Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmids.

BACKGROUND: Efficient gene delivery by synthetic vectors is a major challenge in gene therapy. However, inefficient nuclear delivery of cDNA is thought to be a major limiting step in gene transfer using non-viral vectors. It is commonly thought that, in the cytosol, cDNA has to be released from its vector before importation to the nucleus. The stability of naked cDNA in the cytoplasm is not well established. METHODS: cDNA plasmids, either free or complexed with poly(ethyleneimine) (PEI), were microinjected into the cytoplasm of mammalian cells and their turnover was assessed by fluorescence in situ hybridization (FISH). Incubations of cDNA plasmids in cytosolic extracts were also performed. RESULTS: FISH experiments showed that naked cDNA rapidly fade with time when injected into the cytosol. Fading was not observed when naked cDNA plasmids were injected into the nucleus. Incubation of naked cDNA in a cytosolic fraction isolated from mammalian cells reproduced cDNA degradation as observed in microinjection experiments. Nuclease inhibitors, including aurin tricarboxylic acid or Zn2+, prevented in vitro cDNA degradation. The cytosolic nuclease activity was optimal at physiological pH and physiological Ca2+ concentration. By contrast, it was insensitive to Mg2+ or Na+ concentrations. Finally, cDNA complexation with PEI or addition of oligonucleotides prevented in vitro cDNA degradation. CONCLUSION: Altogether, these experiments suggest that cDNA digestion by cytosolic nucleases occur when the decomplexed transgene is present in the cytosol. We propose that the inefficient transfer of cDNA into the nucleus during transfection with synthetic vectors may result from rapid digestion of naked cDNA by a Ca2+-sensitive cytosolic nuclease.

A randomized controlled trial of chiropractic spinal manipulative therapy for migraine.

OBJECTIVE: To assess the efficacy of chiropractic spinal manipulative therapy (SMT) in the treatment of migraine. DESIGN: A randomized controlled trial of 6 months' duration. The trial consisted of 3 stages: 2 months of data collection (before treatment), 2 months of treatment, and a further 2 months of data collection (after treatment). Comparison of outcomes to the initial baseline factors was made at the end of the 6 months for both an SMT group and a control group. SETTING: Chiropractic Research Center of Macquarie University. PARTICIPANTS: One hundred twenty-seven volunteers between the ages of 10 and 70 years were recruited through media advertising. The diagnosis of migraine was made on the basis of the International Headache Society standard, with a minimum of at least one migraine per month. INTERVENTIONS: Two months of chiropractic SMT (diversified technique) at vertebral fixations determined by the practitioner (maximum of 16 treatments). MAIN OUTCOME MEASURES: Participants completed standard headache diaries during the entire trial noting the frequency, intensity (visual analogue score), duration, disability, associated symptoms, and use of medication for each migraine episode. RESULTS: The average response of the treatment group (n = 83) showed statistically significant improvement in migraine frequency (P < .005), duration (P < .01), disability (P < .05), and medication use (P< .001) when compared with the control group (n = 40). Four persons failed to complete the trial because of a variety of causes, including change in residence, a motor vehicle accident, and increased migraine frequency. Expressed in other terms, 22% of participants reported more than a 90% reduction of migraines as a consequence of the 2 months of SMT. Approximately 50% more participants reported significant improvement in the morbidity of each episode. CONCLUSION: The results of this study support previous results showing that some people report significant improvement in migraines after chiropractic SMT. A high percentage (>80%) of participants reported stress as a major factor for their migraines. It appears probable that chiropractic care has an effect on the physical conditions related to stress and that in these people the effects of the migraine are reduced.

Shoulder pain in elite swimmers.

Much research has investigated shoulder pain which inhibits the performance of elite swimmers. An ever increasing understanding of the epidemiology and aetiology of what has been termed 'swimmer's shoulder' has enabled better treatment, rehabilitation and prevention programs to be implemented. This paper reviews the current research relevant to 'swimmer's shoulder' and the methods of treatment being employed to treat the problem.

Bilateral simultaneous optic nerve dysfunction after periorbital trauma: recovery of vision in association with with chiropractic spinal manipulation therapy.

