Proposed Classification of Auriculotemporal Nerve,Based on the Root System

The topography of the auriculotemporal nerve (ATN) root system is the main criterion of this nerve classification. Previous publications indicate that ATN may have between one and five roots. Most common is a one- or two-root variant of the nerve structure. The problem of many publications is the inconsistency of nomenclature which concerns the terms “roots”, “connecting branches”, or “branches” that are used to identify the same structures. This study was performed on 80 specimens (40 adults and 40 fetuses) to propose a classification based on: (i) the number of roots, (ii) way of root division, and (iii) configuration of interradicular fibers that form the ATN trunk. This new classification is a remedy for inconsistency of nomenclature of ATN in the infratemporal fossa. This classification system has proven beneficial when organizing all ATN variants described in previous studies and could become a helpful tool for surgeons and dentists. Examination of ATN from the infratemporal fossa of fetuses (the youngest was at 18 weeks gestational age) showed that, at that stage, the nerve is fully developed.     

Specificity of the interactions between the Rep proteins and the origins of replication of Staphylococcus aureus plasmids pT181 and pC221

Summary pT181 and pC221 are closely relatedStaphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, Rep, essential for plasmid replication. Former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both Rep proteins to act on either origin. One possible explanation for this difference was based on a previous analysis of the incompatibility expressed by the origin of replication of these plasmids, showing that the origin embedded in therep gene competes for Rep utilization with the origin of a test plasmid and that changes in the sequence of the origin reduce its ability to compete. To avoid this problem, in the present work special hybrids were constructed in which the origin of replication overlapping therep gene was mutationally inactivated, without changing the amino acid sequence of the encoded protein. The level of Rep expression by these hybrids could be varied by taking advantage of what is presently known about the control of Rep synthesis in plasmid pT181. The results of complenentation studies conducted using these hybrids have shown that: (i) at the usual level of expression for a wild-type plasmid each Rep protein can initiate replication strictly from its corresponding origin; (ii) when overproduced, the pT181 RepC protein could also act efficiently on the pC221 origin; a functional pT181 origin present in the same host completely prevented this complementation; (iii) in excess, the RepD protein encoded by pC221 could replicate a plasmid carrying the pT181 origin but could not ensure the hereditary stability of such a plasmid in the absence of another active replication system; (iv) when overproduced both RepC and RepD could act on the origin of replication of three other related plasmids pS194, pC223 and pUB112.     

Cortisol and cortisone production in rat and mouse adrenal incubations

The presence of 17 alpha-hydroxylase in rodent adrenals is debated. The presence in blood of mice of 11-deoxycortisol together with the absence of cortisol is well known. We demonstrated here the in vitro synthesis of 17 alpha-hydroxyprogesterone and cortisol from [3H]progesterone in rat and mouse adrenals. We have shown that these syntheses represented 45 and 28% of those of 11-deoxycorticosterone and corticosterone, respectively, from progesterone. These data clearly suggest the presence of a 17 alpha-hydroxylase activity in vitro in these rodents adrenals. In addition, a noticeable synthesis of cortisol (0.87-1.57% per mg tissue, i.e. 52-64% per incubation flask) from 11-deoxycortisol was also observed and was inhibited by 0.1-0.3 mumol of Metyrapone and SKF 12185. These results allow to underline that the adrenals of rat and mouse, two species commonly used in laboratory experiments, may be used for in vitro investigations on cortisol metabolism from exogenous radioactive precursors.     

Evidence for a new biologic pathway of androstenedione synthesis from 11-deoxycortisol

17-Hydroxyprogesterone is a well-known precursor of androstenedione in adrenal biosynthesis. This study using sheep adrenal incubations demonstrates that 11-deoxycortisol, the precursor of cortisol synthesis, also can be a precursor of androstenedione. Indeed, our data show that androstenedione synthesis is negatively correlated to the synthesis of cortisol and cortisone. This fact allowed us to infer that this new pathway is closely related to the activity of the 11 beta-hydroxylase that is responsible for the synthesis of cortisol. Indeed, when the activity of this enzyme is impaired, 11-deoxycortisol follows the pathway that leads to androstenedione synthesis in the adrenals. This pathway could explain, at least in part, the marked increase of androstenedione observed in congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.     

Amyloid protein of Gerstmann-Sträussler-Scheinker disease (Indiana kindred) is an 11 kd fragment of prion protein with an N-terminal glycine at codon 58.

