Partial characterization of insulin-like growth factor I in primary human lung cancers using immunohistochemical and receptor autoradiographic techniques.

We investigated primary human lung cancers resected surgically or obtained at autopsy. Included were squamous cell carcinoma (SQC) (five cases), adenocarcinoma (ADC) (six cases), large cell carcinoma (LCC) (four cases), and small cell carcinoma (SCC) (two cases). The objective of the study was to search for the presence of insulin-like growth factor I (IGF-I)-like immunoreactivity using immunohistochemical staining and for the localization of IGF-I binding sites, using in vitro quantitative receptor autoradiographic techniques. IGF-I-like immunostaining was present in all cases of SQC, ADC, and LCC, but not in cases of SCC. Strong immunostaining was observed in cases of SQC. On the other hand, ADC and LCC tissues showed a moderate or weak staining. Specific binding sites for IGF-I were present in all cases of SQC, ADC, LCC, and SCC examined. High densities of 125I-IGF-I binding sites were localized in cases of SQC and SCC. Low to high densities of the binding sites were found in LCC. Cases of ADC showed low densities of 125I-IGF-I binding sites. Specific binding obtained at a concentration of 80 pM 125I-IGF-I was competitively displaced by unlabeled IGF-I, with a 50% inhibitory concentration value of 1.84 +/- 0.31 x 10(-10) mol, whereas human insulin was much less potent in displacing the binding. This specificity profile is consistent with characteristics of IGF-I receptors. Scatchard analysis showed the presence of a single class of high affinity binding sites for IGF-I, with a Kd of approximately 1 nmol. Thus, the possibility that IGF-I may play a role in the growth of human lung cancers would have to be considered.

Analysis of the genes encoding immunodominant piroplasm surface proteins of Theileria sergenti and Theileria buffeli by nucleotide sequencing and polymerase chain reaction.

The nucleotide sequences of the cDNAs encoding a 33-kDa piroplasm protein of Theileria sergenti (p33) and a similar protein of Theileria buffeli (p34) were determined. Both of the genes contained an open reading frame of 849 base pairs. The predicted amino acid sequence of p33 and p34, consisting of 283 residues, showed 82% similarity. A transmembrane hydrophobic domain and signal peptides were predicted. The polymerase chain reaction was used to amplify p33/34 genes from the piroplasm DNA of T. sergenti, T. buffeli and Theileria orientalis. Following amplification, p33 and p34 genes were clearly differentiated using the restriction enzymes sites that were not shared between them. These results indicated that p33 and p34 were conserved molecules among these Theileria species, and the genes that encode p33/34 proteins were suitable for discrimination of T. sergenti from T. buffeli/T. orientalis.

Phylogenetic relationships of the benign Theileria species in cattle and Asian buffalo based on the major piroplasm surface protein (p33/34) gene sequences.

In order to examine the taxonomic relationship of Theileria sp. of Asian buffalo to the benign Theileria spp. of cattle, we sequenced and compared the major piroplasm protein (p33/34) genes of these parasites. The two consensus sequences determined for the buffalo parasite were of the same length (852 bp) and showed >80% identity with the sequences of the homologous genes (849 bp) in the cattle parasites. Alignment of the inferred aa sequences with those of Theileria sergenti and Theileria buffeli predicted that there is an insertion of a single residue at the N-terminus in the inferred polypeptide of the buffalo parasite. Phylogenetic analyses based on the aa sequences suggested that Theileria sp. of the Asian buffalo should be classified within the benign Theileria parasite group as a separate species from the cattle parasites. Based on this, we propose a rearrangement of the currently used classification for the benign Theileria species in cattle and Asian buffalo.

Immunohistochemical expression of epidermal growth factor receptors in human adrenocortical carcinoma.

Epidermal growth factor (EGF) receptors were examined immunohistochemically in 64 adrenocortical carcinomas obtained at autopsy, and in 23 adrenocortical adenomas and seven pheochromocytomas obtained during surgery. In the nonneoplastic adrenal gland, EGF receptors were scattered to the zona glomerulosa, zona fasciculata, and zona reticularis. Adrenocortical carcinomas (63 of 64), more than adrenocortical adenomas (10 of 23) or pheochromocytomas (four of seven), stained positively for EGF receptors (P less than .01). The immunoreactivity was limited to the cytoplasm, cell membrane, and chromatin. When the antibody was immunoabsorbed with an excess of immunogen peptide, there was no evidence of immunostaining. The adrenocortical carcinomas could be classified into 16 cases of the well-differentiated type, 33 cases of the moderately differentiated type, and 15 cases of the poorly differentiated type. There was no relationship between histologic grading and staining intensity of the EGF receptors. On the other hand, more than 80% of the cases of adrenocortical carcinomas revealed a moderate to high intensity for EGF receptors. In 62 of the 64 patients, there was already metastases to other organs. We conclude that the expression of EGF receptors is associated with tumor growth and/or metastatic potential in adrenocortical carcinoma.

Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis.

A cDNA expression library prepared from mRNA of Haemaphysalis longicornis (H. longicornis) was screened with a H. longicornis-infested rabbit serum. A cDNA encoding 27/30kDa proteins was cloned and designated P27/30 gene. The predicted amino acid sequence of the P27/30 gene shows a rather high homology (58% amino acid identities and 11% amino acid similarity) with Drosophila melanogaster troponin I clone E2. H. longicornis P27/30 possesses amino acid sequence of actin-binding domains of troponin I at the amino acid residues 128-148, suggesting that H. longicornis P27/30 is a troponin I-like protein. By immunoblot analysis, mouse anti-recombinant P27/30 serum reacted with major constituent protein bands in extracts of adult ticks, and also immunoreacted with muscle, cuticle, gut, and salivary gland in H. longicornis ticks. Moreover, immunohistochemistry using the anti-P27/30 serum showed a strong reactivity in muscle, suggesting that native P27/30 is expressed abundantly in that tissue.

A new neurotological test for detecting cerebellar dysfunction.

The adaptation of the vestibulo-ocular reflex (VOR) was studied in 26 normal subjects and 26 patients with cerebellar lesions, using horizontal vision-reversal prisms. In normal subjects, the adaptation of gain after wearing prisms for one hour was approximately 50% of the VOR value in the dark. In contrast to this, patients with cerebellar lesions showed less adaptation--approximately 20% after a one-hour forced adaptation task. These were type A, higher gain in the initial level and abnormal adaptation (10 cases); type B, higher gain in the initial level and normal adaptation (5 cases); and type C, normal initial level and abnormal adaptation (11 cases). The cases showing the typical type-A responses tended to have severe widespread or midline lesions of the cerebellum. Typical type-B cases had mild cerebellar lesions, and typical type-C cases tended to have lesions restricted to one side of the cerebellum. From these results, it can be speculated that reduction of VOR adaptation occurs when one side of the cerebellum has severe lesions, but it is sufficient to produce a normal vestibulo-ocular reflex if at least half of the cerebellum is intact.

Eye movement analysis system using computerized image recognition.

A new technique for an eye movement analysis system utilizing infrared video recording and a computerized image recognition method is presented. The system consists of an infrared lighting apparatus, a very small infrared video charge-coupled device camera, a video tape recorder, an analogue-digital converter, and microcomputers. This system makes it possible to simultaneously analyze the slow-phase velocity quantitatively not only of the horizontal and vertical but also of the rotatory components of the energy-induced nystagmus. The maximum slow-phase velocity of the rotatory component of energy-induced nystagmus was found to be 4.1 degrees per second on an average in this study.

Extended high-frequency ototoxicity induced by the first administration of cisplatin.

The ototoxicity induced by cisplatin results in high-frequency hearing loss. The sound-pressure thresholds at extended high frequencies of 8 to 20 kHz were measured in 12 patients with head and neck cancer before and after the first administration of cisplatin. Ototoxicity was defined statistically by the newly introduced regression-line analysis. This analysis revealed that the threshold of 1 of the 12 patients increased evenly from 8 to 10 kHz, and the damage to the hearing system of this patient was permanent. The reasons for such a low rate of ototoxicity might be the long-term administration of low doses of cisplatin and the addition of fosfomycin.

Molecular phylogenetic studies on Theileria parasites based on small subunit ribosomal RNA gene sequences.

Classification of Theileria parasites of south-east Asian countries is still ambiguous due to the lack of basic studies, especially their molecular genetic information. In this study, we included 6 known species and 14 unclassified Theileria parasite isolates: Theileria annulata, Theileria parva, Theileria taurotragi, Theileria sergenti, Theileria buffeli, Theileria types Sable, Theileria types A, B, B1, B2, C, D, E, F, G, G1, Theileria type Medan (Indonesia), Theileria type Ipoh (Malaysia) and Theileria type Thong Song (Thailand). Small subunit ribosomal RNA (srRNA) nucleotide sequence data were collected by PCR, cloning and dideoxy sequencing. The srRNA nucleotide sequences were aligned and analyzed by distance methods, maximum parsimony algorithms and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the strength of the different phylogenetic reconstructions. The data indicated that all of the tree-building methods gave very similar results. This study identified two groups of Theileria, the pathogenic and benign groups, which are strongly supported by bootstrap analysis. The analysis also indicated that three subgroups (A, B and C) were generated within the benign Theileria group whereas the classification of Theileria type D and Thong Song is questionable. However, more basic information such as life cycle differences, vectors, modes of transmission, virulent and genetic/sexual compatability is essential for clearer taxonomic definition of the benign Theileria parasites.

