A method for rapid and high-yield production of the tick-borne encephalitis virus E and DIII recombinant proteins in E. coli with preservation of the antigenic properties.

Tick-borne encephalitis virus (TBEV) is a member of the Flavivirus genus and is the main pathogenic arbovirus circulating in Europe, Russia and China. The envelope (E) protein is exposed on the viral surface and is the main antigen that is employed in diagnostic tests based on the detection of protein-specific antibodies from serum samples of infected individuals. The high degree of similarity among the E proteins of flaviviruses can, in some cases, lead to cross-reactivity and false-positive results in serological tests. Increased specificity in the detection of positive sera for different Flavivirus infections is often obtained by using a portion of the E protein, namely, the DIII domain. Different strategies and expression systems have been described for E and DIII protein production. Here, we present the optimization of an easy and fast method for TBEV E and DIII antigen production and partial purification from E. coli inclusion bodies. The antigenic properties of the produced antigens are retained, as validated by ELISAs with anti-TBEV murine sera as well as sera from infected human patients. The potential applications of both proteins as diagnostic reagents were confirmed.

Using archaeal histones for precise DNA fragmentation.

The fragmentation of DNA is a useful procedure for many molecular biology procedures. However, most methods used to fragment DNA are poorly controllable, and cannot be used to create small fragments. We describe a method to generate random DNA fragments of a predictable size to be cloned in expression vectors for the construction of display libraries. The DNA is allowed to form complexes with archaeal histones from Methanothermus fervidus (HMf) and the HMf/DNA core complex is naturally protected from nuclease DNaseI activity, giving rise to DNA fragments of approximately 60 bp and multiples thereof. We found that by varying the wt/wt ratio between DNA and HMf, the concentration of DNA and the incubation time with DNaseI, DNA fragments of desired size can be obtained. This approach should be applicable to the efficient fragmentation of DNA for the construction of phage display polypeptide libraries, as well as any other molecular biology procedures in which small DNA fragments of defined size are required.

The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide.

The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting. We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues. The two cHHs differ in the preprohormone but not in the mature peptide sequence. The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract.

Gonad-inhibiting hormone of the Norway lobster (Nephrops norvegicus): cDNA cloning, expression, recombinant protein production, and immunolocalization.

The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in a neurohemal complex of crustacean eyestalks. The GIH is involved in gonad maturation and plays a more complex role in the control of reproduction and molting. With a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends approaches we determined the cDNA sequence of the Norway lobster Nephrops norvegicus prepro GIH. The open reading frame of 339 bp codes for a polypeptide of 112 amino acids showing 96% identity with the other known GIH of Homarus americanus. The precursor peptide consists of a putative signal peptide of 31 amino acids and a putative mature peptide region of 81. RT-PCR analysis shows that GIH mRNA is expressed mainly in eyestalks, both in female and male; the expression of GIH mRNA also in supraesophageal ganglia suggests the existence of additional GIH-producing neurons besides those of eyestalks. A specific polyclonal antibody was raised against a portion of the mature peptide region obtained through expression in Escherichia coli fused to glutathione-S-transferase. Immunocytochemical studies were carried out by using this antibody in N. norvegicus and in other crustaceans, Munida rugosa and Squilla mantis; these locate GIH in superficial axon terminals of the releasing organ, the sinus gland. The identification of a second GIH sequence in crustaceans allows to hypothesize the occurrence, within the neuropeptide family, of three subfamilies probably involved in different functions: crustacean hyperglycemic hormones, GIHs and molt-inhibiting hormones/mandibular organ-inhibiting hormones.

An antibody to recombinant crustacean hyperglycaemic hormone of Nephrops norvegicus cross-reacts with neuroendocrine organs of several taxa of malacostracan Crustacea.

The crustacean hyperglycaemic hormones (cHHs) are multifunctional neuropeptides that play a central role in the physiology of crustaceans. A partial cDNA coding for cHH of the Norway lobster, Nephrops norvegicus, was cloned; this cDNA was fused to glutathione- S-transferase (GST) to obtain a recombinant fusion protein that was used to raise a rabbit antiserum and to perform a biological assay. The specificity of the purified antibody was demonstrated by means of Western blotting. To validate the specificity of the purified antibody to the cHH of N. norvegicus and its cross-reactivity with other species, we performed standard immunocytochemistry of the eyestalk on: (1) paraffin sections of the decapod species N. norvegicus, Munida rugosa and Astacus leptodactylus and of the stomatopod Squilla mantis; (2) semithin resin sections of N. norvegicus and Palaemon elegans; (3) ultrathin sections of N. norvegicus sinus gland (transmission electron microscopy studies). The pattern of immunoreactivity shown by N. norvegicus eyestalk sections conforms to distribution, relative amount and ultrastructural features of cHH-containing neurons and nerve endings as reported in the previous literature. In all the crustacean species examined, the antibody marks precisely the X organ-sinus gland complex and unspecific staining is completely lacking. In addition, its specific cross-reaction by immunoprecipitation depletes shrimp eyestalk extract of hyperglycaemic activity in an in vivo bioassay. The results obtained show a cHH-specific molecular recognition despite the fact that the species tested belong to systematic groups increasingly remote in the phylogenetic tree. The antibody could be used for advancing our knowledge on cHH activity in a variety of crustacean species, e.g. for monitoring reproductive and stress conditions.

