Ultrastructure and membrane permeability of cultured pancreaticβ-cells exposed to alloxan or 6-hydroxydopamine

Summary Stereological techniques on electron microscopy micrographs were used to evaluate the morphological changes of cultured islet cells that had been exposed to alloxan or 6-hydroxydopamine.Trypan Blue exclusion by cells cultured for 3 days indicated that the cells were 100% viable. Electron microscopy revealed that nearly all of the surviving cultured cells were cells.Exposure to 5 mmol/l alloxan or 1–5 mmol/l 6-hydroxydopamine for 10 or 30 min caused a general swelling of the cultured cells with a concomitant swelling of mitochondria and nuclei. The size of the secretory granules was not affected by the drugs. Only 3–10% of the cells excluded Trypan Blue after exposure to 5 mmol/l alloxan or 6-hydroxydopamine.The data conform with the hypothesis that a primary action of alloxan and 6-hydroxydopamine is at the plasma membrane level of cells.Abbreviations and definitions A _cell Cell profile area (µm²), surface area of one cell section surface - V _n Nuclear volume density (%), number of points over the nucleus divided by the number of points over the total cell area × 100 - V _m Mitochondrial volume density (%), number of points over mitochondria divided by hits over the cytoplasm (points over the cell minus points over the nucleus) × 100 - V _g Granular volume density (%), number of points over granules divided by hits over the cytoplasm × 100     

Complete Sequence of a 93.4-kb Contig from Chromosome 3 of Trypanosoma cruzi Containing a Strand-Switch Region

We have initiated large-scale sequencing of the third smallest chromosome of the CL Brener strain of Trypanosoma cruzi and we report here the complete sequence of a contig consisting of three cosmids. This contig covers 93.4 kb and has been found to contain 20–30 novel genes and several repeat elements, including a novel chromosome 3-specific 400-bp repeat sequence. The intergenic sequences were found to be rich in di- and trinucleotide repeats of varying lengths and also contained several known T. cruzi repeat elements. The sequence contains 29 open reading frames (ORFs) longer than 700 bp, the longest being 5157 bp, and a large number of shorter ORFs. Of the long ORFs, seven show homology to known genes in parasites and other organisms, whereas four ORFs were confirmed by sequencing of cDNA clones. Two shorter ORFs were confirmed by a database homology and a cDNA clone, respectively, and one RNA gene was identified. The identified genes include two copies of the gene for alanine-aminotransferase as well as genes for glucose-6-phosphate isomerase, protein kinases and phosphatases, and an ATP synthase subunit. An interesting feature of the sequence was that the genes appear to be organized in two long clusters containing multiple genes on the same strand. The two clusters are transcribed in opposite directions and they are separated by an ~20-kb long, relatively GC-rich sequence, that contains two large repetitive elements as well as a pseudogene for cruzipain and a gene for U2snRNA. It is likely that this strand switch region contains one or more regulatory and promoter regions. The reported sequence provides the first insight into the genome organization of T. cruzi and shows the potential of this approach for rapid identification of novel genes.     

Effects of mast cell-macrophage interactions on the production of collagenolytic enzymes by metastatic tumor cells and tumor-derived and stromal fibroblasts

Histological examination of the metastatic rat mammary adenocarcinoma line MTLn3 showed that macrophages and mast cells were frequently localized at the tumor periphery in the stromal tissues adjacent to the zones of tumor invasion. The interactions of these host cells with tumor cells and tumor-associated fibroblasts could be important in stimulating the production of extracellular matrix-degrading enzymes that facilitate tumor invasion and metastatic spread. Therefore, we examined the effects of isolated, activated macrophages and mast cells on the secretion of collagenolytic activities by normal fibroblasts, metastatic mammary adenocarcinoma cells and tumor-associated fibroblasts. Medium from activated macrophages or degranulated mast cells stimulated significant increases in production of collagenolytic activities by normal and tumor-associated fibroblasts and MTLn3 tumor cells. Medium from activated macrophages that had been pretreated with medium from degranulated mast cells, however, were less stimulatory to fibroblasts and tumor cell production of collagenolytic activities than medium from degranulated mast cells alone. We also examined the effects of two cytokines, interleukin-1 and tumor necrosis factor-a on activated macrophage- and degranulated mast cell-stimulation of fibroblast and tumor cell collagenolytic activities. The two cytokines alone or in combination stimulated increased production of collagenolytic activities by fibroblasts and tumor cells. Addition of the cytokines to degranulated mast cell products resulted in secretion of higher collagenolytic enzyme activities by normal fibroblasts (but not by tumor-derived fibroblasts or tumor cells) than with degranulated mast cell product-treatment of either target cell alone. Cytokines used in combination with macrophage-conditioned medium were less effective in stimulating fibroblast and tumor cell collagenase activities than cytokines alone. Thus normal infiltrating host cells such as macrophages and mast cells can have profound effects on the production of degradative enzymes by tumor cells and tumor-associated stromal fibroblasts.     

