Carbohydrate side chains of Rauscher leukemia virus envelope glycoproteins are not required to elicit a neutralizing antibody response.

Antisera raised against Rauscher leukemia virus (R-MuLV) contain a preponderance of antibodies against glycoprotein gp70 that are dependent on the presence of carbohydrate side chains for reactivity, as judged by immunoprecipitation or Western blotting. However, the majority of neutralizing antibodies were not dependent on the presence of carbohydrate, as indicated by (i) the ability of deglycosylated R-MuLV to adsorb neutralizing antibody from sera as efficiently as glycosylated R-MuLV and (ii) the ability of deglycosylated R-MuLV to induce neutralizing antibody responses when injected into rabbits. Moreover, a faster response was obtained with deglycosylated R-MuLV than with untreated control virus in the latter experiments. The results indicate that the neutralizing antibodies are a discrete subpopulation of the total antibody response. Furthermore, the carbohydrate moieties appear to afford protection to the virion during infection, rather than serve as a target for neutralization.     

Spontaneous epimerization of (S)-deoxycoformycin and interaction of (R)-deoxycoformycin, (S)-deoxycoformycin, and 8-ketodeoxycoformycin with adenosine deaminase

(R)-Deoxycoformycin (pentostatin), (S)-deoxycoformycin, and 8-ketodeoxycoformycin were compared as inhibitors of calf intestine adenosine deaminase. In contrast to (R)-deoxycoformycin, which had been demonstrated as a tight-binding inhibitor with a dissociation constant of 2.5 X 10(-12) M [Agarwal, R. P., Spector, T., & Parks, R. E., Jr. (1977) Biochem. Pharmacol. 26, 359-367], (S)-deoxycoformycin and 8-ketodeoxycoformycin are slope-linear competitive inhibitors with respect to adenosine. The kinetic constants are 33 microM for inhibition by (S)-deoxycoformycin, 43 microM for 8-ketodeoxycoformycin, and 16 microM for the Km for adenosine. The stereochemistry of carbon 8 of the diazepine ring therefore causes a (1.3 X 10(7]-fold change in the affinity for the enzyme which is specific for the R configuration. This difference is attributed to an induced conformational change which cannot be initiated by the S isomer or the 8-keto analogue of (R)-deoxycoformycin. The studies were complicated by the need to remove traces of tight-binding inhibitor(s) from (S)-deoxycoformycin, since as little as 0.001% of the R isomer causes significant inhibition. The R and S isomers of deoxycoformycin are unstable in neutral or mildly acidic aqueous solutions. Isomerization of the secondary hydroxyl at carbon 8 of the diazepine ring is one of the reactions, resulting in S to R and R to S conversions for deoxycoformycins. Opening of the aglycon is also a major reaction. The tight-binding inhibitor generated from (S)-deoxycoformycin was identified as (R)-deoxycoformycin by high-pressure liquid chromatography, spectroscopy, circular dichroism, and chemical criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional electron microscopic analysis of the chalice form of phosphorylase kinase

Phosphorylase kinase (Mr 1.3 X 10(6], a Ca2+-calmodulin-dependent protein kinase, plays a key role in the initiation of glycogenolysis. After purification on hydroxylapatite, the negatively stained enzyme was used for electron microscopy. In electron micrographs, phosphorylase kinase shows two major molecular forms: a butterfly form (approx. 60%) and a chalice form (approx. 40%). Images of the chalice form of the enzyme were computer-averaged by the method of single particle averaging. The following apparent molecular dimensions were obtained from the averages: total height, 20 nm; maximal width, 18 nm. The chalice form of phosphorylase kinase consists of a major structure termed the cup (11 nm X 18 nm), containing a large accessible cleft, and a minor structure termed the stem (8 nm X 9 nm). A closer examination of the images by averaging of molecular parts revealed two subpopulations of the cup part: a flexed (closed) type and an extended (open) type. The orifice, which can be closed partly by two protrusions (I, I'), is about 6 nm wide when the protrusions are flexed and 9 nm wide when they are extended. It is suggested that the substrates, e.g. phosphorylase b, may be accommodated in the large cleft of the enzyme. While the orientation of the protrusions (I, I') is the most obvious difference between the two types, more structural differences can be detected, suggesting a concerted movement of the protein domains against each other.     

The regulation of''early'' enzymes during the development and dedifferentiation of Dictyostelium discoideum

The specific activities of the enzymes alpha-mannosidase and N-acetylglucosaminidase increase immediately after the initiation of the development of bacterially grown cell cultures of Dictyostelium discoideum. The regulation of these two enzymes was found to be dissociable in the developmental timer mutant, FM-1, which aggregates 4.5 h earlier than wild-type cells due to the absence of the first rate-limiting component of the preaggregative period. The increase in alpha-mannosidase activity occurs in the absence of the first rate-limiting component, but the increase in N-acetylglucosaminidase activity does not. These results indicate the following: (1) the increase in the specific activity of alpha-mannosidase is not related to the timing of subsequent developmental stages; (2) the increase in the specific activity of N-acetylglucosaminidase is not necessary for the subsequent developmental program; and (3) either the increase in the specific activity of N-acetylglucosaminidase is dependent upon progress through the first rate-limiting component, or the increase in this enzyme activity and the first rate-limiting component are both dependent upon an early event for which FM-1 is defective. In addition to early development, we monitored the two enzyme activities during dedifferentiation. The results demonstrate that there is no difference between dedifferentiating wild-type cells and dedifferentiation-defective mutant HI-4 cells. Changes in enzyme specific activity accompanying dedifferentiation are dependent upon the composition of the dedifferentiation-inducing media and are consistent with the levels of these enzymes observed in cells growing in the different nutrient media.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

