Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus

The envelope protein of human immunodeficiency virus (HIV) is synthesized as a polyprotein (gp160) and cleaved intracellularly to a gp120-gp41 heterodimer. In this study, the tryptic-like endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site. The resultant mutant, RIP7/mut10, was found to be indistinguishable from wild-type HIV when analyzed at the level of proviral replication, RNA processing, protein expression, and viral assembly. However, the gp160 polyprotein was not cleaved and the mutated virions were biologically inactive, until and unless they were exposed to limiting concentrations of chymotrypsin. As is the case for other enveloped mammalian viruses, endoproteolytic cleavage of the HIV envelope protein and release of a unique hydrophobic domain appear to be necessary for the full expression of viral infectivity.     

Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells

We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.     

Opsonic activity of antisera to ribosomal vaccine fractions with live and formalinized Pseudomonas aeruginosa

The opsonic capacity of antisera to Pseudomonas aeruginosa ribosomal vaccine fractions was determined by a chemiluminescent technique. Antiserum to a vaccine fraction ("peak A") containing lipopolysaccharide (antiserum A), and antiserum to a vaccine fraction ("peak B"), which did not contain detectable amounts of lipopolysaccharide (antiserum B), were used to opsonify live or formalin-treated bacteria. Polymorphonuclear leukocytes were then stimulated by the opsonified bacteria in the presence of the chemiluminigenic probe, luminol, resulting in the observed chemiluminescence. The data obtained indicated that the antisera had comparable opsonic activity with live (untreated) bacteria. However, antiserum B had far less opsonic activity than did antiserum A when formalinized bacteria were used. Owing to the effects of formaldehyde on protein, these results were interpreted as evidence to suggest that the opsonic activities of the two antisera are dependent on different antigens on the bacterial cell surface. Antiserum A activity is probably dependent on lipopolysaccharide to a great extent, whereas antiserum B activity is most likely dependent primarily on a protein(s).     

Improved Method of Typing Bradyrhizobium japonicum in Soybean Nodules

An improved method for antibiotic resistance recovery of Bradyrhizobium japonicum from soybean (Glycine max (L.) Merr.) nodules that is simple, time saving, and economical was developed. This technique involves the use of two 96-well microtiter plates as a multinodule sterilization chamber and a template and a third plate as a 16-point replicator constructed with steel nails affixed to the plate with epoxy cement. With this system a team of four technicians could type 3,000 nodules per day. This method was useful in assessing strain establishment and interstrain competition when one or more uniquely labeled strains of B. japonicum were inoculated onto either growth-room- or field-grown soybeans. Contamination was low and reproducibility across replicates approached the theoretical upper limit. Simplicity in design and use made this recovery method especially adaptable for field studies in which large numbers of nodules were required to provide a representative statistical sample offering good precision.     

Numerical taxonomic analysis of cross-inoculation patterns of legumes and Rhizobium

Summary A survey was made of published results of tests of the capacity of Rhizobium derived from one legume genus to nodulate plants from other genera. The data were derived from more than 14,000 separate cross-inoculation trials involving species from 165 genera of legumes. Numerical taxonomic techniques were applied to 113 of the genera for which results of substantial cross-infection tests were available. The data were examined using mean character difference coefficients re-expressed as total and positive-only similarity coefficients. The resulting similarity matrices were clustered by the unweighted pair-group method using arithmetic averages. Eighteen affinity groups were defined at the 70% similarity level. With few exceptions, the physiological and cultural behavior of the rhizobia was consistent within the defined groups. Two broad categories were suggested in the numerical taxonomic analysis, and their validity is discussed in regard to the geographic, physiological and cultural characteristics of the legumes and their Rhizobium microsymbionts. The taxonomic and agronomic value of this approach and the new groupings are discussed.     

Factors influencing algal species assemblages on reef and cobble substrata off Ghana

Subtidal algal assemblages were studied on two substrata, rocky reefs and calcareous cobbles. Although the two occur together at comparable depths, their vegetation differs in species composition and richness, and in patterns of plant size, life form, and longevity. The reef bears a species-rich, patchy cover of small filamentous and crustose forms, with occasional clumps of more robust species. The cobbles support a sparse cover of large leafy and dendritic species in addition to many of the smaller species found on the reef. The floristic separation arises from differential establishment and survival of species under conditions of (1) grazing by fish and urchins (on the reef only), and (2) seasonal physical disturbance during storms leading to the removal of most algae (on the cobbles only). Both substrata show a seasonal floristic cycle, but the trend is more pronounced on cobbles. Species do not depart from randomness in their patterns of co-occurrence on individual cobbles or reef fragments. Interspecific competition appears comparatively unimportant in determining species composition on either substratum.     

Theoretical effects of transmembrane electroneutral exchange on membrane potential

Transmembrane electroneutral transport mechanisms [e.g., Na/H exchange, Cl/HCO3 exchange, (K + Cl) cotransport] have recently been identified in a wide variety of cell types. If these exchanges sum to give a net electroneutral Na/K exchange, they may hyperpolarize the membrane potential beyond the value calculated from the Mullins-Noda equation, provided the cell maintains steady state intracellular ionic concentrations. In extreme circumstances, the membrane potential could hyperpolarize beyond the potassium reversal potential. This effect is mediated by the electrogenic Na/K pump. If either Na or K exchanges electroneutrally against a third ion (e.g., Na/Ca exchange), then the exchange may depolarize the membrane potential.     

Genomic hypomethylation and far-5'' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.