Use of plasma rich in growth factors for symptoms of diabetic neuropathy.

Summary: Painful peripheral polyneuropathy is a common complication of diabetes mellitus (DM) and is a significant source of chronic disability and remains a challenging condition with no available disease-modifying treatment. In the present case report, we describe the treatment of a patient featuring painful diabetic neuropathy with perineural injections of autologous plasma rich in growth factors (PRGF). At one-year post-procedure, the patient exhibited improved scores on the neuropathic pain scale and improvement in the activity level. Learning points: Plasma rich in growth factors (PRGF) is an autologous product that can be prepared and administered in a physician's office. PRGF can be infiltrated as a liquid, creating a three-dimensional gel scaffold in the body. PRGF releases growth factors involved in nerve healing. PRGF may be established as a potent alternative treatment of painful diabetic polyneuropathy.

Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG.

The flagellar motor switch in Escherichia coli and Salmonella typhimurium controls swimming behavior by regulating the direction of flagellar rotation. The switch is a complex apparatus composed of at least three proteins--FliG, FliM and FliN. During chemotactic behavior, the switch responds to signals transduced by the chemotaxis sensory signaling system. CheY, the chemotaxis response regulator, is thought to act directly on the switch to induce tumbles in the swimming pattern, but physical interaction of CheY and switch proteins has not been shown. We have undertaken this work to develop the molecular tools to investigate CheY binding to switch proteins, as well as to understand more about the structure and function of the switch. We present here the sequences of the fliG gene and its protein product, the engineering and amplification of fliG by the polymerase chain reaction (PCR) and its subcloning, and the overproduction, purification and determination of the wild-type (wt) level of the FliG protein. The sequence data revealed a 91.8% amino acid (aa) identity between E. coli and S. typhimurium FliG. Engineering and amplifying fliG by PCR allowed convenient cloning into an efficient expression vector. FliG was successfully overproduced and purified to > 98% purity. Polyclonal antibodies (Ab) were generated against purified FliG and used in quantitative Western blots to determine that the wt expression level of fliG results in about 3700 FliG copies per cell. Purified FliG and anti-FliG Ab will be useful for direct biochemical analyses of CheY-switch protein interaction.

Sub-Tenon's anaesthesia: an efficient and safe technique.

AIM: To evaluate sub-Tenon's anaesthesia as an alternative to peribulbar anaesthesia. METHODS: 109 consecutive patients listed for various eye operations (including cataract, trabeculectomy, and vitrectomy) under peribulbar anaesthesia were operated on under sub-Tenon's anaesthesia instead. After topical anaesthesia a buttonhole was fashioned through the conjunctiva and Tenon's capsule 10 mm posterior to the limbus. 1.5 ml of lignocaine 2% was then delivered to the posterior sub-Tenon's space using a blunt cannula. The surgical procedure was performed immediately after the completion of the anaesthetic procedure. Chemosis, conjunctival haemorrhage, degree of akinesia, and pain scoring were analysed. RESULTS: There were no anaesthesia related complications. The administration of the block was painless for 99.1% of the patients. In all, 97.3% reported no pain during surgery. There was no akinesia when assessed just after the completion of the block and akinesia was limited when assessed after surgery. Chemosis and conjunctival haemorrhage were frequent but caused no intraoperative problems. CONCLUSION: Sub-Tenon's anaesthesia is an efficient and safe anaesthetic technique. It is a good alternative to peribulbar anaesthesia.

Surgically induced astigmatism with superior and temporal incisions in cases of with-the-rule preoperative astigmatism.

PURPOSE: To evaluate surgically induced astigmatism (SIA), postoperative astigmatism, and uncorrected visual acuity (UCVA) after cataract surgery with superior corneal, superior scleral, and temporal corneal 4.0 mm sutureless incisions in cases of preoperative with-the-rule (WTR) astigmatism. SETTING: Hopital des Quinze-Vingts, Paris, France. METHODS: This prospective evaluation included patients having phacoemulsification with foldable lens implantation through a 4.0 mm incision. Patients with preoperative WTR astigmatism were randomly assigned to 1 of 3 incisions: superior corneal (Group 1), superior scleral (Group 2), or temporal corneal (Group 3). All patients had autokeratometry preoperatively and postoperatively (1 day, 1 week, 1 month, 1 year). Surgically induced astigmatism using the vector method, postoperative astigmatism, and UCVA (patients whose spherical equivalent was with +/- 1 diopter) were evaluated. RESULTS: Ninety patients were included in the study; there were 30 in each incision group. One year postoperatively, Group 1 had 1.52 diopters (D) of SIA and 1.36 D of postoperative astigmatism; 53.5% of patients had a UCVA of 20/32 or better, Group 2 had 0.69 D of SIA (P < .05) and 0.67 D of postoperative astigmatism (P < .05); 82.7% of patients had a UCVA of 20/32 or better (P < .05). Group 3 had 0.69 D (P > .05), 0.98 D (P < .05), and 79.3% (P > .05), respectively. CONCLUSIONS: In this study, the superior corneal incision produced significant SIA, leading to high postoperative astigmatism and poor UCVA. The scleral and temporal incisions produced minimal SIA and good UCVA.