DNA damage promotes mistyping in the allele specific oligonucleotide probing analysis of forensic samples.

Five polymerase chain reaction (PCR) products which could not be reliably typed by allele-specific oligonucleotide (ASO) probing at the human leukocyte antigen (HLA) DQA1 locus were analyzed by polyacrylamide gel electrophoresis and direct sequencing. The first method revealed the preferential amplification of only one of the two alleles in two cases. Direct sequencing of PCR products allowed unambiguous genetic typing but a high number of artifacts was observed. Several of these artifacts occurred in the sequences recognized by the ASOs. This finding provides an explanation for the mistyping in the ASO probing procedure because Taq polymerase errors both created new genetic specificities and eliminated site-specific polymorphisms. Reversed-phase HPLC-MS of the five forensic templates showed a high degree of DNA damage. These data together indicate that the risk of mistyping when using the ASO probing procedure cannot be neglected in the forensic analysis of damaged DNA samples.

Exposure at the cell surface is required for gas3/PMP22 To regulate both cell death and cell spreading: implication for the Charcot-Marie-Tooth type 1A and Dejerine-Sottas diseases.

Gas3/PMP22 is a tetraspan membrane protein highly expressed in myelinating Schwann cells. Point mutations in the gas3/PMP22 gene account for the dominant inherited peripheral neuropathies Charcot-Marie-Tooth type 1A disease (CMT1A) and Dejerine-Sottas syndrome (DSS). Gas3/PMP22 can regulate apoptosis and cell spreading in cultured cells. Gas3/PMP22 point mutations, which are responsible for these diseases, are defective in this respect. In this report, we demonstrate that Gas3/PMP22-WT is exposed at the cell surface, while its point-mutated derivatives are intracellularly retained, colocalizing mainly with the endoplasmic reticulum (ER). The putative retrieval motif present in the carboxyl terminus of Gas3/PMP22 is not sufficient for the intracellular sequestration of its point-mutated forms. On the contrary, the introduction of a retrieval signal at the carboxyl terminus of Gas3/PMP22-WT leads to its intracellular accumulation, which is accompanied by a failure to trigger cell death as well as by changes in cell spreading. In addition, by substituting the Asn at position 41 required for N-glycosylation, we provide evidence that N-glycosylation is required for the full effect on cell spreading, but it is not necessary for triggering cell death. In conclusion, we suggest that the DSS and the CMT1A neuropathies derived from point mutations of Gas3/PMP22 might arise, at the molecular level, from a reduced exposure of Gas3/PMP22 at the cell surface, which is required to exert its biological functions.

Fidelity of polymerase chain reaction-direct sequencing analysis of damaged forensic samples.

Polymerase chain reaction (PCR) direct sequence analysis was performed on aged forensic samples, six or thirteen years old. This method allowed unambiguous genetic typing, but PCR products from such samples showed several artifacts. Control samples generated sequence ambiguities at a frequency of 1 in 567 bases, but the aged samples had an error frequency about 30-fold higher. In order to study the molecular composition of these aged DNA samples, reversed-phase high performance liquid chromatography (HPLC) was performed. Reduced amounts of the four DNA bases were observed and anomalous peaks were found. These peaks were analyzed by ionization mass spectrometry and identified as molecular products of DNA oxidation. The frequency of sequencing artifacts was found to be proportional to the decay of the PCR templates. Although PCR fidelity is a relevant concern in the forensic analysis of damaged samples, our data indicate that the risk of mistyping is circumventable by sequencing both strands and by performing replicate amplifications from the same PCR template.

Rho-dependent regulation of cell spreading by the tetraspan membrane protein Gas3/PMP22.

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid-induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.

Clinical and pathological correlations in Charcot-Marie-Tooth neuropathy type 1A with the 17p11.2p12 duplication: a cross-sectional morphometric and immunohistochemical study in twenty cases.