Inter-species differentiation of benign Theilerias by genomic fingerprinting with arbitrary primers.

A method was developed to obtain reproducible DNA fingerprints from five distinct purified benign Theileria genomic DNAs by PCR-based amplification. Randomly amplified polymorphic DNA (RAPD) profiles were obtained from 10 randomly designed 12-mers. However, nine of the 10 primers could generate the difference in RAPD-PCR profiles which allowed discrimination of Theileria species. The method has advantage of being simple, fast and sensitive for diagnosis and characterization of the parasites since it does not require prior DNA sequence information to construct species-specific probes or primers. The results are also beneficial for a proper understanding of the epidemiology and designing rational control programmes for Theileriosis in Asian and South-East Asian countries.

Eustachian tube lymphatics.

Serous otitis media is the most common cause of hearing impairment. The role of lymphatic obstruction in the pathogeneis of serous otitis media is significant. A method for removeal of the human Eustachian tube specimen and two techniques for identification of Eustachian tube lymphatic capillaries are described. One involves the antemorten intratympanic installation of Berlin blue. The other utilizes electron microscopy. Lymphatic capillaries cannot be reliably differentiated from blood capillaries with the light microscope. With electron microscopy, lymphatic capillaries can be differentiated from blood capillaries by differences in the basement membrane. The lymphatic capillary has gaps in the basement membrane with large nuclei in the wall. A blood capillary has a continuous basement membrane and sometimes red blood cells be be identified in the lumen. Using these methods, Eustachian tube lymphatic capillaries in the human are described for the first time in this report.

New neurotological test for detecting cerebellar dysfunction. Vestibulo-ocular reflex changes with horizontal vision-reversal prisms.

Adaptation of the vestibulo-ocular reflex (VOR) was studied in 26 normal subjects and 15 patients with cerebellar lesions, using horizontal vision-reversal prisms. In normal subjects, adaptation of gain after wearing prisms for one hour was approximately 50% of the VOR value in the dark. In contrast to this, patients with cerebellar lesions showed less adaptation, approximately 20% after a one-hour forced adaptation task. These cases showed three different types of abnormalities: 1) high gain before wearing prisms and normal adaptation, 2) high gain before wearing prisms and reduction of adaptation, 3) normal gain and reduction of adaptation. From these results it is suggested that observation of the effect of vision-reversal prisms on the VOR may permit the detection of cerebellar lesions of a type or subtlety which escape established tests.

Discrimination of eight chicken Eimeria species using the two-step polymerase chain reaction.

A method was developed for the discrimination of 8 Eimeria species of chickens, i.e., E. acervulina, E. brunetti, E. mitis, E. maxima, E. necatrix, E. praecox, E. tenella, and E. hagani using the 2-step polymerase chain reaction (PCR). In the first PCR, the small subunit ribosomal RNA (srRNA) gene was amplified from the parasite genome using conserved sequences for the Apicomplexa srRNA gene as the primers. The srRNA gene amplified from the parasite genome was discriminated in the second step by random-amplified polymorphic DNA (RAPD) PCR using 10 arbitrary primers. Each arbitrary primer produced species-specific RAPD patterns that provided a simple method for species identification from the srRNA genes of the 8 Eimeria species. This method should be useful for discrimination of the parasite species for diagnosis or epidemiological surveys of chicken coccidiosis.

Theileria sergenti: transformation of zygotes into kinetes in vector ticks, Haemaphysalis (Kaiseriana) longicornis and H. (K.) mageshimaensis.

Invagination or transformation of a zygote of Theileria sergenti into a kinete was examined with Giemsa-stained smears of the midgut of vector ticks, Haemaphysalis longicornis and H. mageshimaensis. Zygotes invaginated almost simultaneously with the moulting of host ticks. They were round and oval, and measured 5.4 to 6.7 microns in diameter on average. The kinetes developing in the zygotes were situated in the periphery of cytoplasm and were spindle- or club-shaped.