Effect of chronic vincristine treatment on mechanical withdrawal response and pre-pulse inhibition in the rat

Chemotherapeutic agents are associated with a number of serious side-effects. In addition to the development of peripheral neuropathy, patients often complain of additional symptoms related to attentional mechanisms. Although a great deal of interest is directed towards understanding the mechanisms underlying the development of peripheral neuropathy, there is a paucity of research that has examined the extent of impairment of attention in animals receiving chemotherapeutic agents. Therefore, the purpose of this experiment was to examine attentional mechanisms using the method of pre-pulse inhibition in animals that were chronically treated with vincristine. Although vincristine treated animals developed signs of peripheral neuropathy, there was no associated alteration of pre-pulse inhibition relative to vehicle treated animals. These results highlight the importance of continuing to develop methodology to model symptom burden in patients receiving chemotherapy.     

Quantitative detection of respiratory Chlamydia pneumoniae infection by real-time PCR

Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C. pneumoniae infection.     

Construction and Characterization of an Isogenic slt-ii Deletion Mutant of Enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga-like toxins (SLT), potent protein synthesis inhibitors. To further dissect the role of SLT-II in the course of disease, we have constructed E. coli TUV86-2, an isogenic SLT-II-negative mutant of EHEC strain 86-24. The slt-ii gene was inactivated by suicide vector mutagenesis. We also isolated derivatives of strain 86-24 that were cured of the phage carrying the toxin genes.     

Human herpesvirus 8 infection among various population groups in southern Israel

OBJECTIVE: To compare the prevalence of antibodies to human herpesvirus 8 (HHV-8) or Kaposi sarcoma-associated herpesvirus among Israeli and Ethiopian subjects. METHODS: Serum samples were obtained from 98 Israeli Jewish students aged 18-30 years, 100 HIV-1-seronegative Ethiopian immigrants to Israel of the same age, and 100 HIV-1-seronegative Ethiopian children 1-12 years old upon their arrival in southern Israel. Plasma samples were obtained from 3 hospitalized patients with multicentric Castleman disease (MCD) as positive controls. All serum samples were tested for antibodies to both latent and lytic antigens. Antibodies to the lytic antigens and the latency-associated nuclear antigen (LANA) of HHV-8 were detected by enzyme linked immunosorbent assay and by immunofluorescence assay. HHV-8 DNA from serum or plasma samples was detected by polymerase chain reaction analysis. RESULTS: Antibodies to HHV-8 LANA were detected in 2.9% of the Israeli subjects aged 18-30 years and in 26% of the Ethiopian subjects from both age groups tested. Antibodies to the lytic antigens were detected in all 3 MCD patients, in 4% of the Ethiopian children, and in 2% of the 18- to 30-year-old Ethiopians. No antibodies to the lytic antigens were detected in the Israeli students. HHV-8 DNA was detected in all 3 MCD patients and in 2 of 4 of the Ethiopian children positive for the lytic antigens. CONCLUSIONS: HHV-8 is highly prevalent in Ethiopian immigrants to Israel as compared with Israeli students. Antibodies to HHV-8 in Ethiopia are acquired before puberty. The results of this study indicate the association of HHV-8 with MCD, as has been documented by many other researchers.     

Gaze-stabilizing deficits and latent nystagmus in monkeys with brief, early-onset visual deprivation: eye movement recordings

The normal development and the capacity to calibrate gaze-stabilizing systems may depend on normal vision during infancy. At the end of 1 yr of dark rearing, cats have gaze-stabilizing deficits similar to that of the newborn human infant including decreased monocular optokinetic nystagmus (OKN) in the nasal to temporal (N-T) direction and decreased velocity storage in the vestibuloocular reflex (VOR). The purpose of this study is to determine to what extent restricted vision during the first 2 mo of life in monkeys affects the development of gaze-stabilizing systems. The eyelids of both eyes were sutured closed in three rhesus monkeys (Macaca mulatta) at birth. Eyelids were opened at 25 days in one monkey and 40 and 55 days in the other two animals. Eye movements were recorded from each eye using scleral search coils. The VOR, OKN, and fixation were examined at 6 and 12 mo of age. We also examined ocular alignment, refraction, and visual acuity in these animals. At 1 yr of age, visual acuity ranged from 0.3 to 0.6 LogMAR (20/40-20/80). All animals showed a defect in monocular OKN in the N-T direction. The velocity-storage component of OKN (i.e., OKAN) was the most impaired. All animals had a mild reduction in VOR gain but had a normal time constant. The animals deprived for 40 and 55 days had a persistent strabismus. All animals showed a nystagmus similar to latent nystagmus (LN) in human subjects. The amount of LN and OKN defect correlated positively with the duration of deprivation. In addition, the animal deprived for 55 days demonstrated a pattern of nystagmus similar to congenital nystagmus in human subjects. We found that restricted visual input during the first 2 mo of life impairs certain gaze-stabilizing systems and causes LN in primates.     

