Transport of substrates and metabolites and their effect on cell metabolism (in butyric-acid and methylotrophic fermentations).

The two components, delta pH and delta psi, of the membrane protonmotive force (delta p) effect and are affected by the transport of many substrates and metabolites. Because the integrity (or restoration) of the delta p requires the expenditure of metabolic energy, such transport processes affect the overall cell bioenergetics. However, the transport or high concentrations of certain substrates and metabolites can have more serious effects on cell metabolism because they partially or completely abolish either or both the delta pH and delta psi. If the cells cannot eventually restore the collapsed component(s) of the delta p, complete growth inhibition and cell death become inevitable. In the butanol/acetone fermentation of Clostridium acetobutylicum, the transport and the presence of key metabolites (acetic and butyric acids, and butanol) have serious and some necessary effects on the delta p. Acetic and butyric acids act as uncouplers of the delta pH, thereby reducing the internal pH. Using other acid uncouplers (such as acetoacetate, which is metabolized by the cells, or FCCP, which is not metabolized by the cells), we found that a lower pHo combined with the metabolic-energy drain of the uncoupling effect and high internal acid concentrations are implicated in the mechanism(s) of solventogenesis. Thus, the production or presence (or both) of the two acids (acetic and butyric) is beneficial to the initiation of solvent production. The transport mechanisms of CH3OH, CH2O, and HCOOH in obligate CH3OH utilizers (methylotrophs) were also discussed in detail. We showed that CH3OH is actively transported by the cells at the expense of metabolic energy and that its transport significantly affects the dynamics of continuous bioreactors. The accumulation of CH2O was found to be driven by the membrane delta p. Finally, formate was accumulated by the delta pH according to the general transport mechanism of short-chain fatty acids. The inhibition of growth by formate was explained by its uncoupling effect on the cells. Growth inhibition by CH3OH appeared to be related to the severe reduction of the membrane delta pH and cell pHi by relatively low CH3OH concentrations.

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. I. Attributes of uncloned virus at different passage levels.

Attempts were made to attenuate prototype dengue (DEN) 4 (H-241) virus. The original viremic human serum was passed once in a susceptible monkey and twice in Aedes albopictus mosquitoes and then serially passed in primary dog kidney (PDK) and African green monkey kidney (GMK) cells. Weekly transfers of undiluted virus were carried to the 50th passage in both primary cell cultures. Biological markers were studied at passages 7, 15, 30 and 50. Parental DEN-4 phenotype characteristics included large plaque formation in LLC-MK2 cells, plaque formation in GMK cells, cytopathic effect in LLC-MK2 cells, growth in human monocyte cultures, growth at 39 degrees C, consistent production of viremia in monkeys and short-incubation neurovirulence in mice. At the seventh passage in both PDK and GMK cell cultures, DEN-4 viruses exhibited reduced plaque-size in LLC-MK2, and failed to plaque in GMK, to produce cytopathic effect in LLC-MK2, or to grow in human monocytes. Serial passage in PDK, as opposed to GMK, resulted in a graduated loss of monkey virulence. Rhesus monkeys inoculated with the PDK 50 strain failed to develop detectable viremia and only 1 of 4 developed an antibody response. Also, replication of PDK 50 was completely shut-off at 39 degrees C. The graduated change in biological properties noted, particularly those in PDK cells, provide a range of potential vaccine candidates for evaluation in human beings.

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. II. Attributes of virus cloned at different dog kidney passage levels.

Uncloned dengue (DEN) 4 (H-241) which had been passaged 15, 30 and 50 times in primary dog kidney (PDK) cells were subjected to two successive terminal dilution procedures. In the first (3Cl), virus was diluted in 10-fold steps in 10 replicate tubes. An infected tube from a dilution row with three or fewer virus-infected tubes was selected for two further passages. In the second (TD3), virus was triple terminal diluted using 2-fold dilution steps and selecting one positive tube out of 10. Both procedures selected virus population which differed from antecedents. Plaque size of PDK 15 was medium, PDK 30, small and PDK 50, pin-point. PDK 19-3Cl were medium and 56-3Cl, 24-TD3, 35-TD3 and 61-TD3 were all small. All cloned virus replication was completely shut-off at 38.5 degrees C; PDK 15 and 30 continued to replicate at this temperature. Uncloned viruses showed a graduated decrease in monkey virulence with PDK passage; cloned viruses were either avirulent for monkeys (19-3Cl, 56-31Cl, 24-TD3 and 35-TD3) or produced revertant large plaque parental-type viremia (35-3Cl and 61-TD3). Those cloned viruses which exhibited temperature sensitivity, reduced monkey virulence and stability after monkey passage may be suitable as vaccine candidates for evaluation in human beings.

Enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to cysticerci of Taenia solium.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to cysticerci of Taenia solium has been developed that employs a pork muscle antigen control for the cysticercus test antigen, somewhat improving the serological distinction between infected and uninfected subjects. Serum antibody to cysticercus was detected in 79% of classical neurocysticercosis patients from Mexico, and in 61% of a group of cysticercosis patients with an unusually rapid invasion of the central nervous system in an endemic focus of disease in Irian Jaya. Antibody was absent in a group of healthy American laboratory personnel, and in residents of a non-endemic region of Papua New Guinea. Additional test on sera from patients with other parasitic diseases showed that cross-reactivity may occur in some patients with schistosomiasis, echinococcosis, and possibly angiostrongyliasis; however, these parasites are not known to cause human infection in Irian Jaya.

Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. III. Reversion to virulence by passage of cloned virus in fetal rhesus lung cells.

Two strains of primary dog kidney-passaged dengue (DEN) 4 (H-241) virus cloned by terminal dilution (PDK 24-TD3 and 35-TD3) were propagated in fetal rhesus lung (FRhL) cells to produce candidate vaccine virus seeds. Both serial passage and prolonged replication of PDK 24-TD3 in FRhL resulted in appearance of medium and large plaques in LLC-MK2 assays. When picked, these plaques proved to contain temperature-resistant, monkey-virulent revertants. Serial passage and prolonged replication of PDK 24-TD3 in LLC-MK2 cells did not result in reversion; but, prolonged replication in PDK cells did. Passage of PDK 35-TD3 in FRhL cells resulted in appearance of medium size plaques which, when picked, yielded temperature sensitive (ts) (38.5 degrees C) viruses of low monkey-virulence. Because of its stability in monkeys and FRhL cells, reduced monkey virulence and ts property. PDK 35-TD3 is a promising candidate for trial in man.

Serological evidence of Hantaan virus infection in the United States.

We found low titers of fluorescent antibodies against Hantaan virus, the etiologic agent of Korean hemorrhagic fever, in sera from 7 of 1,035 patients with febrile illnesses of unknown origin and from 6 of 664 blood donors in the United States. All but 1 of these individuals possessed neutralizing antibodies against Hantaan virus. This was a 31-year-old research technician who had worked with laboratory rodents with virus-induced tumors, but had not traveled abroad, suggesting that infection with Hantaan virus or a closely related agent was locally acquired. However, the precise source of his infection remains unclear.

ELISA antibodies to cysticerci of Taenia solium in human populations in New Guinea, Oceania, and Southeast Asia.

The presence of ELISA antibodies to cysticerci of Taenia solium was surveyed in populations of New Guinea, Micronesia, and several areas of Southeast Asia. It is confirmed that cysticercosis in New Guinea remains limited to the primary Wissel Lakes focus in Irian Jaya, where the disease was introduced by the importation of infected pigs, and that it has not spread to populations east or south of the Wissel Lakes, or to Papua New Guinea. On the island of Bali, Indonesia, 21% of sera were positive from one village where pigs are especially numerous, whereas in Sumatra, Indonesia, only 3%-4% of sera were positive. In Singapore, there was a higher proportion of positive sera among the Chinese (13%) than among the Indian (5%) or Malay (3%) Moslems. From 3 to 13% of sera from populations in Micronesia, Burma, Vietnam, and the Philippines were also found to react with cysticercus antigen. However, the problem of incomplete ELISA specificity raises the possibility that in areas not known to be endemic for T. solium, seropositive results could represent either subclinical infection with cysticerci or crossreactivity to other parasitic infections.

On the theory of gel electrophoresis of DNA: extension and evaluation of the Lumpkin-DèJardin-Zimm model.

Using fundamental concepts of hydrodynamics in porous media, we have rederived the Lumpkin-DèJardin-Zimm (LDZ) model for the gel electrophoresis of reptating, infinitely long, worm-like chains, such as DNA. The force balance provides a constraint for evaluating the correlation among the segment-to-field angles of a given molecular conformation. We have used an approximate analytical expression to account for this correlation in order to apply the present derivation to finite chain lengths. The resulting extended LDZ model predicts a nonlinear variation of electrophoretic mobility (mu) with reciprocal chain length (1/Lc) at low electric field strengths, similar to the one observed. The present derivation is valid only at low electric field strengths, and the predictions of the extended LDZ model fit data for a dimensionless electric field strength, E1*, of less than 1.23. An empirically useful criterion for determining the onset of reptation is also described. The present treatment shows how size-exclusion effects can be included in future theories. Models based on reptation alone are shown to predict a discontinuity in the molecular chain length dependence of mobility at a critical molecular size. Such discontinuities are not observed experimentally.

A lipid inhibitor of dengue virus in human colostrum and milk; with a note on the absence of anti-dengue secretory antibody.

Neutralizing activity against dengue virus types 1--4 was observed in milk samples from 5 non-immune and 29 dengue immune women. Anti-dengue activity in milk and colostrum was found only in the lipid component. The inhibitory activity is directed against the virus and not cell surfaces. When immunoglobulin types IgM,IgA, IgG were isolated from colostrum from dengue immune women, no antibody activity was found. Anti-dengue activity in human milk did not decrease over a period of ten months after delivery.

Ultrastructural studies on dengue virus infection of human lymphoblasts.

