Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes

Abstract Three phyletic groups of Borrelia associated with Lyme disease, B. burgdorferi, B. garinii and group VS461 can be distinguished from each other and other species of Borrelia by Bfa I restriction site polymorphisms in PCR amplified 16S rRNA genes. One strain isolated from an Ixodes pacificus tick in California that was previously unclassifiable was distinguishable from B. burgdorferi by an Mnl I restriction site polymorphism.     

Opportunistic foraging strategy of rainbow lizards at a seaside resort in Togo

Rainbow lizards (Agama agama) are common in suburban areas throughout Africa, and have an opportunistic foraging strategy, with arthropods being the main prey source. In a coastal resort in southern Togo, West Africa, several individuals in a population were observed while feeding regularly upon non-natural human-made food (pizza) and showing a clear preference for a given type of food versus others that were offered (‘four cheeses’ being the preferred one). The fact that all monitored individuals fed upon a same type of pizza suggests that they may have some chemical cues attracting them.     

Characterization of a Small Family (CAIII) of Microsatellite-Containing Sequences with X-Y Homology

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997     

Lymphocyte response to phytohemagglutinin: Intracellular volume and intracellular [K+]

The effect of phytohemagglutinin (PHA) on lymphocytes was examined with respect to free intracellular water volume and intracellular [K⁺]. At a cell concentration of 30 × 10⁶ lymphocytes/ml in modified Hank's Buffered Salt Solution (HBSS) in the presence of 10% human AB serum, addition of PHA at 3 mg/ml resulted in a 24–27% decrease in free intracellular water space within 30 to 60 minutes and a return to control level after three hours. A larger change in intracellular water (44%) was observed under similar conditions in the absence of serum. The absolute intracellular K⁺ content did not change after PHA addition, but the cell water volume decrease arising from PHA addition resulted in a 29% increase in intracellular [K⁺] at 60 minutes. The decrease in lymphocyte water volume induced by PHA was also observed for concanavalin A which stimulates lymphocyte proliferation, but not for wheat germ lectin, an agglutinating agent which is not mitogenic. Thus, volume regulation may be closely associated with the mitogenicity of these compounds.     

Freezing of dCMP aminohydrolase in the activated conformation by glutaraldehyde.

dCMP aminohydrolase, which is an allosteric enzyme, was reacted with glutaraldehyde in the presence of the allosteric activator deoxycytidine-5′-triphosphate and of the competitive inhibitor deoxyadenosine-5′-monophosphate. The isolated modified enzyme is no longer sensitive to the effect of the allosteric ligands and shows kinetics typical of the activated enzyme. Gel electrophoresis demonstrated that glutaraldehyde, under our experimental conditions, does not produce intermolecular cross-links but fixes 80% of the enzyme in a stable hexameric form by intramolecular cross-links.The kinetic and molecular data are explained assuming that glutaraldehyde freezes the enzyme in the activated conformation.     

The reversion of trp frameshift mutations in mut,polA, lig and dnaE mutant strains of Escherichia coli

Mutator mutations mutL25, mutR34, and mutU4 had similar effects on the reversion of 4 trp frameshift mutations of known sequence. The mutation trpE9777, which resulted from the addition of an A–T base-pair to a run of 5 A–T base-pairs, was most strongly reverted by the 4 mutators. Reversion of trpE9777 was also increased by mutation polA1 (DNA polymerase I) and dnaE486 and dnaE511 (DNA polymerase III). No effect was found with the ligase mutations, lig-4 or lig-ts7. Mutations polAex1 and polA107, both deficient in the 5′ → 3′ exonuclease activity of DNA polymerase I, had different mutator effects; the factor increase in reversion of trpE9777 was 28-fold for polAex1, 6-fold for polA107, and 21-fold for polA1. The trpE9777 mutation is a useful indicator of frameshift mutator activity.     

Genetic control of effector cell characteristics: con A-induced suppressor cells carry H-2 I region encoded determinants.

Activation of splenic lymphocytes with Con A leads to the formation of suppressor cells capable of interfering with the activity of several polyclonal B-cell-activating substances. Thus, these suppressor cells, or their products, most probably act directly on B cells. Suppressor cells could be recovered from the effluent cell population of nylon wool columns, and they were absent from the spleens of athymic nude mice. Furthermore, they were absent from the thymus of normal as well as cortison-treated mice. Cortisone treatment did not abolish the formation of Con A-induced suppressor cells in the spleen. Treatment of activated suppressor cells with antisera specific for distinct products of the H-2 I region revealed that they carried I-J cell surface antigens. We conclude that the suppressor cells in our test system, which unlike other Con A-induced suppressor cell populations have a direct effect on B cells, had antigenic characteristics similar to those previously described for I-J carrying suppressor cells.     

