Immunohistochemical organization of the ventral lateral geniculate nucleus in the tree shrew. The ventral lateral geniculate nucleus (vLGN) of the tree shrew (Tupaia belangeri) was differentiated into multiple subdivisions (dorsal cap, intergeniculate leaflet, parvicellular segment, and internal and external magnocellular laminae, the latter being further divisible into a lateral and medial division) on the basis of retinal projections, immunochemistry, and histochemistry. Retinal projections traced with intravitreal injections of wheat germ agglutinin conjugated horseradish peroxidase revealed direct bilateral input to all subregions of the vLGN, except for the internal magnocellular lamina (which received only contralateral input) and the parvicellular segment (which was not retinorecipient). Furthermore, retinal inputs clearly distinguished the relatively heavily retinorecipient intergeniculate leaflet from the less prominently labeled dorsal cap. Immunohistochemical localization of Neuropeptide Y (NPY) perikarya revealed their prominence in the intergeniculate leaflet and the external magnocellular laminae with a concentration along the optic tract. NPY immunoreactive fibers were seen in all but the parvicellular subregion. Gamma amino butyric acid immunoreactivity was seen throughout the vLGN, but was most concentrated in the dorsal cap and the magnocellular laminae, followed by the intergeniculate leaflet. Histochemical studies of cytochrome oxidase and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase localization revealed similar patterns of dense reactivity within the external magnocellular lamina, intergeniculate leaflet and dorsal cap, and somewhat less dense, but substantial reactivity in the internal magnocellular lamina. Within the external magnocellular lamina, cells reactive for cytochrome oxidase were noted in the lateral portion bordering the optic tract, whereas those specific for NADPH-diaphorase were dispersed throughout the lamina. Poor reactivity for both histochemical markers was evident in the parvicellular segment. Overall, the markedly different patterns of retinal input and neurochemical organization between the subdivisions of the tree shrew vLGN suggest their involvement in diverse functions. Furthermore, the basic similarity of the organization of the tree shrew vLGN to that of the taxonomically unrelated ground squirrel may indicate a common mammalian scheme.


Plasma alpha-melanocyte-stimulating hormone, beta-endorphin, met-enkephalin, and natural killer cell activity in vitiligo. The immune system is important in the pathogenesis of vitiligo, and emotional stress has precipitated vitiligo in some patients. Opioid peptides, beta-endorphin, met-enkephalin, and alpha-melanocyte-stimulating hormone (MSH) act as immunomodulators, and their secretion increases during periods of stress. To see whether these three neuropeptides might be related to vitiligo itself or to some alterations of the immune system in patients with vitiligo, we compared circadian variations in their plasma concentrations and natural killer cell activity of peripheral blood lymphocytes in 14 patients with vitiligo with those of 12 healthy subjects. Plasma concentrations of neurohormones were evaluated by radioimmunoassay (immunoradiometric assay for beta-endorphin). Natural killer cell activity (NKCA) was assayed against K562 cells by 51Cr release technique. Data were compared by the Student t test and analyzed by cosinor analysis. The NKCA in vitiligo patients was higher than in controls but had similar circadian rhythm. alpha-MSH had no circadian rhythm in controls or in patients; plasma alpha-MSH levels were the same. Daily met-enkephalin and beta-endorphin oscillations in patients were no longer circadian. beta-Endorphin plasma levels in stable vitiligo were higher than in controls. There were no differences between patients with active vitiligo and normal subjects. Met-enkephalin plasma levels were generally higher in vitiligo patients, especially in the one with active vitiligo, than in controls. In vitiligo there are aberrations in neuropeptide, beta-endorphin, and met-enkephalin secretion. The plasma met-enkephalin level is positively correlated with the aggressiveness of the disease.


Detection with the polymerase chain reaction of human papillomavirus DNA in condylomata acuminata treated in vitro with liquid nitrogen, trichloroacetic acid, and podophyllin. The mechanisms of action for local treatments used against condylomata acuminata are unknown, but most are believed to cause physical destruction of infected tissue. Our purpose was to determine whether liquid nitrogen, trichloroacetic acid (TCA), and podophyllin damage HPV DNA found in condylomata acuminata. Fourteen genital warts were excised from 14 patients and divided. One part was treated with liquid nitrogen, the second and third parts were treated with TCA and podophyllin, respectively, and the remainder served as a control. DNA was then extracted from tissue by proteolytic digestion and amplified by the polymerase chain reaction. Dot blots were performed with the use of radiolabeled consensus and HPV type-specific probes. HPV DNA was amplified and detected in 100% of untreated specimens, in 92% of specimens treated with liquid nitrogen, and in 15% and 7% of specimens treated with podophyllin and TCA, respectively. TCA and podophyllin damage HPV DNA more effectively than does liquid nitrogen.


Posttreatment itraconazole levels in the nail. New implications for treatment in onychomycosis. A problem in the treatment of onychomycosis is the lengthy duration of therapy. The pharmacokinetics of itraconazole suggest a potential for briefer treatment. This study was designed to investigate itraconazole nail kinetics in 39 patients with onychomycosis in relation to their therapeutic outcome. All patients received itraconazole for 3 months at a dose of 100 or 200 mg daily. Itraconazole levels of distal nail clippings were determined during a 6-month posttherapy period. Therapeutic itraconazole concentrations were found in the nail plates of fingernails and toenails for up to 6 months after treatment. Cure of the toenails was observed in 79% of the patients treated with the 200 mg dosage and in 26% of those treated with 100 mg at 6 months after therapy. The data suggest that the drug reaches the nail via incorporation into the matrix and by diffusion from the nail bed and is eliminated with regrowth of the nail after discontinuation of treatment.


Rhinovirus 39 infection in allergic and nonallergic subjects. To determine if individuals with allergic rhinitis are hyperresponsive to upper respiratory tract viral infections, 20 allergic and 18 nonallergic, susceptible, adult volunteers were challenged and infected with rhinovirus type 39 before the pollen seasons. Before challenge and on each of 6 days of cloister, all volunteers were interviewed for symptoms and completed a test battery consisting of evaluations of secretion production by weighed tissues, nasal patency by active posterior rhinomanometry, nasal clearance by the dyed saccharin technique, pulmonary function by spirometry, eustachian tube function by sonotubometry, and middle ear status by tympanometry. The symptomatology and pathophysiology resulting from the rhinovirus infection were consistent with those reported in previous studies with this challenge system. Between-group comparisons revealed no differences in symptom presentation, nasal secretion production, or overall pathophysiologic response. However, for decreased mucociliary clearance rate, increased nasal congestion, eustachian tube dysfunction, and symptoms of sneezing, the allergic group demonstrated an earlier onset compared with that of the nonallergic group. The biologic significance of the differences in onset of dysfunction is tempered by the observation that the temporal pattern of responses in the allergic group was similar with that of nonallergic subjects in previous studies. The results of the present study do not support the hypothesis of a physiologic hyperresponsiveness to rhinovirus type 39 infection in allergic subjects during nonallergy seasons.


Cytomegalovirus retinitis complicated by optic neuropathy: a longitudinal study. Two cases of suspected cytomegalovirus (CMV) retinitis treated with ganciclovir and followed for 18 and 5 months are presented. Each patient developed optic neuropathy and experienced reduced vision despite effective control of their retinitis. In addition to objective ocular findings, subjective visual function changes (perimetry, contrast sensitivity and accommodation) in long term follow up of AIDS patients are reported for the first time. A discussion of CMV, its treatment and complications follows.


Effects of disinfecting solutions recommended by the Centers for Disease Control on Goldmann tonometer biprisms. Twelve new Goldmann biprisms were used to measure the intraocular pressure of one eye of one subject, four times each day for 15 days. Between measurements, each biprism was disinfected by soaking in one of the three solutions recommended by the Centers for Disease Control (CDC) as effective against HIV, or by rubbing with an isopropyl alcohol swab. Intraocular pressure measurements, clarity of the biprism front surface, and ring image were statistically evaluated to determine whether any regimen negatively affected the biprisms. Biprisms soaked in alcohol were unusable after 4 days due to front surface roughness. No clinically significant differences were found in IOP measurements or clarity of the remaining groups, but the ring image was significantly less distorted with hydrogen peroxide treatment. These results suggest that hydrogen peroxide should be used to disinfect Goldmann biprisms.


Cytomegalovirus retinitis in AIDS.

Cytomegalovirus retinitis in AIDS. Cytomegalovirus (CMV) retinitis is the most common retinal opportunistic infection in people with the acquired immune deficiency syndrome (AIDS). Ordinarily, CMV causes little morbidity in an immunocompetent person. However, in the immunosuppressed patient this virus is responsible for progressive retinal destruction with necrosis leading to a devastating loss of vision. This paper will provide the practitioner with a clinical approach to the presentation, diagnosis and management of patients with CMV retinitis secondary to AIDS.


[Hydatidiform mole and its complicated development]. The authors report a series of 260 cases of trophoblastic disease, followed up over a period of 18 years. Supervision was based on clinical findings, biological development, radiology and ultrasound. The outcome was favourable in 80% of cases with 2.7% requiring prophylactic hysterectomy for classifical hydatiform mole. In the other cases (20%) the following was the way the disease developed: 12 cases of persistent trophoblastic activity, 13 cases of invasive mole, 24 cases of choriocarcinoma. Initially the treatment was with chemotherapy and surgery was used as an extra treatment in 14% of cases, which gave encouraging results. The authors regret three deaths in this series following choriocarcinoma. Two of these were after chemotherapy as a sole treatment and one after treatment with chemotherapy and surgery.


Molecular biology of renin. II: Gene control by messenger RNA, transfection and transgenic studies. This review addresses the question of renin gene control by examining information from messenger RNA, transgene, transient expression and DNA-protein binding studies. Although information from messenger RNA studies may be reliable for tissues such as kidney and, in mouse, submandibular gland, which have relatively high expression, more sensitive approaches, e.g. polymerase chain reaction technology, are needed for studies of renin gene regulation in other tissues. Control at the molecular level is particularly uncertain, with conflicting data suggesting that renin promoter activity depends either upon a complex interaction of positive and negative regulatory elements or upon the presence of specific trans-acting factor(s) alone, which act upon putative enhancer(s) in the renin-associated DNA. Only after renin-synthesizing cell lines are studied will a clearer picture emerge. Transgenic work has loosely defined the DNA needed as being less than several kilobases of flanking sequences, with the possibility that sequences in the gene itself, particularly in intron 1, are necessary. Such transgenic work has also shown that the renin gene can cause hypertension. Knowledge of DNA and proteins and the interactions that turn on transcription of the renin gene is still rudimentary and currently represents the major challenge in renin research.