Ultrastructural studies of dengue-2 virus-infected lymphoblastoid Raji cells showed that the virus induced an increase in the size of the rough endoplasmic reticula (RER) and that the replication of the virus was confined to the cisternae of these RER. The proliferating RER formed cytoplasmic inclusions that could be seen by light microscopy. This observation could be used as evidence of a cytopathogenic effect of dengue virus on infected Rajii cells in routine cultures. Accumulation of virions in the infected cells was minimal in comparison with other cell systems, however. Sporadic clusters of mature virions were often seen on the plasma membrane. These extracellular virions were distributed adjacent to the virus-bearing RER and were presumably released virions. Vertical transmission of the virus was evident in mitotic lymphoblasts. The replication pattern of dengue virus in lymphoblastoid cells suggests that efforts should be made to determine whether blast-transformed lymphocytes, numerous in secondary dengue infections, support dengue virus replication in vivo.

Utilization of immunogold labeling to compare the adsorption behavior of fibrinogen, fibronectin and albumin on polymers.

Immunogold labeling followed by scanning electron microscopy (SEM) was used to examine the surface distribution of adsorbed plasma proteins. Adsorption was performed under various conditions on six different polymers; [low density polyethylene (PE), chromic acid-oxidized PE (OXPE), solution grade Biomer (SB), Teflon-(FEP), a laboratory synthesized polyurethane containing some zwitterions (ZW) and a polydimethylsiloxane based polyurethane (ZS) also containing zwitterions]. The proteins used were purified human and canine fibrinogen, fibronectin, and serum albumin. The immunogold staining technique was successful in the labeling of the adsorbed proteins. The adsorbed proteins were distributed differently on the polymers selected. Human and canine fibrinogen were found to cover all surfaces in a dense, uniform fashion. Albumin covered most surfaces in a less uniform fashion and on the zwitterionomers covered only a portion of the surface, leaving large bare patches. Fibronectin appeared to deposit unevenly, forming a network on part of the surface and uniformly coating other parts.

Clinical and laboratory studies on haemorrhagic fever in Burma, 1970-72.

This three-year serologic study of 2 060 children with a clinical diagnosis of haemorrhagic fever, who were admitted to the Children's Hospital and other hospitals in Rangoon, has shown that the etiology of the illness was multiple. Of all these patients, 347 (16.8%) had a dengue infection (96 with primary and 251 with secondary dengue infections), 510 (24.7%) had chikungunya infections, 55 (2.7%) had simultaneous chikungunya and dengue, 263 (12.8%) had influenza A infections, 62 (3.0%) had influenza B, 12 (0.6%) had measles, and there were 811 (39.4%) for whom no etiology could be established. Epidemiological and clinical features and laboratory findings are discussed. Evidence is presented for human infections with all four types of denguevirus in Rangoon.

Dengue carrier culture and antigen production in human lymphoblastoid lines.

Replication of dengue type 2 (D2) viruses was studied in four lymphoblastoid cell lines; Raji, HR1, EB3 and RPMI 6410. The HR1 cell line failed to support D2 growth while the other cell lines showed varying susceptibility. Both D2 strain 16681 and strain New Guinea C (NGC) passaged in LLC-MK2 cells replicated readily in Raji cells, while a high mouse-brain-passaged NGC strain did not. A soluble complement-fixing (SCF) antigen from D2-infected Raji cells showed lines of identity with a D2SCF antigen prepared from infected suckling mouse brains. Electron microscopic studies of a D2 Raji carrier culture showed that virions were located in a membrane-vesicle complex in the cytoplasm of the cells. Precursors of viral particles were more frequently observed in the parts of the vesicle which had rough endoplasmic reticular membranes. Mature virus particles were observed often at the boundary of the other part of the vesicle, which consisted of smooth endoplasmic reticulum. Occasionally, a large crystalloid structure consisting of incomplete viral particles was seen in degenerative cells. Dengue carrier cultures in human lymphoblastoid lines may provide a convenient in vitro system for study of aspects of dengue virus-leukocyte interactions.

Ex vivo platelet deposition on fibronectin-preadsorbed surfaces.

Temporal platelet deposition profiles of canine plasma fibronectin (CPFN) adsorbed to different polymers ex vivo and the in vitro characteristics of CPFN adsorption were studied in an attempt to correlate the two. The maximum platelet deposition (gamma pltmax) obtained at a protein preadsorption time of 30 min was greater than that obtained using an adsorption time of 120 min for all surfaces studied. At 30 min of preadsorption, gamma pltmax was 520,560 and 1230 platelets/1000 micron2 on Biomer, polyethylene (PE) and oxidized PE (OXPE), respectively. In contrast, the platelet deposition at 120 min. of fibronectin preadsorption was about 60 approximately 90 platelets/1000 micron2 on all polymers studied. The surface concentrations of adsorbed CPFN measured using 125I-CPFN, were in the order PE greater than OXPE greater than Biomer. The adsorbed protein concentration increased with increasing adsorption time. The surface distribution of adsorbed CPFN was visualized with antibody-labelled colloidal gold and scanning electron microscopy. The extent of staining was lowest on PE, greater on Biomer, and highest on OXPE, roughly similar to the order of platelet deposition. Platelet deposition ex vivo appears to correlate with the immunogold-stainable-adsorbed protein rather than with the total amount of adsorbed protein.