synovial fluid mononuclear cells (SFMCs) were measured. Expression of pro- and anti-inflammatory cytokines and markers was determined. The SFMCs were dominated by a population of monocytes/macrophages in acute CPP crystal arthritis similar to that in acute gout. Synovial monocytes/macrophages showed the phenotypes of infiltrated monocytes as shown by expression of CD88, C-C chemokine receptor type 2, myeloid-related protein (MRP)8 and MRP14 but not proto-oncogene tyrosine-protein kinase MER. Comparatively, the CD14⁺ cells from patients with acute CPP crystal arthritis had similar high levels of IL-1β and TNF-α production but significantly lower expression of IL-10 and M2 marker (CD163). The monocytes/macrophages had the capacity to produce IL-8 in response to CPP crystals. Proinflammatory features were more dominant in monocytes/macrophages during acute CPP crystal arthritis than those during acute gouty arthritis.
Arthritis ; Arthritis, Gouty ; Calcium Pyrophosphate ; Calcium ; Cytokines ; Gout ; Humans ; Interleukin-10 ; Interleukin-8 ; Macrophages ; Monocytes ; Phenotype ; Phosphotransferases ; Proto-Oncogenes ; Synovial Fluid

STUDY DESIGN: Retrospective study.PURPOSE: This study aimed to investigate the association of surgical intervention with clinical and quality of life (QoL) outcomes in patients who underwent posterior spinal surgery for lumbar spinal stenosis (LSS) with spinal calcium pyrophosphate dihydrate deposition (SCPPD) versus that in those who underwent the surgery for LSS without SCPPD.OVERVIEW OF LITERATURE: Calcium pyrophosphate (CPP)-associated arthritis is one of the most common types of arthritis. The clinical outcomes are well studied in CPP-associated arthritis of the appendicular joints. However, few studies have investigated SCPPD.METHODS: A single-institution database was reviewed. LSS patients were categorized as those who did and did not have SCPPD, based on histologic identification. Clinical presentations and postoperative results were analyzed. Disability and QoL were assessed using the Oswestry Disability Index (ODI) and the 36-item Short-Form Health Survey.RESULTS: Thirty-four patients were enrolled, with 18 patients being allocated to the SCPPD group and 16 being allocated to the non- SCPPD group. Preoperative and postoperative pain scores were not significantly different between the groups (p=0.33 and p=0.48, respectively). The average preoperative ODI score in the SCPPD group was slightly higher than that in the non-SCPPD group (57 vs. 51, p=0.33); however, the postoperative ODI score was significantly lower (15 vs. 43, p=0.01). The postoperative physical function, vitality, and mental health of the SCPPD patients were also significantly improved (p=0.03, p=0.022, and p=0.022, respectively).CONCLUSIONS: Surgical intervention resulted in good clinical outcomes in SCPPD patients. As per our findings, total removal of CPPinvolved tissue is unnecessary. As such, surgery should be performed as indicated according to clinical presentation without considering the presence of CPPD.
Arthritis ; Calcium Pyrophosphate ; Calcium ; Chondrocalcinosis ; Disability Evaluation ; Health Surveys ; Humans ; Joints ; Lumbar Vertebrae ; Mental Health ; Pain, Postoperative ; Quality of Life ; Retrospective Studies ; Spinal Stenosis

Carpal tunnel syndrome (CTS) caused by pseudogout is an uncommon disease. The authors report a 65-year-old female who complained of sudden pain and neurological symptoms on her left hand. Surgical decompression was performed. In the histologic study, a calcium pyrophosphate dihydrate crystal deposit was confirmed. Her pain and neurological symptoms were relieved. Because CTS caused by pseudogout is rare, it is difficult to differentiate it from other diseases. This paper reports an uncommon case of CTS caused by pseudogout.
Aged ; Calcium Pyrophosphate ; Carpal Tunnel Syndrome ; Chondrocalcinosis ; Decompression, Surgical ; Female ; Hand ; Humans ; Median Nerve

BACKGROUND: Calcium pyrophosphate dihydrate deposition disease (CPDD) is a rare disease in the temporomandibular joint (TMJ) space. It forms a calcified crystal mass and induces a limitation of joint movement. CASE PRESENTATION: The calcified mass in our case was occupied in the left TMJ area and extended to the infratemporal and middle cranial fossa. For a complete excision of this mass, we performed a vertical ramus osteotomy and resected the mass around the mandibular condyle. The calcified mass in the infratemporal fossa was carefully excised, and the segmented mandible was anatomically repositioned. Scanning electronic microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) microanalysis was performed to evaluate the calcified mass. The result of SEM/EDS showed that the crystal mass was completely composed of calcium pyrophosphate dihydrate. This result strongly suggested that the calcified mass was CPDD in the TMJ area. CONCLUSIONS: CPDD in the TMJ is a rare disease and is difficult to differentially diagnose from other neoplasms. A histological examination and quantitative microanalysis are required to confirm the diagnosis. In our patient, CPDD in the TMJ was successfully removed via the extracorporeal approach. SEM/EDS microanalysis was used for the differential diagnosis.
Calcium Pyrophosphate ; Calcium ; Chondrocalcinosis ; Cranial Fossa, Middle ; Diagnosis ; Diagnosis, Differential ; Humans ; Joints ; Mandible ; Mandibular Condyle ; Microscopy ; Osteotomy ; Rare Diseases ; Spectrum Analysis ; Temporomandibular Joint