No inhibitory effect of increased serum magnesium upon pressor response to infused calcium. The purpose of this study was to determine whether the pressor effect of infused calcium is inhibited by increased serum magnesium. Intravenous infusions of calcium, magnesium, and calcium+magnesium were performed in 10 subjects and the effect upon haemodynamic variables was investigated. Intra-arterial blood pressure and heart rate were recorded during i.v. infusions of magnesium, calcium and calcium+magnesium. Cardiac output and serum concentration of electrolytes were measured before and after the infusions. Total peripheral vascular resistance (TPVR) was calculated from: mean blood pressure/cardiac output. Magnesium infusion increased serum magnesium, but did not change blood pressure, cardiac output or TPVR. Calcium infusion increased serum calcium and elevated both blood pressure and TPVR with no change in cardiac output. Calcium+magnesium infusion increased both serum magnesium and calcium, showing elevation of blood pressure and TPVR with no change in cardiac output. Changes in haemodynamic variables caused by the calcium+magnesium infusion were similar to those caused by the calcium infusion alone. These results suggest that increased serum calcium elevates blood pressure and TPVR through arterial vasoconstriction by arterial smooth muscle contraction, but increased serum magnesium does not induce arterial vasodilation.


Is atrial natriuretic peptide-guanosine 3',5' cyclic monophosphate coupling a determinant of urinary sodium excretion in essential hypertension? (1) To compare urinary guanosine 3',5' cyclic monophosphate (cyclic GMP) excretion between normotensive subjects and essential hypertensive patients; (2) to determine the influence of changes in sodium intake on urinary cyclic GMP excretion in response to the neutral endopeptidase inhibitor candoxatril in essential hypertensives. (1) Twenty-five normotensive subjects and 25 patients with established essential hypertension not on treatment; (2) Single oral dose of candoxatril in eight patients with essential hypertension after equilibration on a low- or high-sodium diet in a placebo-controlled, double-blind, randomized, crossover study. Blood pressure was measured by ultrasound sphygmomanometry. Atrial natriuretic peptide (ANP) and urinary cyclic GMP were measured by radioimmunoassay. Group comparisons were made using unpaired t-tests and two-way analysis of variance. Plasma ANP was significantly raised in patients with essential hypertension compared with the normotensive group, but there was no difference in urinary cyclic GMP excretion. Plasma ANP increased significantly on the high- compared with low-sodium diet. After candoxatril, there were significant diet-related increases in plasma ANP and urinary sodium excretion up to 6 h after drug administration. There were similar increases in urinary cyclic GMP excretion on both diets, but there were no consistent differences in this excretion between the low- and high-sodium diets. These observations not only point to the importance of ANP-cyclic GMP coupling as a determinant of the natriuretic response to endopeptidase inhibition, but also suggest that the excretion of urinary cyclic GMP can be influenced by other factors in addition to circulating ANP.


Arterial effects of salt restriction in hypertensive patients. A 9-week, randomized, double-blind, crossover study. To investigate the hemodynamic effects of a moderately low-salt diet in a 9-week, randomized, double-blind, crossover study in 20 hypertensive, ambulatory patients. All subjects followed a 9-week, low-salt diet. During this period, they received capsules containing either lactose or salt in 4-week treatment periods, separated by a 1-week washout period. Hemodynamic and biological parameters were evaluated on the day of randomization and at the end of weeks 4 and 9. We defined a low-sodium diet (LSD) as a salt-restriction period with lactose capsules, and a normal-sodium diet (NSD) as a salt-restriction period with capsular salt supplementation. Blood pressure was significantly lower during LSD compared with NSD. This fall in blood pressure was associated with a decrease in peripheral resistance in carotid and forearm circulation. Brachial artery diameter was larger during LSD whereas carotid artery diameter remained unchanged. The changes in brachial artery were: (1) not related to blood pressure changes; (2) positively related to age, and (3) negatively correlated with baseline intracellular sodium content. These results suggest that moderate low-salt restriction is capable of decreasing blood pressure and peripheral resistance in carotid and forearm circulation. The increase in brachial, but not carotid, artery diameter following salt restriction suggests a difference in salt dependence among different arteries.


Regulation of angiotensinogen gene expression by estrogen. Clarification of the role of estrogen in the regulation of angiotensinogen gene expression in multiple tissues. The effect of 17 beta-estradiol (E2; 10 micrograms/100 mg body weight) administration in ovariectomized (OVX) rats upon angiotensinogen messenger RNA (mRNA) levels in multiple tissues was assessed. Confounding ovarian factors were thus removed by studying the animals in the castrate state. Controls consisted of OVX and intact female rats. Adult female Sprague-Dawley rats were ovariectomized and experiments begun 21 days postsurgery. Animals were injected with E2 and studied after 0, 1, 4, and 24 h of treatment. Levels of angiotensinogen mRNA were determined by Northern blot analysis using beta-actin mRNA as an internal standard. A single angiotensinogen mRNA species with molecular size of approximately 1800 bp was observed in rat liver, aorta, kidney, cardiac atria, hypothalamus and whole brain. Little or no angiotensinogen mRNA was identified in the pituitary gland. Angiotensinogen mRNA was most abundant in rat liver, hypothalamus, aorta and progressively less abundant in whole brain, cardiac atria and kidney. A twofold induction of hepatic angiotensinogen mRNA levels in E2-OVX rats was observed by 4h. The angiotensinogen mRNA levels in kidney were threefold higher by 4 h compared with OVX control animals. In aorta, the angiotensinogen mRNA level was also threefold higher by 1 h after E2 treatment. No significant effect of estradiol treatment was observed in cardiac atria although the level of angiotensinogen mRNA was higher in intact female rats compared with OVX controls. These results suggest that estrogen modulates angiotensinogen gene expression in a tissue-specific manner.


The characteristics of erythrocyte Na+ transport systems in normal pregnancy and pregnancy-induced hypertension. To assess the role Na plays in the pathogenesis of pregnancy-induced hypertension (PIH). We assessed Na and K content, the maximum number of ouabain binding sites, Na(+)-Li+ countertransport and Na(+)-K+ cotransport in erythrocytes from women with untreated PIH, normal pregnant women and healthy non-pregnant women. In normal pregnancy, the Na content of erythrocytes decreased, accompanied by the activation of Na excretion systems. In women with PIH, the Na content of erythrocytes and the Na(+)-K+ cotransport activity significantly increased, whilst erythrocyte K content and the maximum number of ouabain binding sites significantly decreased, compared with observations in normal pregnancy. In both normal pregnancy and PIH, there were no differences in Na(+)-Li+ countertransport. These results suggest that the increase of erythrocyte Na content in women with PIH may be contributed to by a reduction in the number of ouabain binding sites, whilst Na(+)-K+ cotransport and Na(+)-Li+ countertransport may compensate for this effect in women with PIH.


Sympathomoderating influence of benazepril in essential hypertension. In essential hypertension, captopril attenuates forearm vasoconstriction reflexly induced by deactivation of cardiopulmonary and arterial baroreceptors, thus exerting a sympathomoderating effect. We investigated whether this is a common effect of angiotensin converting enzyme (ACE) inhibitors. Cardiopulmonary and arterial baroreceptors were deactivated by progressively reducing central venous pressure (CVP) through progressively greater lower body negative pressures in eight untreated mild essential hypertensives on a moderately low-sodium diet (50 mmol/l per day). This deactivation was performed after oral administration of the non-sulphidrylic ACE inhibitor benazepril (10 mg) and placebo according to a double-blind randomized crossover experimental design. After placebo, the reduction in CVP increased forearm vascular resistance (FVR; mean arterial pressure: plethysmographic forearm blood flow ratio). After benazepril, baseline blood pressure (beat-to-beat finger pressure) and FVR were significantly reduced whilst plasma angiotensin II was suppressed and PRA increased (both measured by radioimmunoassay). The FVR increases induced by progressive CVP reduction were less than after placebo administration, and the overall difference was statistically significant. Benazepril did not affect the reflex FVR reduction observed by increasing CVP through leg raising, nor the reflex changes in plasma norepinephrine measured by high-performance liquid chromatography accompanying the changes in FVR. Benazepril attenuates sympathetic vasoconstriction as does captopril. This effect (which is mainly operative during an increased sympathetic drive and exerted through a reduction of adrenoceptor responsiveness) is thus likely to be a class- rather than a compound-related feature.


The influence of ambulatory blood pressure monitoring on the design and interpretation of trials in hypertension. To describe the reproducibility of diastolic blood pressure (DBP) measurement by clinic and ambulatory monitoring and to evaluate the effects of this reproducibility on the design and interpretation of clinical trials in hypertension research. Prospective single-blind study of repeat measurement reproducibility of blood pressure recording. Tertiary referral hospital hypertension clinic. One hundred untreated mild-to-moderate hypertensive subjects taking 1 month of single-blind placebo. A single clinic measurement of DBP was poorly reproducible and the results for single DBP estimates taken out of a daytime ambulatory recording were similar. Average ambulatory DBP was much more reproducible, although this improvement depended upon the averaging of many measurements taken throughout the day. Ambulatory monitoring would decrease antihypertensive trial size by a factor of four or halve the size of detectable DBP difference between treatments. The use of poorly reproducible DBP measurements such as single clinic readings may have led to an underestimation of the risks of minor degrees of blood pressure elevation because of a 'regression dilution' bias. For single clinic DBP readings, we calculate this underestimation to be as much as 69%, and for average ambulatory DBP approximately 20%.