E rosette formation at 37°:A property of mitogen-stimulated human peripheral blood lymphocytes

Peripheral blood lymphocytes from healthy humans formed stable E rosettes with sheep erythrocytes (SRBC) at 37°C after culture with phytohemagglutinin or the divalent cation ionophore A23187. Cells manifesting this phenomenon exhibited “blast” morphology, appeared by 16 hr of culture, increased dramatically in percentage and absolute number by 62 hr, and persisted in large numbers for the duration of culture (182 hr). Unstimulated lymphocytes formed rosettes at 4°C but not at 37°C. Increased “stickiness” due to surface-bound lectin mitogen was not the cause of rosette formation at 37°C.Formation of E rosettes at 37°C has previously been considered a property of lymphocytes less differentiated than the circulating T cell (e.g., thymocytes, leukemic lymphoblasts). The present findings indicate that this property can be “reexpressed” during blastogenesis in culture.This observation also demonstrates technical problems associated with the use of SRBC to quantitate lymphocytes with complement receptors (B cells) by the EAC rosette assay in culture. False positives resulted from 37°C E rosette formation, but this was overcome by replacing the SRBC with guinea pig erythrocytes in the EAC assay.     

Phenyl-substituted aminomethylene-bisphosphonates inhibit human P5C reductase and show antiproliferative activity against proline-hyperproducing tumour cells

In certain cancers, such as breast, prostate and some lung and skin cancers, the gene for the enzyme catalysing the second and last step in proline synthesis, δ¹-pyrroline-5-carboxylate (P5C) reductase, has been found upregulated. This leads to a higher proline content that exacerbates the effects of the so-called proline-P5C cycle, with tumour cells effectively using this method to increase cell survival. If a method of reducing or inhibiting P5C reductase could be discovered, it would provide new means of treating cancer. To address this point, the effect of some phenyl-substituted derivatives of aminomethylene-bisphosphonic acid, previously found to interfere with the catalytic activity of plant and bacterial P5C reductases, was evaluated in vitro on the human isoform 1 (PYCR1), expressed in E. coli and affinity purified. The 3.5-dibromophenyl- and 3.5-dichlorophenyl-derivatives showed a remarkable effectiveness, with IC₅₀ values lower than 1 µM and a mechanism of competitive type against both P5C and NADPH. The actual occurrence in vivo of enzyme inhibition was assessed on myelogenous erythroleukemic K562 and epithelial breast cancer MDA-MB-231 cell lines, whose growth was progressively impaired by concentrations of the dibromo derivative ranging from 10⁻⁶ to 10⁻⁴M. Interestingly, growth inhibition was not relieved by the exogenous supply of proline, suggesting that the effect relies on the interference with the proline-P5C cycle, and not on proline starvation.     

Nusinersen treatment and cerebrospinal fluid neurofilaments: An explorative study on Spinal Muscular Atrophy type 3 patients

The antisense oligonucleotide Nusinersen has been recently licensed to treat spinal muscular atrophy (SMA). Since SMA type 3 is characterized by variable phenotype and milder progression, biomarkers of early treatment response are urgently needed. We investigated the cerebrospinal fluid (CSF) concentration of neurofilaments in SMA type 3 patients treated with Nusinersen as a potential biomarker of treatment efficacy. The concentration of phosphorylated neurofilaments heavy chain (pNfH) and light chain (NfL) in the CSF of SMA type 3 patients was evaluated before and after six months since the first Nusinersen administration, performed with commercially available enzyme-linked immunosorbent assay (ELISA) kits. Clinical evaluation of SMA patients was performed with standardized motor function scales. Baseline neurofilament levels in patients were comparable to controls, but significantly decreased after six months of treatment, while motor functions were only marginally ameliorated. No significant correlation was observed between the change in motor functions and that of neurofilaments over time. The reduction of neurofilament levels suggests a possible early biochemical effect of treatment on axonal degeneration, which may precede changes in motor performance. Our study mandates further investigations to assess neurofilaments as a marker of treatment response.