Construction of a viable BHV1 mutant lacking most of the short unique region

The 8.2 kb HindIII K-fragment of bovine herpesvirus 1 (BHV1) lies entirely within the short unique region of the genome and contains all or parts of 7 coding regions. We have probed this fragment for the presence of nonessential genes by a simple, rapid method of insertional mutagenesis. Our analysis indicates that all the genes present in the K-fragment, except the glycoprotein D gene, are nonessential for replication of BHV1 in tissue culture. After inserting a copy of the glycoprotein D gene into the thymidine kinase locus of BHV1 it was possible to delete the entire HindIII K-fragment and the contiguous 0.36 kb O-fragment in one step. The deletion mutant, which contains 7 kb less BHV1 DNA than wt virus and lacks ORF1, US2, the genes for protein kinase, glycoprotein G, glycoprotein I, and most of glycoprotein E was still replication-competent.     

Effect of frequency ratio on infants' and adults' discrimination of simultaneous intervals

Effects of frequency ratio simplicity on infants' and adults' processing of simultaneous pitch intervals with component sine wave tones were tested. Both infants and adults showed superior performance at detecting a change from a perfect 5th (2:3) to either a tritone (32:45) or a minor 6th (5:8) interval than at detecting the reverse discriminations (minor 6th or tritone to perfect 5th). Similarly, both infants and adults showed superior performance at detecting a change from an octave (1:2) to either a major 7th (8:15) or a minor 9th (15:32) interval than at detecting the reverse discriminations. In combination with previous findings of infants' superior discrimination of tone sequences with prominent perfect 5th intervals, these results suggest that both simultaneous and sequential intervals with simple ratios are easy to process early in development.     

Parvovirus B19 quiescence during the course of human immunodeficiency virus infection in persons with hemophilia

One hundred and seventy patients with major lower limb amputation (MLLA) presenting to The National Prosthetic-Orthotic Centre (NPOC) in Khartoum over a 1-year period were studied. There were 141 males and 29 females giving a M:F ratio of 4.9: 1.0, with mean age of 37 years (range 5-72 years). Forty-one patients (24%) underwent amputation of diabetic septic foot, 30 patients (17.6%) underwent amputation as a result of trauma from road traffic accidents and Madura foot, and war injuries accounted for 29 amputations (17%). One hundred and eleven patients had below knee amputation (BKA), 52 had above knee amputation (AKA) and seven patients had Syme's amputation. Diabetic amputees had higher rate of revisional surgery compared with others because of sepsis and/or flap necrosis. Stump pain was reported by amputees with excessive scarring of the stump and those with undue prominence of bony ends. There are two types of prostheses provided by the NPOC for both BKA and AKA: the peg leg and the conventional prostheses. The Syme's amputees were fitted with either simple hoof or articulated prostheses with solid ankle cushion heel (SACH). The peg leg consists of a leather lined side bearing metal socket connected to a rocker base by side steels. It is used by the country natives as it suits different weather and job conditions, particularly farming, and it can be repaired locally. The urban population use the conventional prostheses which is lighter in weight, can be put on and taken off easily and is cosmetically acceptable. However, these prostheses are more expensive and require frequent repair or replacement. The functional outcome of patient's rehabilitation with the prostheses was significantly affected by the level and indication of amputation. Those with BKA and those amputated because of trauma or Madura foot experienced better functional outcome compared with the diabetics, independent of age. 50% of patients with the AKA and 19% of those with BKA reported poor functional outcome. Surgeons should be more involved with the long-term evaluation of functional outcome in such patients, to offer help if feasible and to modify their technique for future procedures.     