Meeting report of the International Society of Hypertension Conference on Hypertension and Diabetes. Points of agreement: (1) In IDDM, hypertension occurs in patients who have already developed nephropathy, probably in the microalbuminuric phase. (2) Hypertension is an important accelerator of the development of diabetic nephropathy. (3) Hypertension, obesity and NIDDM are often associated, and insulin resistance is commonly observed in all three states. (4) Antihypertensive therapy retards the development of diabetic nephropathy in IDDM and reduces proteinuria in NIDDM. (5) The choice of antihypertensive agent in the diabetic patient must be based upon the efficacy of the drug as well as avoidance of side effects including deleterious influence on glucose, insulin and lipid levels and renoprotection. (6) Carefully conducted long-term comparative trials between different classes of antihypertensive drugs in microalbuminuric IDDM and NIDDM patients are essential. Points of major controversy: (1) Detection of IDDM patients prone to the development of diabetic nephropathy can be performed by measuring specific parameters such as erythrocyte Na(+)-Li+ countertransport activity. (2) Insulin resistance is a pathogenic mechanism rather than purely an association with hypertension and obesity. (3) A certain class of antihypertensive agents--ACE inhibitors--confers a specific renoprotective effect in diabetic nephropathy, in addition to its effects upon systemic blood pressure. (4) Reduction of blood pressure should be considered in the normotensive microalbuminuric diabetic patient. (5) Microalbuminuria is a sufficient 'surrogate endpoint' for the progression of renal failure.


Abrogation of c-MYC protein degradation in human lymphocyte lysates by prior precipitation with perchloric acid. Conventional lysis buffers, though containing cocktails of protease inhibitors, did not prevent the degradation of c-MYC recombinant protein added immediately prior to lysis to cell pellets from human mixed lymphocyte cultures. Treatment of the cells with 4.2% perchloric acid, however, prevented protein degradation and facilitated the detection of c-MYC protein by Western blotting even in unstimulated lymphocytes, where previously it had been reported to be undetectable or barely detectable using this technique. PHA stimulation of lymphocytes induced an approximately six fold increase in measured c-MYC protein within 5 h if cell extracts were prepared using perchloric acid precipitation. However, using conventional lysis buffer the proto-oncogene protein was undetectable until 48-72 h after mitogen addition. Pretreatment with perchloric acid may be useful for Western blotting analysis of protein in other systems where it may be desirable to dispense with the use of toxic protease inhibitors or where these may be incompletely effective.


Mycoplasma contamination in human leukemia cell lines. II. Elimination with various antibiotics. 19 suspension cell lines were treated with antibiotics for elimination of chronic contamination with mycoplasma. We compared the efficiency, cytotoxicity and cross-resistance of the commercially available antibiotics MRA (Mycoplasma Removal Agent, a quinolone derivative and DNA gyrase inhibitor), Ciprobay (ciprofloxacin, also a quinolone derivative and DNA gyrase inhibitor), and BM-cyclin (a combination of tiamulin, a pleuromutilin derivative, and minocycline, a tetracycline derivative, both inhibitors of protein synthesis on ribosomes). Contaminants were eliminated in all 19 cell lines by BM-Cyclin. Only 74% of the cell lines were cleared of contamination by both MRA and Ciprobay. Successful treatment was monitored by three mycoplasma detection assays. Cross-resistance was noted between MRA and Ciprobay in four of the five cell lines not cleared by either reagent. This resistance could, however, be overcome by consecutive exposure to BM-cyclin. Employed at the recommended concentrations, the antibiotics did not cause marked cytotoxicity, but the growth of the cells was affected to various degrees by some antibiotics. The elimination of mycoplasma from chronically contaminated cell lines is an effective alternative to other treatment protocols, but is cost-intensive and time-consuming; lasting damaging effects of the treatments on the eukaryotic cells cannot be excluded. Long-term post-treatment monitoring is mandatory, since contaminants may only be suppressed and then recur.


A quantitative immunocytochemical method for the measurement of islet cell cytoplasmic antibodies. A quantitative immunocytochemical method for the measurement of islet cell cytoplasmic antibodies has been developed. The method employs human or rat pancreas, a protein A-peroxidase/diaminobenzidine secondary antibody system and an independent measurement of islet total and exocrine mean integrated absorbance by scanning microdensitometry. Specific islet cell cytoplasmic antibody binding (islet total-exocrine mean integrated absorbance) was dependent on serum dilution and substrate reaction time. The detection limit was approximately 5 JDF units. Specific islet cell cytoplasmic antibody binding values with human and rat pancreas were similar. Specific islet cell cytoplasmic antibody binding (human pancreas) was greater (p less than 0.001) in sera from patients with newly diagnosed insulin dependent diabetes mellitus (0.119 +/- 0.086, n = 29) compared to normal sera (0.003 +/- 0.008, n = 29). Thus, the method has been validated and may be useful for measuring the blocking effect of potential antigens on specific islet cell cytoplasmic antibody.


Tampons: a novel patient-administered method for the assessment of genital human papillomavirus infection. Assessment of human papillomavirus (HPV) infection usually requires a speculum examination to collect genital specimens. A technique using tampons as a patient-administered method for the collection of specimens was studied by dot blot hybridization (HPV types 6, 11, 16, 18, 31, and 33) and polymerase chain reaction (PCR). Tampons and cervical scrapes were collected from 48 consecutive women attending a dysplasia clinic. Tampons provided a significantly larger pellet volume (P less than .002) and more DNA (P less than .01) than scrapes. There was a close correlation when samples were analyzed for the presence of HPV DNA. Using dot blot hybridization, 8 cervical scrapes (17%) and 9 tampons (19%) were positive for HPV DNA (90% correlation). By PCR, 35 cervical scrapes (73%) and 33 tampons (69%) were positive for HPV DNA (88% correlation). Thus, tampon specimens are an easy method for assessment of genital HPV infection.


Rotavirus-specific helper T cell responses in newborns, infants, children, and adults. An obstacle to developing a successful rotavirus vaccine has been the inability to consistently correlate the humoral immune response with protection against disease. Transplacental transfer of maternal rotavirus-specific antibodies may obscure the capacity to discriminate an active from a passively acquired humoral immune response in infants. In an attempt to circumvent this problem, an assay was developed to detect rotavirus-specific helper T cells among circulating mononuclear cells. Rotavirus-specific lymphoproliferative responses and rotavirus-specific neutralizing antibody titers in blood were determined in 11 mother/newborn pairs at the time of delivery and in 54 infants, children, and adults ranging in age from 16 days to 40 years. Only 1 of 11 infants tested between 16 days and 6 months of age had detectable rotavirus-specific helper T cell activity whereas 8 of 11 had circulating rotavirus-specific neutralizing antibodies. Acquisition of rotavirus-specific helper T cell activity over the first few years of life correlated with the age at which infants and young children are known to be infected with rotavirus. These findings support the hypothesis that detection of rotavirus-specific lymphoproliferative activity in infants may more accurately determine previous exposure to rotavirus than detection of rotavirus-specific antibodies.


Hydrogen peroxide production by Lactobacillus species: correlation with susceptibility to the spermicidal compound nonoxynol-9. Facultative anaerobic lactobacilli were recovered from the vaginas of 96.8% of 63 nonpregnant, healthy, premenopausal women. The predominant species were Lactobacillus jensenii, Lactobacillus acidophilus, and Lactobacillus casei. Of the women, 74.6% had hydrogen peroxide-producing lactobacilli, 22.2% had non-hydrogen peroxide-producing lactobacilli, and 3.2% had no lactobacilli. None of the 68 isolates had catalase activity. Some 68.2% of the isolates were inhibited by concentrations of less than or equal to 1% (wt/vol) of nonoxynol-9 (bactericidal for 73.3% of isolates, bacteriostatic for 26.7%). The remaining 31.8% could grow in all concentrations to 25% (wt/vol) of nonoxynol-9. All of the lactobacilli that were sensitive to nonoxynol-9 produced hydrogen peroxide whereas only 3 of 21 resistant strains were hydrogen peroxide producers. A significant correlation (P less than .001, chi 2 test) was found between hydrogen peroxide production and sensitivity to nonoxynol-9. It is suggested that the vaginal flora of spermicide users could be depleted of hydrogen peroxide-producing lactobacilli, possibly increasing susceptibility to urogenital infection.


Serum levels of lymphokines and soluble cellular receptors in primary Epstein-Barr virus infection and in patients with chronic fatigue syndrome. The immunopathology in primary Epstein-Barr virus (EBV) infections and in chronic fatigue syndrome was studied by examining serum levels of interleukins (IL) and of soluble T cell receptors in serum samples. Serum samples were from patients during and 6 months after primary EBV-induced infectious mononucleosis and from patients with chronic fatigue syndrome and serologic evidence of EBV reactivation. Markers for T lymphocyte activation (soluble IL-2 and CD8) and for monocyte activation (neopterin) were significantly elevated during acute infectious mononucleosis but not in patients with chronic fatigue syndrome. Interferon-alpha, IL-1 beta, and IL-6 levels were not significantly increased in any patient group but inferferon-gamma levels were significantly increased during the acute phase of infectious mononucleosis. The levels of IL-1 alpha were significantly higher than in controls both in patients with infectious mononucleosis and in those with chronic fatigue syndrome. In the latter, the lack of most markers for lymphocyte activation found in patients with infectious mononucleosis makes it less likely that EBV reactivation causes symptoms.


Pancreatic mixed ductal-islet tumors. Is this an entity? Thirty-eight human pancreatic cancer specimens were studied for the reactivity of cancer cells with monoclonal antibodies against insulin, glucagon, somatostatin, pancreatic polypeptide (PP), vasoactive intestinal peptide (VIP), gastrin, calcitonin, and with argyrophilic reactivity. Immunoreactivity with one or several antibodies or argyrophilic reactivity were found in 30 (79%) cases. In 17 cases, the number of endocrine cells was excessive and morphologically consistent with the mixed ductal-islet tumor. Although most immunoreactive cells were located at the base of the malignant glands, some had intraepithelial location and were also present in the invasive portion of cancers, indicating their malignant nature. Endocrine cell proliferation were found in the pancreatic tissue adjacent to the carcinoma in 8 out of 12 specimens examined. In these cases, the immunoreactive cells were either distributed among the acinar cells or ductal cells. More endocrine cells were found in the hyperplastic ducts; however, no correlation was found between the degree of hyperplasia and the occurrence of any type of immunoreactive cells. Although several types of endocrine cells occurred in different pancreatic regions (head, body, and tail), PP cells were restricted to tissues taken from the head of the pancreas. Experimental data and similar observations by other investigators led us to conclude that participation of endocrine cells in ductal-type carcinomas is a general phenomenon and does not justify the classification of these lesions to mixed ductal-islet entity. However, because immunoreactive cells were more common and numerous in well-differentiated carcinomas, they may have some prognostic values.