OBJECTIVE: To discuss the recovery of optic nerve function after chiropractic spinal manipulation in a patient with loss of vision as a result of facial fracture from a fall. CLINICAL FEATURES: In a fall down a stairwell, a 53-year-old woman with migraines fractured her right zygomatic arch, which was later treated surgically. Approximately 3 weeks after the accident, vision in her contralateral eye became reduced to light perception. Electrophysiologic studies revealed that the function of both optic nerves was diminished, the right significantly more than the left. Single photon emission tomography showed pancerebral ischemic foci. INTERVENTION AND OUTCOME: Chiropractic spinal manipulation was used to aid recovery of vision to normal over a course of 20 treatment sessions. At times, significant improvement in vision occurred immediately after spinal manipulation. Progressive recovery of vision was monitored by serial visual field tests and by electrophysiologic studies. Unfortunately, the patient refused a further single photon emission tomographic study when visual recovery was complete. CONCLUSION: This case report adds to previous accounts of progressive and expeditious recovery of optic nerve function in association with spinal manipulation therapy.

The effect of upper cervical or sacroiliac manipulation on hip flexion range of motion.

OBJECTIVES: To compare the effectiveness of an upper cervical manipulation and a manipulation of the sacroiliac joint for increasing hip range of motion. DESIGN: Clinical cohort study. SETTING: Macquarie University Centre for Chiropractic Outpatient Clinic. SAMPLE: Fifty-two randomly chosen university students aged 18 to 34 yr. METHOD: A reliable hand-held dynamometer was used to determine the end point of range of motion before and after the application of a treatment. Three groups of subjects were created: cervical manipulation, sacroiliac manipulation and sham/placebo. Range of motion of the hip in flexion (SLR) was used as the independent variable. RESULTS: The two manipulative treatments resulted in increased flexion range of motion at the hip. Statistical analysis revealed that only the upper cervical manipulation procedure increased hip flexion range of motion significantly. CONCLUSION: The results suggest that manual therapy of the neck may affect hip range of motion in normal adults. Findings such as these may indicate the existence of a link between the cervical spine and the lower extremity.

A dominant negative isoform of the long QT syndrome 1 gene product.

Mutations in the KvLQT1 gene are the cause of the long QT syndrome 1. KvLQT1 gene product is associated with the regulator protein IsK to produce a component of the delayed rectifier K+ current in cardiac myocytes. We identified an N-terminal truncated isoform of the KvLQT1 gene product, referred to as isoform 2. In RNase protection assays, isoform 2 represented 28.1 +/- 0.6% of the total KvLQT1 expression in the human adult ventricle. COS-7 cells injected intranuclearly with KvLQT1 isoform 1 cDNA exhibited a fast-activating K+ current, whereas those injected with a KvLQT1 isoform 1 plus IsK cDNA showed a slow-activating K+ current. Cells injected with KvLQT1 isoform 2 plasmid showed no detectable K+ current. Those injected with a 1/1 isoform 2/isoform 1 ratio showed no detectable K+ current. Those injected with 1/5 and 2/5 ratios showed a K+ current with markedly reduced amplitude. Coexpression of the IsK regulator consistently reduced the dominant negative effects of isoform 2. Our results indicate that KvLQT1 isoform 2 exerts a pronounced negative dominance on isoform 1 channels and that the cardiac KvLQT1 K+ channel complex is composed of at least three different proteins as follows: isoform 1, isoform 2, and IsK.

Wall perimetry in chiropractic.

BACKGROUND: Wall perimetry is a method of examination that led to the initial appreciation of the "tunnel vision information." The visual field loss that wall perimetry indicates generally defines the overall characteristics of the dysfunction associated with 'tunnel vision.' Wall perimetry is an inexpensive, yet sensitive, preliminary screening test for perception abnormality in the outermost periphery of vision. OBJECTIVE: To describe wall perimetry. The term "wall perimetry" denotes a simple preliminary method of examining the visual fields of chiropractic patients. DISCUSSION: The test is described with reference to photographs. The patient stands 1 m from the corner of a room and is instructed to look directly at the junction of the walls, with the head remaining still and gaze fixed. Standing behind the patient, the examiner projects a target of light onto the wall and moves it until it is seen by the subject. CONCLUSION: Chiropractors are encouraged to test appropriate patients by wall perimetry before spinal manipulation therapy. On the basis of existing experience, detection of patients with visual field loss is to be expected. From that point, formal examination of the visual fields using kinetic or static perimetry apparatus is recommended to further verify the "tunnel vision" discovery.