Nonlinear estimation of the parameters of Monod kinetics that best describe mineralization of several substrate concentrations by dissimilar bacterial densities

The kinetics of mineralization of a wide range of concentrations of benzoate, glucose, and benzylamine by Pseudomonas sp., Salmonella typhimurium, and microorganisms in acclimated sewage was studied. The treatment of initial substrate concentration and population density as independent variables in nonlinear regression analysis permitted the estimation of a single value for each of the parameters of Monod kinetics that best described the mineralization of substrate at each concentration by the pure cultures and the sewage microflora. One value for each of the parameters of Monod kinetics was used for each of the three compounds to produce theoretical curves which lay close to the observed data on mineralization. Statistically significant differences existed in the values of the parameters of Monod kinetics that best described mineralization in cultures differing only in initial substrate concentration and cell density. However, for the compounds tested, the variance left by analyses using one value for each parameter of Monod kinetics was less than double the unexplained variance left by individual analyses of the data from each treatment. Although significant, this increase is small compared with the amount of variance that could be explained using only one value for each parameter of Monod kinetics.     

An observation of episodic feeding and growth of larval Leiostomus xanthurus in the northern Gulf of Mexico

Four cruises were conducted in the northern Gulf of Mexico overtwo spawning seasons of the sciaenid fish Leiostomus xanthurus.On only one occasion did unusually high densities of larvaeand their principal microzooplanktonic foods co-occur. Peakdensities of larvae and microzooplankton were observed in athin lens of cool surface water that characterized a hydrographicdiscontinuity, and all larvae contained high numbers of foodorganisms in their guts. Instantaneous exponential growth ratesestimated from measurements of otolith growth increments, indicatedaccelerated growth on the day that larvae were collected. Alaboratory experiment verified that larval L. xanthurus respondsto an increased ration with accelerated growth that is detectableon otoliths. Together these data suggest that the spatial distributionof L. xanthurus larvae and their microzooplanktonic food ispatchy and that interactions of larvae and microzooplanktonmay be episodic.     

Elastic properties of the hind foot of the Donkey, Equus asinus

The elastic properties of the hind feet of Donkeys, and of tendons removed from the feet, have been investigated by methods similar to those used in a previous study of the forefoot. The elastic strain energy stored in the foot, during a trotting step, is calculated to be approximately the optimum which would minimize the work required of the muscles in this gait.     

Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression

Vascular smooth muscle cells (VSMC) in the rat mesenteric artery show specific immunofluorescent staining with antisera against purified human uterine myosin (ASMM) but not human platelet myosin (APM). However, in primary cultures produced by enzymatic dissociation of this vessel, VSMC stain specifically with both ASMM and APM within 5 h after plating and throughout growth to confluence (4-10 d). In confluent cultures, APM staining remains bright while ASMM staining is reduced in intensity in most cells. In contrast, cellular myosin content, determined by quantitative SDS PAGE, is comparable in confluent and growing cultures. Immunoprecipitation of high salt extracts of cultured VSMC with ASMM and APM yields myosins with the same mobilities on SDS PAGE. When serial, exhaustive precipitations are performed with one antiserum, followed by reprecipitation with the other, myosin in subconfluent and confluent VSMC cultures is exhaustively precipitated by either antiserum, thus indicating complete immunological cross- reactivity. These results might be explained by synthesis of a new myosin isoform reactive with both ASMM and APM. However, the development of APM staining in cultured VSMC did not require protein synthesis. Therefore, it is more likely that the changes in immunofluorescent staining observed in vitro reflect conformational alterations, perhaps related to cytoskeletal rearrangements. These changes in myosin antigenic expression may be relevant to the problem of VSMC phenotypic modulation both in vitro and in vivo.     

Kinetics of mineralization of phenols in lake water

The kinetics of mineralization of phenol and p-nitrophenol in lake water was determined at concentrations from 200 pg/ml to 5 micrograms/ml. The mineralization data were fit by nonlinear regression to equations for 14 kinetic models that describe patterns of biodegradation by nongrowing cells or by microorganisms growing on either the test chemical or other organic substrates. The kinetics od mineralization of phenol in water samples collected in July was best described by first-order models for 0.5 ng of phenol per ml; by Monod-without-growth, logistic, and logarithmic models for 1.0 and 2.0 ng/ml and 5.0 ng/ml to 1.0 micrograms/ml, respectively, if it is assumed that the mineralizing population uses phenol as the sole carbon source for growth; by models (for phenol at concentrations of 2.0 ng/ml to 1.0 micrograms/ml) that assume that the phenol-mineralizing populations do not grow or grow logarithmically or logistically on uncharacterized carbon compounds but metabolize the phenol when present at levels below and above Km, respectively, for that compound; and by a logarithmic model at 5.0 micrograms/ml. Under the test conditions, usually less than 10% of the phenol C that was metabolized was incorporated into microbial cells or retained by other particulate material in the water at substrate concentrations of 10 ng/ml or less, and the percentage increased at higher substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)