Identification of the substrate and pseudosubstrate binding sites of phosphorylase kinase gamma-subunit

Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).     

Suppression and the upward spread of masking

The purpose of this study is to clarify the role of suppression in the growth of masking when a signal is well above the masker in frequency (upward spread of masking). Classical psychophysical models assume that masking is primarily due to the spread of masker excitation, and that the nonlinear upward spread of masking reflects a differential growth in excitation between the masker and the signal at the signal frequency. In contrast, recent physiological studies have indicated that upward spread of masking in the auditory nerve is due to the increasing effect of suppression with increasing masker level. This study compares thresholds for signals between 2.4 and 5.6 kHz in simultaneous and nonsimultaneous masking for conditions in which the masker is either at or well below the signal frequency. Maximum differences between simultaneous and nonsimultaneous masking were small (< 6 dB) for the on-frequency conditions but larger for the off-frequency conditions (15-32 dB). The results suggest that suppression plays a major role in determining thresholds at high masker levels, when the masker is well below the signal in frequency. This is consistent with the conclusions of physiological studies. However, for signal levels higher than about 40 dB SPL, the growth of masking for signals above the masker frequency is nonlinear even in the nonsimultaneous-masking conditions, where suppression is not expected. This is consistent with an explanation based on the compressive response of the basilar membrane, and confirms that suppression is not necessary for nonlinear upward spread of masking.     

A bipartite membrane-binding signal in the human immunodeficiency virus type 1 matrix protein is required for the proteolytic processing of Gag precursors in a cell type-dependent manner

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4(+) T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4(+) T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.     

The flavonoid constituents of two Polypodium species (Calaguala) and their effect on the elastase release in human neutrophils

Five flavonoid compounds were isolated from two Polypodium species (P.decumanum and P.triseriale) with the common name Calaguala. Structure elucidation was carried out using different NMR techniques and revealed the presence of one new glycoside (kaempferol 3-O-beta-D-xylopyranosyl-(1-2)-beta-D-arabinopyranoside) (1), two known flavonoid glycosides, rutin and kaempferol 3-O-alpha-D-arabinopyranoside (2,3), the trimeric proanthocyanidin, selligueain (4), and the coumarinic acid derivative, melilotoside (5). The compounds were tested for their activity in PAF induced exocytosis in human neutrophils but none of the compounds showed PAF specific activity. Instead, they showed more general effects on the neutrophil including inhibition of the spontaneous elastase release (5) and potentiation of the release induced by PAF (1). Selligueain was found to inhibit the proteolytic enzyme, elastase in vitro.     

Reduced retrograde labeling of diencephalic-projecting neurons in the gracile nucleus of the monkey following removal of dorsal column input

As part of an anatomical investigation of neuronal responses to deafferentation of the dorsal column nuclei by transection of the dorsal spinal columns, the uptake and retrograde transport of HRP by thalamic projection cells in the dorsal column nuclei was studied. The ventrobasal thalamus of 13 macaque monkeys was injected bilaterally with HRP at periods ranging from 3 to 364 days following intended unilateral transection of fasciculus gracilis at a mid- to upper thoracic level. The density of labeled cells in the gracile nuclei ipsilateral to complete lesions of fasciculus gracilis was compared with the density of labeled cells in the contralateral gracile nuclei that were fully innervated or partially denervated by an incomplete lesion. Also, the density of labeled cells in the fully innervated cuneate nuclei was compared. In general, there was a reduction in density of labeled cells in the gracile nuclei ipsilateral to complete lesions, without a corresponding decrement in labeled cells in the cuneate nuclei on that side. This result confirms effects on spinal motoneurons and on thalamocortical projection cells in the lateral geniculate nucleus following deafferentation. However, attempts to define a time course for the reduction in transport by lemniscal projection cells revealed an effect that was dramatic in some animals, partial in others, and not demonstrable in the remainder, without a clear relationship to time after surgery. This result is related to a literature which describes a variety of morphological, biochemical, electrophysiological, and behavioral effects of deafferentation which appear to wax and wane with time after neuronal injury.     

The identification of Mycoplasma mycoides mycoides LC as the aetiological agent of balanoposthitis and vulvovaginitis in sheep in South Africa

Clinical ulcerative balanoposthitis and vulvovaginitis was experimentally reproduced in 14 sheep infected with a Mycoplasma mycoides mycoides LC field strain, isolated from the Straussheim Dorper stud. The study encompassed a series of field observations, a therapeutic trial and experimental investigations. A wide range of bacteria and various mycoplasma spp., but no viruses, were isolated from a large number of infected animals.     

Handball injuries. An epidemiological and socioeconomic study

A total of 570 injuries in handball players were prospectively registered in a well-defined geographical area of 124,321 inhabitants. The incidence of handball injuries was 46/10,000 inhabitants/year and in females (61/10,000/year) double that for males (31/10,000/year). Sixty-two percent of the injuries were distortions and sprains and 12% were fractures. A total of 7% were hospitalised and the loss of income because of sick leave was in total $3870. Sixty-eight percent of the injured handball players did not play for more than one week. Surprisingly, 8% of the minor injuries resulted in a risk leave of more than six days. Five percent of these injured quit a tournament of training after sustaining a handball injury.