Further observations of stage-specific effects seen after short-term hypophysectomy in the rat.

Although hypophysectomy has been a popular tool to study the effects of hormone deprivation as well as concomitant or subsequent hormone supplementation, there is relatively little morphological information available on the structural manifestation of pituitary removal on the testis. In the report, changes, in addition to those previously reported after short-term (6 days) hypophysectomy in the rat (Russell and Clermont, 1977), are described. Membrane-bound vacuoles (primarily) appeared within the basal region of the Sertoli cell at approximately the level of Sertoli-Sertoli junctions. In stages VIII through XI elongating spermatids were abnormal and manifested manchette indentation of the nucleus, a variety of other abnormal head shapes, acrosomal breaks and enlargement of the subacrosomal space. These defects were interpreted as the effect of declining hormonal levels in stage VII on spermatids that had survived the stage VII hormone sensitivity known to occur with severe hormone depletion. Abnormalities in the flagellum involving the mitochondrial sheath and fibrous sheath were detected. Preleptotene spermatocytes degenerated and could be identified in the process of doing so near the base of the seminiferous epithelium. The contact of preleptotene spermatocytes with the basal lamina was also significantly reduced. The results show that both Sertoli cell and germ cell abnormalities were present although germ cell abnormalities could be a secondary consequence of lack of appropriate stimulation of the Sertoli cell. Degeneration of basal compartment germ cells shows that germ cells other than those located in the adluminal compartment are vulnerable to hormonal withdrawal. The question of how hormone effects are mediated in the testis at midcycle to produce these effects is discussed.     

DNA binding and I kappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide

The sequence and biochemical properties of the product of the cloned cDNA for the p65 subunit of nuclear factor kappa B (NF-kappa B) have been determined. The cDNA has an open reading frame of 549 amino acids capable of encoding a 60 kd protein. NF-kappa B p65 contains an amino-terminal region of 320 amino acids with extensive similarity to the oncogene c-rel and lesser similarity to NF-kappa B p50. In vitro translated p65 forms a DNA-binding complex with NF-kappa B p50, and the binding of this complex can be specifically inhibited by purified I kappa B. Progressive carboxy-terminal deletions of p65 show that, contrary to previous assumptions, p65 does include a DNA-binding domain that in vivo might become activated only through hetero-oligomerization with p50. DNA binding by truncated p65 is inhibited by I kappa B, thus mapping the I kappa B interaction domain to the rel-homologous region and suggesting that I kappa B exerts its inhibitory effect upon NF-kappa B primarily through interaction with p65.     

Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role

A cDNA encoding the cell-cell adhesion molecule E-cadherin was transfected into highly invasive epithelial tumor cell lines of dog kidney or mouse mammary gland origin. Transfectants with a homogeneously high expression of E-cadherin showed a reproducible loss of activity in two types of in vitro invasion assays. Invasiveness of these transfectants could be reinduced specifically by treatment with anti-E-cadherin antibodies. In vivo, they formed partly differentiated tumors, instead of fully undifferentiated tumors. Alternatively, a plasmid encoding E-cadherin-specific anti-sense RNA was introduced into noninvasive ras-transformed cells with high endogenous E-cadherin expression. The resulting down-regulation, albeit partial, rendered the cells invasive. These data provide direct evidence that E-cadherin acts as an invasion suppressor molecule.     

Isolation and characterization of conditional adherent and non-type 1 fimbriated Salmonella typhimurium mutants

Mutations in the genes encoding the type 1 fimbriae of Salmonella typhimurium were isolated by selecting for the deletion of Tn10 inserted adjacent to the chromosomal fim+ genes and screening for the loss of mannose-sensitive haemagglutination (HA) activity. S. typhimurium strains with Tn10 insertions in ahp were hypersensitive to peroxides, and tetracycline-sensitive derivatives of ahp::Tn10 mutants displayed two fim mutant phenotypes. The predominant class of fim mutants did not synthesize type 1 fimbriae. A second type of fim mutant synthesized type 1 fimbriae and exhibited a conditional lipoic acid requirement for HA. A fim-lip conditional mutant synthesized type 1 fimbriae when grown in Mueller-Hinton broth but the haemagglutinating activity of the fimbriae was dependent upon the addition of lipoic acid to the growth medium. Independently isolated lip mutations did not demonstrate a similar pleiotropic effect on HA. Western blots of fimbriae extracted from a fim-lip conditional mutant that was grown under permissive and restrictive conditions indicated the presence of 33 and 36.6 kDa proteins in HA+ fimbriae that were absent in HA- fimbriae. The HA+ phenotype of both conditional and non-fimbriated mutants was restored by transformation with cloned genes encoding S. typhimurium type 1 fimbriae.     