Dissociation between the effects of somatostatin (SS) and octapeptide SS-analogs on hormone release in a small subgroup of pituitary- and islet cell tumors

The effects of somatostatin (SS-14 and/or SS-28) and of the three octapeptide SS-analogs that are available for clinical use (octreotide, BIM-23014 and RC-160) on hormone release by primary cultures of 15 clinically nonfunctioning pituitary adenomas (NFA), 7 prolactinomas, and 2 insulinomas were investigated. In the pituitary adenoma cultures, a comparison was made with the effects of the dopamine (DA) agonists bromocriptine and/or quinagolide. In 5 NFAs, 2 prolactinomas and 1 insulinoma somatostatin receptor (subtype) expression was determined by ligand binding studies and by in situ hybridization to detect sst1, sst2, and sst3 messenger RNAs (mRNAs). Four NFA cultures did not secrete detectable amounts of alpha-subunit, FSH, and/or LH. In the other cultures, hormone and/or subunit release was inhibited by DA-agonists (10 nM) in 9 of 11, by SS (10 nM) in 7 of 11, and by octapeptide SS-analogs (10 nM) in 3 of 10 cultures. In three NFA cultures, hormone release was sensitive to SS but not to SS-analogs. In all cultures, except for one, DA-agonists were the most effective in inhibiting hormone release. In the prolactinoma cultures, PRL release was inhibited by DA-agonists (10 nM) in 7 of 7, by SS in 4 of 4, and by octapeptide SS-analogs in 3 of 7 cultures. A dissociation between the effects of SS and SS-analogs was found in 3 cases. In the cultures sensitive to both bromocriptine and SS-28, bromocriptine was the most potent compound in 2 out of 4 cultures. In the 2 other cultures, both compounds were equally effective. In 2 insulinoma cultures, insulin release was inhibited by SS, and by octapeptide SS-analogs in only one. The presence or absence of an inhibitory effect by octreotide was in all cases in parallel with the presence or absence of the inhibitory effect by BIM-23014 and RC-160. Autoradiographic studies using [125I-Tyr0]SS28 showed specific binding in 4 of 5 NFAs, 1 of 2 prolactinomas, and 1 of 1 insulinoma. Specific [125I-Tyr3]octreotide binding was found in 2 of 5 NFAs, in 1 of 2 prolactinomas, and in the insulinoma. Two NFAs showed binding of SS28, but not of the sst2.5 specific ligand octreotide. The tumors showed variable sst1 and/or sst3 mRNA expression, whereas no sst2 expression was found. In conclusion, a dissociation between the inhibitory effects of SS on the one hand and of the octapeptide SS-analogs octreotide, BIM-23014 and RC-160 on the other hand, is observed in a small subgroup of NFAs, prolactinomas, and insulinomas, suggesting that novel sst subtype specific SS-analogs might be of benefit in the treatment of selected patients with somatostatin receptor positive secreting tumors not responding to octapeptide SS-analogs. However, in the majority of NFAs and prolactinomas, DA-agonists were equally or more effective than SS in the suppression of tumoral secretion products.     

Placental trophoblasts resist infection by multiple human immunodeficiency virus (HIV) type 1 variants even with cytomegalovirus coinfection but support HIV replication after provirus transfection

Whether cell-free human immunodeficiency virus type 1 (HIV-1) can productively infect placental trophoblasts (which in turn could transmit the virus into the fetal circulation) is controversial but essential to know for the evaluation of alternative routes (such as cell-mediated infection or trophoblast damage). We have addressed infection factors such as cell purity, source, culture methods, and activation states as well as virus variant and detection methods to conclusively determine the outcome of trophoblast challenge by free virus. Pure (> 99.98%) populations of trophoblasts from 11 different placentas were challenged at a multiplicity of infection (MOI) as high as 6 with five different HIV-1 variants, three of which are non-syncytium-forming, macrophage-tropic isolates from infected infants, with and without coinfection with cytomegalovirus; these preparations were monitored for productive infection for up to 3 weeks after challenge by five different criteria, the most sensitive of which were cocultivation with target cells that can detect virus at an MOI of 10(-7) and HIV DNA PCR that detects 30 virus copies per 10(5) cells. Infection was never detected. However, molecularly cloned T-cell (pNL4-3)- and macrophage (pNLAD8)-tropic provirus plasmids, when transfected into primary trophoblasts, yielded productive infections, indicating that trophoblasts do not suppress late-stage virus replication and assembly. Because of the purity of the trophoblast preparations, the extended length of the infection culture period, the number of trophoblast preparations and virus types examined, the sensitivity of the bioassays and molecular detection assays, and the observations that trophoblasts can support virus replication from provirus, the results of this study strongly argue that free virus cannot infect primary villous trophoblasts.     