[An immunohistochemical study on three aldolase isozymes in human lung cancer]. We investigated three aldolase isozymes (aldolase A, B, and C) in human lung cancer by using an indirect peroxidase labeled antibody method. We used 27 tissue samples obtained at surgical operations which were fixed in periodate-lysine-4% paraformaldehyde (PLP) solution, and embedded in optimum cutting temperature (OCT) compound. They were 11 adenocarcinomas, 9 squamous cell carcinomas, 3 large cell carcinomas, 3 small cell carcinomas, and 1 adenosquamous carcinoma. Aldolase A and C expressed intensely positive stainings in the cytoplasm of cancer cells compared with normal lung tissues, and its positivities were 81% respectively. However, Aldolase B showed almost negative staining, and its positivities were only 41%. These rates had no relation to the histological types or pathological stages of lung cancers, and suggested that human lung cancer contained increased levels of aldolase A, and C.


Preloading of micromolar intracellular Ca2+ during capacitation of Sicyonia ingentis sperm, and the role of the pHi decrease during the acrosome reaction. In studying the mechanism controlling the sperm acrosome reaction (AR) in the marine shrimp Sicyonia ingentis, intracellular Ca2+ and pH were measured using the fluorescent indicators Fura-2 and Fluo-3 for Ca2+, and SNARF-1 for pH. Capacitated sperm possessed an apparent resting Ca2+ concentration of 1-2 microM which remained constant upon induction of the AR with egg water. Uncapacitated sperm had extremely low Ca2+ levels and did not respond to egg water. These results suggest that, while in other species the Ca2+ is elevated to micromolar levels during initiation of the AR, S. ingentis sperm are preloaded with Ca2+ during capacitation and the trigger for the AR is downstream of the Ca2+ increase. The notion that Ca2+ influx is not involved at the actual time of the AR in capacitated S. ingentis sperm is supported by the inability of Ca2+ ionophore A23187 to induce the AR and the ineffectiveness of Ca2+ channel antagonists to block egg water-induced AR. Measurements of capacitated sperm pH showed a significant decrease during the first 10-15 min of the AR, which did not correlate temporally to either acrosomal exocytosis (at 5 min post-induction) or filament formation (after 45 min). Inhibition of egg protease activity required for induction of filament formation did not inhibit the pH drop, indicating that intracellular acidification is not the final trigger for filament formation, although it may be required prior to action of the protease.


Characterization of a genetic variant of human hepatitis A virus. Human isolates of hepatitis A (HAV) are a single serotype; however, recent genetic surveys using limited nucleotide sequencing have provided evidence that more than one genotype is responsible for HAV infection in different parts of the world (Jansen et al. [1990]: Proc Natl Acad Sci USA 87:2867-2871; Robertson et al. [1991] J Infect Dis 163:286-292). One of these genotypes was originally isolated from Panamanian owl monkeys (strain PA21), but has subsequently been found associated with human cases of HAV from Sweden in 1979 (H-122) and the United States of America in 1976 (GA76). The nucleic acid sequence of the exposed capsid polypeptide region of GA76 differs from other human HAV sequences by approximately 20%, yet differs by only 2.4% when compared with P1 sequence of the PA21 strain. The 20% nucleic acid variability between GA76 and other human HAV results in limited amino acid changes (3%), while a comparison with PA21 revealed only four homologous amino acid substitutions within VP2, VP3, and VP1 polypeptides. HAV infected stool specimens from Nepal and northern India during 1989 and 1990 were found to contain virus whose genetic makeup was related to the PA21 and GA76 isolates. This genotype of HAV appears to be circulating in some parts of the world where HAV is hyperendemic, and is a potential cause of hepatitis A infection within a susceptible population.


Increased detection of HPV 16 virus in invasive, but not in early cervical cancers. Human papilloma virus type 16 (HPV 16) DNA is found in about 50% of cervical squamous cell carcinomas (SCCs), and this association has raised the possibility of a causal role for HPV 16 in cervical carcinogenesis. We have tested this hypothesis by assaying a series of biopsies (n = 119) ranging from normal mucosa to infiltrating SCC with the PCR-technique for the presence of HPV 16 DNA. While HPV 16 DNA was detected in 50% of our cases with invasive SCC, the incidence of HPV 16-positive samples was about 10% in all other biopsies ranging from normal mucosa to cases of carcinoma in situ. HPV 16 therefore appears to be involved in late tumor promotion but not in early tumor development.


The diagnostic significance of the polymerase chain reaction and isoelectric focusing in herpes simplex virus encephalitis. In this study, serum and CSF samples of 55 neurological patients have been examined to confirm the diagnosis of herpes simplex virus encephalitis (HSVE). Different methods were applied, including serological titer evaluations, determination of intrathecally-produced HSV-specific antibodies by isoelectric focusing with affinity immunoblotting (IEF), as well as HSV-specific ELISA and HSV-specific polymerase chain reaction (PCR). The results of IEF and PCR have been compared and contrasted to develop general directions for virological diagnosis of HSVE. Of 14 patients suffering from clinically diagnosed HSVE, HSVE was confirmed in 12 cases by the demonstration of PCR or IEF positivity. A HSV-specific CNS infection could be excluded in 2 of these 14 patients. In 17 patients suffering from non-HSVE, PCR and IEF results were negative. Twenty-four patients, suffering from other neurological diseases, serving as a control group, were PCR- and HSV-IEF-negative. The study indicated that there are two possibilities for unequivocal demonstration of HSV-specific CNS involvement: first, performance of PCR especially in the acute phase of disease and in suspicious relapses, and second, performance of HSV-specific IEF for determination of intrathecally synthesized HSV-specific antibodies. It is suggested that these two methods should be introduced in routine diagnosis of viral encephalitis.


Changing seroepidemiological patterns of cytomegalovirus infection in children in Taiwan from 1984 to 1989. The prevalence of cytomegalovirus (CMV) antibody was studied in 966 children in 1984 and 927 children in 1989. The overall prevalence rate of CMV antibody was 59% for children in 1984 and 46% for children in 1989 (P less than 0.05). In both study years, the prevalence rate of CMV antibody was about 70% in infants under 6 months of age, declined to a trough between the ages of 6 and 12 months, and then increased to 40-50% between 1 and 4 years of age. The rate of CMV antibody for children above 4 years in 1984 increased steadily with age and reached 82% by 12 years. In contrast, the prevalence rate in 1989 remained at the level of 40-50% from age 4 to 10 years. It was followed by a sharp increase after 10 years of age and reached 84% at 12 years old. The seropositive rate in each of the 5-, 6-, 8-, 9-, and 10-year-old groups was higher in 1984 than that in 1989. These observations indicated that the prevalence rate of CMV antibody is decreasing in children. This change may be related to various socioeconomic factors, especially the less crowded family conditions in recent years.


Recoverability of Rift Valley fever and sandfly fever Sicilian viruses from infected Phlebotomus papatasi (Diptera: Psychodidae) trapped in various oils. We studied the effects of various oils used to trap sand flies on the recovery of virus from infected adult Phlebotomus papatasi. Both Rift Valley fever and sandfly fever Sicilian viruses were readily recovered from virus-inoculated specimens held at 26 degrees C on mineral, olive or castor oil-soaked sheets for up to 12 h. However, after 50 h on oil-soaked paper, significantly greater titers were recovered from sand flies trapped with mineral oil than from sand flies trapped with either of the other oils. This indicates that sand flies trapped on oil-soaked paper would be suitable for virus isolation attempts and that mineral oil had the least effect on virus recovery.


Neurological risk profile in organic erectile impotence. Thirty men who presented with erectile impotence to the urological department underwent a thorough urological, angiological, and neurological examination with complementary neurophysiological tests of somatosensory and sympathetic and parasympathetic function. Most had vascular and neurological abnormalities. Clinical findings and electrophysiological tests for autonomic dysfunction had the highest yield of abnormal results. Nerve conduction studies and pudendal nerve somatosensory evoked potentials were far less informative. The lack of correlation between vascular and general neurological abnormalities emphasises that patients must be screened for both vascular and neurological dysfunction to prevent unrewarding vascular operation in impotent men.


Levodopa induced ON-OFF motor fluctuations in Parkinson's disease related to rhythmical masticatory jaw movements. The motor disturbance in Parkinson's disease affects all voluntary movement, including innate rhythmical processes such as gait, breathing, and chewing. While there are good descriptions and pathophysiological hypotheses of the changes in gait less is known about the way masticatory movements are affected. By means of a three-dimensional optoelectronic recording technique the differences were investigated in mandibular movement displacement, velocity, and masticatory cycle duration during levodopa induced OFF and ON states. Recordings were made before ordinary morning medication and one hour after medication with 200 mg levodopa and a decarboxylase inhibitor (madopar). There were no differences before and after medication in the opening and closing duration of the masticatory cycle, but a significantly decreased occlusal level phase duration in the ON state was seen. Mandibular velocity and mandibular movement amplitude were significantly greater after medication. Thus the changes in dopamine transmission selectively influenced parts of the masticatory cycle but not the fundamental rhythmical pattern.


Establishment and characterization of a human primitive neuroectodermal tumor cell line from the cerebral hemisphere. The primitive neuroectodermal tumors (PNET) comprise a class of malignant nervous system neoplasms that afflict children. These tumors consist of cells that are morphologically identical to the primitive neuroepithelial cells normally seen in early stages of neural embryogenesis, supporting the notion that PNET result from a disturbance in the process of normal neuronal or glial differentiation. In the central nervous system, PNET occur most commonly in the cerebellum (medulloblastomas), but only occasionally in the cerebral hemispheres. We report here the establishment and characterization of a new human cell line (PFSK) derived from a PNET from the cerebral hemisphere of a child. The growth characteristics of PFSK cells were typical of an immortalized, transformed cell line. Cytogenetic and molecular genetic studies showed that three different sublines were present. In one of these sublines, sequences from chromosome 17 had been lost during establishment in culture. Immunocytochemical studies showed that PFSK cells expressed nestin, an intermediate filament protein normally expressed by neuroepithelial stem cells during neurulation. The PFSK cells did not express antigens typically found in terminally differentiated neurons or glia, indicating that this tumor cell line might represent neuroepithelial stem cells prior to commitment to a neuronal or glial lineage.


