Genetic modifiers of the Kv beta2-null phenotype in mice.

Shaker-type potassium (K+) channels are composed of pore-forming alpha subunits associated with cytoplasmic beta subunits. Kv beta2 is the predominant Kv beta subunit in the mammalian nervous system, but its functions in vivo are not clear. Kv beta2-null mice have been previously characterized in our laboratory as having reduced lifespans, cold swim-induced tremors and occasional seizures, but no apparent defect in Kv alpha-subunit trafficking. To test whether strain differences might influence the severity of this phenotype, we analyzed Kv beta2-null mice in different strain backgrounds: 129/SvEv (129), C57BL/6J (B6) and two mixed B6/129 backgrounds. We found that strain differences significantly affected survival, body weight and thermoregulation in Kv beta2-null mice. B6 nulls had a more severe phenotype than 129 nulls in these measures; this dramatic difference did not reflect alterations in seizure thresholds but may relate to strain differences we observed in cerebellar Kv1.2 expression. To specifically test whether Kv beta1 is a genetic modifier of the Kv beta2-null phenotype, we generated Kv beta1.1-deficient mice by gene targeting and bred them to Kv beta2-null mice. Kv beta1.1/Kv beta2 double knockouts had significantly increased mortality compared with either single knockout but still maintained surface expression of Kv1.2, indicating that trafficking of this alpha subunit does not require either Kv beta subunit. Our results suggest that genetic differences between 129/SvEv and C57Bl/6J are key determinants of the severity of defects seen in Kv beta2-null mice and that Kv beta1.1 is a specific although not strain-dependent modifier.

Redistribution of microtubules and Golgi apparatus in herpes simplex virus-infected cells and their role in viral exocytosis.

Earlier studies have shown that the Golgi apparatus was fragmented and dispersed in herpes simplex virus 1-infected Vero and HEp-2 cells but not in human 143TK- cells, that the fragmentation and dispersal required viral functions expressed concurrently with or after the onset of DNA synthesis (G. Campadelli-Fiume, R. Brandimarti, C. Di Lazzaro, P. L. Ward, B. Roizman, and M. R. Torrisi, Proc. Natl. Acad. Sci. USA 90:2798-2802, 1993), and that in 143TK- cells, but not Vero or HEp-2 cells, infected with viral mutants lacking the UL20 gene virions were glycosylated and transported to extracellular space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991; E. Avitabile, P. L. Ward, C. Di Lazzaro, M. R. Torrisi, B. Roizman, and G. Campadelli-Fiume, J. Virol. 68:7397-7405, 1994). Experiments designed to elucidate the role of the microtubules and of intact or fragmented Golgi apparatus in the exocytosis of virions showed the following. (i) In all cell lines tested (Vero, 143TK-, BHK, and Hep-2) microtubules underwent fragmentation particularly evident at the cell periphery and then reorganized into bundles which circumvent the nucleus. This event was not affected by inhibitors of viral DNA synthesis. We conclude that redistribution of microtubules may be required but is not sufficient for the fragmentation and dispersal of the Golgi apparatus. (ii) In all infected cell lines tested, nocodazole caused fragmentation and dispersal of the Golgi and a far more extensive depolymerization of the microtubules than was seen in untreated, infected Vero or HEp-2 cells. Taxol precluded the depolymerization of the microtubules and fragmentation of the Golgi in both infected cell lines. Neither nocodazole nor taxol affected the exocytosis of infectious virus from Vero, HEp-2, or 143TK- cells infected with wild-type virus. We conclude that the effects of nocodazole or of taxol are dominant over the effects of viral infection in the cell lines tested and that viral exocytosis is independent of the organization of microtubules or of the integrity of the Golgi apparatus. Lastly, the data suggest that herpes simplex viruses have evolved an exocytic pathway for which the UL20 protein is a component required in some cells but not others and in which this protein does not merely compensate for the fragmentation and dispersal of the Golgi apparatus.

Fusion and duplication variants of pancreatic duct system. Clinical and pancreatographic evaluation.