Hydrolysis of soyabean by papain immobilized on wood by radiation polymerization

Summary Papain was immobilized on wood chips by radiation polymerization without substantial loss of enzyme activity. The immobilized papain was used to hydrolyse soyabean meal and found to be stable upto 6 cycles of operation. Maximum hydrolysis occurred with 15% (W/V) immobilized matrix.     

Theory of chaperonin action: inertial model for enhancement of prokaryotic Rubisco assembly.

We have performed a computational simulation of the aggregation and chaperonin-dependent reconstitution of dimeric prokaryotic ribulose bisphosphate carboxylase/oxygenase (Rubisco), based on the data of P. Goloubinoff et al. (1989, Nature 342, 884-889) and P. V. Viitanen et al. (1990, Biochemistry 29, 5665-5671). The aggregation is simulated by a set of 12 differential equations representing the aggregation of the Rubisco folding intermediate, Rubisco-I, with itself and with aggregates of Rubisco-I, leading up to dodecamers. Four rate constants, applying to forward or reverse steps in the aggregation process, were included. Optimal values for these constants were determined using the ellipsoid algorithm as implemented by one of us (Ecker, J.G. & Kupferschmid, M., 1988, Introduction to Operations Research, Wiley, New York, pp. 315-322). Intensive exploration of simpler aggregation models did not identify an alternative that could simulate the data as well as this one. The activity of the chaperonin in this system was simulated by using this aggregation model, combined with a model similar to that proposed by Goloubinoff et al. (1989). The model assumes that the chaperonin can bind the folding intermediate rapidly, and that the chaperonin complex releases the Rubisco molecule slowly, permitting time for its spontaneous folding while interacting with the chaperonin. This is followed by self-association of the folded Rubisco monomer to yield the active dimeric Rubisco. A modification of the model that simulates temperature effects was also constructed. The most important results we obtained indicate that the chaperonin-dependent reconstitution of Rubisco can be simulated adequately without invoking any catalysis of folding by the chaperonin. In addition, the simulations predict values for the association rate constant of Rubisco-I with the chaperonin, and other variables, that are subject to experimental verification.     

Tumour invasion: effects of cell adhesion and motility

Metastasis is the major cause of failure in cancer therapy. Recent studies of the molecular cell biology of the metastatic process have provided new insights into the mechanisms of cell-cell adhesion, cell-substrate adhesion and cell motility that underly invasion by tumour cells. In this review, Van Roy and Mareel discuss the role of proteins with invasion-promoting and invasion-suppressing functions in metastasis.     

Sugars fermented byBifidobacterium infantis ATCC 27920 in relation to growth and α-galactosidase activity

Summary The ability of Bifidobacterium infantis ATCC 27 920 to ferment glucose, galactose, lactose, melibiose and raffinose was investigated with respect to -galactosidase (-d-galactoside galactohydrolase, E.C. 3.2.1.22). The sugars were tested at three concentrations: 0.5, 1.0 and 2.0%. The growth of B. infantis was slower on glucose compared with the other sugars. The highest specific growth rate was observed on melibiose followed by lactose. High cell numbers could be rapidly obtained on galactose-containing sugars. For each carbohydrate, enzyme activity was maximal at the end of the exponential phase and the highest specific -galactosidase activities were recorded on the two -1,6 galactosaccharides (melibiose and raffinose: 3.0 and 4.5 nkat · 10⁹ colony-forming units, respectively).Contribution no. 186 from the Food Research and Development Centre Offprint requests to: D. Roy