Astroglial growth factors in normal human brain and brain tumors: comparison with embryonic brain. Aqueous extracts of 18-day embryonic chicken brains, 15-day embryonic and adult rat brains and human brain tumors, as well as control histologically-normal adult human brain taken from around brain tumors or around arteriovenous malformations each stimulated the growth of cultured chick astrocytes. Eight mitogenic fractions were separated reproducibly by Bio-Gel P-10 molecular seive chromatography. They had apparent molecular weights (M.W.) of 24, 17, 12, 9, 5, 2.8, 1.4 and 1.2 kD. The activity of each fraction was concentration dependent. The fractions did not appear to be artifactually derived by proteolysis from a larger mitogen since (i) protease inhibitors were added at the time of homogenization to prevent degradation, (ii) protease treatment did not produce large quantities of the lower molecular weight fractions, (iii) incubation of brain extracts for up to four hours at 30 degrees C did not alter the activity of the various mitogenic fractions and (iv) addition of albumin to inhibit protease activity similarly did not change the profile of the factors. In contrast, treatment with protease reduced the activity of all the factors although those with M.W. of 5 and 1.2 kD were inactivated more slowly than the others. The various fractions were stable when rechromatographed. This suggested they were not chance aggregates derived artifactually during extraction but rather might have physiological and pathological roles. The activities of each mitogenic fraction were significantly higher in brain extracts from embryonic rats than in those from adult rats. In brain extracts of rat and chicken embryos the fractions of lower M.W. 5 kD to 1.2 kD were relatively abundent. In contrast in brain extracts from adult rats the predominant mitogenic fractions had apparent M.W. of 24, 17 and 12 kD. In histologically normal adult human brain taken from around the tumors or around arteriovenous malformations the 5 kD fraction was present in small amounts and the fractions of lower molecular weight were present in very small amounts. In human glial brain tumors there was a preponderance of the 5 kD activity and more of the 2.8 and 1.4 kD activity fractions than in histologically normal adult human brain. But there was relatively less activity in the 24 and 17 kD fractions. The growth factor profile of human meningiomas was quite different from that of histologically normal human brain or human glial brain tumors. The fraction from meningiomas that was most mitogenic for astrocytes had a molecular weight of 12 kD.(ABSTRACT TRUNCATED AT 400 WORDS)


In-vitro and in-vivo wear profile of composite resins. The aim of this study was to evaluate in-vitro wear by two types of slurry of 10 commercial composite resin materials (seven hybrid and three microfilled composites). There was great variation in the in-vitro wear pattern by two types of slurry. The wear rate of microfilled composites was greater than that of hybrid composites, and a negative correlation was observed between wear rate and Knoop hardness values among all materials when hydroxyapatite slurry was used. In contrast, the wear rate of hybrid composites was greater than that of microfilled composites when abraded by green carborundum slurry. These abraded surfaces were compared with SEM micrographs of in-vivo composites surface after 4 years of service. The profile of in-vivo wear surfaces was found to be similar to that of in-vitro wear surfaces abraded with hydroxyapatite slurry.


Oral signs and symptoms in 160 Greek HIV-infected patients. One hundred and sixty HIV-infected Greek patients were prospectively examined and the oral signs and symptoms were recorded. At the time of oral examination, 76 patients were asymptomatic seropositive, 47 were in ARC stage and 37 had AIDS. One or more oral findings were recorded in 90.6% of the patients while a total of 33 different lesions were observed. The more common oral lesions (highly suspicious) were candidiasis (61%), hairy leukoplakia (24%), periodontitis (19%), necrotizing gingivitis (11%) and Kaposi's sarcoma (12%). In addition some unclassified lesions or symptoms (xerostomia 26%, burning mouth syndrome 19%, patchy depapillated tongue 16%, hairy tongue 10%, exfoliative cheilitis 4%), were common while submandibular and cervical lymph node enlargement were found in 49% of the patients. Interestingly in 16 patients (10%) the suspicion of HIV infection was exclusively based on oral lesions. Our findings show that oral signs and symptoms are common and some times early manifestations of HIV infection and it is in association to those reported in previous studies.


Manipulation of phototransductive membrane turnover by crab photoreceptors in vitro: effects of two protein kinase activators, SC-9 and phorbol ester in the presence of a protein phosphatase inhibitor, okadaic acid. 1. Retinae of crabs, Leptograpsus variegatus, held on a 12:12 h light-dark cycle were prepared for culture in vitro shortly before light-off. After an hour in darkness to permit the assembly of "night" rhabdoms, retinae were exposed to various combinations of drugs: 1 microM okadaic acid (OKA); 60 microM SC-9; 10 microM phorbol, 12,13-diacetate (PDA). 2. The effects of the specific protein phosphatase inhibitor, OKA, are confirmed as light-dependent. Rhabdom sizes were not compromised by OKA, nor by either of the two protein kinase activators, SC-9 or PDA when each was deployed alone in darkness. 3. In combination with OKA, PDA induced demolition of rhabdoms by abnormal macropinocytosis of microvillar membranes. 4. Combined with OKA, SC-9 induced a transient reduction of rhabdoms, followed by overgrowth to abnormal sizes. Overgrowth was blocked by the transcription inhibitor actinomycin D. 5. Disparate consequences of combining OKA with SC-9 or PDA imply that more than one protein kinase C may be involved.


Binding capacities and affinities at mu-, delta- and kappa-opioid sites in membrane suspensions from guinea-pig brain regions. Binding capacities and affinities (KD) of ligands at mu-, delta- and kappa-opioid binding sites were determined by selective labelling techniques together with analysis of saturation curves in seven regions of the guinea-pig brain. The kappa-sites predominated over the other sites in most regions and were 90% of the total in the cerebellum; binding capacities at kappa-sites were highest in the cortex, intermediate in the cerebellum, striatum and mesencephalon and lowest in the diencephalon, hippocampus and pons-medulla. At the mu-sites, binding capacities were highest in the diencephalon and mesencephalon, with intermediate levels in the pons-medulla, cortex and striatum, and low levels in the hippocampus and cerebellum. The highest binding capacity at the delta-sites was in the striatum, intermediate in the cortex, diencephalon and hippocampus, low in the mesencephalon and pons-medulla and not detectable in the cerebellum. No regional differences in binding affinities were found at mu-, delta- and kappa-sites with [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin (KD = 1.10-2,61 nM), [3H]-[D-Ala2,D-Leu5]enkephalin (KD = 0.81-1.94 nM) and [3H]-(-)-bremazocine (KD = 0.083-0.185 nM). Thus in guinea-pig brain there are regional differences in opioid binding capacity and in the distribution of mu-, delta- and kappa-sites, but not in binding affinities.


Site-specific anti-c-erb A antibodies recognizing native thyroid hormone receptors: their use to detect the expression and localization of alpha and beta c-erb A proteins in rat liver. The cell-specific expression and tissue distribution of c-erbA proteins alpha and beta is still unknown. To address this problem, we prepared anti-peptide antibodies directed against epitopes of human (h) c-erbA, specific for the alpha or beta form of thyroid hormone receptors. The cDNAs coding for h c-erbA beta 1, alpha 1 and alpha 2 were transcribed and the mRNAs were translated in vitro in the presence of 35S-methionine, and then their reactivity with the antisera was evaluated. The antiserum anti-beta 62-81 immunoprecipitated only the beta 1 receptor. The antiserum anti-alpha 144-162 determined precipitation of both alpha 1 and alpha 2 proteins but not of the beta 1 receptor. Anti-alpha 2 431-451 produced a selective precipitation of alpha 2, and had no effect on alpha 1 or beta 1 receptor. In order to study the interaction of the antibodies with native T3 receptor we evaluated the binding of antibodies to rat liver T3 receptors by Sephacryl S300 chromatography: both antisera anti-beta 62-81 and anti-alpha 144-162 caused a partial shift of the labeled T3-receptor complex to a higher molecular form, while the antibody directed against c-erbA alpha 2 did not produce any significant shift. The anti-peptide antibodies were then immunopurified by affinity chromatography and used to immunolocalize the different forms of c-erb A proteins in adult and fetal rat liver, by a sensitive immunohistochemical technique. All 3 antibodies stained mainly the nuclei of the majority of adult liver cells. No staining was detectable when the original antiserum was deprived of anti-peptide antibodies by running through the affinity columns or when the antibodies were pre-absorbed with the homologous peptide. No significant staining was present in the liver from rat fetus.


Carbohydrate involvement in sperm-egg interaction in the chicken. The solubilized perivitelline layer (PL) of the chicken ovum contains one or more components which behave in a manner analogous to sperm-receptors. Electrophoretic analyses of solubilized PL confirmed the existence of three major glycoproteins having apparent molecular weights of 33,000, 53,500 and greater than 200,000. The role of carbohydrate in sperm receptor activity was evaluated by extensive deglycosylation of the solubilized PL with trifluoromethanesulfonic acid (TFMS). Sperm receptor activity as measured by an in vitro competition assay was extremely sensitive to TFMS. Pretreatment of spermatozoa with solubilized PL exposed only to TFMS buffers inhibited sperm attachment and digestion of intact PL by 81% (9.67 +/- 1.76 sperm/mm2) as compared with the controls (i.e., no pretreatment of spermatozoa with solubilized PL; 51.33 +/- 6.24 sperm/mm2), a value similar to that observed with spermatozoa exposed to untreated solubilized PL (0.17 +/- 0.17 sperm/mm2). In comparison, PL treated with TFMS inhibited sperm attachment and digestion of intact PL by less than 26% (38.33 +/- 5.88 sperm/mm2) as compared with the control. These data indicate that removal of both N- and O-linked oligosaccharides from components of the chicken PL results in elimination of its sperm receptor activity.


Postnatal short stature, microcephaly, severe syndactyly of hands and feet, dysmorphic face, and mental retardation: a new syndrome? We report on a 2 year old boy with an apparently previously undescribed multiple congenital anomaly/mental retardation syndrome characterised by postnatal short stature, postnatal microcephaly, dysmorphic face, syndactyly 2/5 of the hands and 1/4 of the feet, and brachymesophalangy of fingers 2 and 5.


Small cell lung carcinoma in a patient with Sotos syndrome: are genes at 3p21 involved in both conditions? A 22 year old female with Sotos syndrome and a small cell lung carcinoma is described. This case is of interest not only because of the somatic growth pattern, atypical of Sotos syndrome, but also because of the association with a rare tumour. Of significance is the possible role of mutations at 3p21 in the aetiology of Sotos syndrome and tumour development.