A polarized salivary cell monolayer useful for studying transepithelial fluid movement in vitro.

There are no reported, convenient in vitro models for studying polarized functions in salivary epithelial cells. Accordingly, we examined three often-used salivary cell lines for their ability to form a polarized monolayer on permeable, collagen-coated polycarbonate filters. Only the SMIE line, derived from rat submandibular gland, had this ability. The SMIE cell monolayer exhibited junctional complexes, with a tight-junction-associated protein, ZO-1, localized to cell-cell contact areas. The Na+/K+-ATPase alpha1-subunit was detected predominantly in the basolateral membranes, while the Na+/H+ exchanger isoform 2 appeared primarily in the apical membranes. Using adenovirus-mediated cDNA transfer, SMIE cells were shown to be capable of routing marker proteins (beta-galactosidase +/- a nuclear targeting signal, alpha1-antitrypsin, aquaporin-1) to appropriate locations. Furthermore, this salivary cell monolayer provided a convenient tool for studying aquaporin-1-mediated, osmotically directed, transepithelial fluid movement in vitro. Thus, SMIE cells appear to be a useful experimental model with which to study some polarized functions in a salivary epithelial cell line.

A study of two stretching techniques for improving hip flexion range of motion.

OBJECTIVES: To compare the effectiveness of a spinal (suboccipital) stretching technique and a peripheral stretching technique. DESIGN: Clinical cohort study. SETTING: Macquarie University Centre for Chiropractic Outpatient Clinic. METHOD: A reliable hand-held dynamometer was used to determine the end point of range of motion (ROM) before and after the application of a treatment. Three groups of subjects were treated: cervical stretch, hip stretch and sham/placebo. ROM of the hip in flexion (straight leg raise) was used as the independent variable. SAMPLE: Sixty randomly allocated university students aged between 18 and 35 yr. RESULTS: The two stretching treatments resulted in increased flexion ROM at the hip. Statistical analysis revealed that only the sub-occipital stretching procedure increased hip flexion ROM significantly. CONCLUSION: Manual therapy of the neck may have a role to play in the treatment of extraspinal, lower-limb musculoskeletal conditions.

The effect of sacroiliac manipulation on hip flexion range of motion.

Chiropractors claim to be able to influence sites far removed from the point of application of spinal adjustment. Little scientific research has, however, been conducted showing conclusively that the spine and associated structures have an influence on distal function. Demonstration of such influence on distal tissues would aid in the scientific validation of Chiropractic by other health professionals and facilitate treatment of peripheral injuries such as hamstring strains. This study aimed to investigate the effect of a manipulation of the sacroiliac joint on the mechanical function of the hip joint. The results demonstrate that the sacroiliac joint manipulation did not statistically alter the range of motion of the hip joint.

Novel isoforms of synexin in Xenopus laevis: multiple tandem PGQM repeats distinguish mRNAs in specific adult tissues and embryonic stages.

Synexin (annexin VII) is a calcium-dependent, phospholipid-binding and membrane fusion protein in the annexin gene family, which forms calcium channels and may play a role in exocytotic secretion. We report here the cloning and characterization of five novel isoforms of cDNAs encoding Xenopus synexin from brain, oocyte and stage 24 cDNA libraries. The most prevalent Xenopus synexin has 1976 bp of cDNA sequence, which contains a 1539 bp open reading frame of 512 amino acids encoding a 54 kDa protein. This Xenopus protein is 6 kDa larger than the previously reported human and mouse synexins with which it shares approx. 73% identity in the C-terminal region and approx. 44% identity in the N-terminal region. Further studies with PCR revealed the molecular basis of the substantial divergence in the Xenopus synexin's N-terminal domain. The domain equivalent to the mammalian tissue-specific cassette exon occurs at a different position and is variable in size and sequence. The most interesting observation relates to the occurrence of different forms of synexin due to the varying numbers of tandem PGQM repeats that are expressed differently in different adult tissues and embryonic stages. For these reasons we have labelled this set of unique isoforms annexin VIIb, referring to mammalian forms, which lack the PGQM tandem repeats, as annexin VIIa. In spite of these differences from annexin VIIa, the form of recombinant annexin VIIb with three PGQM repeats was found to be catalytically active. We interpret these results to indicate that the actual calcium and phospholipid binding sites are conserved in Xenopus, and that the variations observed between members of the synexin gene family in the regulatory domain clearly point towards the tissue- and stage-specific roles of individual members, possibly involving the exocytotic process.