Clinical,hematologic, and immunologic effects of interleukin-10 in humans

We conducted a double-blind, placebo-controlled study to investigate the safety, pharmacokinetics, and immunological properties of interleukin-10 (IL-10) administration in healthy humans. Volunteers received a single intravenous bolus injection of recombinant human IL-10 (1, 10, or 25g/kg) or placebo. Cytokine production in whole blood and peripheral blood mononuclear cells (PBMC) was assessed before and 3, 6, 24, and 48 hr after the injection. Peak serum concentrations of IL-10 (15±1.1, 208±20.1, and 505±22.3 ng/ml) occurred after 2–5 min for 1, 10, and 25g/kg IL-10, respectively. The terminal-phase half-life was 3.18 hr. A transient leukocytosis (24–63% above baseline) was observed 6 hr after injection, which coincided with a dose-dependent decrease (12–24%) in neutrophil superoxide generation. There was a marked inhibition (60–95%) of endotoxin-induced IL-6 production from whole blood in each group receiving IL-10. Production of IL-8 in endotoxin-stimulated blood was reduced in the 10g/kg group. In PBMC stimulated with phytohemagglutinin and phorbol ester, there was a decrease (72–87%) in interferon- (IFN) production 6 hr after IL-10 with a return to pre-IL-10 levels after 24 hr. This reduction was only partially associated with a decrease in the number of CD2-bearing cells. We conclude that IL-10 administration into humans is without significant side effects, and a single injection reducesex vivo production of IL-6, IL-8, and IFN.     

Discharge patterns and recruitment order of identified motoneurons and internuclear neurons in the monkey abducens nucleus

1. Single neurons in the abducens nucleus were recorded extracellularly in alert rhesus macaques trained to make a variety of eye movements. An abducens neurons was identified as a motoneuron (MN) if its action potentials triggered an averaged EMG potential in the lateral rectus muscle. Abducens internuclear neurons (INNs) that project to the oculomotor nucleus were identified by collision block of spontaneous with antidromic action potentials evoked with a stimulating electrode placed in the medial rectus subdivision of the contralateral oculomotor nucleus. 2. All abducens MNs and INNs had qualitatively similar discharge patterns consisting of a burst of spikes for lateral saccades and a steady firing whose rate increased with lateral eye position in excess of a certain threshold. 3. For both MNs and INNs the firing rates associated with different, constant eye positions could be described accurately by a straight line with slope, K (the eye position sensitivity in spikes.s-1.deg-1), and intercept, T (the eye position threshold for steady firing). For different MNs, K increased as T varied from more medial to more lateral values. In contrast, the majority of INNs already were active for values of T more medial than 20 degrees and showed little evidence of recruitment according to K. 4. During horizontal sinusoidal smooth-pursuit eye movements, both MNs and INNs exhibited a sinusoidal modulation in firing rate whose peak preceded eye position. From these firing rate patterns, the component of firing rate related to eye velocity, R (the eye velocity sensitivity in spikes.s-1.deg-1.s-1), was determined. The R for INNs was, on average, 78% larger than that for MNs. Furthermore, R increased with T for MNs, whereas INNs showed no evidence of recruitment according to R. If, as in the cat, the INNs of monkeys provide the major input to medial rectus MNs and if simian medial rectus MNs behave like our abducens MNs, then recruitment order, which is absent in INNs, must be established at the MN pool itself. 5. Unexpectedly, the R of MNs decreased with the frequency of the smooth-pursuit movement. Furthermore, the eye position sensitivity, K, obtained during steady fixations was usually less than that determined during smooth pursuit. Therefore, conclusions about the roles of MNs and premotor neurons based on how their R and K values differ must be viewed with caution if the data have been obtained under different tracking conditions.(ABSTRACT TRUNCATED AT 400 WORDS)