STUDY DESIGN: Prospective study. PURPOSE: To assess the prevalence of periodontoid calcification and its associations with acute cervical pain. OVERVIEW OF LITERATURE: Calcium pyrophosphate dihydrate (CPPD) deposition disease is a common rheumatological disorder that occurs especially in elderly patients. Although CPPD crystals induce acute arthritis, these crystals are not usually symptomatic. Calcification surrounding the odontoid process (periodontoid calcification) has been reported to induce inflammation, resulting in acute neck pain. This disease is called crowned dens syndrome. Whether calcification induces inflammation or whether the crystals are symptomatic remains unclear. METHODS: The prevalence of periodontoid calcification at the atlas transverse ligament was examined by computed tomography of the upper cervical spine in patients suspected of brain disease but no cervical pain (control group, n=296), patients with pseudogout of the peripheral joints but no cervical pain (arthritis group, n=41), and patients with acute neck pain (neck pain group, n=22). Next, the correlation between the prevalence of periodontoid calcification and symptoms was analyzed. RESULTS: In the control group, 40 patients (13.5%) showed periodontoid calcification with no significant difference in the prevalence with gender. The prevalence of calcification increased significantly with age (p=0.002). In the arthritis group, 26 patients (63.4%) reported periodontoid calcification. In the neck pain group, 14 patients (63.6%) reported periodontoid calcification. Multiple logistic regression analysis by age and group revealed that higher age, inclusion in the arthritis group, and inclusion in the neck pain group significantly affected the prevalence of calcification. CONCLUSIONS: Our results cumulatively suggest that periodontoid calcification is an aging-related reaction and that calcification per se does not always cause neck pain. Periodontoid calcification was observed more frequently in patients with pseudogout of the peripheral joints and in those with acute neck pain than in asymptomatic control patients.
Aged ; Arthritis ; Brain Diseases ; Calcium Pyrophosphate ; Chondrocalcinosis ; Crowns ; Humans ; Inflammation ; Joints ; Ligaments ; Logistic Models ; Neck Pain ; Odontoid Process ; Prevalence ; Prospective Studies ; Spine

Calcium pyrophosphate dihydrate deposition disease most commonly presents with acute arthritis of the peripheral joints. Infrequently, a mass effect of this disease can cause axial symptoms, such as spinal stenosis, radiculopathy, or myelopathy. Herein, we report on the first Korean case of acute arthritis in the lumbar facet joint due to calcium pyrophosphate dihydrate crystal deposition disease. A 73-year-old female presented with acute fever, severe lumbago, and knee arthralgia, 11 days after partial parathyroidectomy. Plain radiographs showed multiple chondrocalcinosis, while a bone scan, computed tomography, and magnetic resonance imaging showed right L5-S1 facet arthritis. In synovial fluid from the facet and knee joints, positively birefringent calcium pyrophosphate dihydrate crystals were observed under polarized light microscopy. Under the diagnosis of acute calcium pyrophosphate dihydrate crystal arthritis (formerly known as 'pseudogout') in the facet joint, an intra-articular triamcinolone injection was administered, which resulted in dramatic improvement of the symptoms within 24 hours.
Aged ; Arthralgia ; Arthritis* ; Calcium Pyrophosphate* ; Calcium* ; Chondrocalcinosis ; Diagnosis ; Female ; Fever ; Humans ; Joints ; Knee ; Knee Joint ; Low Back Pain ; Magnetic Resonance Imaging ; Microscopy, Polarization ; Parathyroidectomy ; Radiculopathy ; Spinal Cord Diseases ; Spinal Stenosis ; Synovial Fluid ; Triamcinolone ; Zygapophyseal Joint*

Calcium pyrophosphate dihydrate crystal deposition disease is associated with an acute mono- or pauciarthritis, termed "pseudogout" in elderly patients, involving a large joint (including the knees, ankles) or a chronic arthropathy manifesting as mild joint pain and stiffness. Pseudogout is a crystal-deposition disease of peripheral joints, usually encountered in elderly patients. However, acute presentation of pseudogout around the odontoid process comprises a "crowned-dens" appearance, and requires contemplation of differential diagnoses. We recently experienced a case of pseudogout in the cervical spine presenting with fever and acute neck pain that was successfully treated with a colchicine and low-dose oral steroid. We reported this case with a review of the relevant literature.
Aged ; Arthralgia ; Calcium Pyrophosphate ; Chondrocalcinosis* ; Colchicine ; Diagnosis, Differential ; Fever ; Humans ; Joints ; Knee ; Neck Pain ; Neck* ; Odontoid Process ; Spine