The aim of this study was to assess the incidence of fusion and duplication variants of the pancreatic duct system and their clinical significance. A total of 650 endoscopic retrograde cholangiopancreatography were reviewed; 485 cases with satisfactory imaging of the pancreatic ducts were included in the study. Anatomic variants were observed in 48 patients (9.9%), fusion variants were 54.1% of the cases (22 pancreas divisum and 4 functional divisum), and duplication variants were 45.8% (13 bifurcations of the main pancreatic duct, 4 loop, 2N-shape, 3 ring). Clinical indications to endoscopic cholangiopancreatography were idiopathic acute pancreatitis (33.3%), suspected chronic pancreatitis (18.7%), unexplained abdominal pain (14.5%), suspected pancreatic mass (10.4%), chronic hyperamylasemia (6.2%), and acute biliary pancreatitis (16.6%). Except for acute biliary pancreatitis (significantly more frequent in duplication variants), no statistical difference was observed between the groups with anatomical variants concerning clinical features.

[Echography in the monitoring of bone callus in fractures treated with external fixation]. / L'ecografia nel monitoraggio del callo osseo nelle fratture trattate con fissatori esterni.

The authors report on the use of US in the evaluation of osteogenesis in the fractures treated with external fixation. From April 1991 to October 1992, fifty patients with diaphyseal fractures were submitted to real-time US examinations, with 7.5-5 MHz probes, mainly on longitudinal scans. The evolutive stage of bone callus was studied and, in the presence of periosteal bone bridge, its length and height were measured. In all but 4 patients US, unlike radiology, allowed the signs of osteogenesis to be detected as early as from day 10 on and the peculiar bilobate pattern known as primary callus response was clearly demonstrated. This stage is of fundamental importance because it allows, when periosteal callus is absent, direct intervention on the dynamic process, by varying the external fixation-bone complex so as to make it more flexible. In 80% of patients periosteal apposition was observed on day 24. Delayed osteogenesis depended on the patient's age and on fracture type. Such pathologic signs as hyperstimuli, pseudoarthrosis and/or hematoma persistence were quite unfrequent findings. Even though the study of US semiology is still in progress, our results are in substantial agreement with literature data and emphasize the role of US in the early evaluation of the bone callus and its evolution.

Monoclonal antibodies to gp100 inhibit penetration of human herpesvirus 6 and polykaryocyte formation in susceptible cells.

We report the derivation and properties of a monoclonal antibody (MAb 2E4) which neutralizes human herpesvirus 6 (HHV-6). MAb 2E4 precipitated from lysates of infected cells a glycosylated polypeptide 100,000 in apparent molecular weight and minor components of 80,000, and 32,500. The predominant reactive protein after a pulse was the 100,000-molecular-weight peptide designated as gp100. The smaller polypeptides appeared in the precipitate predominantly after a chase. MAb 2E4 neutralized HHV-6 infectivity in the presence and in the absence of complement, and it inhibited the penetration of virus into the cells. Addition of MAb 2E4 as late as 6 h postinfection inhibited the formation of large polykaryocytes typical of HHV-6-infected cells.

Origin of unenveloped capsids in the cytoplasm of cells infected with herpes simplex virus 1.

In cells infected with herpes simplex viruses the capsids acquire an envelope at the nuclear membrane and are usually found in the cytoplasm in structures bound by membranes. Infected cells also accumulate unenveloped capsids alone or juxtaposed to cytoplasmic membranes. The juxtaposed capsids have been variously interpreted as either undergoing terminal deenvelopment resulting from fusion of the envelope with the membrane of the cytoplasmic vesicles or undergoing sequential envelopment and deenvelopment as capsids transit the cytoplasm into the extracellular space. Recent reports have shown that (i) wild-type virus attaches to but does not penetrate cells expressing glycoprotein D (G. Campadelli-Fiume, M. Arsenakis, F. Farabegoli, and B. Roizman, J. Virol. 62:159-167, 1988) and that (ii) a mutation in glycoprotein D enables the mutant virus to productively infect cells expressing the wild-type glycoprotein (G. Campadelli-Fiume, S. Qi, E. Avitabile, L. Foa-Tomasi, R. Brandimarti, and B. Roizman, J. Virol. 64:6070-6079, 1990). If the unenveloped capsids in the cytoplasm result from fusion of the cytoplasmic membranes with the envelopes of viruses transiting the cytoplasm, cells infected with virus carrying the mutation in glycoprotein D should contain many more unenveloped capsids in the cytoplasm inasmuch as there would be little or no restriction in the fusion of the envelope with cytoplasmic membranes. Comparison of thin sections of baby hamster kidney cells infected with wild-type and mutant viruses indicated that this was the case. Moreover, in contrast to the wild-type parent, the mutant virus was not released efficiently from infected cells. The conclusion that the unenveloped capsids are arrested forms of deenveloped capsids is supported by the observation that the unenveloped capsids were unstable in that they exhibited partially extruded DNA.