In a cross-sectional, clinical, and morphometric analysis we assessed the correlation between the clinical and pathological evolution of disease in 20 unrelated patients of various ages affected by Charcot-Marie-Tooth neuropathy type 1A (CMT1A) with the 17p11.2p12 (peripheral myelin protein 22, PMP22) duplication. The severity of neurologic deficits and slowing of motor conduction velocity at the median nerve did not vary significantly with the patients' age. The amount of demyelination was significantly higher below 15 years than in older age groups; in contrast, myelinated fiber and onion bulb densities were similar at all ages. The results indicate that in duplicated CMT1A, the pathological process develops early in life and progresses little during the course of the disease. Younger patients had lower g-ratio values, suggesting that the trigger of demyelination in early years could be a hypermyelination, resulting from PMP22 overexpression. Yet none of the 20 patients examined had immunohistochemical evidence of altered PMP22 expression. The early onset and development of the disorder make it difficult to detect PMP22 overdosage in nerve biopsies.

Phage display of Bacillus thuringiensis CryIA(a) insecticidal toxin.

The display of proteins or peptides on the surface of filamentous phages or phagemids has been shown to be a very powerful technology for the rescue of specific binders from large combinatorial libraries, as well as to select derivatives of known proteins with altered binding properties. The Bacillus thuringiensis (Bt) crystal proteins are a large family of insecticidal toxins which bind to receptors found on the brush border of larval midgut cells, different crystal toxins having different larval specificities. Here we describe the display of different CryIA(a) toxin regions on the surface of phagemids using the display vector pHEN1, the purpose being the identification of toxin sequences suitable for mutagenesis and selection using phage display. We show that CryIA(a) domain II, in which the receptor binding activity is located, is efficiently displayed as well as being secreted as soluble protein into the periplasm of bacterial cell. This forms the basis of a simple means for the modification of toxin specificity and the selection of toxin proteins with novel or expanded host ranges.

Apoptotic phenotype induced by overexpression of wild-type gas3/PMP22: its relation to the demyelinating peripheral neuropathy CMT1A.

Although the Gas3/PMP22 protein is expressed at highest levels in differentiated Schwann cells, its presence, albeit at lower levels, in non-neuronal tissues and in NIH-3T3 growth-arrested fibroblasts argues for a more general function of this protein that is uncoupled to myelin structure. We show that gas3/PMP22 overexpression in NIH-3T3 growing cells leads to an apoptotic-like phenotype, which is suppressed by antioxidants and characterized by typical membrane blebbing, rounding up, and chromatin condensation, but with no evidence of DNA fragmentation. REF-52 fibroblasts seem to be completely refractive to gas3/PMP22 overexpression. Recently, several point mutations of the human gas3/PMP22 gene have been associated with Charcot-Marie-Tooth type 1A (CMT1A), a common hereditary demyelinating neuropathy. When gas3/PMP22 point mutations (L16P, S79C, T118M, and G150D) are similarly overexpressed in NIH-3T3 cells, the induced apoptotic-like phenotype as compared to the wild-type is significantly reduced. Both of the dominant mutations (L16P, S79C) for CMT1A behave as dominant negatives with respect to the wild type, whereas T118M, the only recessive mutant described, behaves as recessive under the same coexpression experiments. These data suggest a role for altered Schwann cell apoptosis in the pathogenesis of CMT1A.

Structure, function, and chromosome mapping of the growth-suppressing human homologue of the murine gas1 gene.

We describe the isolation, growth-suppressing activity, and chromosomal localization of the human homologue of the murine growth-arrest-specific gene gas1. Overexpression of h-gas1 is able to block cell proliferation in the A549 lung carcinoma and the T24 bladder carcinoma cell lines. No effect was observed when h-gas1 was introduced into the osteosarcoma cell line SAOS-2 and into the adenovirus-type-5 transformed cell line 293. This finding is related to our previous evidence that simian virus 40-transformed NIH 3T3 cells are also refractory to murine gas1 overexpression, suggesting that the retinoblastoma and/or p53 gene products have an active role in mediating the growth-suppressing effect of gas1. We also show that h-gas1 is on chromosome 9q21.3-22.1, in a region considered to be a fragile site. Altogether, the results raise the possibility that h-gas1 may be a target for genetic alterations leading to its inactivation in tumor cells.

Sequence of human GAS3/PMP22 full-length cDNA.

We have cloned the human growth arrest-specific gene GAS3 cDNA that encodes a transmembrane glycoprotein, the peripheral myelin protein 22 (PMP22). GAS3/PMP22 is implicated in Charcot-Marie-Tooth disease type 1A, an inherited peripheral neuropathy.