Palpable unilateral sacral prominence as a clinical sign of lower limb anisomelia: a pilot study.

OBJECTIVE: To examine the role of oblique-axis sacral torsion in the presence of true or apparent short leg. DESIGN: Prospective blind trial of eighteen subjects. SETTING: A suburban chiropractic practice, a hospital and a university campus. SUBJECTS: Of 33 subjects selected, 27 completed the study, 8 exhibited elimination criteria, and 1 X-ray was nondiagnostic. MAIN RESULTS: 3 x 3 contingency table showed the presence of unilateral sacral prominence as statistically significant (p > .001) in subjects with LLI. The incidence of unilateral sacral prominence on the short leg side was also significant (p > .05). Weighted kappa confirmed both (alpha > .001). CONCLUSIONS: Oblique-axis sacral torsion may play a role in intrapelvic adaptation to anisomelia of the lower limb; further examination of this prospect is warranted.

Cervical radiculopathy: a case for ancillary therapies?

OBJECTIVE: To present two cases, one of a patient with a radicular syndrome and another of a patient with a pseudoradicular syndrome. CLINICAL FEATURES: A 45-yr-old man visited one chiropractic clinic complaining of a "pinched nerve" in his neck, with pain and paresthesia in his left hand. He reported that these symptoms began after a work accident 1 month before, when he lifted a heavy object. Radiographs revealed disk space thinning at C4-5, C5-6 and C6-7. CT scans revealed foraminal narrowing with a minor disk bulging at the level of C5-6 and a large disk protrusion at C7-T1. The second patient is a 60-yr-old man with left shoulder and cervical spine pain. The patient stated that the shoulder pain felt like an ache and had begun 2 wk earlier when he had awakened with pain in the shoulder and a stiff neck. X-ray evaluation revealed a moderate level of degenerative change at the the C4-5, C5-6 and C7-T1 region. CONCLUSION: This article identifies the similarities and variations between two syndromes receiving chiropractic intervention that included ancillary therapy. Certain conditions, including cervical radiculopathy, seem to respond well to chiropractic spinal manipulative therapy. However, in other conditions with similar symptomatology, appropriate referral may be necessary for the condition to respond. Alternatively, adjunctive or ancillary therapy may be indicated to improve the effect of the chiropractic intervention.

N-Methyl-D-aspartate receptor subunits NR1 and NR2C are overexpressed in the inferior colliculus of audiogenic mice.

Some non-DBA2 Albino Swiss mice exhibit noise induced epileptic seizures during a short period of postnatal development. Because N-methyl-D-aspartate (NMDA) glutamate ionotropic receptors are involved in the occurrence of audiogenic seizures, we investigated by in situ hybridization methods, the expression of the different subunits (NR1, NR2A, NR2B, NR2C) of this receptor in the central nucleus of the inferior colliculus (IC), a main relay of the auditory pathways. At postnatal day 20, the NR2C subunit is highly expressed in the IC of convulsive mice, while in non-convulsive mice a slight signal is only found for NR1, NR2A, and NR2B. In adult mice, the NR1 and NR2A signals are observed while the NR2B signal is almost undetectable. The audiogenic susceptibility may be related to the transient expression of the NR2C subunit during a brief neonatal period during which synaptic reorganization happens.

Effect of protein synthesis inhibitors on synexin levels and secretory response in bovine adrenal medullary chromaffin cells.