Gerstmann-Sträussler-Scheinker (GSS) disease is a familial neurological disorder pathologically characterized by amyloid deposition in the cerebrum and cerebellum. The GSS amyloid is immunoreactive to antisera raised against the hamster prion protein (PrP) 27-30. This is a proteinase K-resistant glycoprotein of 27-30 kd that is derived from an abnormal isoform of a neuronal glycoprotein of 33-35 kd designated PrPSc and is a molecular marker of amyloid fibrils isolated from animals with scrapie and humans with related disorders. We have purified and characterized proteins extracted from amyloid plaque cores isolated from two patients of the Indiana kindred of GSS disease. We found that the major component of GSS amyloid is an 11 kd degradation product of PrP, whose N-terminus corresponds to the glycine residue at position 58 of the amino acid sequence deduced from the human PrP cDNA. In addition, amyloid fractions contained larger PrP fragments with apparently intact N-termini and amyloid P component. These findings suggest that the disease process leads to proteolytic cleavage of PrP, generating an amyloidogenic peptide that polymerizes into insoluble fibrils. The N-terminal cleavage of PrP in GSS disease occurs at a tryptophan-glycine peptide bond identical to that cleaved by proteinase K in vitro to generate PrP 27-30 from hamster PrPSc at codon 90. Since no mutations of the structural PrP gene have been found in the Indiana family of GSS disease, it is conceivable that factors other than the primary structure of PrP play a crucial role in the process of amyloid formation and the development of clinical neurologic dysfunction.     

Suppressive effect of cyclosporin A on the development of Leishmania tropica-induced lesions in genetically susceptible BALB/c mice

The effect of cyclosporin A (CyA) application on the development of cutaneous lesions was analyzed in genetically susceptible BALB/c mice infected s.c. with Leishmania tropica promastigotes. Daily i.p. injections of CyA, beginning 2 days before or at the day of the infection, dose dependently inhibited the development of parasite-induced lesions; no effect on the lesions was observed, however, if CyA application was started 14 days after the infection. Cessation of CyA administration after having successfully suppressed the cutaneous lesions for a period of 42 days, resulted in the development of lesions within 3 days. CyA had no inhibitory effect on lesions developing in L. tropica infected hypothymic BALB/c nu/nu mice. CyA or CyA-containing mouse serum did not directly affect the viability and the growth rate of L. tropica promastigotes, suggesting that the effect of the agent was imposed on the cells participating in the formation of the cutaneous lesions. Quantitative analysis of the cell distribution in the spleens of infected mice revealed that CyA markedly suppressed the infection-associated numerical increase of splenocytes. Within the Thy-1+ lymphocyte compartment, CyA had its most pronounced effect on the Lyt-1+ T lymphocyte subset. Early in the disease, the frequency of splenic cells proliferating in response to L. tropica antigen in vitro was clearly inhibited by CyA; in the later stages of the infection, however, this effect could not be observed, indicating the presence of L. tropica-inducible T cells being relatively resistant to CyA. Taken together, our findings indicate that CyA reversibly inhibits or delays the parasite-induced expansion of Lyt-1+ splenic T lymphocytes, and thus suppresses the biological function of those T cells that are instrumental for the formation of cutaneous lesions in L. tropica-infected BALB/c mice.     

Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.     

Composite Technique for Regional Neurochemical Studies: Measurement of Energy and Neurotransmitter Metabolites in Single Tissue Sample

A combined method is described for the determination of various metabolites from a single tissue sample of the brain. It comprises a quick inactivation of cerebral enzymes by microwave irradiation, easy separation of the desired brain regions, and perchloric acid extraction of tissue substances, which are assayed either by specific enzymatic techniques or by HPLC with electrochemical detection. The obtained values of most energy and neurotransmitter metabolites in the brain are in agreement with those reported using other methods. However, this technique, in contrast to the brain freezing in vitro or freeze-blowing, provides a more efficient procedure for rapid arrest of cerebral metabolism even in the deep brain structures and is therefore suitable for detection of early changes particularly those occurring in experimental pathological conditions such as ischemia.     

Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication

Transgenic (Tg) mice expressing both Syrian hamster (Ha) and mouse (Mo) prion protein (PrP) genes were used to probe the mechanism of scrapie prion replication. Four Tg lines expressing HaPrP exhibited distinct incubation times ranging from 48 to 277 days, which correlated inversely with HaPrP mRNA and HaPrPC. Bioassays of Tg brain extracts showed that the prion inoculum dictates which prions are synthesized de novo. Tg mice inoculated with Ha prions had approximately 10(9) ID50 units of Ha prions per gram of brain and less than 10 units of Mo prions. Conversely, Tg mice inoculated with Mo prions synthesized Mo prions but not Ha prions. Similarly, Tg mice inoculated with Ha prions exhibited neuropathologic changes characteristic of hamsters with scrapie, while Mo prions produced changes similar to those in non-Tg mice. Our results argue that species specificity of scrapie prions resides in the PrP sequence and prion synthesis is initiated by a species-specific interaction between PrPSc in the inoculum and homologous PrPC.     

Successful synthesis of a glial-specific blood–brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin

Successful manual synthesis of the TD2.2 peptide acting as a blood–brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.