The quantification of fluorescent emission from biological samples using analysis of polarization. The quantification of fluorescent emission from biological specimens can only be carried out in cellular regions where the relationship between fluorophore concentration and fluorescent emission is linear. Using a confocal scanning laser microscope, we show that quantification of fluorescent emission from biological samples labelled with fluorescein and fluorescein analogues mounted in a viscous medium can be readily achieved. Where the distribution of fluorophore is highly localized, for example in cells labelled for immunofluorescence analysis, we demonstrate that analysis of fluorescence depolarization can identify regions in which fluorophore concentration exceeds the range in which the relationship to fluorescent emission is linear. We also demonstrate that, under the conditions examined, depth-dependent effects, fading and quenching are either small enough to be ignored or can be corrected for mathematically when quantifying fluorescent emission.


Control of segregation of chromosomal DNA by sex factor F in Escherichia coli. Mutants of DNA gyrase subunit A suppress letD (ccdB) product growth inhibition. The letA (ccdA) and letD (ccdB) genes, located just outside the sequence essential for replication of the F plasmid, apparently contribute to stable maintenance of the plasmid. The letD gene product acts to inhibit partitioning of chromosomal DNA and cell division of the host bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product. To identify the target of the letD gene product, temperature-sensitive growth-defective mutants were screened from bacterial mutants that had escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant. Of nine mutants analysed, three mutants were shown, by phage P1-mediated transduction and complementation analysis, to have mutations in the gyrA gene and the other six in the groE genes. The nucleotide sequence revealed that one of the gyrA mutants has a base change from G to A at position 641 (resulting in an amino acid change from Gly to Glu at position 214) of the gyrA gene. The mutant GyrA proteins produced by these gyrA(ts) mutants were trans-dominant over wild-type GyrA protein for letD tolerance. The wild-type GyrA protein, produced in excess amounts by means of a multicopy plasmid, overcame growth inhibition of the letD gene product. These observations strongly suggest that the A subunit of DNA gyrase is the target of the LetD protein.


Unblock of the slow inward current induces the arrhythmogenic transient inward current in isolated guinea-pig myocytes. This study investigated whether abrupt changes in extracellular Ca2+ concentration or washout of the Ca2+ antagonists Mn2+ or verapamil, could induce transient inward current (ITI) in enzymatically disaggregated guinea-pig myocytes. Single electrode voltage-clamp techniques were used. ITI was elicited upon repolarization to various voltage steps from an activating step to +20 mV. The holding potential was -80 mV. Slow inward current (ICa) was induced by steps to -10 mV. Continuous exposure to either 2.5 or 6.0 mM Ca2+ did not induce ITI; however, following exposure of cells to 0.5 mM Ca2+ for 20 min which decreased ICa, return to 2.5 or 6.0 mM Ca2+ induced ITI. ITI could be observed for 10 to 20 min following sudden elevations of Ca2+. Similar effects also were seen when Ca2+ was increased from 2.5 to 6.0 mM. Exposure to 2.0 mM Mn2+ or 2.0 microM verapamil blocked ICa. Washout of either blocker induced ITI, particularly in 6.0 mM Ca2+. Peak ITI occurred upon repolarization at c. -70 mV; a reversal potential could not be demonstrated. Thus, abrupt changes in Ca2+ influx, produced either by sudden changes in external Ca2+ or by washout of Ca2+ antagonists, induced ITI with characteristics similar to those described for ITI induced by toxic concentrations of cardiac glycosides.


The need for clinical awareness of polymorphous low-grade adenocarcinoma: a review. Adenocarcinomas of the salivary glands are a heterogeneous group both in terms of histomorphology and clinical course. One recently described adenocarcinoma, polymorphous low-grade adenocarcinoma (solely of minor salivary gland occurrence), is appropriately named. This neoplasm has a wide spectrum of histology and can resemble tumors such as pleomorphic adenoma and adenoid cystic carcinoma; both aspects can lead to diagnostic problems for the pathologist. As well, despite an infiltrative growth pattern, which frequently results in perineural invasion, this tumor has a low local recurrence rate and a remarkable infrequency of local or distant metastasis. In order to provide appropriate therapy for polymorphous low-grade adenocarcinoma, oral and head and neck surgeons must be familiar with this neoplasm.


Organ- and age-specific replication of polyomavirus in mice. A novel organ- and age-specific pattern of polyomavirus DNA replication in mice is described. Two broadly defined classes of response to polyomavirus infection were observed: class I organs (mammary gland, bone, and skin) responded with high levels of replication in neonate mice and moderate levels in adults; class II organs (kidney, liver, and lung) responded with high levels in neonates and very low levels in adults. Thus, aging affected replication in all organs, and organ specificity was superimposed on this age-related decrease. We argue that the organ- and age-specific pattern likely reflects in part the activities of a multiplicity of general or tissue-specific, age-dependent transcription factors, which modulate viral replication or viral transcription or both. Interestingly, the majority of tumors in mice infected as neonates or as immunoincompetent adults originate in class I organs, suggesting that the ability to replicate in adult tissues is an important factor controlling polyomavirus oncogenesis. From the analysis of the infection process in adult mammary glands, a novel mode of polyomavirus infection emerged which contrasts with that derived from observations of tissue culture systems. A nonproductive infection was seen, characterized by very low levels of live virus (in the range of 10(-4) PFU per cell) and maintenance of the viral genome in an unintegrated, moderately replicating state. Maintenance of the viral genome was accomplished without integration into host cell DNA in all three tumor-prone organs, both prior to as well as beyond oncogenesis.


Enhancer dependence of polyomavirus persistence in mouse kidneys. We previously showed that alterations in the enhancer sequence of polyomavirus DNA can alter both the level and the organ specificity of viral DNA replication during the acute phase of infection of newborn mice (R. Rochford, B. A. Campbell, and L. P. Villarreal, J. Virol. 64:476-485, 1990). In this study, we examined whether these enhancer sequence alterations can also affect polyomavirus replication during the persistent phase of infection in vivo. After infection of newborn mice with a mixture of three enhancer variants, the individual organs could select for enhancer-specific viral DNA replication during both the acute and the persistent phases of infection. Contrary to expectations, the ability of some variants to establish a high-level acute infection in some organs (e.g., the pancreas) did not necessarily lead to a persistent infection in those organs. Thus, enhancers can affect acute and persistent infections differently. In addition, some enhancer variants tended to establish a high-level persistent infection in the kidneys immediately following an acute infection; however, in all cases considerable histopathology was associated with these elevated long-term infections, and these mice were always runty. A persistent infection in the kidneys thus appears able to exist in two distinguishable states, a high-level pathological state and a low-level nonpathological state, which can be affected by the viral enhancer sequence.


Murine retrovirus-induced spongiform encephalomyelopathy: host and viral factors which determine the length of the incubation period. A molecular clone of wild mouse ecotropic retrovirus CasBrE (clone 15-1) causes a spongiform neurodegenerative disease with a long incubation period, greater than or equal to 6 months. This virus infects the central nervous system (CNS) at low levels. In contrast, a chimeric virus, FrCasE, containing env and 3' pol sequences of 15-1 in a Friend murine leukemia virus background, infects the CNS at high levels and causes a rapid neurodegenerative disease with an incubation period of only 16 days. With both viruses, the induction of neurologic disease is dependent on inoculation during the perinatal period. Since the length of the incubation period of this disease appears to be a function of the relative level of CNS infection, we have attempted to identify the viral and host factors which determine the relative level of virus infection of the CNS. It was previously shown that the CNS is susceptible to infection only during the perinatal period (M. Czub, S. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 65:2539-2544, 1991). Here we have found that the susceptibility of the CNS wanes progressively or gradually as a function of the age of the host, this age-dependent resistance being complete by 12 to 14 days of age. Utilizing a group of chimeric viruses, we found that the relative level of CNS infection achieved after inoculation of mice at 1 day of age was a function of the kinetics of virus replication and spread in peripheral organs. Viruses which reached peak viremia titers early (5 to 7 days of age) infected the CNS at high levels, and viruses which reached peak titers later infected the CNS at lower levels. Among the group of viruses examined in the current study, the kinetics of peripheral virus replication and spread appeared to be influenced primarily by sequences within the R-U5-5' leader region of the viral genome. These results suggested that the relative level of CNS infection was determined very early in life and appeared to be a function of a dynamic balance between the kinetics of virus replication in the periphery and a progressively developing restriction of virus replication in the CNS.


Determinants of substrate recognition by poliovirus 2A proteinase. Poliovirus proteinase 2A (2Apro) is autocatalytically released from the viral polyprotein by cleavage in cis of a Tyr-Gly dipeptide at its own amino terminus, resulting in separation of the P1 structural and P2-P3 nonstructural protein precursors. A second Ty-Gly dipeptide within 3D polymerase is cleaved by 2Apro in trans, but this is not essential for viral proliferation. The mechanism which limits cleavage to only 2 of the 10 Tyr-Gly dipeptides within the poliovirus polyprotein has not been characterized. We have therefore undertaken a systematic mutational analysis of the VP1-2A site to elucidate determinants of substrate recognition by 2Apro. The P2 and P1' positions are important determinants for cis cleavage of this site, whereas a variety of substituents could be tolerated at the P2', P1, and P3 positions. The requirements for trans cleavage of this site were more stringent. We found that the 2Apro of coxsackievirus type A21 and rhinoviruses 2 and 14 have stringent requirements similar to those of poliovirus 2Apro for cleavage in trans.


Simian virus 40 T antigen activates the late promoter by modulating the activity of negative regulatory elements. Late promoter activity measured before viral DNA replication results from a complex involvement of negative and positive cis-acting elements located both in the enhancer and in the 21-bp repeats. GC motifs located within the 21-bp repeats act in cooperation with sequences overlapping the early TATA box to down-regulate the late promoter activity. Analysis of insertion mutants indicates that the late promoter might be negatively regulated at least partially by the early promoter machinery. The GTI motif located within the enhancer as well as the GC motifs lose the ability to down-regulate the late promoter in the presence of T antigen. Results obtained with tsA58 protein indicate that two different domains of T antigen are involved in the negative autoregulation of the early promoter activity and in the release of the down-regulation of the late promoter by the GC motifs.


