The subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase

Incubation of subcellular fractions isolated from rat cerebral cortex with [-³²P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes.A preliminary account of part of this work has been published (1).     

Effect of bovine somatotropin on reproductive function in lactating dairy cows

Several recent experiments have reported that chronic treatment with bovine somatotropin (bST) increased the number of days open without affecting the services per conception. The physiological basis for these effects was examined. Eleven lactating Holstein cows received daily injections of bST (40 mg) and 10 received daily injections of vehicle. Treatment was initiated between 32 and 85 d post partum and continued for up to 180 d. Eight of 11 bST-treated cows experienced at least one period of extended ovarian acyclicity during treatment. Only 3 of 10 control cows did so (P = 0.05). Concentrations of progesterone during luteal phases were lower in bST-treated cows than in controls (P = 0.06). Baseline concentrations of LH were suppressed in bST-treated cows compared with those of controls (P < 0.04). Neither the pulse frequency of LH nor the expression of estrous behavior was affected by bST (P > 0.30). These results indicate that chronic administration of a high dose of bST can reduce reproduction performance by promoting ovarian acyclicity.     

IV. A model system for mass spectrometric sequencing of orthophthalaldehyde peptide derivatives

Presented is a pilot project describing a new strategy for mass spectrometric peptide sequencing. Using orthophthalaldehyde (OPA), two peptides were derivatized and their fluorescent adducts isolated using reversed phase high performance liquid chromatography. The OPA-peptides were permethylated and the derivatized molecules subjected to direct probe mass spectral analysis. The spectra obtained from the OPA-peptides was analogous to those observed for standard $\text{N}$-acyl permethyl derivatives enabling the complete sequence of the peptides to be determined.     

A mechanism for maintaining an up-to-date GenBank(R)database via Usenet

In this paper, we describe an automated system for distributingupdates to the GenBank nucleic acid sequence database, usingthe Usenet news system as the underlying transport mechanism.Our system allows new loci to be distributed as soon as thesequences are available, over existing networks, using existingUsenet software and infrastructure currently available on awide range of computer systems.     

SARS-CoV-2-specific T cells associate with inflammation and reduced lung function in pulmonary post-acute sequalae of SARS-CoV-2

As of January 2022, at least 60 million individuals are estimated to develop post-acute sequelae of SARS-CoV-2 (PASC) after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While elevated levels of SARS-CoV-2-specific T cells have been observed in non-specific PASC, little is known about their impact on pulmonary function which is compromised in the majority of these individuals. This study compares frequencies of SARS-CoV-2-specific T cells and inflammatory markers with lung function in participants with pulmonary PASC and resolved COVID-19 (RC). Compared to RC, participants with respiratory PASC had between 6- and 105-fold higher frequencies of IFN-γ- and TNF-α-producing SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells in peripheral blood, and elevated levels of plasma CRP and IL-6. Importantly, in PASC participants the frequency of TNF-α-producing SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells, which exhibited the highest levels of Ki67 indicating they were activity dividing, correlated positively with plasma IL-6 and negatively with measures of lung function, including forced expiratory volume in one second (FEV¹), while increased frequencies of IFN-γ-producing SARS-CoV-2-specific T cells associated with prolonged dyspnea. Statistical analyses stratified by age, number of comorbidities and hospitalization status demonstrated that none of these factors affect differences in the frequency of SARS-CoV-2 T cells and plasma IL-6 levels measured between PASC and RC cohorts. Taken together, these findings demonstrate elevated frequencies of SARS-CoV-2-specific T cells in individuals with pulmonary PASC are associated with increased systemic inflammation and decreased lung function, suggesting that SARS-CoV-2-specific T cells contribute to lingering pulmonary symptoms. These findings also provide mechanistic insight on the pathophysiology of PASC that can inform development of potential treatments to reduce symptom burden.