Transient rigid lens-induced striae in keratoconus

We have observed spontaneous development of striae and an enhanced visibility of them after placing rigid gas permeable contact lenses on six patients suspected of having early keratoconus. These fine folds subside upon removal of the rigid lens. This observation assists clinicians with the differential diagnosis of irregular astigmatism when the definitive biomicroscopic signs of keratoconus are either not present or are so subtle that their presence is questionable. Although these striae may not occur in all patients suspected of having early keratoconus, we find that observation of the posterior cornea in the presence of a rigid lens will often confirm the diagnosis of keratoconus. We have not seen similar striae in patients whose clinical presentation is not consistent with early keratoconus.     

Parathyroid hormone--related protein (PTHrP) is an epidermal growth factor-regulated secretory product of human prostatic epithelial cells

Integral membrane proteins have one or more transmembrane alpha-helical domains and carry out a variety of functions such as enzyme catalysis, transport across membranes, transducing signals as receptors of hormones and growth factors, and energy transfer in ATP synthesis. These transmembrane domains are not mere structural units anchoring the protein to the lipid bilayer but seem to-contribute in the overall activity. Recent findings in support of this are described using some typical examples-LDL receptor, growth factor receptor tyrosine kinase, HMG-CoA reductase, F0-ATPase and adrenergic receptors. The trends in research indicate that these transmembrane domains participate in a variety of ways such as a linker, a transducer or an exchanger in the overall functions of these proteins in transfer of materials, energy and signals.     

Refractory giant cell arteritis with spinal cord infarction

We report an elderly patient with aggressive steroid-refractory giant cell arteritis manifesting as myelopathy and bilateral visual loss while on treatment. Pathologically, spinal cord infarction was observed and was due to extensive necrotizing granulomatous arteritis of spinal arteries. Spinal cord damage in giant cell arteritis is rare. One prior autopsy report of spinal cord infarction in giant cell arteritis did not identify vasculitic changes in the spinal arteries.     

Glucocorticoids may enhance oxygen radical-mediated neurotoxicity

Modern populations are constantly exposed to a variety of compounds in the workplace and the environment that promote formation of reactive oxygen species (ROS) within susceptible tissues. Due to its high oxygen consumption, the brain may be particularly vulnerable to oxidative damage and degeneration. Agents that impact cellular oxidative homeostasis would therefore be expected to alter the toxicity of ROS generating compounds. We are testing this hypothesis using endogenous stress hormones, glucocorticoids, to perturb neuronal homeostasis, and adriamycin to generate ROS. Glucocorticoids (GCs) are hormones secreted by the adrenals in response to stress, and are also prescribed clinically to control inflammatory and autoimmune disorders in millions of people annually. Therefore, high GC levels may not be uncommon in individuals exposed to low levels of toxic compounds. Also, GCs appear to act on cellular pathways relevant to ROS as seen by their potentiation of neurodegeneration following insults such as stroke, hypoglycemia and seizure. Using rat primary neuronal culture, we determined neuronal susceptibility to adriamycin toxicity by cell counting (using MAP-2 staining). Dichlorofluorescein fluorescence confirmed ROS generation after adriamycin administration. Physiological levels of GCS (up to mM concentrations) in the culture media exacerbated both adriamycin toxicity and ROS generation. We hypothesize that GCs may exacerbate the toxicity of three neurotoxins whose mechanisms of action overlap GC pathways.