Binding of EBNA-1 to DNA creates a protease-resistant domain that encompasses the DNA recognition and dimerization functions. The Epstein-Barr virus nuclear antigen EBNA-1 is essential for replication of the viral DNA during latency. EBNA-1 binds as a dimer to palindromic recognition sequences within the plasmid origin of replication, ori-P. In this study, proteinase K susceptibility has been used to further characterize the DNA-binding domain of EBNA-1. Limited protease digestion of EBNA-1 (amino acids 408 to 641) generated a smaller DNA-binding species that had a degree of inherent protease resistance. When EBNA-1 was preincubated with a specific DNA probe, the protease resistance of the smaller binding species increased 100-fold, suggesting that the conformation of EBNA-1 changes on binding. The protease-resistant species comprised an 18-kDa polypeptide that was further cleaved at high levels of protease to 11- and 5.4-kDa products. A model of the proposed protease-resistant domain structure is presented. Constructions carrying serial, internal deletions across the 18-kDa domain were created. Each of the deletions perturbed dimerization ability and abolished DNA binding. These studies suggest that the DNA-binding and dimerization motifs of EBNA-1 lie within a conformationally discrete domain whose overall integrity is necessary for EBNA-1-DNA interaction.


Interaction of rotavirus particles with liposomes. We have studied the interactions of purified viral particles with liposomes as a model to understand the mechanism of entry of rotavirus into the cell. Liposomes, made from pure as well as mixed lipids, that contained encapsulated self-quenching concentrations of the fluorophore carboxyfluorescein (CF) were used. Rotavirus-liposome interactions were studied from the fluorescence dequenching of CF resulting from its release to the bulk solution. Purified infectious double-shelled virus particles induced a concentration- and temperature-dependent release of CF. The rate and extent of CF release was maximum between pH 7.3 and 7.6. The removal of outer structural proteins VP4 and VP7 from virus, which results in the formation of single-shelled particles, prevented virus interaction with liposomes. Rotavirus particles with uncleaved VP4 did not interact with liposomes, but treatment in situ of these particles with trypsin restored the interaction with the liposomes and resulted in CF dequenching. Our data support the view that rotavirus enters the cell through direct penetration of the plasma membrane. In contrast, adenovirus, the only other nonenveloped virus studied by this method, shows the optimum rate of marker release from liposomes at around pH 6 (R. Blumenthal, P. S. Seth, M. C. Willingham, and I. Pastan, Biochemistry 25:2231-2237, 1986). The interaction between rotavirus and liposomes is sensitive to specific divalent metal ions, unlike the adenovirus-liposome interaction, which is independent of them.


Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication. We have localized a cis-acting sequence that promotes initiation of lytic-phase DNA replication (oriLyt) within the HindIII D fragment of the human cytomegalovirus (HCMV) AD169 genome and investigated its sequence requirements by testing the ability of plasmid constructs to mediate DNA replication in a transient transfection-plus-infection assay. Replication of plasmids containing HCMV oriLyt required at least the virus-specified DNA polymerase activity supplied by HCMV infection of transfected cells and was autonomous in that it did not result from recombination with the virus genome. Progeny molecules in the transient assay were high-molecular-weight tandem oligomers, which is consistent with predictions of a rolling-circle model. Experiments testing subclones of HindIII-D defined a core 2.4-kbp region containing elements required for oriLyt function that extended rightward from around 1.0 kbp upstream of UL57 near the middle of the long unique component of the virus genome. Sequences flanking this core also were needed for full activity. The defined region contains at least four clustered sets of repeated sequence elements identical to or candidate counterparts of elements present in the corresponding cytomegalovirus Colburn lytic-phase replication origin. These elements are novel in that they apparently do not correspond to previously characterized motifs. Also present are multiple copies of elements similar to known binding sites for the transcription factors ATF/CREB, MLTF/USF, and Sp1. Preliminary deletion analysis suggests that multiple components within the boundaries of oriLyt cooperate to enable initiation of HCMV lytic-phase DNA synthesis.


Efficacy of inactivated whole-virus and subunit vaccines in preventing infection and disease caused by equine infectious anemia virus. We report here on a series of vaccine trials to evaluate the effectiveness of an inactivated equine infectious anemia virus (EIAV) whole-virus vaccine and of a subunit vaccine enriched in EIAV envelope glycoproteins. The inactivated vaccine protected 14 of 15 immunized ponies from infection after challenge with at least 10(5) 50% tissue culture-infective doses of the homologous prototype strain of EIAV. In contrast, it failed to prevent infection in any of 15 immunized ponies that were challenged with the heterologous PV strain. Levels of PV virus replication and the development of disease, however, were significantly reduced in 12 of the 15 ponies so challenged. The subunit vaccine prevented infection from homologous challenge in four of four ponies tested but failed to prevent infection in all four challenged with the PV strain. Two of the four subunit vaccinates had more severe symptoms of equine infectious anemia than nonimmunized ponies infected in parallel. Both vaccines stimulated EIAV-specific cell-mediated immunity. The in vitro lymphoproliferative response was shown to be mediated by T lymphocytes and appeared to be indistinguishable from that induced by EIAV infection. Significant differences were observed in the in vivo lymphocyte responses following challenge with the two virus strains. While peripheral blood mononuclear cells from the inactivated virus vaccinates were equally stimulated by both the prototype and PV strains, the subunit vaccinates challenged with PV exhibited lower levels of spontaneous proliferation and serine esterase activity. This diminished cellular response to PV was correlated with more severe clinical disease in the same ponies. These studies demonstrate for the first time that both an EIAV inactivated whole-virus vaccine and a viral envelope glycoprotein-based subunit vaccine can provide protection against rigorous challenge levels of homologous virus but are unable to protect against similar challenge levels of a heterologous virus. Moreover, the data demonstrate that protection can be achieved in the absence of detectable levels of virus-specific neutralizing antibody in the vaccine recipients at the time of virus challenge. While vaccine-induced virus-specific cell-mediated immune responses were detected, their role in conferring protection was not obvious. Nevertheless, protection from disease appeared to be correlated with the induction of high levels of serine esterase activity following challenge. A significant observation is that while the whole-virus vaccine was usually capable of preventing or markedly moderating disease in the PV-infected ponies, the subunit vaccine appeared to have a high potential to enhance the disease induced by PV infection.(ABSTRACT TRUNCATED AT 400 WORDS)


Epstein-Barr virus enters B cells and epithelial cells by different routes. Epstein-Barr virus (EBV) infects two cell types, B lymphocytes and epithelial cells. Electron microscopic studies have shown that the virus fuses with the lymphoblastoid cell line Raji but is endocytosed into thin-walled non-clathrin-coated vesicles in normal B cells before fusion takes place. To compare early interactions of EBV with epithelial cells and B cells, a fluorescence dequenching assay of fusion was employed, using virus labeled either with the pH-insensitive probe octadecyl rhodamine B chloride (R18) or with 5(N-octadecanoyl) aminofluorescein (AF), which loses emission intensity at a pH below 7.4. Fusion of virus labeled with R18 could be monitored with B cells, Raji cells, and epithelial cells. Lowering the extracellular pH or pretreatment of cells with ammonium chloride or methylamine had no effect on these measurements. In contrast, fusion of virus labeled with AF could be measured with Raji cells and epithelial cells, but not with normal B cells unless cells were previously treated with ammonium chloride. Fusion of virus with normal B cells was inhibited with chlorpromazine, chloroquine, and sodium azide, but none of these reagents had any effect on fusion with Raji or epithelial cells. These results suggest that entry of EBV into nonpolarized suspensions of epithelial cells occurs by fusion at the cell surface, that EBV may be incapable of fusing with normal B cells unless it has first been endocytosed, and that pH appears to be irrelevant to either event. A combination of the two probes, R18 and AF, may have general use for determining the sites of entry of enveloped viruses that fuse in a pH-independent manner.


Two amino acid substitutions within the capsid are coordinately required for acquisition of fibrotropism by the lymphotropic strain of minute virus of mice. Nucleotide changes at both codons 317 and 321 in the VP2 capsid gene of the immunosuppressive strain of the murine parvovirus minute virus of mice, MVM(i), are required to create a virus capable of growing in A9 fibroblasts. This double mutant virus, ILB1, has growth characteristics very similar to those of the prototype fibrotropic strain MVM(p) in both single- and multiple-round infections of fibroblasts and is about 100-fold better at infecting fibroblasts than MVM(i). When only one nucleotide position is changed, either in codon 317 (as in ILB2) or in codon 321 (as in ILB3), the resulting viruses are less than twice as efficient as their parent MVM(i) at infecting fibroblasts. In the restrictive infection of A9 cells by the single mutants and MVM(i), gene expression and DNA replication were markedly reduced compared with ILB1 infection of the same cells or compared with infections of permissive hybrid cells by each of the viruses. This suggests that restriction acts predominantly at an early step in the infection. Since the phenotypes of ILB2 and ILB3 are essentially indistinguishable in restrictive infections, it is most likely that the individual loci affect the same step in the viral life cycle. The dramatic increase in fibroblast infectivity shown by ILB1 indicates a synergistic interaction between these two amino acid residues in the same rate-limiting process in fibroblast infection.


Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants. Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.


Characterization of a herpes simplex virus sequence which binds a cellular protein as either a single-stranded or double-stranded DNA or RNA. Earlier we reported that herpes simplex virus 1 DNA contains a sequence which binds a host protein in a sequence-specific manner as either a single-stranded or a double-stranded DNA or RNA and that this sequence is located in a transcriptional unit whose RNA traverses the origin of viral DNA replication (OriSRNA) (R.J. Roller, L. McCormick, and B. Roizman, Proc. Natl. Acad. Sci. USA 86:6518-6522, 1989). The protein reacts with both DNA and RNA in band-shift assays and protects the single-stranded RNA sequence from digestion by RNase. We report that the minimal cognate sequence required for these interactions consisted of [N(GTGGGTGGG)2(N less than or equal to 10)]. The ninemer repeat sequence was located at nucleotides -1 to -18 relative to the transcription initiation of the major species of OriSRNA. The activity of the cognate sequence required at least three guanines between thymines and tolerates the insertion of additional thymines, but it was inactivated by the insertion of adenines or by the substitution of some of the guanines with cytosines in one repeat. Replacement of the 10 3' nucleotides has no effect on binding activity, whereas deletion of these sequences abolished it. Among the related sequences with no demonstrable binding activity were some telomeric sequences which interact with known cognate proteins. The electrophoretic mobility of the herpes simplex virus cognate sequences in nondenaturing gels suggests that they may be able to form higher-order structures, but the conditions under which they were formed were different from the optimal conditions for binding the protein. UV light cross-linking studies of labeled RNA-protein complexes following digestion with RNases indicated that the electrophoretic mobility of the protective activity corresponded to that of a protein with an apparent molecular weight of 100,000.