The effects of the protein synthesis inhibitors actinomycin D and cycloheximide on the cellular content of the calcium binding protein synexin, and on the secretory response of cultured bovine adrenal medullary chromaffin cells were determined. Both protein synthesis inhibitors produced a slow decrease in the cellular synexin content. The synexin level was reduced by 50% after 133 h of incubation in the presence of 2 micrograms/ml actinomycin D or 5 micrograms/ml cycloheximide. However, this was partly due to an artefactual stabilization of synexin, since metabolic labelling of synexin with [35S]methionine showed that the half-time of degradation was only 40 h. The secretory response of chromaffin cells was quickly diminished in the presence of protein synthesis inhibitors. Catecholamine secretion induced by membrane depolarization or barium stimulation of intact cells, or by calcium stimulation of digitonin-permeabilized cells was decreased by 77-82% after 24 h of incubation in the presence of 5 micrograms/ml cycloheximide. These results suggest that, in addition to synexin, at least one or more proteins with a shorter half-time of degradation than synexin are involved in the secretory response of adrenal chromaffin cells.

Analysis of zygapophyseal joint cracking during chiropractic manipulation.

OBJECTIVES: To determine if there is a relationship between the side of head rotation and the side of joint crack during "diversified" rotatory manipulation of the cervical spine. DESIGN: Randomized experimental study. SETTING: Macquarie University, Centre for Chiropractic, Summer Hill, New South Wales. SUBJECTS: Fifty asymptomatic subjects were recruited from the students and staff of the above college. INTERVENTION: Single, unilateral "diversified," high velocity, low amplitude, rotatory thrust technique. MAIN OUTCOME MEASURES: Joint crack sound wave analysis of digital audio tape (DAT) recordings, taken from two skin mounted microphones positioned on either side of the cervical spine. RESULTS: All 50 subjects exhibited at least one audible joint crack sound during manipulation. Forty-seven subjects (94%) exhibited cracking on the ipsilateral side to head rotation (95% confidence interval, 83.5% to 98.7%). One subject exhibited joint cracking on the contralateral side only, while two subjects exhibited bilateral joint crack sounds. There was a statistically significant lower rate of exclusively ipsilateral joint cracking in subjects with a history of neck trauma (80% vs. 100%, p = .023). CONCLUSIONS: This research suggests that during the "diversified" rotatory manipulation of the cervical spine utilized in this study, there is a higher occurrence of the joint crack on the ipsilateral side to head rotation.

Enhancement of norepinephrine biosynthesis by ascorbic acid in cultured bovine chromaffin cells.

Ascorbic acid donates electrons to dopamine beta-monooxygenase during the hydroxylation of dopamine to norepinephrine in vitro. However, the possible role of ascorbic acid in norepinephrine biosynthesis in vivo has not been defined. We therefore investigated the effect of newly accumulated ascorbic acid on catecholamine biosynthesis in cultured bovine adrenal chromaffin cells. Cells supplemented for 3 h with ascorbic acid accumulated 9-fold more ascorbic acid than found in control cells. Under these conditions, the cells loaded with ascorbate were found to double the rate of norepinephrine biosynthesis from [14C]tyrosine compared to control. By contrast, the amounts present of [14C] 3,4-dihydroxyphenylalanine and [14C]dopamine synthesized from [14C]tyrosine were unaffected by the preloading of ascorbic acid. Ascorbate preloaded cells incubated with [3H]dopamine also showed a similar increase in the rate of norepinephrine formation, without any change in dopamine transport into the cells. Thus, these data were consistent with ascorbate action at the dopamine beta-monooxygenase step. In order to determine if ascorbate could interact directly with dopamine beta-monooxygenase localized within chromaffin granules, we studied whether isolated chromaffin granules could accumulate ascorbic acid. Ascorbic acid was not transported into chromaffin granules by an uptake or exchange process, despite coincident [3H]dopamine uptake which was Mg-ATP dependent. These data indicate that ascorbic acid does augment norepinephrine biosynthesis in intact chromaffin cells, but by a mechanism that might enhance the rate of dopamine hydroxylation indirectly.

Further evidence for the existence of opiate binding sites on neurosecretory LHRH mediobasal hypothalamic terminals.

Opiate binding sites as well as LHRH and SRIF content were evaluated in mediobasal hypothalamus (MBH) from normal male rats and animals subjected to anterolateral deafferentation of the hypothalamus. A 87 and 44% depletion of LHRH and SRIF content respectively and a 50% decrease in the number of specific opiate binding sites was observed in the deafferented MBH. Dopa-decarboxylase activity remained unchanged. These data indicate that presynaptic opiate binding sites are present on LHRH and SRIF mediobasal hypothalamic nerve endings.