A chemotactic signaling surface on CheY defined by suppressors of flagellar switch mutations.

CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN. Genetic analysis of Escherichia coli has identified fliG and fliM as genes in which mutations occur that allele specifically suppress cheY mutations, indicating interactions among these gene products. We have generated a class of cheY mutations selected for dominant suppression of fliG mutations. Interestingly, these cheY mutations dominantly suppressed both fliG and fliM mutations; this is consistent with the idea that the CheY protein interacts with both switch gene products during signaling. Biochemical characterization of wild-type and suppressor CheY proteins did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization. These data indicate that suppressor CheY proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation. Physical mapping of suppressor amino acid substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch.

Specificity and affinity of binding of phosphate-containing compounds to CheY protein.

1H- and 31P-n.m.r. have been used to study the interaction of the bacterial chemotaxis protein, CheY, with ATP and a variety of other phosphates in the presence and absence of bivalent metal ions. In the metal-bound conformation, CheY will bind nucleotide phosphates and phosphates in general, while in the metal-free conformation CheY loses its affinity for phosphates. In the presence of low concentrations of nitroxide-spin-labelled ATP (SL-ATP), specific proton resonances of metal-bound CheY are suppressed, indicating that ATP binds to a specific site on this metal-bound form of the protein. These studies also show that the same resonances are affected by the binding of SL-ATP and Mn2+, indicating that the phosphate- and metal-binding sites are close to each other and to Asp-57 (the site of phosphorylation in CheY). 1H- and 31P-n.m.r. studies using ATP, GTP, TTP, UTP, ADP, AMP and inorganic phosphates show that the binding is not specific for adenine, and does not involve the base directly, but is mediated primarily by the phosphate groups. Experiments with a phosphorylation mutant (Asp-13-->Asn) suggest that the observed phosphate binding and activation of CheY by phosphorylation may be related. Our results indicate that the conformational change and charge interactions brought about by the binding of a metal ion at the active site are required for CheY to interact with a phosphate. These studies also demonstrate the utility of spin-label-induced relaxation in conjunction with two-dimensional-n.m.r. measurements for exploring ligand-binding sites.

Light Regulation of Peridinin-Chlorophyll a-Protein (PCP) Complexes in the Dinoflagellate, Glenodinium sp. : Use of Anti-Pcp Antibodies to Detect Pcp Gene Products in Cells Grown in Different Light Conditions.

As a step toward developing the tools needed to study the molecular bases of light regulation of gene expression in dinoflagellates, light-harvesting peridinin-chlorophyll a-protein (PCP) complexes from Glenodinium sp. were purified and used to generate anti-PCP antibodies. Affinity purified anti-PCP antibodies were isolated from the crude anti-PCP antiserum resulting in improved specificity of immune reactions. The affinity purified anti-PCP antibodies were shown to react strongly and specifically with all major isoforms of PCP complexes in Glenodinium sp. cells, and were used to assess qualitative changes in the levels of PCP gene products in cells grown under different light conditions. Western blot analysis revealed a two- to three-fold increase in detectable PCP apoprotein in low light compared to high light grown cells. In vitro translation reactions supplied with total RNA from high and low light grown Glenodinium sp. cultures also showed an approximate twofold increase in translatable PCP mRNAs in low light grown cells as determined by immunoprecipitation of the primary translation products with affinity purified anti-PCP antibodies. In addition, PCP apoproteins appear to be encoded as larger pre-proteins, since the major immunoprecipitated products from in vitro translation are 23 and 22 kilodaltons, while mature PCP apoproteins are 15.5 kilodaltons. The parallel increases in PCP apoprotein and translatable PCP mRNAs indicate that light regulation of PCP complexes occurs at the level of PCP mRNA abundance.

Characterization of two full-length cDNA sequences encoding for apoproteins of peridinin-chlorophyll a-protein (PCP) complexes.

Characterizations are presented for RNA, 2 cDNA libraries, and 2 full-length cDNA sequences encoding for photosynthetic light-harvesting peridinin-chlorophyll a-protein (PCP) in the dinoflagellate Heterocapsa pygmaea. Subsequent analyses of the PCP system also indicate that (1) it is represented by multiple nuclear encoded genes, (2) a subset of mRNAs encoding for PCP apoproteins are regulated by growth irradiance, (3) PCP preproteins are larger than the mature apoproteins, and (4) PCP cDNA clones sequenced thus far contain a conserved region but are not identical. Results are discussed in the context of photoadaptation in dinoflagellates.

Analytical performance of the Tandem-R free PSA immunoassay measuring free prostate-specific antigen.

The analytical performance of the Tandem-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA-alpha 1-antichymotrypsin was determined to be < 1%. The minimum-detectable concentration was < 0.05 microgram/L. The within-run and between-day CVs were < or = 5% for samples with > 0.3 microgram/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.