Reticuloendotheliosis type C and primate type D oncoretroviruses are members of the same receptor interference group. The reticuloendotheliosis viruses (REVs), originally isolated from avian species, constitute a group of retroviruses which are more closely related to mammalian retroviruses than to other avian retroviruses. The envelope glycoproteins of members of the REV group display a striking amino acid sequence identity with a group of primate oncoretroviruses which belong to a single receptor interference group and include all of the type D and some type C primate oncoretroviruses. Members of the REV group also have a broad host range which covers most avian cells and some mammalian cells, including those of simian and human origin. In view of this broad host range and the envelope sequence similarities, we investigated the cross-interference pattern between REV and primate virus groups to determine whether they utilized the same receptor. Superinfection experiments using a vector virus containing an Escherichia coli lacZ gene showed that reticuloendotheliosis and simian oncoretroviruses constitute a single receptor interference group on both human and canine cells and indicate that the viruses bind to the same receptor to initiate infection. These results suggest that this receptor binding specificity has been maintained over a wide range of retroviruses and may be responsible for the broad spread of these retroviruses between different orders of vertebrates.


Equine infectious anemia virus gene expression: characterization of the RNA splicing pattern and the protein products encoded by open reading frames S1 and S2. The utilization of predicted splice donor and acceptor sites in generating equine infectious anemia virus (EIAV) transcripts in fetal donkey dermal cells (FDD) was examined. A single splice donor site identified immediately upstream of the gag coding region joins the viral leader sequence to all downstream exons of spliced EIAV transcripts. The predominant 3.5-kb transcript synthesized in EIAV-infected FDD cells appears to be generated by a single splicing event which links the leader sequence to the first of two functional splice acceptor sites near the 5' end of the S1 open reading frame (ORF). The translation products encoded by the 3.5-kb transcript were examined by producing in vitro transcripts from a cDNA corresponding to this RNA followed by in vitro translation in wheat germ extracts. These transcripts directed the synthesis of three proteins: the virus trans-activator protein (EIAV Tat) encoded by ORF S1, a protein of unknown function encoded by ORF S2, and the virus envelope glycoprotein. When transfected into FDD cells, this cDNA also directed expression of EIAV Tat. Amino-terminal sequence analysis of the in vitro-synthesized S1 protein supports the suggestion that translation of EIAV Tat is initiated at a CUG codon within the virus leader region. Both in vitro-synthesized S2 protein and synthetic peptides corresponding to S2 are shown to react positively with sera obtained from EIAV-infected horses, providing the first direct evidence of expression of this protein in infected animals.


Mutations within the env gene of Mason-Pfizer monkey virus: effects on protein transport and SU-TM association. By deletion mutagenesis analyses, we have examined the contribution of the immunosuppressive peptide (ISP) region within the transmembrane (TM) protein of Mason-Pfizer monkey virus to viral maturation and infectivity. Deletion of the entire region (mutant D105) results in the production of an Env precursor that is transport defective and therefore unable to be processed to mature glycoproteins. This mutation results in the release of noninfectious virions devoid of surface glycoproteins. A second deletion that removes the most highly conserved 11 amino acids of the ISP (mutant D33) does not affect the production, transport, or processing of the Env precursor yet produces virions that are noninfectious. The mutation was shown to cause the loss of interaction between the surface (SU) and TM proteins and result in the efficient shedding of gp70 into the culture medium. The released gp70 protein was biologically active and could still bind with high specificity to susceptible target cells. Since the ISP domain may represent an area of contact between SU and TM, it could provide an additional explanation for the amino acid sequence homology observed within this region of a variety of retroviruses.


In vitro transactivation of baculovirus early genes by nuclear extracts from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells. Nuclear extracts, prepared from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells during a time course of infection, were analyzed for activation of early gene transcription and for late gene transcription. The templates used in the in vitro transcription assays contained promoters for baculovirus genes that have been classified as immediate early, delayed early, and late. The promoters were derived from the baculovirus 39K, p26, gp64, and DNA polymerase genes. In addition, the adenovirus major late promoter was included in these studies. We found that transcription from promoters classified as immediate early or delayed early was accurately initiated by using extracts from uninfected cells. Furthermore, transcription from all early promoters tested was found to be transactivated by nuclear extracts prepared at 4 and 8 h postinfection. However, baculovirus enhancer-dependent transcriptional activation was not observed in tests with templates containing the hr5 enhancer sequence. Transcription from baculovirus late promoters was also not observed. A decline in transcription by nuclear extracts prepared from cells late in infection was associated with the presence of DNase activity.


Late promoter of human papillomavirus type 8 and its regulation. Human papillomavirus type 8 (HPV8) belongs to the HPV types associated with skin carcinomas of patients with epidermodysplasia verruciformis (EV). Its noncoding regulatory sequences (NCR) were shown to drive the expression of the reporter gene chloramphenicol acetyltransferase (cat) in transient assays with human epithelial cells (HT3 cells). This constitutive activity could be enhanced by coexpression of the HPV8 transactivator protein E2. The analysis of 5' deletions of the NCR showed that the EV-specific sequence motif M33 and the neighboring AP1 site are essential for the promoter activity, whereas 44 nucleotides located immediately upstream of M33 are strongly inhibitory. The same effects were observed in simian virus 40-immortalized fetal keratinocytes (SV61 cells) and spontaneously immortalized skin keratinocytes (HaCaT cells). By using primer extension and RNase protection analyses two promoters could be identified within the HPV8 NCR. A nested set of weak signals, corresponding to start sites between positions 175 to 179, represented the previously described E6 promoter. The vast majority of transcripts was initiated at position 7535 and shown to undergo processing at an NCR-internal splice donor (positions 1 to 8). The promoter P7535 is similar to late promoters of other skin-associated papillomaviruses as far as localization, transcript structure, and sequence characteristics are concerned. To confirm that P7535-initiated transcripts proceed indeed to the L1 gene for the major capsid protein, viral mRNAs from an HPV8-induced lesion of a patient with EV were characterized by RNase protection and sequence analysis of polymerase chain reaction-amplified cDNAs. The NCR leader (positions 7535 to 4) appeared in two messages with three exons each. The third exon started with the second ATG codon of L1 in both cases; the short central exons from the 3' part of the early coding region were defined by a common splice acceptor site (position 3303) and different splice donor sites (positions 3443 and 3704).


Identification and characterization of a coronavirus packaging signal. Previously, a mouse hepatitis virus (MHV) genomic sequence necessary for defective interfering (DI) RNA packaging into MHV particles (packaging signal) was mapped to within a region of 1,480 nucleotides in the MHV polymerase gene by comparison of two DI RNAs. One of these, DIssF, is 3.6 kb in size and exhibits efficient packaging, whereas the other, DIssE, which is 2.3 kb, does not. For more precise mapping, a series of mutant DIssF RNAs with deletions within this 1,480-nucleotide region were constructed. After transfection of in vitro-synthesized mutant DI RNA in MHV-infected cells, the virus product was passaged several times. The efficiency of DI RNA packaging into MHV virions was then estimated by viral homologous interference activity and by analysis of intracellular virus-specific RNAs and virion RNA. The results indicated that an area of 190 nucleotides was necessary for packaging. A computer-generated secondary structural analysis of the A59 and JHM strains of MHV demonstrated that within this 190-nucleotide region a stable stem-loop of 69 nucleotides was common between the two viruses. A DIssE-derived DI DNA which had these 69 nucleotides inserted into the DIssE sequence demonstrated efficient DI RNA packaging. Site-directed mutagenic analysis showed that of these 69 nucleotides, the minimum sequence of the packaging signal was 61 nucleotides and that destruction of the secondary structure abolished packaging ability. These studies demonstrated that an MHV packaging signal was present within the 61 nucleotides, which are located on MHV genomic RNA 1,381 to 1,441 nucleotides upstream of the 3' end of gene 1.


Pathogenesis of feline leukemia virus T17: contrasting fates of helper, v-myc, and v-tcr proviruses in secondary tumors. A naturally occurring feline thymic lymphosarcoma (T17) provided the unique observation of a T-cell antigen receptor beta-chain gene (v-tcr) transduced by a retrovirus. The primary tumor contained three classes of feline leukemia virus (FeLV) provirus, which have now been characterized in more detail as (i) v-tcr-containing recombinant proviruses, (ii) v-myc-containing recombinant proviruses, and (iii) apparently full-length helper FeLV proviruses. The two transductions appear to have been independent events, with distinct recombinational junctions and no sequence overlap in the host-derived inserts. The T17 tumor cell line releases large numbers of FeLV particles of low infectivity; all three genomes are encapsidated, but passage of FeLV-T17 on feline fibroblast and lymphoma cells led to selective loss of the recombinant viruses. The oncogenic potential of the T17 virus complex was, therefore, tested by infection of neonatal cats with virus harvested directly from the primary T17 tumor cell line. A single inoculation of FeLV-T17 caused persistent low-grade infection culminating in thymic lymphosarcoma and acute thymic atrophy, which was accelerated by coinfection with the weakly pathogenic FeLV subgroup A (FeLV-A)/Glasgow-1 helper. Molecularly cloned FeLV-tcr virus (T-31) rescued for replication by a weakly pathogenic FeLV-A/Glasgow-1 helper virus was similarly tested in vivo and induced thymic atrophy and thymic lymphosarcomas. Most FeLV-T17-induced tumors manifested either v-myc or an activated c-myc allele and had undergone rearrangement of endogenous T-cell antigen receptor beta-chain genes, supporting the proposition that the oncogenic effects of c-myc linked to the FeLV long terminal repeat are targeted to a specific window in T-cell differentiation. However, neither the FeLV-T17-induced tumors nor the T-31 + FeLV-A-induced tumors contained clonally represented v-tcr sequences. Only one of the FeLV-T17-induced tumors contained detectable v-tcr proviruses, at a low copy number. While v-tcr does not have a readily transmissible oncogenic function, a more restricted role is not excluded, perhaps involving antigenic peptide-major histocompatibility complex recognition by the T-cell receptor complex. Such a function could be obscured by the genetic diversity of the outbred domestic cat host.