Molecular cloning and immunological analysis of immunodominant piroplasm surface proteins of Theileria sergenti and T. buffeli.

cDNA libraries of Theileria sergenti and T. buffeli piroplasms were constructed in lambda gt11 and screened with rabbit anti-piroplasm sera. A major antigen of T. sergenti (33 kDa) and that of T. buffeli (34 kDa) was identified from the recombinant phages by using recombinant antigen-selected monospecific antibodies. The reactivities of the cloned proteins with rabbit antisera, infected calf sera and mouse monoclonal antibody suggested that the 33 and 34 kDa proteins expressed species-common and species-specific epitopes. The DNA probes from these recombinant clones showed species-specific hybridizations in Southern blotting with genomic DNA from piroplasms. These results indicate that the Japanese T. sergenti can be distinguishable from the Australian T. buffeli with regard to a polymorphism of the major immunodominant proteins of piroplasm.

Development of Theileria sergenti schizonts in the lymph node of experimentally infected cattle.

Schizogony of Japanese Theileria sergenti of cattle was studied by light and electron microscopy. Schizonts were detected in the draining lymph node between 4 and 8 days after sporozoite inoculation. Macroschizonts (the phase of nuclear division having invaginations) were formed 6 days after inoculation. Subsequently, microschizonts (the phase of merozoite formation displaying rosette-like appearance) were observed 8 days after inoculation. Multiple infections of a host cell with sporozoites were suggested to occur since different stages of schizonts were simultaneously detected in the same cell. Host cells of schizonts were considerably enlarged by parasitism. However, morphological characteristics of the developmental stages of T. sergenti schizonts resembled those of malignant Theileria species (e.g. T. parva). Schizogony of T. sergenti observed in this study seems to be the primary generation.

Histological observations on the schizonts in cattle infected with Japanese Theileria sergenti.

Histological observations were performed on the schizonts of Japanese Theileria sergenti in three calves necropsied 8 and 10 days after application of nymphal Haemaphysalis longicornis infected with T. sergenti. In all the three calves, schizonts were observed in the cytoplasm of huge cells formed in the drainage lymph nodes, liver and spleen. The huge cells were 50 to 200 microns in diameter. Schizonts had granular appearance and an irregular shape and were 1 to 7 microns in diameter. Ultrastructurally, schizonts had more than one nuclei and were formed in the unit enlarged cell. Schizonts showed a specific reaction against anti-T. sergenti anti serum, therefore, it was concluded that the schizonts were those of T. sergenti histologically.

Plasma high density lipoprotein in anemic cattle infected with Theileria sergenti.

Changes in plasma concentration of lipid composition were analysed in cattle with anemia due to Theileria sergenti infection. Plasma levels of phospholipids, cholesterol, free cholesterol, high density lipoprotein cholesterol, and vitamin E decreased to 40-67% of the pre-infection levels, corresponding to the decrease of PCV due to the infection. However, no definite changes were detected in plasma level of triglyceride. By gradient centrifugation, it was confirmed that lipid components, other than triglyceride, occur in high density lipoprotein (HDL) and these decreases lowered the HDL value. There was no correlation between this phenomenon and liver function. As similar changes in lipid composition were also observed in phlebotomized calves, it was considered that this phenomenon might partially depend on the acceleration of erythropoiesis as a reaction to anemia caused by T. sergenti infection.

Experimental transmission of Babesia ovata oshimensis n. var. of cattle in Japan by Haemaphysalis longicornis.

Transovarial transmission of a newly isolated large intraerythrocytic parasite, Babesia sp. 1 by Haemaphysalis longicornis was experimentally demonstrated. Larvae of H. longicornis were transovarially infected with the parasite by feeding as adults on the calf which had been experimentally infected with B. sp. 1. Piroplasms of B. sp. 1 were observed in peripheral blood of the calf which was infested with the parasite-infected larvae. Based on the transmissibility of the parasite with vector ticks, this parasite was suggested to be a variety of B. ovata. Thus, we propose a new variety name for B. sp. 1 as B. ovata oshimensis n. var.

DNA polymorphism of srRNA gene among Eimeria tenella strains isolated in Japan.

DNA polymorphism in twelve starains of Eimeria tenella isolated from various places in Japan was examined using 1.l kb small subunits ribosomal RNA amplified by PCR. Genetic variation was evaluated by random amplification of polymorphic DNA (RAPD) analysis. DNA fingerprint patterns were grouped into two, indicating that at least two DNA polymorphisms exist in Japanese E. tenella strains.